U.S. patent application number 10/858869 was filed with the patent office on 2005-03-31 for jab1 as a prognostic marker and a therapeutic target for human cancer.
Invention is credited to Claret, Francois.
Application Number | 20050069918 10/858869 |
Document ID | / |
Family ID | 34958587 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050069918 |
Kind Code |
A1 |
Claret, Francois |
March 31, 2005 |
JAB1 as a prognostic marker and a therapeutic target for human
cancer
Abstract
Methods of diagnosing and prognosticating the development of
human cancers, such as breast cancer, colon cancer, and pancreatic
cancer, are provided. The diagnostic and prognostic methods include
the detection and/or quantifying of the amount of expression of
JAB1 in human cells, particularly in relation to the amount of p27
or c-Jun. In addition, methods for reducing the expression of JAB1
protein in cells and inhibiting its interaction with p27 or c-Jun,
for example, are provided.
Inventors: |
Claret, Francois; (Houston,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
34958587 |
Appl. No.: |
10/858869 |
Filed: |
June 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60474048 |
May 29, 2003 |
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/7.23 |
Current CPC
Class: |
C12Q 2600/112 20130101;
C12Q 1/6886 20130101; G01N 33/57415 20130101; G01N 33/57434
20130101; G01N 33/57449 20130101; G01N 2333/4704 20130101; C12Q
2600/118 20130101; G01N 33/57438 20130101; G01N 33/57419 20130101;
G01N 33/57426 20130101; G01N 33/57496 20130101; C12Q 2600/136
20130101; G01N 33/57423 20130101; G01N 2500/02 20130101; C07K 14/47
20130101; C12Q 2600/106 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Goverment Interests
[0002] This invention was made with U.S. Government support from
the National Cancer Institute/National Institutes of Health Grant
number 1RO1CA90853-01A1. The U.S. Government may have certain
rights in this invention.
Claims
What is claimed is:
1. A method of diagnosing, or determining a prognosis for, cancer
in an individual, the method comprising assessing the JAB1 level in
one or more cancer cells, or cells suspected of being cancerous, of
the individual, wherein the cancer is breast cancer, non-Hodgkins
lymphoma, colon cancer, prostate cancer, pancreatic cancer, or lung
cancer.
2. The method of claim 1, wherein the one or more cells is obtained
from a sample from the individual.
3. The method of claim 1, wherein the assessing step comprises
comparing the JAB1 level the cell to the JAB1 level in a
non-cancerous sample.
4. The method of claim 3, wherein the non-cancerous sample is
obtained from the individual.
5. The method of claim 3, wherein when the JAB1 expression level is
higher in the cell compared to the JAB1 expression level in the
non-cancerous sample, the cell is cancerous.
6. The method of claim 1, wherein the assessing step is further
defined as assessing the ratio of JAB1/p27 level in the cell.
7. The method of claim 6, wherein when the ratio of JAB1/p27
comprises high JAB1 expression and low p27 expression, the cell is
cancerous.
8. The method of claim 7, wherein when the ratio of JAB1/p27
expression is greater than 1, the cell is cancerous.
9. The method of claim 6, wherein the assessing step is further
defined as assessing the ratio of JAB1 polypeptide/p27
polypeptide.
10. The method of claim 6, wherein the assessing step is further
defined as assessing the ratio of JAB1 polynucleotide/p27
polynucleotide.
11. The method of claim 1, wherein the assessing step provides
information of the stage of the cancer.
12. The method of claim 11, wherein when the JAB1 level is greater
in the cell compared to the JAB1 level in a non-cancerous sample,
the cell is an advanced stage cancer cell.
13. The method of claim 11, wherein the assessing step is further
defined as determining the subcellular localization of JAB1, p27,
or both.
14. The method of claim 13, further defined as identifying the
ratio of JAB1 expression in the nucleus compared to the
cytoplasm.
15. The method of claim 6, wherein the assessing step provides
information of the stage of the cancer.
16. The method of claim 15, wherein when the ratio of JAB1/p27
comprises high JAB1 expression and low p27 expression, the sample
comprises advanced stage cancer.
17. A method of treating cancer, comprising: diagnosing or
determining prognosis for a cancer in accordance with any one of
claims 1-17; determining an appropriate therapy based on said
diagnosis or determination of prognosis; and treating a patient
with the therapy.
18. The method of claim 17, wherein the treating step comprises
delivering a JAB1-inhibiting agent to the individual.
19. The method of claim 18, wherein the JAB1-inhibiting agent is
delivered directly to one or more cancerous cells of the
individual.
20. The method of claim 18, wherein the JAB1-inhibiting agent
comprises a polynucleotide, a polypeptide, a peptide, a small
molecule, or a mixture thereof.
21. The method of claim 20, wherein the JAB1-inhibiting agent
comprises a polynucleotide.
22. The method of claim 21, wherein the polynucleotide comprises
antisense JAB1 sequence.
23. The method of claim 22, wherein the antisense JAB1
polynucleotide is further defined as being comprised on a vector,
said polynucleotide being operably linked to a promoter suitable
for regulation of the polynucleotide in said cancer cell.
24. The method of claim 23, wherein the vector is a viral vector or
a non-viral vector.
25. The method of claim 24, wherein the viral vector comprises an
adenoviral vector, a retroviral vector, an adeno-associated viral
vector, a lentiviral vector, or a herpesviral vector.
26. A method for identifying the stage of a cancer in an
individual, comprising: assessing the ratio of JAB1/p27 level in a
cancer cell from the individual; and determining the stage of the
cancer based on said assessment.
27. The method of claim 26, wherein when the ratio of JAB1/p27 is
high, the stage of the cancer is late stage.
28. A method for monitoring treatment of a cancer for an
individual, said cancer characterized by a high JAB1/p27 ratio,
comprising: determining the JAB1/p27 ratio in at least one
cancerous cell from a tissue from the individual prior to the
treatment; administering the treatment to the individual; and
determining the JAB1/p27 ratio in a cell from the tissue from the
individual subsequent to the treatment.
29. The method of claim 28, wherein the treatment comprises
delivering a JAB1-inhibiting agent to the individual.
30. The method of claim 28, wherein when the determination of the
JAB1/p27 ratio in a cell from the individual subsequent to the
treatment is lower than the JAB1/p27 ratio in the cancerous cell
from the individual prior to the treatment, said treatment is
efficacious for the cancer.
31. A method of treating cancer in an individual, comprising
delivering a JAB1-inhibiting agent to the individual.
32. The method of claim 31, wherein the cancer is further defined
as comprising a high JAB1/p27 level.
33. The method of claim 31, wherein the JAB1-inhibiting agent is
delivered directly to the cancer.
34. The method of claim 31, wherein the delivery comprises
intratumoral injection, microinjection, electroporation, liposomal
delivery, by catheter, or a combination thereof.
35. The method of claim 31, wherein the JAB1-inhibiting agent
comprises a polynucleotide, a polypeptide, a peptide, a small
molecule, or a mixture thereof.
36. The method of claim 31, wherein the JAB1-inhibiting agent
comprises a polynucleotide.
37. The method of claim 36, wherein the polynucleotide comprises
antisense JAB1 sequence.
38. The method of claim 37, wherein the antisense JAB1
polynucleotide is further defined as being comprised on a vector,
said polynucleotide being operably linked to a promoter suitable
for regulation of the polynucleotide in a cancerous cell.
39. The method of claim 38, wherein the vector is a viral vector or
a non-viral vector.
40. The method of claim 39, wherein the viral vector comprises an
adenoviral vector, a retroviral vector, an adeno-associated viral
vector, a lentiviral vector, or a herpesviral vector.
41. A method of screening for a JAB1-inhibiting agent, comprising:
providing a JAB1 polypeptide; providing a p27 polypeptide; and
providing a test compound, wherein when the test compound inhibits
binding of the JAB1 polypeptide to the p27 polypeptide, said test
compound is said JAB1-inhibiting agent.
42. The method of claim 41, wherein binding of the JAB1 polypeptide
to the p27 polypeptide is detected by color, fluorescence,
radioactivity, or particle emission.
43. The method of claim 41, further comprising the step of
delivering the JAB1-inhibiting agent to an individual having
cancer.
44. A kit, housed in a suitable container, comprising a
JAB1-inhibiting agent identified by the method of claim 41.
45. A composition comprising a JAB1-inhibiting agent.
46. The composition of claim 45, wherein the agent is a
polynucleotide, polypeptide, peptide, small molecule, or a mixture
thereof.
47. The composition of claim 46, wherein the polynucleotide
comprises antisense JAB1 sequence.
48. The composition of claim 46, wherein the peptide comprises a
p27-binding domain on JAB1.
49. The composition of claim 46, wherein the peptide comprises a
c-Jun-binding domain on JAB1.
50. The composition of claim 45, comprised in a pharmaceutically
acceptable diluent.
51. A method of treating cancer in an individual, comprising
delivering to the individual a viral vector comprising JAB1
siRNA.
52. The method of claim 51, wherein the viral vector comprises an
adenoviral vector, a retroviral vector, a lentiviral vector, a
herpesviral vector, or an adeno-associated viral vector.
53. A composition comprising a viral vector including antisense
JAB1 sequence.
54. The composition of claim 53, wherein the antisense JAB1
sequence comprises JAB1 siRNA.
55. The composition of claim 53, wherein the viral vector comprises
an adenoviral vector, a retroviral vector, a lentiviral vector, a
herpesviral vector, or an adeno-associated viral vector.
56. The composition of claim 55, wherein the viral vector is an
adenoviral vector.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application 60/474,048 filed May 29, 2003, which is incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to methods of
diagnosing, prognosticating and treating human cancers, as well as
assaying for therapeutic agents for treating human cancers. More
specifically, the invention regards JUN activation binding protein
1 (JAB1)-associated embodiments for cancer diagnosis and therapy,
such as breast cancer.
BACKGROUND OF THE INVENTION
[0004] Cancer can be caused by a wide variety of genetic
abnormalities, such as hereditary or non-hereditary mutations.
However, many of the known genetic causes of cancer are caused by
mutations in or overexpression of genes that belong to a class
encoding proteins having similar functional characteristics. For
example, cell cycle regulatory genes are a class of genes that have
been found to be mutated or differentially expressed in some
cancers.
[0005] Cell cycle regulatory genes include genes that encode for
cyclins and cyclin-dependent kinases (CDKs). Different cyclins are
expressed and degraded at different stages of the eukaryotic cell
cycle. Cyclins are positive cell cycle regulators, as they bind and
activate CDKs, which contribute to the progression of the cell
cycle from one stage of the cell cycle to the next stage of the
cell cycle. CDK inhibitors are negative cell cycle regulators that
bind cyclin-CDK complexes and inhibit the activity of CDKs in the
cyclin-CDK complexes.
[0006] One example of a CDK inhibitor gene is the p27 gene, which
encodes p27 protein, also known as p27.sup.Kip1. The p27 protein
inhibits the activity of CDKs that are involved in the G1 to S
phase transition of the cell cycle, contributing to G1 arrest of
the cell cycle and prevention of unregulated, abnormal cell
division.
[0007] While mutations in the p27 gene have not been found to date
in human tumors, the level of p27 protein in many human tumors has
been found to be below the level of p27 protein typically found in
healthy human tissue. As the level of p27 protein in cells is
regulated primarily at the post-transcriptional level, it is
believed that factors that contribute to the degradation of p27
protein play a key role in regulating the level of p27 protein in
cells.
[0008] HER-2 protein is a protein that is believed to be involved
in the degradation of p27 protein. An inverse relationship between
the amount of HER-2 protein and the amount of p27 protein has been
found in primary breast tumor samples. Overexpression of HER-2
protein in a breast cancer cell line resulted in reduced levels of
p27 protein in the cell line (Yang, et al., Journal of Biological
Chemistry 275:24735-24739 (2000)).
[0009] Methods of using the HER-2 gene or protein in breast cancer
diagnostics and therapeutic agents for breast cancer have been
proposed (see, e.g., U.S. Pat. No. 6,251,601 and Herceptin.RTM.
antibody, available from Genentech, San Francisco, Calif.).
However, overexpression of HER-2 protein via gene amplification of
the HER-2 gene has been found to date in only approximately 25% of
breast cancer patients. Furthermore, one study has shown that less
than half of the HER-2 overexpressing breast cancer patients in the
study responded to HER-2 antibody-based treatment (Vogel, et al.,
Journal of Clinical Oncology 20: 719-726 (2002); Baselga J et al.,
Seminars in Oncology, Vol 26(4): Suppl. 12 pp 78-83, 1999; Slamon
D. J., et al., The New England Journal of Medicine, Vol 344
pp783-792, 2001; Vogel C. L et al, Journal of Clinical Oncology,
Vol 20, pp 719-726, 2002.).
[0010] In vitro studies of JAB1 protein (also referred to as CSN5
or p38.sup.JAB1) demonstrate that JAB1 protein contributes to the
degradation of p27 protein, as overexpression of JAB1 protein in
cultured cell lines resulted in decreased levels of p27 protein in
the cell lines (see, Tomoda, et al., Nature 398:160-165 (1999), and
Tomoda, et al., Journal of Biological Chemistry 277:2302-2310
(2002)).
[0011] To date, JAB1 protein has been studied in several types of
human cancer (see, Tsuchida, et al., Jpn J Cancer Res. 93:1000-6
(2002), and Shen, et al., International Journal of Oncology
17:749-754 (2000)). In one study, it was found that the amount of
JAB1 protein was inversely related to the amount of p27 protein in
tumors of ovarian carcinomas; however, no correlation was found
between JAB1 protein overexpression and the histological
characteristics of the tumors, such as the stage of the patients'
cancer and the grade of the tumors (Sui, et al., Clinical Cancer
Research 7:4130-4135 (2001)). Shen et al. (2000) describe high
levels of p27 in neuroblastomas, particularly in differentiated
tumors. Localization of subcellular JAB1 expression was determined
to be in both the nucleus and cytoplasm of undifferentiated and
differentiating tumors, whereas predominantly nuclear localization
was identified in differentiated tumor cells.
[0012] In another study, no difference was found between JAB1
protein levels in human pituitary tumors, such as corticotroph
tumors or other pituitary adenomas, compared to normal pituitary
tissue; however, a small but significant increase in JAB1 protein
was detected in pituitary carcinomas compared to normal pituitary
tissue (Korbonits, et al., Journal of Clinical Endocrinology and
Metabolism 87:2635-2649 (2002)). The same study also examined p27
protein levels in human pituitary tumors. Low p27 protein levels
were found in corticotroph adenomas and pituitary carcinomas. The
study concluded that the low p27 protein levels found in
corticotroph adenomas were not caused by JAB1 overexpression, as
JAB1 overexpression was not found in corticotroph adenomas. Thus,
it appears that JAB1 protein levels are elevated in some tumors in
certain tissues, but are not elevated in tumors in other tissues,
and that low p27 protein levels in cancer cells are not always
correlated with a high level of JAB1 protein expression.
[0013] Previous studies have found that low p27 protein levels in
breast tumors are often correlated with a poor prognosis and
survival rate (Catzavelos, et al. (1997) Nat. Med. 3:227-230,
Porter, et al. (1997) Nat. Med. 3:222-225). However, the
investigations described herein indicate that JAB1 protein
expression is frequently found in breast tumor cells and that JAB1
protein expression is prognostic of a lower survival rate and a
lower progression-free survival rate. As discussed above,
embodiments described herein also indicate that JAB1 protein levels
and p27 protein levels are often inversely related in breast
carcinomas and in T-cell lymphoma.
[0014] EP0856582 describes an inhibitor of the transcription factor
activator protein-1 and a DNA encoding same. In particular
embodiments, the inhibitor is the exportin protein.
[0015] United States Patent Application Publications US
20020156012; US 20030166243; and US 20030153097 relate to the
peptidase activity of the JAB subunit or JAM domain. Compositions
comprising the JAM domain are disclosed therein, and in some
embodiments they are utilized to screen for agents that affect the
peptidase activity. These agents may be further utilized for
rational drug design. In specific embodiments, a screen entails in
part the contact of a target protein to a JAB subunit, and there is
also provided amelioration of a pathologic condition by modulating
the activity and agents directed thereto.
[0016] United States Patent Application Publication US 20030148954
describes agents for modulating AP-1 mediated gene expression, such
as those comprising an internalization moiety and a peptide from
the intracellular domain of Notch-1 or an analog or peptidomimetic
thereof.
[0017] There remains a need for biological markers that can be used
as a basis for diagnosing and prognosticating different types of
cancer, as well as a need for therapeutic agents for treating such
cancers.
BRIEF SUMMARY OF THE INVENTION
[0018] The human JAB1 (Jun activation domain-binding protein 1)
protein was originally identified as a binder of the c-Jun
activation domain. JAB1 protein selectively binds and activates
several members of the Jun family of proteins. Jun proteins
heterodimerizeu with Fos proteins to form the transcription factor
AP-1, the activity of which is believed to play a role in cellular
transformation and invasion in cancer.
[0019] As noted above, JAB1, aside from being an AP-1-coactivator,
is involved in degradation of the cyclin-dependent kinase inhibitor
p27. The present inventors examined JAB1 and p27 protein expression
in the exemplary invasive breast carcinomas, colon cancer, and
pancreatic cancer, for example, and identified the association of
this expression with clinical outcome. JAB1 was detected
immunohistochemically in the vast majority of tumors, with breast
carcinomas showing high JAB1 expression and reduced or absent p27
levels. Tumors with high p27 expression were rarely positive for
JAB1. Furthermore, all tested patients with JAB1-negative tumors
had no evidence of relapse or disease progression at a median
follow-up of 70 months. Immunoblotting showed strong JAB1
expression in breast carcinoma samples but not in paired normal
breast epithelial samples, and JAB1 upregulation paralleled
HER2/neu overexpression. Targeted overexpression of JAB1 by
regulated adenovirus in breast cancer cell lines also reduced p27
levels by accelerating its degradation. Thus, the JAB1/p27 ratio is
a novel indicator of aggressive, high-grade tumor behavior, and
control of JAB1 provides a novel target for cancer therapy.
[0020] Thus, aspects of the invention provide methods of detecting
the expression of JAB1 protein and methods of altering the
expression of JAB1 protein in human cells. Aspects of the invention
also provide compositions that express JAB1 protein in cells or
alter the endogenous expression of JAB1 protein in cells. In
addition, aspects of the invention include methods for screening
for agents that alter JAB1 expression.
[0021] In one particular aspect of the invention, the ratio of JAB1
to another gene product is utilized for diagnosis, prognosis, or
therapeutic applications for cancer. The other gene product may be
of any kind such that in conjunction with the assessment of JAB1
expression it can be indicative of particular presence of disease
and/or particular stage of disease. In specific embodiments, the
other gene product is one in which JAB1 indirectly or directly is
associated with, such as, for example, in a complex with JAB1. In
particular embodiments, the other gene product binds to JAB1, such
as directly to JAB1. In a specific embodiment, the other gene
product is considered a JAB1 target. Specific examples of other
gene products include p27, c-Jun, p53, cyclin D1, or any gene
product involved in the COP9 signalosome. In particular aspects of
the invention, the binding of JAB1 to p27 is involved with the
disparity of their expression levels, given that JAB1 causes the
translocation of p27 from the nucleus to the cytoplasm, decreasing
the amount of p27.sup.Kip1 in the cell by accelerating its
degradation (Tomoda et al., 1999). In additional aspects, JAB1 is
also associated with subcellular transport of other targets.
[0022] Thus, as JAB1 protein is involved in the degradation of p27
protein, and p27 protein activity is important in protecting a cell
from becoming cancerous, JAB1 protein expression levels and/or the
ratio of JAB1 expression levels to p27 expression levels are useful
for detecting certain types of cancer and for prognosticating the
progression of certain cancers. In addition, altering the
expression of JAB1 is useful in treating certain cancers.
[0023] In a specific aspect, the ratio of JAB1 to p27, which is
provided for exemplary purposes given the variety of gene products
that may be utilized in association with JAB1, is determined. The
levels of JAB1 and p27 may be determined based on their respective
chromosome amplification, RNA levels or polypeptide levels, for
example. In particular embodiments, the expression levels of JAB1
and p27 are determined as polypeptide levels, and the respective
levels are considered in association with one another, such as in
the form of a ratio JAB1/p27. In particular aspects of the
invention, a ratio comprising high levels of JAB1 to low levels of
p27 is indicative of cancer, and in particular embodiments it is
indicative of particular stages of cancer. In other embodiments,
the ratio provides information regarding a predisposition to
developing cancer and/or a predisposition to developing aggressive
cancer, which may be considered metastatic or invasive cancer.
[0024] In specific embodiments, the term "high" refers to
expression levels that are detectably higher (increased) in cancer
tissue or tissue suspected to be cancerous compared to normal
tissue, such as adjacent normal tissue (paired tissue). The term
"low" refers to expression levels that are detectably lower in
cancer tissue or tissue suspected to be cancerous compared to
normal tissue, such as adjacent normal tissue (paired tissue).
These terms may refer to JAB1 expression alone, p27 alone, or to
the ratio of JAB1/p27, for example. The assessment of levels may be
qualitative or quantitative. That is, an unknown sample compared to
a known standard may be assessed as having positive or negative
expression in relation to one another. Alternatively, a percentage
of positive cells may be represented quantitatively, wherein, for
example, numbers of cells are counted and scored for expression
level. In a specific embodiment, the tissue or cells for comparison
which are considered normal (non-cancerous) are obtained from or
are derived from the individual for which the cancerous tissue is
obtained.
[0025] In accordance with known statistical assessments (Kouvaraki
et al., 2003), a labeling index may be employed that provides a
continuous variable. In specific embodiments, the labeling index
for JAB1 and p27 is utilized as being indicative of expression
states. For example, a labeling index may be considered high if it
is greater than 50%, and the labeling index may be considered low
if it is lower than 50%. In other specific embodiments, expression
of JAB1 being higher than that of p27 for a particular tissue is
indicative of cancer and/or indicative of a particular stage of
cancer, such as late stage. Thus, in specific aspects of the
invention, the JAB1/p27 ratio is utilized as a marker for cancer.
In other embodiments, the ratio of JAB1 expression in the nucleus
compared to the JAB1 expression in the cytoplasm of a cell is
determined. When the ratio is high (greater than about 1), the
presence of cancer is identified.
[0026] In particular embodiments, JAB1 being at high levels and p27
being at low levels corresponds to a detection of cancer in at
least one cell or tissue. In further embodiments, a high JAB1/p27
ratio corresponds to detecting particular stages of cancer. For
example, a high JAB1/p27 ratio may be indicative of late stages of
cancer, which may be further defined as aggressive cancer, invasive
cancer, or metastatic cancer. For example, in the exemplary form of
cancer being breast cancer, a high JAB1/p27 ratio may be indicative
of stage II, stage III, or stage IV cancer, for example. The
JAB1/p27 ratio may correlate with progression of cancer, such as
there being a low ratio for normal or benign tissue, and there
being a higher ratio for hyperplasias, increasing to the highest
ratios for metastatic, invasive cancer. In further specific
embodiments, the JAB1/p27 ratio is utilized for determining a
cancer treatment regimen.
[0027] In other embodiments, there is a correlation between JAB1
localization in the nucleus with stage progression, such as the
exemplary cancer being breast cancer. In specific embodiments,
Stage I may be represented by a ratio of JAB1 nuclear vs.
cytoplasmic staining at about 3.0; Stage II may be represented by a
ratio of JAB1 nuclear vs. cytoplasmic staining at about 4.5; and
Stage III may be represented by a ratio of JAB1 nuclear vs.
cytoplasmic staining at about 5. Thus, by determining JAB1 levels
and its subcellular localization (nuclear versus cytoplasmic, for
example), there is an indicator of tumor progression.
[0028] In other aspects of the invention, the level and/or
localization of expression of JAB1, including high nuclear
localization levels, and/or the JAB1/p27 ratio is utilized as a
means to monitor therapy for cancer associated with high levels of
JAB1, including high nuclear localization levels, or high levels of
the JAB1/p27 ratio, respectively. That is, the level or
localization of expression or ratio may be determined prior to
therapy, such as upon or following diagnosis, and this level is
monitored during therapy to observe the efficacy of the treatment.
If the level of JAB1 expression or JAB1/p27 ratio continues to be
high following treatment, an alternative treatment may be
employed.
[0029] In additional aspects of the invention, blocking the
interaction between JAB1 and p27 provides a mechanism for therapy
of cancer. In particular embodiments, inhibiting this interaction
facilitates arrest of cells and stops tumor growth. An agent to
block the interaction may be of any kind, such as, for example, a
polynucleotide, such as an RNA, a polypeptide, a peptide, a small
molecule, and so forth. For example, the agent may be an inhibitor
or ligand that binds to a pocket on JAB1 that is responsible at
least in part for binding to its respective target, such as
p27.
[0030] RNA agents include antisense RNA, such as RNAi or siRNA, for
example. Peptide inhibitors may be of any kind, but in specific
embodiments, they comprise at least part of a binding region
between JAB1 and a gene product to which it binds, such as p27. For
example, the peptide inhibitor may comprise at least part of the
JAB1 sequence that binds p27, or the peptide inhibitor may comprise
at least part of the p27 sequence that binds JAB1. In particular
embodiments, a peptide inhibitor or any polypeptide or peptide
further comprises a protein transduction domain, such as, for
example, HIV Tat (Schwarze et al., 1999) or synthetic derivatives
thereof (Ho et al., 2001).
[0031] Screens for agents that inhibit the binding of JAB1 to its
target, such as p27, may be of any suitable kind. Multiple screens
may be used in succession to narrow a pool of candidate inhibitors.
In specific aspects of the invention, an in vitro screen may
include ELISA, such as to monitor by dye visualization the absence
of binding of p27 to JAB1 in the presence of a potential inhibitor.
An example of an in vivo screen is a cell-based assay, in which
binding of JAB1 to p27 in the presence of potential inhibitors is
visualized from within the cell, such as by fluorescence or X-ray.
Another screen utilizes the p27 binding domain of JAB1 and/or the
JAB1 binding domain of p27, for example, immobilized to a substrate
such that when a potential inhibitor binds the immobilized domain,
the binding is visualized, such as by presence or absence of color,
fluorescence, or radioactivity, for example. Finally, another
screen that may be utilized is a two-hybrid screen wherein the JAB1
binding domain of p27 or the p27 binding domain of JAB1 are used as
bait to identify peptides or polypeptides that bind at least in
part thereto.
[0032] In a particular aspect of the invention, there is a method
of diagnosing cancer or determining a prognosis for cancer in an
individual, comprising assessing the JAB1 level in a cell of the
individual. In a particular aspect of the invention, there is a
method of diagnosing cancer or determining a prognosis for cancer
in an individual, comprising obtaining a sample from the
individual; assessing the JAB1 level in the sample; and diagnosing
or prognosticating cancer based on the assessment, wherein the
cancer is breast cancer, non-Hodgkins lymphoma, Hodgkins lymphoma,
colon cancer, prostate cancer, pancreatic cancer, or lung cancer.
The breast cancer may be ductal carcinoma or lobular carcinoma.
[0033] In a specific embodiment, the cancer is HER2 positive. The
assessing step may comprise comparing the JAB1 level in the sample
from the individual to the JAB1 level in a non-cancerous sample,
such as a non-cancerous standard, including a known standard. In
specific embodiments, the non-cancerous sample may be obtained from
the individual, such as from a distant region of the same tissue or
from adjacent normal tissue, for example. In particular aspects of
the invention, when the JAB1 expression level is higher in the
sample from the individual compared to the JAB1 expression level in
the non-cancerous sample, the sample from the individual comprises
at least one cancerous cell, and the diagnosis may thus be
considered positive for cancer.
[0034] In a particular aspect of the invention, the assessing step
may be further defined as assessing the ratio of JAB1/p27 level in
a sample from an individual. When the ratio of JAB1/p27 comprises
high JAB1 expression and low p27 expression, the sample may be
considered to comprise at least one cancerous cell. This may be
further defined as when the ratio of JAB1/p27 expression is greater
than 1, the sample comprises at least one cancerous cell. This may
be further defined as when there is high JAB1 expression and low
p27 expression compared to that identified in normal cells,
preferably from the same or analogous tissue, the sample comprises
at least one cancerous cell.
[0035] In a particular aspect of the invention, the assessing step
may be further defined as assessing the subcellular localization of
JAB1 in a sample from an individual, such as the nuclear
localization. When the ratio of nuclearly localized JAB1 compared
to cytoplasmically localized JAB1 is high, the sample may be
considered to comprise at least one cancerous cell. This may be
further defined as when the ratio of nuclearly localized JAB1 to
cytoplasmically localized JAB1 is greater than at least 1, the
sample comprises at least one cancerous cell. This may be further
defined as when the ratio of nuclearly localized JAB1 to
cytoplasmically localized JAB1 is greater than at least about 3,
the sample comprises at least one cancerous cell and is at least in
stage III, for breast cancer embodiments.
[0036] The assessing step may be further defined as assessing the
ratio of JAB1 polypeptide/p27 polypeptide and/or assessing the
ratio of JAB1 polynucleotide/p27 polynucleotide. Although any
suitable means of assessing the levels of JAB1 and p27 may be
employed, in specific embodiments the assessing comprises
hybridization, western blotting, ELISA, immunohistology, polymerase
chain reaction, or a combination thereof. The hybridization may
include in situ hybridization, such as with radioactive or
non-radioactive means, including by fluorescence. Polymerase chain
reaction methods may include RT-PCR and/or real time PCR.
[0037] Samples to be obtained from individuals may be of any kind,
so long as they provide information as to the level or localization
of expression of JAB1 and/or p27, particularly for cancer diagnosis
and prognosis. In specific embodiments, the sample comprises at
least one cancer cell, and although the sample may be from any
tissue, in specific embodiments the sample comprises a biopsy,
nipple aspirate, blood, urine, saliva, or feces.
[0038] In particular aspects of the invention, the diagnosing or
prognosticating step provides information of the stage of the
cancer and also may facilitate determining a therapy based on the
stage of the cancer. In specific aspects, when the JAB1 level is
greater in the sample from the individual compared to a
non-cancerous sample, the sample from the individual comprises
advanced stage cancer, or when the ratio of JAB1/p27 comprises high
JAB1 expression and low p27 expression, the sample comprises
advanced stage cancer.
[0039] In specific embodiments, the diagnosing and prognosticating
methods described herein further comprise the step of treating the
cancer, although treatments provided herein may be administered to
an individual with cancer following diagnosis and/or prognosis
through another means. Treatment may comprise delivering a
JAB1-inhibiting agent to the individual, such as by delivering the
JAB1-inhibiting agent directly to the cancerous cell, and this
delivery may comprise microinjection, electroporation, liposomal
delivery, by catheter, or a combination thereof, although other
suitable means may be utilized.
[0040] In a particular aspect of the invention, a JAB1-inhibiting
agent comprises a polynucleotide, a polypeptide, a peptide, a small
molecule, or a mixture thereof. In specific aspects, the
polynucleotide comprises antisense JAB1 sequence, which may be
further defined as being comprised on a vector, wherein the
polynucleotide is operably linked to a promoter suitable for
regulation of the polynucleotide in the cancerous cell. The vector
may be a viral vector, such as an adenoviral vector, a retroviral
vector, an adeno-associated viral vector, a lentiviral vector, or a
herpesviral vector, or it may be a non-viral vector, such as a
plasmid. In some embodiments, the therapeutic composition is
encapsulated in a cell, and one or more of the cells are delivered
to the individual, such as directly to the tumor.
[0041] In specific aspects of the invention, antisense JAB1
polynucleotides comprise a hairpin structure having a duplex
portion and a loop portion, wherein the duplex portion is about 10
base pairs (bp) to about 50 bp in length or about 15 bp to about 33
bp in length. Particular antisense JAB1 polynucleotides may
comprise RNAi or siRNA compositions.
[0042] In additional aspects of the invention, there is a method
for identifying the stage of a cancer in an individual, comprising
obtaining a cancerous sample from the individual; assessing the
ratio of JAB1/p27 level in the sample; and determining the stage of
the cancer based on the assessment. In a specific embodiment, when
the ratio of JAB1/p27 is high, the stage of the cancer is late
stage.
[0043] In further aspects of the invention, there is a method for
monitoring treatment of a cancer for an individual, wherein the
cancer is characterized by a high JAB1/p27 ratio, comprising
determining the JAB1/p27 ratio in a cancerous sample from a tissue
from the individual prior to the treatment; administering the
treatment to the individual; and determining the JAB1/p27 ratio in
a sample from the tissue from individual subsequent to the
treatment. The treatment may comprise delivering a JAB1-inhibiting
agent to the individual. When the determination of the JAB1/p27
ratio in a sample from the individual subsequent to the treatment
is lower than the JAB1/p27 ratio in the cancerous sample from the
individual prior to the treatment, the treatment may be efficacious
for the cancer.
[0044] In another aspect of the invention, there is a method of
screening for a JAB1-inhibiting agent, comprising providing a JAB1
polypeptide; providing a p27 polypeptide; and providing a test
compound, wherein when the test compound inhibits binding of the
JAB1 polypeptide to the p27 polypeptide, the test compound is the
JAB1-inhibiting agent. The method may further comprise the step of
delivering the JAB1-inhibiting agent to an individual having
cancer. The method may occur in vitro or in vivo. The providing of
the JAB1 polypeptide may be further defined as providing a
polynucleotide that encodes the JAB1 polypeptide, and the providing
of the p27 polypeptide may be further defined as providing a
polynucleotide that encodes the p27 polypeptide. Binding of the
JAB1 polypeptide to the p27 polypeptide may be detected by any
suitable means, although in particular aspects the detection
comprises color detection, radioactivity detection, or fluorescence
detection.
[0045] In specific embodiments, particular JAB1 regions are
utilized in a screen. For example, agents that bind to the
p27-binding domain (SEQ ID NO:13 or SEQ ID NO:11) of JAB1 are
identified and utilized as inhibitors of JAB1 binding to p27.
Agents that bind the c-Jun-binding domain (SEQ ID NO:12) may
similarly be employed to inhibit JAB1 binding to c-Jun.
[0046] In an additional aspect of the invention, there is a kit,
housed in a suitable container, comprising reagents to detect JAB1
or p27 localization or levels and/or comprising one or more
JAB1-inhibiting agents identified by any suitable method, such as
those exemplary methods described herein.
[0047] In another aspect of the invention, there is a composition
comprising a JAB1-inhibiting agent, which may be, for example, a
polynucleotide, polypeptide, peptide, small molecule, or mixture
thereof. In specific embodiments, the peptide comprises SEQ ID
NO:11, SEQ ID NO:12, or SEQ ID NO:13. The composition may be
comprised in a pharmaceutically acceptable diluent. The composition
may comprise antisense JAB sequence, or the composition may
comprise at least part of a p27-binding domain of JAB1 or a
c-Jun-binding domain of JAB1.
[0048] In an additional aspect of the invention, there is a method
of enhancing therapy for cancer in an individual, comprising
delivering to the individual a JAB1-inhibiting agent. In specific
embodiments, the cancer comprises breast cancer. In additional
specific embodiments, the therapy comprises a humanized antibody to
the receptor HER2. The JAB1-inhibiting agent comprises a JAB1
antisense polynucleotide, in some embodiments, and in particular
embodiments the JAB1 antisense polynucleotide comprises a viral
vector having JAB1 siRNA, such as an adenoviral vector. In other
embodiments, the JAB1-inhibiting agent comprises activity that
inhibits binding of JAB1 to p27.
[0049] In specific aspects of the invention, there is a method of
diagnosing cancer in an individual comprising obtaining a sample
from the individual; measuring a level of JAB1 expression in the
sample; and diagnosing the likelihood of cancer occurrence. The
method may further comprise comparing the level of JAB1 expression
in the sample to a pre-determined standard level of JAB1
expression. The method may also further comprise comparing the
level of JAB1 expression in the sample to a second sample taken
from another tissue from the individual. In specific embodiments,
the measuring step is performed by dot blotting, Southern blotting,
western blotting, ELISA, immunohistology, sandwich blotting,
immunohistochemistry, polymerase chain reaction, RT-PCR, or
non-radioactive in situ hybridization (NISH), for example. The
method may also further comprise the steps of measuring a level of
p27 in the sample; and determining a ratio of the level of JAB1
expression in the sample to the level of p27 expression in the
sample. In particular embodiments, the method further comprises
comparing the ratio of the level of JAB1 expression in the sample
to the level of p27 expression in the sample to a pre-determined
standard ratio of the level of JAB1 expression in the sample to the
level of p27 expression.
[0050] In an additional aspect of the invention, there is a method
for determining whether disease, such as breast cancer or
non-Hodgkins lymphoma, is progressing in an individual, comprising
obtaining a first sample from the individual at t=1; measuring a
level of JAB1 expression in the first sample; obtaining a second
sample from the individual at t=2, where t=2 is later than t=1;
measuring a level of JAB1 expression in the second sample; and
comparing the level of JAB1 expression in the first sample to the
level of JAB1 expression in the second sample, wherein if the level
of JAB1 expression in the first sample is higher than or the same
as level of JAB1 expression in the second sample, disease is not
progressing; and wherein if the level of JAB1 expression in the
first sample is lower than level of JAB1 expression in the second
sample, disease is progressing. Measuring steps may be performed by
dot blotting, Southern blotting, western blotting, ELISA,
immunohistology, or sandwich blotting.
[0051] In another aspect, there is a method of determining efficacy
of treatment in an individual with a disease comprising obtaining a
first sample from the individual at t=1; measuring a level of JAB1
expression in the first sample; treating the individual with a
treatment at t=2, wherein t=2 is later than t=1; obtaining a second
sample from the individual at t=3, where t=3 is later than t=2;
measuring a level of JAB1 expression in the second sample; and
comparing the level of JAB1 expression in the first sample to the
level of JAB1 expression in the second sample, wherein if the level
of JAB1 expression in the first sample is higher than or the same
as level of JAB1 expression in the second sample, the treatment is
efficacious; and wherein if the level of JAB1 expression in the
first sample is lower than level of JAB1 expression in the second
sample, the treatment is not efficacious. The measuring steps are
performed by dot blotting, Southern blotting, western blotting,
ELISA, immunohistology, or sandwich blotting.
[0052] In an additional aspect of the invention, there is an assay
for selecting treatments effective for inhibiting a level of JAB1
expression in a cell, comprising constructing a JAB1 expression
vector; transforming the cell with the JAB1 expression vector;
measuring a level of JAB1 expression in the cell at t=1; treating
the cell with a treatment at t=2, wherein t=2 is later than t=1;
measuring a level of JAB1 expression in the cell at t=3, where t=3
is later than t=2; and comparing the level of JAB1 expression in
the cell at t=1 to the level of JAB1 expression in the cell at t=3,
wherein if the level of JAB1 expression in the cell at t=1 is
higher than or the same as level of JAB1 expression in the cell at
t=3, the treatment is efficacious; and wherein if the level of JAB1
expression in the cell at t=1 is lower than level of JAB1
expression in the cell at t=3, the treatment is not efficacious.
The measuring steps are performed by dot blotting, Southern
blotting, western blotting, ELISA, immunohistology, or sandwich
blotting. In a specific embodiment, the cell is selected from
normal human tissue or cancerous tissue.
[0053] In an additional aspect of the invention, there is a method
for treating cancer in an individual comprising treating the
individual with an efficacious treatment identified using the
methods described herein. In another aspect of the invention, there
is a method for treating a proliferative disease in an individual
comprising inhibiting the expression of JAB1 in a cell, such as a
proliferating cell, in the individual. The inhibition of the
expression of JAB1 in the cell may be accomplished by treating the
proliferating cells with an antisense agent complementary to a JAB1
gene.
[0054] In a specific embodiment, an antisense agent is
complementary to at least part of GenBank Accession No.
NM.sub.--006837 (SEQ ID NO:9), for example, which can be obtained
at the World Wide Web site of the National Center for Biotechnology
Information. Other exemplary sequences include GenBank Accession
Nos.: U65928 (SEQ ID NO:8); BC001859; (SEQ ID NO:14); BC007272 (SEQ
ID NO:15); and BC001187 (SEQ ID NO:16). In a specific embodiment,
the treating step is accomplished by delivering the antisense agent
to the proliferating cells by direct transformation of the
proliferating cells with the antisense agent, or microinjection,
electroporation, or liposomal delivery of the antisense agent. The
inhibition of expression of JAB1 in the proliferating cells may be
accomplished by treating the cancer cells with an RNAi agent
complementary to a JAB1 polynucleotide. In a specific embodiment,
the RNAi agent is delivered directly to the proliferating cells.
The RNAi agent may be chemically modified to increase a half-life
and stability of the RNAi agent in the proliferating cells. The
RNAi agent may be delivered to the proliferating cells by direct
transformation of the proliferating cells with the RNAi agent, or
by microinjection, electroporation, by catheter or liposomal
delivery of the RNAi agent.
[0055] In specific embodiments, an RNAi agent is expressed in the
proliferating cells, such as from a viral vector or a non-viral
vector. Particular viral vectors include, for example, adenoviral
vectors, retroviral vectors, adeno-associated viral vectors,
lentiviral vectors, or herpesviral vectors. Particular non-viral
vectors include plasmids. The vector preferably comprises a
promoter operably linked to a sequence to be transcribed, such as
the siRNA, and the promoter is preferably suitable for expression
in a mammalian cell. However, the promoter may be an RNA polymerase
I, RNA polymerase II, or RNA polymerase III promoter. In specific
embodiments, the promoter for the vector is tissue-specific, such
as specific for the tissue type of cancer cell being treated. For
example, when treating breast cancer, a tissue-specific breast
cancer promoter may be employed. Examples of breast cancer-specific
promoters include human alpha-lactalbumin (ALA) or ovine
beta-lactoglobulin (BLG) promoters, for example. In particular, the
promoter may be a U6 promoter, H1 promoter, 7SL promoter, human Y
promoter, human MRP-7-2 promoter, Adenovirus VA1 promoter, human
tRNA promoter, 5S ribosomal RNA promoter, or a functional hybrid or
a combination of any of these promoters, for example. In specific
embodiments, the viral vector comprises a terminator.
[0056] In specific embodiments of the invention, an RNAi agent is
transcribed as a hairpin structure with a duplex portion and a loop
portion. The duplex portion of the hairpin structure may be 10 to
50 bp in length. The duplex portion of the hairpin structure may be
15 to 33 bp in length. In embodiments wherein the proliferative
disease is cancer, the cancer may be breast cancer, such as ductal
carcinoma or lobular carcinoma, or it may be lymphoma, such as, for
example, non-Hodgkins lymphoma.
[0057] In one embodiment, siRNAs and antisense constructs that
reduce the expression of JAB1 protein in human cells are provided.
The siRNAs and antisense constructs may be used in methods of
treating human cancers. In another embodiment, a recombinant
adenovirus that expresses JAB1 protein is provided.
[0058] In a further embodiment, JAB1 protein is detected and/or
quantitated in human tissue samples in order to diagnose and/or
prognosticate the development of human cancers. In one aspect, the
detection or quantitation of JAB1 protein is used in methods of
diagnosing cancer, prognosticating a cancer survival rate, and/or
prognosticating the development of cancer. In specific embodiments,
the cancer can be any cancer, such as breast cancer, non-Hodgkins
lymphoma, colon cancer, prostate cancer, pancreatic cancer, or lung
cancer, for example.
[0059] In specific embodiments the present invention encompasses
any type of cancer, such as solid tumors and leukemias, including:
apudoma, choristoma, branchioma, malignant carcinoid syndrome,
carcinoid heart disease, carcinoma (e.g., Walker, basal cell,
basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs
2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary,
scirrhous, bronchiolar, bronchogenic, squamous cell, and
transitional cell), histiocytic disorders, leukemia (e.g., B cell,
mixed cell, null cell, T cell, T-cell chronic, HTLV-II-associated,
lymphocytic acute, lymphocytic chronic, mast cell, and myeloid),
hystiocytosis malignant, Hodgkin disease, immunoproliferative
small, non-Hodgkin lymphoma, plasmacytoma, reticuloendotheliosis,
melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma,
fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma,
mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing
sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma,
chordoma, cranio-pharyngioma, dysgerminoma, hamartoma,
mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma,
odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma,
adenoma, cholangioma, cholesteatoma, cylindroma,
cystadenocarcinoma, cystadenoma, granulosa cell tumor,
gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig
cell tumor, papilloma, Sertoli cell tumor, theca cell tumor,
leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma,
rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma,
medulloblastoma, meningioma, neurilemmoma, neuroblastoma,
neuroepithelioma, neurofibroma, neuroma, paraganglioma,
paraganglioma nonchromaffin, angiokeratoma, angiolymphoid
hyperplasia with eosinophilia, angioma sclerosing, angiomatosis,
glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma,
hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma,
pheochromocytoma, pinealoma, carcinosarcoma, chondrosarcoma,
cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma,
leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma,
myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma,
sarcoma (e.g., Ewing, experimental, Kaposi, and mast cell),
neoplasms (e.g., bone, breast, digestive system, colorectal, liver,
pancreatic, pituitary, testicular, orbital, head and neck, central
nervous system, acoustic, pelvic, respiratory tract, and
urogenital), neurofibromatosis, and cervical dysplasia, and other
cells that have become immortalized or transformed. In specific
embodiments, however, the present invention is directed to methods
and compositions concerning breast, lymphoma, colon, prostate,
pancreatic, and lung cancer.
[0060] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated that the conception and
specific embodiment disclosed may be readily utilized as a basis
for modifying or designing other structures for carrying out the
same purposes of the present invention. It should also be realized
that such equivalent constructions do not depart from the invention
as set forth in the appended claims. The novel features which are
believed to be characteristic of the invention, both as to its
organization and method of operation, together with further objects
and advantages will be better understood from the following
description when considered in connection with the accompanying
figures. It is to be expressly understood, however, that each of
the figures is provided for the purpose of illustration and
description only and is not intended as a definition of the limits
of the present invention
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] So that the manner in which the above recited features of
the present invention can be understood in detail, a more
particular description of the invention, briefly summarized above,
may be had by reference to embodiments, some of which are
illustrated in the appended drawings. It is to be noted, however,
that the appended drawings illustrate only typical embodiments of
this invention and are therefore not to be considered limiting of
its scope, for the invention may admit to other equally effective
embodiments.
[0062] FIGS. 1A and 1B demonstrate survival rates JAB1-negative
breast tumors vs. JAB1-positive breast tumors. FIG. 1A is a graph
showing the progression-free survival (PFS) rates of breast cancer
patients having JAB1-positive breast tumors or JAB1-negative breast
tumors. FIG. 1B is a graph showing the overall survival (OS) rates
of breast cancer patients having JAB1-positive breast tumors or
JAB1-negative breast tumors.
[0063] FIG. 2A shows JAB1 and p27 expression in normal breast
tissue and breast tumors. Western blots of paired samples of
normeoplastic breast tissue (N) and tumor tissue (T) immunoblotted
against JAB1 or vinculin (used as a loading control). JAB1 and p27
expression in normal breast tissue and breast tumors. Western blots
of paired samples of noneoplastic breast tissue (N) and tumor
tissue (T) immunoblotted against JAB1, and HER2, with vinculin used
as a loading control. FIG. 2B shows JAB1 and p27 expression in
normal breast tissue and breast tumors. Western blots of paired
samples of noneoplastic breast tissue (N) and tumor tissue (T)
immunoblotted against JAB1, and HER2, with vinculin used as a
loading control.
[0064] FIGS. 3A and 3B show a western blot of four breast cancer
cell lines transfected with Ad-JAB1-Myc and a chart showing the
Phosphorlmager quantitation of the proteins detected on the western
blot, respectively.
[0065] FIGS. 4A-4I show an exemplary in vivo mouse tumor model
wherein JAB1 promotes cell cycle progression through S-phase and
induces tumorigenesis. FIG. 4A shows immunoblot analysis of cell
lysates from parental NIH-3T3 cells and stable NIH-3T3 clones
expressing Myc-JAB1 (Clones, #C1-4) showing various levels of
exogenous Myc-JAB1 levels (Top panel). Myc-JAB1 expression
corresponds to a decreased level of p27 in the stable clones but
not in the control parental cells (Bottom panel). FIG. 4B shows the
stable expression of Myc-JAB1 in 3T3 cells increased cellular
proliferation as measured by [.sup.3H]-thymidine incorporation. The
increase in thymidine incorporation was directly proportional to
the expression of exogenous Myc-JAB1 in the various stable clones.
For thymidine incorporation, 1.times.10.sup.5 cells of each
JAB1-Myc clones and parental 3T3 cells were plated in six wells of
a 24-well plate. After 24 h, the media was changed to serum-free
DMEM medium and incubated at 37.degree. C. for 24 h. The media was
aspirated and replaced with DMEM with Serum, containing 1 mCFiml
.sup.3H-thymidine (Amersham Biosciences, Piscataway, N.J., USA) and
incubated at 37.degree. C. for 1 h. Cells were washed twice with
PBS and solubilized in 200 mM NaOH. Counts per minute were
determined in a Liquid Scintillation Beta Analyzer (Packard
Instruments Co., Meridan, Conn., USA). FIG. 4C shows morphology of
parental NIH-3T3 cells and NIH-3T3-JAB1#C4. The Stable clone
3T3-JAB1#C4, expressing high levels of Myc-JAB1 exhibited
morphologic transformation compared to control cells. They were
spindle-shaped and display highly refractile morphology, with long
protrusions and pseudopodia. A representative clonal population of
cells photographed under phase-contrast microscopy is shown, with
the inbox showing a single cell. FIG. 4D shows that exogenous JAB1
expression promotes S-phase progression as measured by
Bromodeoxyuridine (BrdU) incorporation and propidium idodide (PI)
staining. The parental cells and stable clones were serum starved
for 24 hours and then replaced with DMEM with serum and labeled
with Brdu for 45 minutes. The cells were then stained with
fluorescent anti-Brdu antibodies and PI for Flow-cytometry
analysis. FIG. 4E shows that JAB1 promotes growth in soft-agar.
Stable clones #C3 and C4 along were parental control cells were
plated in soft agar. After 1 week and 2 weeks plates were stained
with crystal violet and foci formation were counted, and an average
is shown. Summary of tumorigenesis assay (F) is shown. FIG. 4F
shows that exogenous JAB1 expression induced tumorigenesis in nude
mice. Stable clones, #C3, C4 and control cells (NIH-3T3) were
injected s.c (6.times.10.sup.6 cells) into 6-week old female nude
mice (BALB/C). Five mice were used for each cell line. After 35
days mice developed tumors >10 mm only with clones C3 and C4 but
not with control injected clone. Pictures of each mice are shown at
35 and 42 days post-injection. FIG. 4G shows JAB1 expression
promotes tumor development in nude mice. Mice were injected as in
FIG. 4F, and tumor formation was scored weekly. In FIG. 4H, there
is JAB1 and p27 immunostaining in mouse normal and tumor tissues.
Mice-bearing JAB1 tumors (in FIG. 4F) were isolated and
paraffin-embedded tissue sections obtained and stained with
monoclonal antibodies for JAB1 or p27 and counterstained with
hematoxylin. Representative tissue sections of the
immunohistochemical analysis show low JAB1 expression and high p27
expression in normal tissue while the inverse was seen in tumor
tissues. In FIG. 41, there is a column chart representing the
immunostaining above, indicating the relationship between JAB1 and
p27 positive staining in normal and tissue samples. Three hundred
positive and negative cells were counted in each of three fields
for JAB1 and p27 in normal and four tumor tissue samples; the
percent positive staining is shown.
[0066] FIG. 5 is a plot of average tumor volume vs. time for JAB1
expression-induced tumors in nude mice.
[0067] FIGS. 6A and 6B show JAB1 and p27 levels in normal,
hyperplastic-benign and invasive-neoplastic lesions of human breast
tissue samples. JAB1 levels increase with tumorigenicity,
correlating with a decrease in p27. In FIG. 6A, there is
immunohistochemical staining of a breast tumor progression array
for JAB1 and p27. JAB1 levels are low in normal tissue and increase
with tumorigenesis. In FIG. 6B, the percent of cells staining
positive for either JAB1 or p27 were quantified and graphed.
[0068] FIG. 7 depicts a western blot of four exemplary ALCL cell
lines.
[0069] FIG. 8 shows JAB1 and p27 expression in ovarian cancer
(tissue array).
[0070] FIG. 9 shows JAB1 and p27 expression in colon carcinoma
(tissue array).
[0071] FIG. 10 shows the sequence of an exemplary double stranded
siRNA (including the exemplary SEQ ID NO:3 and SEQ ID NO:4) used in
a specific embodiment.
[0072] FIGS. 11A-11C show that depletion of endogenous JAB1 with
either antisense (AS) or siRNA but not control siRNA, promotes
p27-increased stability and leads to G1-arrest. In FIG. 11A,
expression of antisense JAB1 increased the endogenous level of p27.
HeLa cells were transfected with a tetracycline-inducible (Tet-Off
system) antisense JAB1. Cell lysates were immunoblotted with JAB1
and p27 antibodies Quantification of the immunoblots is shown on
the right. In FIG. 11B, there is depletion of JAB1 by siRNA oligos
in HeLa cells. Cells were transfected with siRNA targeting JAB1
(JAB1 siRNA) or a scrambled sequence (Control siRNA). Forty-eight
hours after transfection, cell lysates were prepared and were
subjected to western blotting analysis using anti-JAB1, anti-p27,
anti.Cyclin A, anti-pRb and anti-actin antibodies. For kinase assay
(last panel), Cyclin A was immunoprecipated from cell lysates and
analyzed for cyclinA/Cdk2-associated activity using Histone 1B as a
substrate. In FIG. 11C, knockdown of endogenous JAB1 expression
decreases the S-phase progression in cell cycle and increases 5 G1
cells. Hela cells transfected with JAB1 siRNA and Control siRNA.
Progression through S-phase was measured with anti-Brdu fluorescent
antibodies and propidium idodide (PI) staining for Flow cytometery
analysis.
[0073] FIG. 12 shows silencing with adenoviral vector expressing
JAB1siRNA (Ad-JAB1siRNA). In FIG. 12A, there is a schematic of
pSIREN Adeno strategy (Adeno-X viral DNA, BD-Pharmingen). In FIG.
12B, inhibition of endogenous JAB1 with Ad-JAB1 siRNA but not with
control Ad-LUCsiRNA, increases p27 expression levels. HeLa cells
were transduced (MOI 50) with either Luciferase-RNAi pSIREN Shuttle
vector or JAB1-RNAi pSIREN Shuttle vector. Cells were harvested 48
hours post-transfection and analyzed by western blotting analysis
using both anti-JAB1 and anti-p27 antibodies.
[0074] FIGS. 13A-13B demonstrate that depletion of JAB1 by siRNA
adenovirus causes accumulation of p27kip1 and induces G1 arrest in
MDA-MB 231 breast carcinoma cells. In FIG. 13A, MDA-MB 231 cells
were transduced with adenoviruses driven JAB1 siRNA, or Luciferase
siRNA as a control, at MOI 50. Forty eight hours after, protein
lysates were prepared and immunobloted with an anti-JAB1, anti-p27
and anti-Cyclin A antibodies. Anti-.beta. actin was used as a
loading control. SiRNA ablation of JAB1 increases the steady-state
level of p27Kip1 protein and decreased cyclin A levels. In FIG.
13B, siRNA ablation of JAB1 induces G1 arrest. Cells were treated
same as in FIG. 13A, and cell cycle profile was determined by
propidium iodine staining and FACS.
[0075] FIG. 14 demonstrates that siRNA ablation of JAB1 causes
p27kip1 accumulation and prevents S-phase re-entry in Karpas 299
T-cells lymphoma. In FIG. 14A, knockdown of JAB1 protein levels by
siRNA increases the steady-state level of p27 protein, decreases
cyclin A and phopho-Rb levels. Karpas 299 cells were transfected
with p-Siren JAB1 siRNA or luciferase siRNA as a control (5 .mu.g
each). Lysates were immunoblotted 48 h after with the indicated
antibodies. In FIG. 14B, siRNA ablation prevents S-phase re-entry.
Karpas 299 cells were treated as in FIG. 14A, and progression
through S-phase was measured with anti-BrdU fluorescent antibodies
and FACS 48 hr after. Forty-six % of control siRNA-treated cells
were in S-phase compared to 15% with siRNA JAB1.
[0076] FIG. 15 shows Jab1 amplification in T-cells lymphoma and
breast carcinoma. Two-color FISH analysis of Jab1 (green spectrum)
and chromosome 8 centromeric (8p11.1-q11.1) (CEP8) (orange
spectrum) in lymphoma (Karpas 299 T-cells (top left) and aggressive
breast carcinoma (MDA-MD 231 cells) (bottom, left) and compare to
normal cells (right). Nuclei were counterstained with
4',6-diamidino-2-phenylindole (blue). Sporadic cancer cells with
increased jab1 copy numbers are shown in each case.
[0077] FIG. 16 depicts an exemplary ELISA protocol with JAB1
monoclonal antibodies.
[0078] FIGS. 17A-17D show delineation of the JAB1-JUN interaction
domain and JAB1-p27 interaction domain. In FIG. 17A, there is a
schematic representation of JAB1 full length (FL) and JAB1
N-terminal and JAB1 C-terminal sequential deletion constructs
(.DELTA.N and AC, respectively). Mapping of both JAB1-c-jun and
JAB1-p27 interacting domains is depicted. The table summarizes the
results of the below in vitro binding assays of various JAB1
recombinant protein to GST-c-Jun and GST-p27. In FIG. 17B, there is
in vitro expression analysis of JAB1 full length and C- and
N-terminal deletion deletions mutants. Using TnT coupled
reticulocyte lysate system (Promega) full length and deletion
mutants were in vitro translated and [.sup.35S]-Methionine
labelled. Ten percent of labeled products (input) were separated on
SDS-PAGE. Gel was then fixed (in 50% methanol, 10% acetic acid),
and dried. An autoradiography is shown. In FIG. 19C, for in vitro
binding assay recombinant JAB1 and JAB1 deletion mutants prepared
as in FIG. 17B were incubated with either Glutathione-S-transferase
(GST) alone or GST-p27 fusion protein that was immobilized on
glutathione agarose. The results of the binding assay show all
N-terminal but not C-terminal deletion mutants of JAB1 binding to
p27, indicating p27 binds to JAB1 (299-334 amino acids). In FIG.
17D, there is deletion analysis of JAB1-c-Jun interaction domain.
Methods are the same as in FIG. 17C with recombinant JAB1 and JAB1
deletion mutants incubated with either Glutathione-S-transferase
(GST) alone or GST-c-Jun (1-79 amino acids) fusion protein that was
immobilized on glutathione agarose. JAB1 interaction domain on
c-Jun was mapped to 1-67 amino acids (Claret et al., Nature, 1996).
The results of the binding assay show all C-terminal but not
N-terminal deletion mutants of JAB1 bind to c-Jun, indicating c-Jun
binds to JAB1 at its N-terminus between 49-96 amino acids of
JAB1.
[0079] FIGS. 18A-18B show a small region of JAB1 is sufficient for
interaction with p27. Recombinant proteins were bacterially
expressed and purified as Glutathione-S-transferase (GST) alone or
fused to p27 (GST-p27). Results of a GST (lane 1) or GST-p27 (lanes
3-7) pull-down experiments with in vitro .sup.35S-methionine
labeled JAB1 (full length) is shown. The effect of increasing
concentrations (0, 0.08, 0.4, 2, 10 and 20 .mu.g/ml) of synthetic
JAB1-peptide #1 corresponding to JAB1-p27 binding-domain (18 amino
acid residues) (Bottom panel, lanes 2-7) or to a peptide with
scrambled sequence (control) was examined (Top panel, lanes 2-7).
Reactions were incubated 1 hr at room temperature and washed 5
times and bound proteins to glutathione-Sepharose beads were loaded
onto an SDS-PAGE. Autoradiogram is shown after .sup.35S
exposure.
[0080] FIG. 19 illustrates nuclear vs. cytoplasmic localization of
JAB1 correlating with stage of human breast cancer.
[0081] FIGS. 20A-20C demonstrate characterization of the JAB1
promoter region and its transcriptional start site. In FIG. 20A, 1,
2 and 3 kb upstream of the mRNA start site have been amplified by
PCR, and the JAB1 promoter regions were predicted by using Proscan
V1.7. Primers were designed to amplify 1, 2, and 3 kb upstream of
the ATG. In FIG. 20B, PCR amplification products of the predicted
regions are identified on the agarose gel. In FIG. 20C, there is
the transcriptional start site of the Jab1 gene.
[0082] FIG. 21 provides the JAB1 promoter sequence and the
corresponding transcription factor binding sites, as well as the
transcription start site at +1.
[0083] FIG. 22 shows JAB1 and p27 expression in normal and
neoplastic pancreas. JAB1 and p27 immunostainings are shown (brown
stainings). Three different type of cells comprise the pancreas:
ductal, acinar and islet cells. Normal ducts are negative for JAB1
and positive for p27 stainings (Left panel). Neoplastic carcinoma
arise from the duct cells that are positive for JAB1 and negative
for p27 (Right panel). Brown staining is specific iimmunostainings.
(-): negative; (+): Positive stainings.
[0084] FIG. 23 shows p27 and JAB1 expression in lymphoma types
including: Hodgkin's Lymphoma; high grade non-Hodgkin's lymphomas
(ALCL, DLBCL and Burkitt); intermediate grade non-Hodgkin's
lymphomas (MCL and follicular lymphoma); and low grade
non-Hodgkin's lymphomas (CLL/SLL).
[0085] FIG. 24 shows that JAB1 bypasses Herceptin-mediated G1
arrest in breast cancer cells.
DETAILED DESCRIPTION OF THE INVENTION
[0086] I. The Present Invention
[0087] In keeping with long-standing patent law convention, the
words "a" and "an" when used in the present specification in
concert with the word comprising, including the claims, denote "one
or more."
[0088] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, and so forth which are within the
skill of the art. Such techniques are explained fully in the
literature. See e.g., Sambrook, Fritsch, and Maniatis, MOLECULAR
CLONING: A LABORATORY MANUAL, Second Edition (1989),
OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984), ANIMAL CELL
CULTURE (R. I. Freshney, Ed., 1987), the series METHODS IN
ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR
MAMMALIAN CELLS (J. M. Miller and M. P. Calos eds. 1987), HANDBOOK
OF EXPERIMENTAL IMMUNOLOGY, (D. M. Weir and C. C. Blackwell, Eds.),
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, R. Brent, R.
E. Kingston, D. D. Moore, J. G. Siedman, J. A. Smith, and K.
Struhl, eds., 1987), CURRENT PROTOCOLS IN IMMUNOLOGY (J. E.
coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W.
Strober, eds., 1991); ANNUAL REVIEW OF IMMUNOLOGY; as well as
monographs in journals such as ADVANCES IN IMMUNOLOGY. All patents,
patent applications, and publications mentioned herein, both supra
and infra, are hereby incorporated herein by reference.
[0089] In view of the unpredictable expression and significance of
JAB1 protein in human cancers, the expression of JAB1 protein in
several types of cancer, including breast cancer, non-Hodgkins
lymphoma, colon cancer, pancreatic cancer, and others was studied.
The present invention provides methods of diagnosing and
prognosticating the development of human cancer, as well as methods
for reducing the expression or altering the subcellular
localization of JAB1 protein in cells to treat certain cancers. The
present invention also provides assays that may be employed to
screen for compounds that affect JAB1 expression.
[0090] In particular aspects of the invention, the level of JAB1
expression is diagnostic for cancer. When the level of JAB1 is high
in a sample suspected of comprising at least one cancer cell
compared to the level in a normal cell from the same or similar
tissue, this is indicative of cancer. In particular, the ratio of
JAB1 level to the level of one of its targets may be indicative of
cancer. Although a variety of targets may provide diagnostic
molecular indicia in conjunction with JAB1, such as c-Jun, p53, and
cyclin D1, in specific and illustrative embodiments the ratio of
JAB1 to p27 is utilized. A skilled artisan recognizes that although
in specific embodiments there are p27-associated embodiments
described herein, these methods and reagents also apply to any
suitable target that provides diagnostic and/or prognostic cancer
information, but for the sake of brevity only the present inventors
herein focus on p27 aspects.
[0091] To facilitate the diagnosis and/or prognosis of cancer,
there are provided herein methods of identifying the stage of a
cancer in an individual, such as by determining the level and/or
subcellular localization of JAB1 and/or the ratio of JAB1/p27 in a
sample from the individual. When the level and/or nuclear
localization of JAB1 and/or the ratio of JAB1/p27 is high in the
sample, the stage of the cancer is thus determined. In specific
aspects, when the level of JAB1 and/or the ratio of JAB1/p27 is
high, the stage of the cancer is a late stage or advanced stage,
such as being metastatic or invasive cancer.
[0092] In particular embodiments, cancer treatments may be
monitored via assessment of JAB1 localization and/or levels, such
as assessment of JAB1/p27 ratio levels. The level and/or
localization of JAB1 or its ratio to p27 is determined prior to
treatment, a treatment is delivered to the individual, and
thereafter the level and/or localization of JAB1 or its ratio to
p27 is determined. The treatment may or may not target JAB1,
although in particular embodiments the treatment modulates JAB1
expression, such as by decreasing it. If the level of JAB1
expression or its ratio to p27 is reduced following the treatment,
the cancer treatment may be considered efficacious.
[0093] Treatments that target JAB1 are employed herein. The
treatment may comprise reducing the expression level or
localization of JAB1 itself or it may comprise inhibiting the
binding of JAB1 to p27, or a combination of the treatments may be
used. In specific embodiments, JAB1 expression is reduced by
employing antisense JAB1 sequence to target the JAB1 transcripts.
Treatments-may also comprise inhibiting the binding of JAB1 to p27,
such as by delivering agents that inhibit the binding of JAB1 to
its target, p27. These may be polypeptides, peptides, or small
molecules, for example. JAB1-inhibiting agents may directly or
indirectly affect the binding of JAB1 to its target, but in
specific embodiments the agents interfere physically with the
binding of JAB1 to its target domain on p27. Specifically, an agent
may bind JAB1 and inhibit its binding to p27, or the agent may bind
p27 and inhibit its binding to JAB1.
[0094] In vitro and in vivo screens to identify JAB1-inhibiting
agents are described herein. In specific embodiments, the screens
employ providing JAB1 and p27 polypeptides, and the interference of
their respective binding to each other is assayed in the presence
of a test compound. When the binding is inhibited, the test
compound is useful as a JAB1-inhibiting agent and may be utilized
in cancer treatment methods. Assessment of the binding of JAB1 and
p27 may be by any suitable means, although in specific embodiments
colorimetric, radioactive, or fluorescent methods are utilized.
[0095] Kits for the diagnosis, prognosis, and/or treatment of
cancer are provided herein, particularly those reagents suitable
for detection of JAB1 and p27 levels and/or those kits comprising
JAB1-inhibiting agents suitable for cancer treatment.
[0096] II. Diagnostic Uses
[0097] JAB1 nucleic acid, antibody, and/or polypeptide compositions
may be used to analyze patient samples for a level of JAB1 and/or
p27 expression associated with a disease state or predisposition to
a disease state. In a first step, patient sample is obtained.
Samples, as used herein, include biological fluids such as semen,
blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid
and the like; organ or tissue-derived fluids; or derivatives and
fractions of such fluids. The cells may be dissociated, in the case
of solid tissues, or tissue sections may be analyzed. Once patient
samples are obtained, the level or localization of JAB1 expression
in the sample is assessed. Optionally, the level of expression of a
target, such as p27, is assessed. Assessing JAB1 and/or p27
expression can be performed in various ways well known in the art.
For example, the level of JAB1 expression may be determined by
assaying the amount of JAB1 mRNA present in a patient sample by
Southern blotting, dot blots, or other such techniques (see, e.g.,
Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory
Manual (1982 and recent editions)).
[0098] Alternatively, levels of expression for JAB1 in patient
samples can be determined by assaying the amount of JAB1 protein
present in the patient samples, such as wherein antibodies specific
for JAB1 are used for detecting the JAB1 protein and/or respective
antibodies are utlized for p27. Assays used to detect levels of
JAB1 protein in a sample derived from a host are well-known to
those of skill in the art and include western blot analysis, ELISA
assays, "sandwich" assays and radioimmunoassays (see, e.g., Coligan
et al., Current Protocols in immunology 1(2), Chapter 6, (1991)),
for example. Generally, western blotting is a technique for
blotting proteins onto nictrocellulose, nylon or other transfer
membrane after the proteins have been resolved by gel
electrophoresis. The proteins can be detected by one of several
methods, including autoradiography (if labeled), or through binding
to labeled, .sup.125I-labeled or enzyme-linked antibodies, lectin
or other specific binding agents, for example.
[0099] An ELISA assay may be employed, which initially comprises
preparing an antibody specific to the JAB1 antigen, preferably a
monoclonal antibody. Next, a reporter antibody typically is
prepared against the monoclonal antibody. To the reporter antibody
is attached a detectable reagent such as a radioactive moiety,
fluorescent moiety or horseradish peroxidase enzyme, for example. A
sample is removed from the host and incubated on a solid support,
e.g., a polystyrene dish, binding the proteins in the sample. Any
free protein binding sites on the dish are then blocked by
incubating with a non-specific protein, such as bovine serum
albumen or milk proteins. Next, the monoclonal antibody is
incubated in the dish during which time the monoclonal antibodies
attach to any JAB1 proteins from the sample attached to the
polystyrene dish. Unbound monoclonal antibody is washed out with
buffer. The reporter antibody is then placed in the dish resulting
in binding of the reporter antibody to any monoclonal antibody
bound to JAB1. Unattached reporter antibody is then washed out,
JAB1 protein is then detected and the amount of JAB1 protein
present in a given volume of patient sample is compared against a
standard curve.
[0100] A "sandwich" assay is similar to an ELISA assay and may also
be used in the invention. In a "sandwich" assay, JAB1 is passed
over a solid support and allowed to bind to antibody attached to
the solid support. A second antibody is then allowed to bind to the
JAB1. A third antibody specific to the second antibody is labeled
and is passed over the solid support, and binding to the second
antibody is detected.
[0101] Alternatively, detection may utilize staining of cells or
histological sections, performed in accordance with conventional
methods. Generally in such techniques, cells are permeabilized to
stain cytoplasmic molecules. The antibodies of interest are added
to the cell sample and incubated for a period of time sufficient to
allow binding to the epitope, usually at least about 10 minutes.
Again, the antibody may be labeled with radioisotopes, enzymes,
fluorophores, chemiluminophores, or other labels for direct
detection. Alternatively, a second stage antibody or reagent is
used to amplify the signal. Such reagents are well known in the
art. For example, the primary antibody may be conjugated to biotin,
with horseradish peroxidase-conjugated avidin added as a second
stage reagent. Alternatively, the secondary antibody may be
conjugated to a fluorescent compound, e.g. fluorescein, rhodamine,
Texas red, etc. Final detection typically uses a substrate that
undergoes a color change in the presence of the peroxidase. The
absence or presence of antibody binding may be determined by
various methods, including flow cytometry of dissociated cells,
microscopy, radiography, scintillation counting, etc.
[0102] Once the levels of expression for JAB1 in the patient sample
is determined, the sample JAB1 expression level may be compared to
the JAB1 expression level in normal samples, such as samples of
undiseased tissue from the patient or to standardized levels of
expression established in a population, for example. If the nuclear
localization and/or level of expression for JAB1 in the patient
sample is higher than the respective nuclear localization or level
of expression for JAB1 in a normal sample or an established
standard, a disease state may be diagnosed, and further diagnostic
procedures may be administered and/or appropriate therapeutic
measures may be taken. On the other hand, if the level of
expression for JAB1 in the patient sample is lower than or
substantially the same level as the level of expression for JAB1 in
a normal sample or an established standard, a disease state may not
be diagnosed.
[0103] In an alternative aspect to the method of using JAB1
expression for diagnosing a disease state as described above, the
levels of expression of both JAB1 and p27 are measured. A ratio of
JAB1 expression to p27 expression is determined for the patient
sample and is then compared to the JAB1/p27 ratio from a normal
sample of undiseased tissue from the patient or to a standardized
level of expression established in a population, for example. It
should be noted that a ratio of expression does not need to be made
before comparison, since the level of JAB1 expression and the level
of p27 expression in the patient sample may each be compared
separately to the level of JAB1 expression and the level of p27
expression in the normal sample or standard. In any case, if the
ratio of the level of expression of JAB1 to p27 in the patient
sample is higher than the ratio of the level of expression of JAB1
to p27 in the normal sample or an established standard, a disease
state may be diagnosed and appropriate further action can be taken.
On the other hand, if the ratio of the level of expression of JAB1
to p27 in the patient sample is lower than or substantially the
same as the ratio of the level of expression of JAB1 to p27 in the
normal sample or an established standard, a disease state may not
be diagnosed and appropriate further action or no action may be
taken.
[0104] III. Disease Prognosis
[0105] In addition and related to diagnostic methods, the present
invention provides embodiments for prognostic methods. Two
exemplary aspects of this embodiment are provided. A patient sample
may be obtained at a time=1. Next, the level of JAB1 expression or
the ratio of JAB1 expression to p27 expression for this first
sample is determined. As described above, the sample can be of
virtually any biological origin and the level of JAB1 expression
(or the ratio of JAB1 expression to p27 expression) can be
performed in any one of many different methods well known in the
art. At time=2, another sample is obtained from the same patient,
and the level JAB1 expression (or the ratio of JAB1 expression to
p27 expression) for this second sample is determined. Next, the
level of expression of JAB1 or the ratio of JAB1 expression to p27
expression of the first and second samples may be compared.
[0106] Similar to the diagnosis methods described supra, if the
nuclear localization or overall level of expression of JAB1 or the
ratio of the level of expression of JAB1 to p27 in the second
sample is higher than the nuclear localization or level of
expression of JAB1 or the ratio of the level of expression of JAB1
to p27 in the first sample, the prognosis would indicate an
increasing state of disease, such as a poorer prognosis, and the
necessity for appropriate intervention. On the other hand, if the
level of expression of JAB1 or the ratio of the level of expression
of JAB1 to p27 in the second sample is lower than or is
substantially the same as the level of expression of JAB1 or the
ratio of the level of expression of JAB1 to p27 in the first
sample, the prognosis would indicate a stabilized or decreasing
state of disease, and is a more faborable prognosis compared to
that for a higher level of JAB1 expression or ratio of
JAB1/p27.
[0107] In another embodiment, a patient sample is obtained from an
individual at a time=1. Next, the level of JAB1 expression or the
ratio of JAB1 expression to p27 expression for this first sample is
determined. As described above, the sample can be of virtually any
biological origin and the level of JAB1 expression (or the ratio of
JAB1 expression to p27 expression) can be performed in any one of
many different ways well known in the art. Next, the individual is
treated with a therapeutic at time=2. At time=3, another patient
sample is obtained from the individual, and the level JAB1
expression (or the ratio of JAB1 expression to p27 expression) for
this second sample is determined. The level of expression of JAB1
or the ratio of JAB1 expression to p27 expression of the first and
second samples are compared. As with the method described above, if
the level of expression of JAB1 or the ratio of the level of
expression of JAB1 to p27 in the second sample is higher than the
level of expression of JAB1 or the ratio of the level of expression
of JAB1 to p27 in the first sample, it would indicate that the
therapeutic is not effective. On the other hand, if level of
expression of JAB1 or the ratio of the level of expression of JAB1
to p27 in the second sample is lower than or is substantially the
same as the level of expression of JAB1 or the ratio of the level
of expression of JAB1 to p27 in the first sample, it would indicate
that the therapeutic is effective.
[0108] Levels of expression of JAB1 and/or of JAB1 compared to p27
may identify tumor progression, aggressiveness, or invasiveness of
the tumor, and/or it may be indicative of the stage of the cancer,
which may provide prognosis for an individual. For example, when
the JAB1 and/or JAB1/p27 level is increased compared to normal
tissue, the stage of the cancer may be identified, and the higher
the increase in expression level there is, the later the stage the
cancer may be. In particular embodiments, an increased level of
JAB1 or JAB1/p27 ratio may identify a late stage cancer, such as a
metastatic cancer. For the particular embodiment of breast cancer,
an increased JAB1 expression or JAB1/p27 ratio may identify the
cancer as at least at stage II, such as stage III or IV.
[0109] Reagents useful for the diagnostic and prognostic methods of
the present invention may be conveniently provided in kit form.
Thus, the present invention encompasses kits that comprise JAB1
polypeptides, antibodies, and polynucleotides. In one embodiment,
the kit comprises one or more of the following exemplary components
in a suitable container: (1) one or more JAB1 polynucleotides
(e.g., oligonucleotide primers or probes corresponding to the JAB1
cDNA sequence and capable of amplifying the target polynucleotides,
or siRNA) or fragments thereof; (2) anti-JAB1 antibodies, which may
be polyclonal or monoclonal; (3) JAB1 polypeptides or fragments
thereof, optionally coated on a solid surface (such as a slide,
multiple well plate, or test tube) for use as a standard or
control; (4) a JAB1 polynucleotide (e.g., for use as positive
controls in assays); (5) fluorescent or non-radioactive JAB1
oligonucleotide or probe corresponding to the JAB1 genomic sequence
that can be used for in situ hybridization (FISH or NISH); (6)
other necessary reagents or buffers, (7) and tubes or multiple well
plates. Instructions for carrying out the detection methods of the
invention and optionally calibration curves can also be
included.
[0110] IV. Screening Assays
[0111] In yet another aspect, the present invention contemplates a
method of screening candidate substances for their ability to
affect or modulate JAB1 expression to thereby affect or modulate
the growth, proliferation, or nonproliferation of cells, such as
cancer cells.
[0112] A. Screening for Modulators of the Protein Function
[0113] The present invention further comprises methods for
identifying modulators of the function of JAB1, such as the
exemplary function of JAB1 interaction with a target, for example
p27. These assays may comprise random screening of large libraries
of candidate substances; alternatively, the assays may be used to
focus on particular classes of compounds selected with an eye
towards structural attributes that are believed to make them more
likely to modulate the function of JAB1.
[0114] By function, it is meant that one may assay for the binding
of JAB1 to its target, such as p27; one may assay the ability of
JAB1 to function in a COP9 signalosome; one may assay for the
ability of JAB1 to translocate a target subcellularly, such as
between the nucleus and cytoplasm; or one may assay for any
function of JAB1 that directly or indirectly affects proliferation
of a cell in which it resides.
[0115] In specific embodiments, to identify a JAB1 modulator one
generally will determine the binding of JAB1 to its target in the
presence and absence of the candidate substance, a modulator
defined as any substance that alters the binding. For example, a
method generally comprises:
[0116] (a) providing a candidate modulator;
[0117] (b) admixing the candidate modulator with an isolated
compound or cell, or a suitable experimental animal;
[0118] (c) measuring one or more characteristics of the compound,
cell or animal in step (b); and
[0119] (d) comparing the characteristic measured in step (c) with
the characteristic of the compound, cell or animal in the absence
of said candidate modulator,
[0120] wherein a difference between the measured characteristics
indicates that said candidate modulator is, indeed, a modulator of
the compound, cell or animal.
[0121] Assays may be conducted in cell free systems, in isolated
cells, or in organisms including transgenic animals.
[0122] It will, of course, be understood that all the screening
methods of the present invention are useful in themselves
notwithstanding the fact that effective candidates may not be
identified. The invention provides methods for screening for such
candidates, not solely methods of finding them.
[0123] 1. Modulators
[0124] As used herein the term "candidate substance" refers to any
molecule that may potentially inhibit or enhance JAB1 activity. The
candidate substance may be a protein or fragment thereof, a small
molecule, or even a nucleic acid molecule. It may prove to be the
case that the most useful pharmacological compounds will be
compounds that are structurally related to the respective binding
domain of JAB1, such as the p27-binding domain of JAB1. Using lead
compounds to help develop improved compounds is know as "rational
drug design" and includes not only comparisons with known
inhibitors and activators, but predictions relating to the
structure of target molecules.
[0125] The goal of rational drug design is to produce structural
analogs of biologically active polypeptides or target compounds. By
creating such analogs, it is possible to fashion drugs that are
more active or stable than the natural molecules; that have
different susceptibility to alteration; or that may affect the
function of various other molecules. In one approach, one would
generate a three-dimensional structure for a target molecule, or a
fragment thereof. This could be accomplished by x-ray
crystallography, computer modeling or by a combination of both
approaches.
[0126] It also is possible to use antibodies to ascertain the
structure of a target compound activator or inhibitor. In
principle, this approach yields a pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein
crystallography altogether by generating anti-idiotypic antibodies
to a functional, pharmacologically active antibody. As a mirror
image of a mirror image, the binding site of anti-idiotype would be
expected to be an analog of the original antigen. The anti-idiotype
could then be used to identify and isolate peptides from banks of
chemically or biologically-produced peptides. Selected peptides
would then serve as the pharmacore. Anti-idiotypes may be generated
using the methods described herein for producing antibodies, using
an antibody as the antigen.
[0127] On the other hand, one may simply acquire, from various
commercial sources, for example, small molecule libraries that are
believed to meet the basic criteria for useful drugs in an effort
to "brute force" the identification of useful compounds. Screening
of such libraries, including combinatorially-generated libraries
(e.g., peptide libraries), is a rapid and efficient way to screen
large number of related (and unrelated) compounds for activity.
Combinatorial approaches also lend themselves to rapid evolution of
potential drugs by the creation of second, third and fourth
generation compounds modeled of active but otherwise undesirable
compounds.
[0128] Candidate compounds may include fragments or parts of
naturally-occurring compounds, or may be found as active
combinations of known compounds, which are otherwise inactive. It
is proposed that compounds isolated from natural sources, such as
animals, bacteria, fungi, plant sources, including leaves and bark,
and marine samples may be assayed as candidates for the presence of
potentially useful pharmaceutical agents. It will be understood
that the pharmaceutical agents to be screened could also be derived
or synthesized from chemical compositions or man-made compounds.
Thus, it is understood that the candidate substance identified by
the present invention may be peptide, polypeptide, polynucleotide,
small molecule inhibitors or any other compounds that may be
designed through rational drug design starting from known
inhibitors or stimulators.
[0129] Other suitable modulators include antisense molecules,
ribozymes, and antibodies (including single chain antibodies), each
of which would be specific for the target molecule. Such compounds
are described in greater detail elsewhere in this document. For
example, an antisense molecule that bound to a translational or
transcriptional start site, or splice junctions, would be ideal
candidate inhibitors.
[0130] In addition to the modulating compounds initially
identified, the inventors also contemplate that other sterically
similar compounds may be formulated to mimic the key portions of
the structure of the modulators. Such compounds, which may include
peptidomimetics of peptide modulators, may be used in the same
manner as the initial modulators.
[0131] An inhibitor according to the present invention may be one
that exerts its inhibitory or activating effect upstream,
downstream or directly on JAB1. Regardless of the type of inhibitor
or activator identified by the present screening methods, the
effect of the inhibition or activator by such a compound results
in, for example, inhibition of JAB1 binding to its target or
reduction in expression of JAB1 as compared to that observed in the
absence of the added candidate substance.
[0132] 2. In Vitro Assays
[0133] A quick, inexpensive and easy assay to run is an in vitro
assay. Such assays generally use isolated molecules, can be run
quickly and in large numbers, thereby increasing the amount of
information obtainable in a short period of time. A variety of
vessels may be used to run the assays, including test tubes,
plates, dishes and other surfaces such as dipsticks or beads.
[0134] One example of a cell free assay is a binding assay. While
not directly addressing function, the ability of a modulator to
bind to a target molecule in a specific fashion is strong evidence
of a related biological effect. For example, binding of a molecule
to a target may, in and of itself, be inhibitory, due to steric,
allosteric or charge-charge interactions. The target may be either
free in solution, fixed to a support, expressed in or on the
surface of a cell. Either the target or the compound may be
labeled, thereby permitting determining of binding. Usually, the
target will be the labeled species, decreasing the chance that the
labeling will interfere with or enhance binding. Competitive
binding formats can be performed in which one of the agents is
labeled, and one may measure the amount of free label versus bound
label to determine the effect on binding.
[0135] A technique for high throughput screening of compounds is
described in WO 84/03564. Large numbers of small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. Bound polypeptide is detected by
various methods.
[0136] 3. In Cyto Assays
[0137] The present invention also contemplates the screening of
compounds for their ability to modulate JAB1 expression in cells.
Various cell lines can be utilized for such screening assays,
including cells specifically engineered for this purpose. For
example, cells transfected with JAB1, such as cells comprising a
labeled JAB1 polypeptide or a JAB1 polynucleotide encoding a
detectable JAB1 polypeptide may be employed. The detectabiltiy of
JAB1 may comprise color or fluorescence, for example.
[0138] Depending on the assay, culture may be required. The cell is
examined using any of a number of different: physiologic assays.
Alternatively, molecular analysis may be performed, for example,
looking at protein expression, mRNA expression (including
differential display of whole cell or polyA RNA) and others. In
specific embodiments, those of skill in the art may refer to in
cyto assays as in vivo assays.
[0139] 4. In Vivo Assays
[0140] In vivo assays may involve the use of various animal models,
including non-human transgenic animals that have been engineered to
have specific defects, or carry markers that can be used to measure
the ability of a candidate substance to reach and effect different
cells within the organism. Due to their size, ease of handling, and
information on their physiology and genetic make-up, mice are a
preferred embodiment, especially for transgenics. However, other
animals are suitable as well, including rats, rabbits, hamsters,
guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs,
cows, horses and monkeys (including chimps, gibbons and baboons).
Assays for modulators may be conducted using an animal model
derived from any of these species, and mammals are preferred.
[0141] In such assays, one or more candidate substances are
administered to an animal, and the ability of the candidate
substance(s) to alter one or more characteristics, as compared to a
similar animal not treated with the candidate substance(s),
identifies a modulator. The characteristics may be any of those
discussed above with regard to the function of a particular
compound (e.g., enzyme, receptor, hormone) or cell (e.g., growth,
tumorigenicity, survival), or instead a broader indication such as
behavior, anemia, immune response, etc.
[0142] Thus, the present invention in some embodiments provides
methods of screening for a candidate substance that reduces JAB1
expression or inhibits binding of JAB1 to a target, such as p27. In
some specific embodiments, the present invention is directed to a
method for determining the ability of a candidate substance to
inhibiting binding of JAB1 to a target, generally including the
steps of administering a candidate substance to an animal; and
determining the ability of the candidate substance to reduce the
binding.
[0143] Treatment of these animals with test compounds will involve
the administration of the compound, in an appropriate form, to the
animal. Administration will be by any route that could be utilized
for clinical or non-clinical purposes, including but not limited to
oral, nasal, buccal, or even topical. Alternatively, administration
may be by intratracheal instillation, bronchial instillation,
intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous injection. Specifically contemplated routes are
systemic intravenous injection, regional administration via blood
or lymph supply, or directly to an affected site.
[0144] Determining the effectiveness of a compound in vivo may
involve a variety of different criteria. Also, measuring toxicity
and dose response can be performed in animals in a more meaningful
fashion than in in vitro or in cyto assays.
[0145] Thus, creens for agents that inhibit the binding of JAB1 to
its target, such as p27, may be of any suitable kind. Multiple
screens may be used in succession to narrow a pool of candidate
inhibitors. In specific aspects of the invention, an in vitro
screen may include ELISA, such as to monitor by dye visualization
the absence of binding of p27 to JAB1 in the presence of a
potential inhibitor. An example of an in vivo screen is a
cell-based assay, in which binding of JAB1 to p27 in the presence
of potential inhibitors is visualized from within the cell, such as
by fluorescence or X-ray. Another screen utilizes the p27 binding
domain of JAB1 and/or the JAB1 binding domain of p27, for example,
immobilized to a substrate such that when a potential inhibitor
binds the immobilized domain, the binding is visualized, such as by
presence or absence of color or fluorescence, for example. Finally,
another screen that may be utilized is a two-hybrid screen wherein
the JAB1 binding domain of p27 or the p27 binding domain of JAB1
are used as bait to identify peptides or polypeptides that bind at
least in part thereto.
[0146] B. Two-Hybrid Screen
[0147] In yet another embodiment, proteins that interact with JAB1
may be identified by using a yeast two-hybrid system or a
co-immunoprecipitation assay. The yeast two-hybrid system may be
used to identify new protein targets for pharmaceutical
intervention, determine the specific residues involved in a given
protein-protein interaction, and find compounds that modulate
protein interactions. The yeast two-hybrid system can also be used
to identify previously unknown proteins that interact with a target
protein by screening a two-hybrid library. The yeast two-hybrid
system is outlined in U.S. Pat. No. 5,283,173 (incorporated herein
by reference), and: is a technique well known to those of skill in
the art. Briefly, the method is designed to detect an interaction
between a first test protein and a second test protein, in vivo,
using reconstitution of the activity of a transcriptional
activator. Two chimeric proteins that express hybrid proteins are
prepared. The first hybrid protein contains the DNA-binding domain
of a transcriptional activator fused to the first test protein,
while the second hybrid protein contains a transcriptional
activation domain fused to the second test protein. If the two test
proteins interact, the two domains of the transcriptional activator
are brought into close proximity, resulting in the transcription of
a marker gene that contains a binding site for the DNA-binding
domain. An assay can be performed to detect activity of the marker
gene.
[0148] All yeast two-hybrid systems share a set of common elements:
1) a plasmid that directs the synthesis of a "bait"; the bait is a
known protein which is fused to a DNA binding domain, 2) one or
more reporter genes ("reporters") with upstream DNA binding sites
for the bait, and 3) a plasmid that directs the synthesis of
proteins fused to activation domains and other useful moieties
("activation tagged proteins" or "prey"). All current systems
direct the synthesis of proteins that carry the activation domain
at the amino terminus of the fusion, facilitating the expression of
open reading frames encoded by cDNAs. DNA binding domains used in
the yeast two-hybrid systems include the native E. coli LexA
repressor protein (Gyuris et al., 1993), and the GAL4 protein
(Chien et al., 1991). Some reporter genes that may be utilized in
the yeast system included HIS3, LEU2, and lacZ.
[0149] Although most two-hybrid systems use yeast, mammalian
variants may also be utilized. In one system, interaction of
activation tagged VP16 derivatives with a Gal4-derived bait drives
expression of reporters that direct the synthesis of Hygromycin B
phosphotransferase, Chloramphenicol acetyltransferase, or CD4 cell
surface antigen (Fearon et al., 1992). In another system,
interaction of VP16-tagged derivatives with Gal4-derived baits
drives the synthesis of SV40 T antigen, which in turn promotes the
replication of the prey plasmid, because the plasmid carries a SV40
origin (Vasavada et al., 1991).
[0150] Protein-protein interactions may also be studied by using
biochemical techniques such as cross-linking,
co-immunoprecipitation, and co-fractionation by chromatography,
which are well known to those skilled in the art. The
co-immunoprecipitation technique consists of (i) generating a cell
lysate; (ii) adding an antibody to the cell lysate; (iii)
precipitating and washing the antigen; and (iv) eluting and
analyzing the bound proteins (Phizicky and Fields, 1995). The
antigen used to generate the antibody can be a purified protein, or
a synthetic peptide coupled to a carrier. Both monoclonal and
polyclonal antibodies can be utilized in co-immunoprecipitation, or
alternatively, a protein can be used which carries an epitope tag
recognized by a commercially available antibody.
[0151] In specific embodiments of the present invention, JAB1 two
hybrid baits identify enolase and glucose-6-phosphate dehydrogenase
as interactors.
[0152] C. Screening for Modulators of JAB1 Expression
[0153] In some aspects of the invention, compositions that modulate
JAB1 expression are screened. In specific embodiments, these
modulators decrease JAB1 expression, although in alternative
embodiments the modulators increase JAB1 expression. Particular
characteristics that may be screened for include the ability of the
modulator to indirectly or directly decrease JAB1 expression, such
as by negatively affecting transcription of JAB1 or translation of
a JAB1 message. In particular embodiments, the modulator physically
binds to a JAB1 transcript, thereby inhibiting completion of its
transcription, targeting the transcript for degradation, or
inhibiting the translation of the transcript into a polypeptide.
The physical binding may comprise hybridization of the modulator to
the transcript through at least some complementary sequences.
[0154] An exemplary method of screening candidate substances for
their ability to modulate JAB1 expression may comprise the steps of
constructing or obtaining a JAB1 expression vector; transfecting
cells of interest with the JAB1 expression vector; assaying for
JAB1 expression level or the ratio of expression of JAB1 to p27 in
the transfected cells at time=1; treating the transfected cells
with a therapeutic agent at time=2; assaying for JAB1 expression
level or the ratio of expression of JAB1 to p27 at time=3;
comparing the expression level of JAB1 or the ratio of expression
of JAB1 to p27 at time=1 and time=3; and determining the efficacy
of the therapeutic agent to modulate JAB1 expression. The present
invention also provides a recombinant cell line suitable for use in
the exemplary method. The candidate therapeutic agent identified to
modulate JAB1 expression according to a screening assay described
herein may have utility in the treatment of proliferative
disorders, particularly cancer, such as breast cancer or lymphoma.
In addition, by choosing specific cell types for transfection, the
screening method can be used to identify the types of cancers that
are affected by JAB1 expression, and which may be treated by
altering JAB1 expression levels.
[0155] Transformation or transfection techniques are well known in
the art and can be found generally in Maniatis, Fritsch and
Sambrook, Molecular Cloning: A Laboratory Manual. In general, when
the host is a eukaryote, methods of transfection of DNA include
calcium phosphate co-precipitates; or conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or transduction using viral-based
vectors. To monitor transfection efficiency, the cells may be
cotransformed with a DNA molecule encoding a gene for a selectable
phenotype, such as the herpes simplex thymidine kinase, green
fluorescent protein and the like.
[0156] The transformed JAB1 cells used in certain of the assays
according to the present invention may be any cells of interest
including any cells from normal human tissues such as liver, heart,
kidney, skin, prostate, and the like. Also, the transformed JAB1
cells used in aspects of the present invention may be those from
solid tumors and leukemias, including: apudoma, choristoma,
branchioma, malignant carcinoid syndrome, carcinoid heart disease,
carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce,
ductal, Ehrlich tumor, in situ, Krebs 2, Merkel cell, mucinous,
non-small cell lung, oat cell, papillary, scirrhous, bronchiolar,
bronchogenic, squamous cell, and transitional cell), histiocytic
disorders, leukemia (e.g., B cell, mixed cell, null cell, T cell,
T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocytic
chronic, mast cell, and myeloid), hystiocytosis malignant, Hodgkin
disease, immunoproliferative small, non-Hodgkin lymphoma,
plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma,
chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell
tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma,
myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma,
adenofibroma, adenolymphoma, carcinosarcoma, chordoma,
cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma,
mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma,
teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma,
cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,
cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,
hidradenoma, islet cell tumor, Leydig cell tumor, papilloma,
Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma,
myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma,
ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma,
neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma,
neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma,
angiolymphoid hyperplasia with eosinophilia, angioma sclerosing,
angiomatosis, glomangioma, hemangioendothelioma, hemangioma,
hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,
lymphangiosarcoma, pheochromocytoma, pinealoma, carcinosarcoma,
chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma,
hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma,
lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma,
rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and
mast cell), neoplasms (e.g., bone, breast, digestive system,
colorectal, liver, pancreatic, pituitary, testicular, orbital, head
and neck, central nervous system, acoustic, pelvic, respiratory
tract, and urogenital), neurofibromatosis, and cervical dysplasia,
and other cells that have become immortalized or transformed.
[0157] With respect to a representative method for the preparation
of a transgenic mouse, cloned recombinant or synthetic DNA
sequences or DNA segments encoding a JAB1 gene product are injected
into fertilized mouse eggs (e.g. an embryo). The injected eggs are
implanted in pseudo pregnant females and are grown to term to
provide transgenic mice whose cells express a JAB1 gene product.
Because the expression of JAB1 may be deleterious to the animal,
JAB1 expression in the chimera or transgenic offspring produced by
germ line transmission of the DNA sequence may be established
through incorporation of the JAB1 gene under the control of an
inducible promoter. The expression of the JAB1 protein is then
induced by treatment of the chimera or transgenic offspring thereof
with the inducing agent.
[0158] V. Therapeutics
[0159] A particularly important aspect of the present invention is
treatment of cancers characterized by increased JAB1 expression
compared to expression in normal tissue or a high ratio of JAB1/p27
compared to a normal cell or tissue. JAB1-inhibiting agents may be
provided to inhibit the binding of JAB1 to a target, or the
expression of JAB1 itself may be useful as a therapeutic target. In
specific embodiments, gene therapy vectors and substances that
inhibit expression of JAB1 are used as therapeutics to treat
cancer. An individual with cancer at least in part resulting
directly or indirectly from a high level of JAB1 expression or
otherwise characterized by a high level of expression or an
abnormal ratio of JAB1 expression to p27 expression is identified.
One or more JAB1 expression-reducing agents is administered to the
individual.
[0160] In particular embodiments, a therapeutic composition
comprises a composition identified by any suitable screen, such as
a screen described herein. Exemplary JAB1-inhibiting substances
including antisense and RNAi agent expression constructs of the
present invention, which may be used in the treatment of cancer,
including solid tumors, breast cancers, pituitary carcinomas,
lymphomas, prostate, pancreatic, colon, and lung, for example. In
conjunction with the inventive therapy described herein, there may
be additional cancer therapy provided to an individual, such as
prior to the JAB1-associated treatment, during the JAB1-associated
treatment, or subsequent to the JAB1-associated treatment.
Additional cancer therapies include chemotherapy, hormone therapy,
drug therapy, radiation, surgery, gene therapy, or immunotherapy,
for example.
[0161] Therapeutics that inhibit expression of JAB1 can be
identified by the screening assays described herein. The method of
administration shall depend on the nature of the therapeutic, and a
skilled artisan is aware of methods and reagents suitable for
determining same. For example, a JAB1 nucleic acid can be used as a
tool for gene therapy in humans to treat cancer. Exemplary gene
therapy methods, including liposomal transfection of nucleic acids
into host cells, are described in U.S. Pat. Nos. 5,279,833;
5,286,634; 5,399,346; 5,646,008; 5,651,964; 5,641,484; and
5,643,567, the contents of each of which are herein incorporated by
reference. Exemplary JAB1 nucleic acids from which therapeutic
antisense RNAs may be derived include GenBank Accession No. U65928
(SEQ ID NO:8) from the World Wide Web site of the National Center
for Biotechnology Information or the following GenBank Accession
Nos.: NM.sub.--006837 (SEQ ID NO:9); BC001859; (SEQ ID NO:14);
BC007272 (SEQ ID NO:15); and BC001187 (SEQ ID NO:16). For any
embodiments of the invention wherein a JAB1 polypeptide is
utilized, an example of such may be obtained from GenBank No.
NP.sub.--006828 (SEQ ID NO:10).
[0162] In one aspect of a gene therapy embodiment, antisense RNAs
against JAB1 provide treatment of the cancer caused by JAB1
expression. Therapeutic methods utilizing antisense
oligonucleotides have been described in the art, for example in
U.S. Pat. Nos. 5,627,158 and 5,734,033, the contents of each of
which are herein incorporated by reference. The antisense RNAs can
be delivered to the tumor or cancer cells directly or may be
expressed in the cells by a gene therapy vector. Antisense gene
therapy vectors include promoters, terminators and possibly other
genetic elements for expression. Exemplary promoters, terminators
and the like are described herein infra in the discussion of RNAi
agents.
[0163] In general, the specific hybridization of an antisense RNA
with its target nucleic acid interferes with the normal function of
the target nucleic acid. The functions of RNA that may be
interfered with include all vital functions such as, for example,
translocation of the RNA to the site of protein translation,
translation of protein, splicing of the RNA to yield one or more
mRNA species, and catalytic activity which may be engaged in or
facilitated by the RNA. The overall effect of such interference
with target nucleic acid function is modulation of the expression
of the JAB1 protein. In the context of the present invention,
inhibition of JAB1 is the preferred form of modulation of gene
expression and mRNA is the preferred target.
[0164] "Targeting" an antisense compound to a particular nucleic
acid is a multistep process. The process usually begins with the
identification of a nucleic acid sequence whose function is to be
modulated, in this case, the target is a nucleic acid molecule
encoding JAB1. The targeting process also includes determination of
a site or sites within the JAB1 gene for the antisense interaction
to occur such that the desired effect--inhibition of expression of
the protein--will result. A preferred intragenic site may be the
region encompassing the translation initiation or termination codon
of the open reading frame (ORF) of the gene.
[0165] Alternatively, the ORF or. "coding region," which is the
region between the translation initiation codon and the translation
termination codon, is also a region which may be targeted
effectively. Other target regions include the 5' untranslated
region (5'UTR), the portion of an mRNA in the 5' direction from the
translation initiation codon including nucleotides between the 5'
cap site and the translation initiation codon of the mRNA, and the
3' untranslated region (3'UTR), the portion of an mRNA in the 3'
direction from the translation termination codon including
nucleotides between the translation termination codon and 3' end of
an mRNA. The 5' cap of an mRNA comprises an N7-methylated guanosine
residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage. The 5' cap region of an mRNA is considered to
include the 5' cap structure itself as well as the first 50 or so
nucleotides adjacent to the cap. The 5' cap region may also be a
preferred target region. Also, mRNA splice sites, i.e., intron-exon
junctions, also may be preferred target regions, and are
particularly useful in situations where aberrant splicing is
implicated in disease, or where an overproduction of a particular
mRNA splice product is implicated in disease.
[0166] Once one or more target sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect. "Hybridization"means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. It is understood in the art that the sequence of
an antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired;
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and in the case of in vitro assays, under
conditions in which the assays are performed.
[0167] A. Antisense RNA, RNAi, and siRNA
[0168] In one aspect of the present invention, interfering RNAs are
used. RNA interference (RNAi) is a phenomenon describing
double-stranded (ds)RNA-dependent gene specific
post-transcriptional silencing. Initial attempts to harness this
phenomenon for experimental manipulation of mammalian cells were
foiled by a robust and nonspecific antiviral defense mechanism
activated in response to long dsRNA molecules; however, the field
was significantly advanced upon the demonstration that synthetic
duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in
mammalian cells without invoking generic antiviral defense
mechanisms. As a result, small-interfering RNAs (siRNAs) have
become powerful tools to dissect gene function. The chemical
synthesis of small RNAs to be delivered directly to cells is one
avenue that has produced promising results; on the other hand,
numerous groups have also sought the development of DNA-based
vectors capable of generating siRNA within cells.
[0169] The sequences for the RNAi agents, such as the siRNAs, are
selected based upon the genetic sequence of the target JAB1 nucleic
acid sequence; and preferably are based on regions of target
nucleic acid sequences that are conserved. As oncogenes are known
to mutate rapidly, selection of conserved sequences is likely to
preserve the efficacy of the RNAi over time.
[0170] In general, inhibition of target sequences by RNAi requires
a high degree of sequence homology between the target sequence and
the sense strand of the RNAi molecules. In some embodiments, such
homology is higher than about 70%, and may be higher than about
75%. Preferably, homology is higher than about 80%, and is higher
than 85% or even 90%. More preferably, sequence homology between
the target sequence and the sense strand of the RNAi is higher than
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
[0171] In addition to selecting the RNAi sequences having a high
degree of homology to conserved regions of a JAB1 target sequence,
selection of the RNAi sequences may be based on other factors.
Despite a number of attempts to devise selection criteria for
identifying sequences that are effective for RNAi based on features
of the desired target sequence (e.g., percent GC content, position
from the translation start codon, thermodynamic pairing criteria),
it is presently not possible to predict with much degree of
confidence which of the myriad possible candidate RNAi sequences
that correspond to a desired segment of the JAB1 sequence will, in
fact, elicit an RNA silencing response. Instead, individual
specific candidate RNAi polynucleotide sequences typically are
generated and tested.
[0172] There is no particular limitation in the length of the RNAi
agents of the present invention as long as they are effective in
inhibiting JAB1 expression. The RNAi agents can be, for example, 10
to 50 bp in length, preferably 12 to 40 bp in length, and are more
preferably 15 to 33 bp in length. The double-stranded RNA portions
of RNA is may be completely homologous, or may contain non-paired
portions due to sequence mismatch (the corresponding nucleotides on
each strand are not complementary), bulge (lack of a corresponding
complementary nucleotide on one strand), and the like. Such
non-paired portions can be tolerated to the extent that they do not
significantly interfere with RNAi duplex formation or efficacy. In
embodiments wherein siRNAs are employed, these molecules are
preferably about 21 nt in length.
[0173] The termini of an RNAi agents according to the present
invention may be blunt or cohesive (overhanging) as long as the
RNAi effectively silences the JAB1 target gene. The cohesive
(overhanging) end structure is not limited only to a 3' overhang,
but a 5' overhanging structure may be included as long as the
resulting RNAi is capable of inhibiting the expression of JAB1. In
addition, the number of overhanging nucleotides may be any number
as long as the resulting RNAi agent is capable of inducing the RNAi
effect. For example, if present, the overhang may consist of 1 to 8
nucleotides, preferably it consists of 2 to 4 nucleotides.
[0174] As stated, RNAi polynucleotide sequences (RNAi agents) may
be delivered directly to the cell or tissue, or may be expressed in
the cell by an expression vector. Various chemical modifications
have been made to short-interfering RNAs (siRNAs) for direct
delivery into a cell or tissue to stabilize and optimize the
biochemical properties required for RNA interference (RNAi).
Modifications at the 2'-position of pentose sugars in siRNAs showed
the 2'-OHs were not required for RNAi, indicating that RNAi
machinery does not require the 2'-OH for recognition of siRNAs and
catalytic ribonuclease activity of RNA-induced silencing complexes
(RISCs) does not involve the 2'-OH of guide antisense RNA. However,
2' modifications increased the persistence of RNAi as compared with
wild-type siRNAs.
[0175] RNAi also has been induced with chemical modifications that
stabilize the interactions between A-U base pairs, demonstrating
that these types of modifications may enhance mRNA targeting
efficiency in allele-specific RNAi. Modifications altering the
structure of the A-form major groove of antisense siRNA-mRNA
duplexes abolished RNAi, suggesting that the major groove of these
duplexes is required for recognition by activated RISC. Comparative
analysis of the stability and RNAi activities of
chemically-modified single-stranded antisense RNA and duplex siRNA
suggested that some catalytic mechanism(s) other than siRNA
stability were linked to RNAi efficiency. In addition, modified or
mismatched ribonucleotides incorporated at internal positions in
the 5' or 3' half of the siRNA duplex, as defined by the antisense
strand, shows that the integrity of the 5' half, but not the 3'
half, of the siRNA structure is important for RNAi, highlighting
the asymmetric nature of siRNA recognition for initiation of
unwinding.
[0176] In addition, it has been found that RNA duplexes containing
either phosphodiester or varying numbers of phosphorothioate
linkages are remarkably stable during prolonged incubations in
serum. Treatment of cells with RNA duplexes containing
phosphorothioate linkages leads to selective inhibition of gene
expression. RNAi also tolerates the introduction of
2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides.
Introduction of LNA nucleotides also increases substantially the
thermal stability of modified RNA duplexes without compromising the
efficiency of RNAi. Other modifications are known in the art and
are currently in development.
[0177] As an alternative to directly delivering antisense or RNAi
agents to cells, the antisense or RNAi agents may be genetically
engineered as part of a gene therapy vector where the vector is
used to transform the cancer cells and express the antisense or
RNAi agents in the cells. The constructs into which the antisense
or RNAi agent is inserted and used for high efficiency transduction
and expression of the RNAis in various cell types preferably are
derived from viruses and are compatible with viral delivery.
Generation of the construct can be accomplished using any suitable
genetic engineering techniques well known in the art, including
without limitation, the standard techniques of PCR, oligonucleotide
synthesis, restriction endonuclease digestion, ligation,
transformation, plasmid purification, and DNA sequencing. The
construct preferably comprises, for example, sequences necessary to
package the antisense or RNAi agent expression construct into viral
particles and/or sequences that allow integration of the antisense
or RNAi agent expression construct into the target cell genome. The
viral construct also may contain genes that allow for replication
and propagation of virus, though in preferred embodiments such
genes will be supplied in trans. Additionally, the viral construct
may contain genes or genetic sequences from the genome of any known
organism incorporated in native form or modified. For example, the
preferred viral construct comprises sequences useful for
replication of the construct in bacteria.
[0178] The construct also may contain additional genetic elements.
The types of elements that may be included in the construct are not
limited in any way and may be chosen by one with skill in the art.
For example, additional genetic elements may include a reporter
gene, such as one or more genes for a fluorescent marker protein
such as GFP or RFP; an easily assayed enzyme such as
beta-galactosidase, luciferase, beta-glucuronidase, chloramphenical
acetyl transferase or secreted embryonic alkaline phosphatase; or
proteins for which immunoassays are readily available such as
hormones or cytokines. Other genetic elements that may find use in
embodiments of the present invention include those coding for
proteins which confer a selective growth advantage on cells such as
adenosine deaminase, aminoglycodic phosphotransferase,
dihydrofolate reductase, hygromycin-B-phosphotransferase, or those
coding for proteins that provide a biosynthetic capability missing
from an auxotroph. If a reporter gene is included along with the
antisense or RNAi agent expression cassette, an internal ribosomal
entry site (IRES) sequence can be included. Preferably, the
additional genetic elements are operably linked with and controlled
by an independent promoter/enhancer.
[0179] A "promoter" or "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase in a cell and initiating
transcription of a polynucleotide or polypeptide coding sequence
such as messenger RNA, ribosomal RNAs, small nuclear of nucleolar
RNAs or any kind of RNA transcribed by any class of any RNA
polymerase I, II or III. In some embodiments, promoters of variable
strength may be employed, but preferably for expression of JAB1 in
cells, strong promoters are used. Use of strong promoters (such as
a Pol III-type promoter) not only expresses JAB1 at a high level,
but may synergistically work to inhibit cancer cell progression by
taxing the cell, by, e.g., depleting the pool of available
nucleotides or other cellular components needed for transcription
of other genes. For JAB1 promoter embodiments, see FIG. 20.
[0180] In addition, tissue-specific or cell-specific promoters may
be employed. The term "tissue specific" as it applies to a promoter
refers to a promoter that is capable of directing selective
expression of a nucleotide sequence of interest to a specific type
of tissue (e.g., liver) in the relative absence of expression of
the same nucleotide sequence of interest in a different type of
tissue (e.g., brain). Such tissue specific promoters include
promoters such as lck, myogenin, or thyl. The term "cell-specific"
as applied to a promoter refers to a promoter which is capable of
directing selective expression of a nucleotide sequence of interest
in a specific type of cell in the relative absence of expression of
the same nucleotide sequence of interest in a different type of
cell within the same tissue (see, e.g., Higashibata, et al., J.
Bone Miner. Res. Jan 19(1):78-88 (2004); Hoggatt, et al., Circ.
Res., Dec. 91(12):1151-59 (2002); Sohal, et al., Circ. Res. Jul
89(1):20-25 (2001); and Zhang, et al., Genome Res. Jan 14(1):79-89
(2004)). The term "cell-specific" when applied to a promoter also
means a promoter capable of promoting selective expression of a
nucleotide sequence of interest in a region within a single tissue.
Alternatively, promoters may be constitutive or regulatable.
Additionally, promoters may be modified so as to possess different
specificities.
[0181] The term "constitutive" when made in reference to a promoter
means that the promoter is capable of directing transcription of an
operably linked nucleic acid sequence in the absence of a stimulus
(e.g., heat shock, chemicals, light, etc.). Typically, constitutive
promoters are capable of directing expression of a coding sequence
in substantially any cell and any tissue. The promoters used to
transcribe the JAB1 RNAi species preferably are constitutive
promoters, such as the promoters for ubiquitin, CMV, .beta.-actin,
histone H4, EF-1 alfa or pgk genes controlled by RNA polymerase II,
or promoter elements controlled by RNA polymerase I. In preferred
embodiments, promoter elements controlled by RNA polymerase III are
used, such as the U6 promoters (U6-1, U6-8, U6-9, e.g.), H1
promoter, 7SL promoter, the human Y promoters (hY1, hY3, hY4 (see
Maraia, et al., Nucleic Acids Res 22(15):3045-52 (1994)) and hY5
(see Maraia, et al., Nucleic Acids Res 24(18):3552-59 (1994)), the
human MRP-7-2 promoter, Adenovirus VA1 promoter, human tRNA
promoters, the 5s ribosomal RNA promoters, as well as functional
hybrids and combinations of any of these promoters.
[0182] Alternatively in some embodiments it may be optimal to
select promoters that allow for inducible expression of the JAB1
RNAi species. A number of systems for the inducible expression
using such promoters are known in the art, including but not
limited to the tetracycline responsive system and the lac
operator-repressor system (see WO 03/022052 A1; and US 2002/0162126
A1), the ecdyson regulated system, or promoters regulated by
glucocorticoids, progestins, estrogen, RU-486, steroids, thyroid
hormones, cyclic AMP, cytokines, the calciferol family of
regulators, or the metallothionein promoter (regulated by inorganic
metals).
[0183] As stated, the JAB1 RNAi coding regions of the RNAi
expression vector are operatively linked to terminator elements. In
one embodiment, the terminators comprise stretches of four or more
thymidine residues. In another embodiment, the terminator elements
used are matched to the promoter elements from the gene from which
the terminator is derived. Such terminators include the SV40 poly
A, the Ad VA1 gene, the 5S ribosomal RNA gene, and the terminators
for human t-RNAs. In addition, promoters and terminators may be
mixed and matched, as is commonly done with RNA pol II promoters
and terminators.
[0184] The termini of an RNAi species according to the present
invention may be blunt or cohesive (overhanging) as long as the
RNAi effectively silences the target gene. The cohesive
(overhanging) end structure is not limited only to a 3' overhang,
but a 5' overhanging structure may be included as long as the
resulting RNAi is capable of inducing the RNAi effect. In addition,
the number of overhanging nucleotides may be any number as long as
the resulting RNAi is capable of inducing the RNAi effect. For
example, if present, the overhang may consist of 1 to 8
nucleotides, preferably it consists of 2 to 4 nucleotides.
[0185] B. Delivery Systems
[0186] Any delivery system suitable in the art may be employed to
provide to a cancer cell of an individual a therapeutic composition
in accordance with the present invention. Vectors may be utilized,
including viral or non-viral vectors. One vector that may be useful
comprises one or more encapsulated cells expressing the therapeutic
compound (an siRNA, for example), which could be used also as
implant in solid tumor (e.g. for brain tumor, spinal cord, etc.) to
treat cancer.
[0187] A viral delivery system based on any appropriate virus may
be used to deliver the antisense or RNAi agent expression
constructs of the present invention. In addition, hybrid viral
systems may be of use. The choice of viral delivery system will
depend on various parameters, such as the tissue targeted for
delivery, transduction efficiency of the system, pathogenicity,
immunological and toxicity concerns, and the like. Given the
diversity of cancers and proliferative disease that are amenable to
interference by the antisense or RNAi agent expression constructs
of the present invention, it is clear that there is no single viral
system that is suitable for all applications. When selecting a
viral delivery system to use in the present invention, it is
important to choose a system where the antisense or RNAi agent
expression construct-containing viral particles are preferably: 1)
reproducibly and stably propagated; 2) able to be purified to high
titers; and 3) able to mediate targeted delivery (delivery of the
antisense or RNAi agent expression construct to the tissue or organ
of interest without widespread dissemination).
[0188] In general, the five most commonly used classes of viral
systems used in gene therapy can be categorized into two groups
according to whether their genomes integrate into host cellular
chromatin (oncoretroviruses and lentiviruses) or persist in the
cell nucleus predominantly as extrachromosomal episomes
(adeno-associated virus, adenoviruses and herpesviruses). This
distinction is an important determinant of the suitability of each
vector for particular applications; non-integrating vectors can,
under certain circumstances, mediate persistent gene expression in
non-proliferating cells, but integrating vectors are the tools of
choice if stable genetic alteration needs to be maintained in
dividing cells, particularly in the present invention where the
target cells are rapidly proliferating cancer cells.
[0189] For example, in one embodiment of the present invention,
viruses from the Parvoviridae family are utilized. The Parvoviridae
is a family of small single-stranded, non-enveloped DNA viruses
with genomes approximately 5000 nucleotides long. Included among
the family members is adeno-associated virus (AAV), a dependent
parvovirus that by definition requires co-infection with another
virus (typically an adenovirus or herpesvirus) to initiate and
sustain a productive infectious cycle. In the absence of such a
helper virus, AAV is still competent to infect or transduce a
target cell by receptor-mediated binding and internalization,
penetrating the nucleus in both non-dividing and dividing
cells.
[0190] Once in the nucleus, the virus uncoats and the transgene is
expressed from a number of different forms--the most persistent of
which are circular monomers. AAV will integrate into the genome of
1-5% of cells that are stably transduced (Nakai, et al., J. Virol.
76:11343-349 (2002). Expression of the transgene can be
exceptionally stable and in one study with AAV delivery of Factor
IX, a dog model continues to express therapeutic levels of the
protein 4.5 years after a single direct infusion with the virus.
Because progeny virus is not produced from AAV infection in the
absence of helper virus, the extent of transduction is restricted
only to the initial cells that are infected with the virus. It is
this feature which makes AAV a non-preferred gene therapy vector
for the present invention. However, unlike retrovirus, adenovirus,
and herpes simplex virus, AAV appears to lack human pathogenicity
and toxicity (Kay, et al., Nature. 424: 251 (2003) and Thomas, et
al., Nature Reviews Genetics 4:346-58 (2003)).
[0191] Typically, the genome of AAV contains only two genes. The
"rep" gene codes for at least four separate proteins utilized in
DNA replication. The "cap" gene product is spliced differentially
to generate the three proteins that comprise the capsid of the
virus. When packaging the genome into nascent virus, only the
Inverted Terminal Repeats (ITRs) are obligate sequences; rep and
cap can be deleted from the genome and be replaced with
heterologous sequences of choice. However, in order produce the
proteins needed to replicate and package the AAV-based heterologous
construct into nascent virion, the rep and cap proteins must be
provided in trans. The helper functions normally provided by
co-infection with the helper virus, such as adenovirus or
herpesvirus mentioned above, also can be provided in trans in the
form of one or more DNA expression plasmids. Since the genome
normally encodes only two genes it is not surprising that, as a
delivery vehicle, AAV is limited by a packaging capacity of 4.5
single stranded kilobases (kb). However, although this size
restriction may limit the genes that can be delivered for
replacement gene therapies, it does not adversely affect the
packaging and expression of shorter sequences such as RNAi nucleic
acids.
[0192] However, technical hurdles must be addressed when using AAV
as a vehicle for antisense or RNAi agent expression constructs. For
example, various percentages of the human population may possess
neutralizing antibodies against certain AAV serotypes. However,
since there are several AAV serotypes, some of which the percentage
of individuals harboring neutralizing antibodies is vastly reduced,
other serotypes can be used or pseudo-typing may be employed. There
are at least eight different serotypes that have been
characterized, with dozens of others which have been isolated but
have been less well described. Another limitation is that as a
result of a possible immune response to AAV, AAV-based therapy may
only be administered once; however, use of alternate, non-human
derived serotypes may allow for repeat administrations.
Administration route, serotype, and composition of the delivered
genome all influence tissue specificity.
[0193] Another limitation in using unmodified AAV systems with the
antisense or RNAi agent expression constructs is that transduction
can be inefficient. Stable transduction in vivo may be limited to
5-10% of cells. Yet, different methods are known in the art to
boost stable transduction levels. One approach is utilizing
pseudotyping, where AAV-2 genomes are packaged using cap proteins
derived from other serotypes. One group of investigators
exhaustively pseudotyped AAV-2 with AAV-1, AAV-3B, AAV-4, AAV-5,
and AAV-6 for tissue culture studies. The highest levels of
transgene expression were induced by virion which had been
pseudotyped with AAV-6; producing nearly 2000% higher transgene
expression than AAV-2. Thus, the present invention contemplates use
of a pseudotyped AAV virus to achieve high transduction levels,
with a corresponding increase in the expression of the RNAi
multiple-promoter expression constructs.
[0194] Another viral delivery system useful with the
multiple-promoter RNAi expression constructs of the present
invention is a system based onviruses from the family Retroviridae.
Retroviruses comprise single-stranded RNA animal viruses that are
characterized by two unique features. First, the genome of a
retrovirus is diploid, consisting of two copies of the RNA. Second,
this RNA is transcribed by the virion-associated enzyme reverse
transcriptase into double-stranded DNA. This double-stranded DNA or
provirus can then integrate into the host genome and be passed from
parent cell to progeny cells as a stably-integrated component of
the host genome.
[0195] In some embodiments, lentiviruses are the preferred members
of the retrovirus family for use in the present invention.
Lentivirus vectors are often pseudotyped with vesicular stomatitis
virus glycoprotein (VSV-G), and have been derived from the human
immunodeficiency virus (HIV), the etiologic agent of the human
acquired immunodeficiency syndrome (AIDS); visan-maedi, which
causes encephalitis (visna) or pneumonia in sheep; equine
infectious anemia virus (EIAV), which causes autoimmune hemolytic
anemia and encephalopathy in horses; feline immunodeficiency virus
(FIV), which causes immune deficiency in cats; bovine
immunodeficiency virus (BIV) which causes lymphadenopathy and
lymphocytosis in cattle; and simian immunodeficiency virus (SIV),
which causes immune deficiency and encephalopathy in non-human
primates. Vectors that are based on HIV generally retain <5% of
the parental genome, and <25% of the genome is incorporated into
packaging constructs, which minimizes the possibility of the
generation of reverting replication-competent HIV. Biosafety has
been further increased by the development of self-inactivating
vectors that contain deletions of the regulatory elements in the
downstream long-terminal-repeat sequence, eliminating transcription
of the packaging signal that is required for vector
mobilization.
[0196] Reverse transcription of the retroviral RNA genome occurs in
the cytoplasm. Unlike C-type retroviruses, the lentiviral cDNA
complexed with other viral factors--known as the pre-initiation
complex--is able to translocate across the nuclear membrane and
transduce non-dividing cells. A structural feature of the viral
cDNA--a DNA flap--seems to contribute to efficient nuclear import.
This flap is dependent on the integrity of a central polypurine
tract (cPPT) that is located in the viral polymerase gene, so most
lentiviral-derived vectors retain this sequence. Lentiviruses have
broad tropism, low inflammatory potential, and result in an
integrated vector. The main limitations are that integration might
induce oncogenesis in some applications. The main advantage to the
use of lentiviral vectors is that gene transfer is persistent in
most tissues or cell types.
[0197] A lentiviral-based construct used to express the antisense
or RNAi agent agents preferably comprises sequences from the 5' and
3' LTRs of a lentivirus. More preferably the viral construct
comprises an inactivated or self-inactivating 3' LTR from a
lentivirus. The 3' LTR may be made self-inactivating by any method
known in the art. In a preferred embodiment, the U3 element of the
3' LTR contains a deletion of its enhancer sequence, preferably the
TATA box, Spl and NF-kappa B sites. As a result of the
self-inactivating 3' LTR, the provirus that is integrated into the
host ell genome will comprise an inactivated 5' LTR. The LTR
sequences may be LTR sequences from any lentivirus from any
species. The lentiviral-based construct also may incorporate
sequences for MMLV or MSCV, RSV or mammalian genes. In addition,
the U3 sequence from the lentiviral 5' LTR may be replaced with a
promoter sequence in the viral construct. This may increase the
titer of virus recovered from the packaging cell line. An enhancer
sequence may also be included.
[0198] Adenoviruses are non-enveloped viruses containing a linear
double-stranded DNA genome. While there are over 40 serotype
strains of adenovirus--most of which cause benign respiratory tract
infections in humans--subgroup C serotypes 2 or 5 are predominantly
used as vectors. The adenovirus life cycle normally does not
involve integration into the host genome, rather it replicates as
episomal elements in the nucleus of the host cell and consequently
there is no risk of insertional mutagenesis. The wildtype
adenovirus genome is approximately 35 kb of which up to 30 kb can
be replaced with foreign DNA. There are four early transcriptional
units (E1, E2, E3 and E4), which have regulatory functions, and a
late transcript, which codes for structural proteins. Progenitor
vectors have either the E1 or E3 gene inactivated, with the missing
gene being supplied in trans either by a helper virus, plasmid or
by an integrated gene in a helper cell genome. Second generation
vectors additionally use an E2a temperature sensitive mutant or an
E4 deletion. The most recent "gutless" vectors contain only the
inverted terminal repeats (ITRs) and a packaging sequence around
the transgene, all the necessary viral genes being provided in
trans by a helper virus.
[0199] Adenoviral vestors are very efficient at transducing target
cells in vitro and in vivo, and can be produced at high titres
(>10.sup.11/ml). With the exception of one study that showed
prolonged transgene expression in rat brains using an E1 deletion
vector, transgene expression in vivo from progenitor vectors tends
to be transient. Following intravenous injection, 90% of the
administered vector is degraded in the liver by a non-immune
mediated mechanism. Thereafter, an MHC class I restricted immune
response occurs, using CD8+ CTLs to eliminate virus infected cells
and CD4+ cells to secrete IFN-alpha which results in
anti-adenoviral antibody. Alteration of the adenoviral vector can
remove some CTL epitopes; however, the epitopes recognized differ
with the host MHC haplotype. The remaining vectors, in those cells
that are not destroyed, have their promoter inactivated and
persisting antibody prevents subsequent administration of the
vector.
[0200] Approaches to avoid the immune response involving transient
immunosupressive therapies have been successful in prolonging
transgene expression and achieving secondary gene transfer. A less
interventionist method has been to induce oral tolerance by feeding
the host UV inactivated vector. However, it is more desirable to
manipulate the vector rather than it is to manipulate the host
through immunosuppression. Although only replication deficient
vectors are used, viral proteins are expressed at a very low level,
which are then presented to the immune system. The development of
vectors containing fewer genes--culminating in the "gutless"
vectors which contain no viral coding sequences--has resulted in
prolonged in vivo transgene expression in liver tissue. However,
the initial delivery of DNA packaged within adenovirus
proteins--the majority of which will be degraded and presented to
the immune system--may still cause problems for clinical
trials.
[0201] Until recently, the mechanism by which the adenovirus
targeted the host cell was poorly understood. Tissue-specific
expression was therefore only possible by using cellular
promoter/enhancers, e.g., the myosin light chain 1 promoter or the
smooth muscle cell SM22a promoter, or by direct delivery to a local
area. Uptake of the adenovirus particle has been shown to be a
two-stage process involving an initial interaction of a fiber coat
protein in the adenovirus with a cellular receptor or receptors,
which include the MHC class I molecule and the
coxsackievirus-adenovirus receptor. The penton base protein of the
adenovirus particle then binds to the integrin family of cell
surface heterodimers allowing internalization via receptor mediated
endocytosis. Most cells express primary receptors for the
adenovirus fiber coat protein, however internalization is more
selective. Methods of increasing viral uptake include stimulating
the target cells to express an appropriate integrin and conjugating
an antibody with specificity for the target cell type to the
adenovirus. However, the use of antibodies increases the production
difficulties of the vector and the potential risk of activating the
complement system.
[0202] Another virus that may be used as a basis for a viral
delivery vector in the present invention is the Herpes simplex
virus-1. HSV-1 is a double-stranded DNA virus with a packaging
capacity of 40 kb, or up to 150 kb (helper dependent). HSV-1 has
strong tropism for neurons, but also has a high inflammatory
potential. HSV-1 is maintained episomally. Replication defective
HSV-1 vectors generally are produced by deleting all, or a
combination, of the five immediate-early genes (ICP0, ICP4, ICP22,
ICP27 and ICP47), which are required for lytic infection and
expression of all other viral proteins. Unfortunately, the ICP0
gene product is both cytotoxic and required for high level and
sustained transgene expression. As such, the production of
non-toxic quintuple immediate-early mutant vectors is a trade-off
against efficient and persistent transgene expression. An HSV-1
protein that is activated during latency has recently be shown to
complement mutations in ICP0 and overcome the repression of
transgene expression that occurs in the absence of ICP0.
Substitution of this protein in place of ICP0 might facilitate
efficient transgene expression without cytotoxicity in non-neuronal
cells. Long-term expression can be achieved in the nervous system
by using one of the HSV-1 neuron-specific latency-activated
promoters to drive transgene expression.
[0203] Other viral or non-viral systems known to those skilled in
the art may be used to deliver the antisense or RNAi agent
expression cassettes of the present invention to cells of interest,
including but not limited to gene-deleted adenovirus-transposon
vectors that stably maintain virus-encoded transgenes in vivo
through integration into host cells (see, Yant, et al., Nature
Biotech. 20:999-1004 (2002)); systems derived from Sindbis virus or
Semliki forest virus (see Perri, et al, J. Virol. 74(20):9802-07
(2002)); systems derived from Newcastle disease virus or Sendai
virus; or mini-circle DNA vectors devoid of bacterial DNA sequences
(see Chen, et al., Molecular Therapy. 8(3):495-500 (2003)). In
addition, hybrid viral systems may be used to combine useful
properties of two or more viral systems.
[0204] To deliver a viral-based antisense or RNAi agent expression
construct into target cells, the expression construct first must be
packaged into viral particles. Any method known in the art may be
used to produce infectious viral particles whose genome comprises a
copy of the viral antisense or RNAi agent expression construct. For
example, certain methods utilize packaging cells that stably
express in trans the viral proteins that are required for the
incorporation of the viral antisense or RNAi agent expression
construct into viral particles, as well as other sequences
necessary or preferred for a particular viral delivery system (for
example, sequences needed for replication, structural proteins and
viral assembly) and either viral-derived or artificial ligands for
tissue entry. In such a method, an antisense or RNAi agent
expression cassette is ligated to a viral delivery vector and the
resulting viral antisense or RNAi agent expression construct is
used to transfect packaging cells. The packaging cells then
replicate viral sequences, express viral proteins and package the
viral antisense or RNAi agent expression constructs into infectious
viral particles (step 420). The packaging cell line may be any cell
line that is capable of expressing viral proteins, including but
not limited to 293, HeLa, A549, PerC6, D17, MDCK, BHK, bing cherry,
phoenix, Cf2Th, or any other line known to or developed by those
skilled in the art. One packaging cell line is described, for
example, in U.S. Pat. No. 6,218,181.
[0205] Alternatively, a cell line that does not stably express
necessary viral proteins may be co-transfected with two or more
constructs to achieve efficient production of functional particles.
One of the constructs comprises the viral antisense or RNAi agent
expression construct, and the other plasmid(s) comprises nucleic
acids encoding the proteins necessary to allow the cells to produce
functional virus (replication and packaging construct) as well as
other helper functions. This method utilizes cells for packaging
that do not stably express viral replication and packaging genes.
In this case, the antisense or RNAi agent expression construct is
ligated to the viral delivery vector and then co-transfected with
one or more vectors that express the viral sequences necessary for
replication and production of infectious viral particles. The cells
replicate viral sequences, express viral proteins and package the
viral antisense or RNAi agent expression constructs into infectious
viral particles.
[0206] The packaging cell line or replication and packaging
construct may not express envelope gene products. In these
embodiments, the gene encoding the envelope gene can be provided on
a separate construct that is co-transfected with the viral
antisense or RNAi agent expression construct. As the envelope
protein is responsible, in part, for the host range of the viral
particles, the viruses may be pseudotyped. As described supra, a
"pseudotyped" virus is a viral particle having an envelope protein
that is from a virus other than the virus from which the genome is
derived. One with skill in the art can choose an appropriate
pseudotype for the viral delivery system used and cell to be
targeted. In addition to conferring a specific host range, a chosen
pseudotype may permit the virus to be concentrated to a very high
titer. Viruses alternatively can be pseudotyped with ecotropic
envelope proteins that limit infection to a specific species (e.g.,
ecotropic envelopes allow infection of, e.g., murine cells only,
where amphotropic envelopes allow infection of, e.g., both human
and murine cells.) In addition, genetically-modified ligands can be
used for cell-specific targeting.
[0207] After production in a packaging cell line, the viral
particles containing the antisense or RNAi agent expression
cassettes are purified and quantified (titered). Purification
strategies include density gradient centrifugation, or, preferably,
column chromatographic methods.
[0208] In a further embodiment of the invention, expression
constructs, vectors, polypeptides, or peptides may be entrapped in
a liposome. Liposomes are vesicular structures characterized by a
phospholipid bilayer membrane and/or an inner aqueous medium.
Multilamellar liposomes have multiple lipid layers separated by
aqueous medium. They form spontaneously when phospholipids are
suspended in an excess of aqueous solution. The lipid components
undergo self-rearrangement before the formation of closed
structures and/or entrap water and/or dissolved solutes between the
lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an
expression construct complexed with Lipofectamine (Gibco BRL).
[0209] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful (Nicolau and Sene,
1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al.
(1980) demonstrated the feasibility of liposome-mediated delivery
and/or expression of foreign DNA in cultured chick embryo, HeLa and
hepatoma cells.
[0210] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinatin virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and/or promote cell
entry of liposome-encapsulated DNA (Kaneda et al., 1989), for
example. In other embodiments, the liposome may be complexed and/or
employed in conjunction with nuclear non-histone chromosomal
proteins (HMG-1) (Kato et al., 1991). In yet further embodiments,
the liposome may be complexed and/or employed in conjunction with
both HVJ and HMG-1. In other embodiments, the delivery vehicle may
comprise a ligand and a liposome.
[0211] In another embodiment of the present invention, the direct
introduction of a JAB1 inhibiting protein ligand into a diseased
tissue is contemplated to provide a therapeutic effect. Such a JAB1
inhibiting ligand may be identified by the screening methods of the
present invention described herein. This therapeutic method
comprises administering to a subject a therapeutic composition
which comprises a JAB1 inhibiting ligand in amount effective to
decrease JAB1-mediated biological activity in the subject. In
specific embodiments, polypeptide or peptide compositions are
delivered in liposomes.
[0212] In one embodiment, a polypeptide for use in such a JAB1
inhibiting ligand composition comprises no more than about 100
amino acid residues, preferably no more than about 60 residues,
more preferably no more than about 30 residues. Peptides may be
considered as having fewer than about 30 residues and can be linear
or cyclic. Additionally, the JAB1-inhibiting ligand can be in any
of a variety of forms of peptide derivatives, that include amides,
conjugates with proteins, cyclized peptides, polymerized peptides,
analogs, fragments, chemically modified peptides, and the like
derivatives.
[0213] C. Pharmaceutical Compositions
[0214] Pharmaceutical compositions of the present invention
comprise an effective amount of one or more JAB1-inhibiting agents
dissolved or dispersed in a pharmaceutically acceptable carrier.
The phrases "pharmaceutical or pharmacologically acceptable" refers
to molecular entities and compositions that do not produce an
adverse, allergic or other untoward reaction when administered to
an animal, such as, for example, a human, as appropriate. The
preparation of an pharmaceutical composition that contains at least
one JAB1-inhibiting agent or additional active ingredient will be
known to those of skill in the art in light of the present
disclosure, as exemplified by Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, incorporated herein by
reference. Moreover, for animal (e.g., human) administration, it
will be understood that preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards. As used herein, the term
"JAB1-inhibiting agent" refers to an agent that modulates, such as
by reducing, expression of JAB1 or that inhibits the activity of a
JAB1 polypeptide, such as by inhibiting binding of JAB1 to a
target, including p27.
[0215] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated
herein by reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the
pharmaceutical compositions is contemplated.
[0216] The JAB1-inhibiting agent may comprise different types of
carriers depending on whether it is to be administered in solid,
liquid or aerosol form, and whether it need to be sterile for such
routes of administration as injection. The present invention can be
administered intravenously, intradermally, transdermally,
intrathecally, intraarterially, intraperitoneally, intranasally,
intravaginally, intrarectally, topically, intramuscularly,
subcutaneously, mucosally, orally, topically, locally, inhalation
(e.g., aerosol inhalation), injection, infusion, continuous
infusion, localized perfusion bathing target cells directly, via a
catheter, via a lavage, in cremes, mouthwashes, in lipid
compositions (e.g., liposomes), or by other method or any
combination of the forgoing as would be known to one of ordinary
skill in the art (see, for example, Remington's Pharmaceutical
Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein
by reference).
[0217] Any of the JAB1 inhibiting ligands of the present invention
may be used in the form of a pharmaceutically acceptable salt or
inorganic acids such as trifluoroacetic acid (TFA), hydrochloric
acid (HCl), hydrobromic acid, perchloric acid, nitric acid,
thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic
acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,
malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic
acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or
the like. HCl and TFA salts are particularly preferred.
[0218] Alternatively, suitable bases capable of forming salts may
be used with the peptides of the present invention and include
inorganic bases such as sodium hydroxide, ammonium hydroxide,
potassium hydroxide and the like; and organic bases such as mono-
di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl
amine, methyl amine, dimethyl amine and the like), and optionally
substituted ethanolamines (e.g. ethanolamine, diethanolamine and
the like).
[0219] A JAB1 inhibiting ligand of the present invention can be
synthesized by any of the techniques that are known to those
skilled in art. If the ligand is a polypeptide, synthetic chemistry
techniques, such as a solid-phase Merrifield-type synthesis, are
preferred for reasons of purity, antigenic specificity, freedom
from undesired side products, ease of production and the like.
[0220] The JAB1 inhibiting ligands and/or gene therapy vectors as
described above are adapted for administration as a pharmaceutical
compositions. Formulation and dose preparation techniques have been
described in the art, for example, those described in U.S. Pat. No.
5,326,902 issued to Seipp et al.; U.S. Pat. No. 5,234,933 issued to
Marnett et al.; and PCT Publication WO 93/25521 of Johnson et al.,
the entire contents of each of which are herein incorporated by
reference.
[0221] The therapeutic agents of the present invention may be
administered systemically or parenterally, for example. The doses
to be administered are determined depending upon age, body weight,
symptom, the desired therapeutic effect, the route of
administration, the nature of the therapeutic agent and the
duration of the treatment etc. In a human adult, the doses per
person per administration are generally between 1 mg and 500 mg, by
oral administration, up to several times per day, and between 1 mg
and 100 mg, by parenteral administration up to several times per
day. Since the doses to be used depend upon various conditions, as
mentioned above, there may be a case in which doses are lower than
or greater than the ranges specified above.
[0222] A composition of the present invention that is to be
administered parenterally typically is in dosage unit formulations
containing standard, well-known nontoxic physiologically acceptable
carriers, adjuvants, and vehicles as desired. The term "parenteral"
as used herein includes intravenous, intramuscular, intra-arterial
injection, or infusion techniques. Injectable preparations, for
example sterile injectable aqueous or oleaginous suspensions, are
formulated according to the known art using suitable dispersing or
wetting agents and suspending agents. The sterile injectable
preparation can also be a sterile injectable solution or suspension
in a nontoxic parenterally acceptable diluent or solvent, for
example, as a solution in 1,3-butanediol.
[0223] Among the acceptable vehicles and solvents that may be
employed in conjunction with the therapeutic agents are water,
Ringer's solution, and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland, fixed oil
can be employed including synthetic mono- or di-glycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables. Preferred carriers include neutral
saline solutions buffered with phosphate, lactate, Tris, and the
like.
[0224] Solid compositions for oral administration include
compressed tablets, pills, dispersible powders, capsules, and
granules. In such compositions, one or more of the active
substance(s) is or are, admixed with at least one inert diluent
(lactose, mannitol, glucose, hydroxypropylcellulose,
microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium
metasilicate alminate, etc.). The compositions may also comprise,
as is normal practice, additional substances other than inert
diluents: e.g. lubricating agents (magnesium stearate, etc.),
disintegrating agents (cellulose, calcium glycolate etc.), and
agents that assist in dissolving (glutamic acid, aspartic acid,
etc.) or stabilizing (lactose etc.). The tablets or pills may, if
desired, be coated with gastric or enteric material (sugar,
gelatin, hydroxypropylcellulose or hydroxypropylmethyl cellulose
phthalate, etc.). Capsules include soft ones and hard ones.
[0225] Liquid compositions for oral administration include
pharmaceutically-acceptable emulsions, solutions, suspensions,
syrups and elixirs. In such compositions, one or more of the active
substance(s) is or are admixed with inert diluent(s) commonly used
in the art (purified water, ethanol etc.). Besides inert diluents,
such compositions may also comprise adjuvants (wetting agents,
suspending agents, etc.), sweetening agents, flavoring agents,
perfuming agents and preserving agents. Other compositions for oral
administration include spray compositions which may be prepared by
known methods and which comprise one or more of the active
substance(s). Spray compositions may comprise additional substances
other than inert diluents: e.g. preserving agents (sodium sulfite,
etc.), isotonic buffer (sodium chloride, sodium citrate, citric
acid, etc.). For preparation of such spray compositions, for
example, the method described in U.S. Pat. No. 2,868,691 or
3,095,355 may be used.
[0226] In further embodiments, the present invention may concern
the use of a pharmaceutical lipid vehicle compositions that include
a JAB1-inhibiting agent, one or more lipids, and an aqueous
solvent. As used herein, the term "lipid" will be defined to
include any of a broad range of substances that is
characteristically insoluble in water and extractable with an
organic solvent. This broad class of compounds are well known to
those of skill in the art, and as the term "lipid" is used herein,
it is not limited to any particular structure. Examples include
compounds which contain long-chain aliphatic hydrocarbons and their
derivatives. A lipid may be naturally occurring or synthetic (i.e.,
designed or produced by man). However, a lipid is usually a
biological substance. Biological lipids are well known in the art,
and include for example, neutral fats, phospholipids,
phosphoglycerides, steroids, terpenes, lysolipids,
glycosphingolipids, glycolipids, sulphatides, lipids with ether and
ester-linked fatty acids and polymerizable lipids, and combinations
thereof. Of course, compounds other than those specifically
described herein that are understood by one of skill in the art as
lipids are also encompassed by the compositions and methods of the
present invention.
[0227] One of ordinary skill in the art would be familiar with the
range of techniques that can be employed for dispersing a
composition in a lipid vehicle. For example, the JAB1-inhibiting
agent may be dispersed in a solution containing a lipid, dissolved
with a lipid, emulsified with a lipid, mixed with a lipid, combined
with a lipid, covalently bonded to a lipid, contained as a
suspension in a lipid, contained or complexed with a micelle or
liposome, or otherwise associated with a lipid or lipid structure
by any means known to those of ordinary skill in the art. The
dispersion may or may not result in the formation of liposomes.
[0228] The actual dosage amount of a composition of the present
invention administered to an animal patient can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. Depending upon the dosage and the
route of administration, the number of administrations of a
preferred dosage and/or an effective amount may vary according tot
he response of the subject. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0229] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein.
Naturally, the amount of active compound(s) in each therapeutically
useful composition may be prepared is such a way that a suitable
dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half-life,
route of administration, product shelf life, as well as other
pharmacological considerations will be contemplated by one skilled
in the art of preparing such pharmaceutical formulations, and as
such, a variety of dosages and treatment regimens may be
desirable.
[0230] In other non-limiting examples, a dose may also comprise
from about 1 microgram/kg/body weight, about 5 microgram/kg/body
weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about
200 microgram/kg/body weight, about 350 microgram/kg/body weight,
about 500 microgram/kg/body weight, about 1 milligram/kg/body
weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about
100 milligram/kg/body weight, about 200 milligram/kg/body weight,
about 350 milligram/kg/body weight, about 500 milligram/kg/body
weight, to about 1000 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 5
mg/kg/body weight to about 100 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0231] In addition, the JAB1 inhibiting ligands and/or gene therapy
vectors of the present invention may be used in combination with
other treatment modalities, such as chemotherapy, surgical
intervention, cryotherapy, hyperthermia, radiation therapy, and the
like.
[0232] VI. Transgenic Animals
[0233] It is also contemplated to be within the scope of the
present invention to prepare a transgenic non-human animal that
expresses JAB1. The term "transgene" refers to exogenous genetic
material which does not naturally form part of the genetic material
of an animal to be genetically altered but can be incorporated into
the germ and/or somatic cells of that animal by standard transgenic
techniques. The term "transgenic" refers to cells, tissues,
embryos, fetuses or animals which carry one or more transgenes. The
term "chimeric" refers to an embryo, fetus or animal which consists
of two or more tissues of different genetic composition.
[0234] Techniques for the preparation of transgenic animals are
known in the art. Exemplary techniques are described in U.S. Pat.
No. 5,489,742 (transgenic rats); U.S. Pat. Nos. 4,736,866,
5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice);
U.S. Pat. No. 5,573,933 (transgenic pigs); U.S. Pat. No. 5,162,215
(transgenic avian species) and U.S. Pat. No. 5,741,957 (transgenic
bovine species), the entire contents of each of which are herein
incorporated by reference.
EXAMPLES
[0235] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventor to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
Example 1
JAB1 and p27 Expression Profiles in Breast Tumors
[0236] Breast tumor samples were obtained from a study group of 53
women with invasive breast carcinoma. The women were 35 to 90 years
old and had a mean age of 63.2.+-.13.3 years and a median age of
65. None of the women had a family history of breast cancer. The
patients had not undergone any chemotherapy or radiotherapy before
surgery. Patient selection was based on the availability of
archived paraffin blocks for immunohistochemical studies, which are
described below. Six cases (11%) were stage I, 28 (53%) were stage
II, 13 (25%) were stage III, and 6 (11%) were stage IV. Five tumors
(9%) were grade 1, 29 (55%) were grade 2, and 19 (36%) were grade
3. The tumors were surgically staged according to the American
Joint Committee on Cancer's tumor-nodes-metastasis system and
graded according to the Nottingham modification of the
Bloom-Richardson system. All of tumors excepted for one had a
maximum diameter larger than 1 cm. 47 of the breast cancer
carcinomas were ductal carcinomas, 3 were lobular carcinomas, and 3
were mixed invasive carcinomas. The cut-off level for considering a
tumor estrogen- or progesterone-receptor positive was 10
finol/mg.
[0237] Consecutive sections were cut from each tumor specimen and
processed for immunohistochemical analysis with the LSAB+kit
available from DAKO as described below. The following monoclonal
antibodies were used in the immunohistochemical analysis of the
tumor sections: JAB1 antibody from clone 4D11D8, available from
Zymed, San Francisco, Calif., at a dilution of 1:400; p27 antibody
from DAKO clone SX53K8, available from DAKO, Carpinteria, Calif.,
at a dilution of 1:200; and a Ki-67 antibody, MIB-1, available from
Immunotech, Westbrook, Me., at a dilution of 1:100. The specificity
of the JAB1 antibody was tested in normal tonsil tissue samples by
competition with a specific JAB1 peptide and a non-specific
peptide.
[0238] The sections from the tumor specimens were fixed in buffered
formalin and embedded in paraffin. 5-.mu.m thick paraffin-embedded
sections were mounted on poly-L-lysine-coated slides, dewaxed in
xylene, rehydrated in a graded series of ethanol, and incubated for
15 minutes with 0.3% hydrogen peroxide. For the unmasking of the
JAB1 and p27 antigens, the sections were incubated in plastic
Coplin jars containing preheated target retrieval solution, Dako
Catalog #S1699, and heated for 35 minutes in a household vegetable
steamer (Model Sunbeam 4713/5710, 900 W, available from
Sunbeam-Oster) and allowed to cool at room temperature for at least
15 minutes. After incubation in endogenous protein blocking
solution, Dako Catalog #X0909, the sections were incubated with the
monoclonal antibodies at the aforementioned dilutions at 4.degree.
C. overnight. The sections were washed three times with pH 8.0
TBS/0.05% Tween-20 for 5-10 minutes. The sections were then
incubated with biotin-conjugated secondary antibody, Dako Catalog
#K0690, for 30 minutes at room temperature. The sections were then
incubated with streptavidin-horseradish peroxidase complex, Catalog
#K0690, for 20 minutes. 3,3-diaminobenzidine tetrahydro-chloride
(DAB) was used as the chromogen and hematoxylin was used as the
counterstain, according the LSAB+kit instructions. To eliminate
false-positive staining, i.e., background staining, a control assay
was performed in which each immunostaining-assay step was
sequentially eliminated. Tissue sections stained with
immunoglobulin G isotype, available from Dako, were used as
negative controls in all immunostainings.
[0239] Evaluation of all immunostained slides was performed
independently by a pathologist who counted at least 1000 tumor
cells in 10 representative high-power fields. Epithelial cells of
the adjacent normal breast ducts were used as internal positive
controls for JAB1 and p27 expression. Cells were considered
JAB1-positive when nuclear staining of JAB1 protein was detected,
and cells were considered p27-positive when nuclear staining of p27
protein was detected. Serial tissue sections from the same areas of
the breast were used to examine JAB1, p27, and Ki-67 protein
expression levels. Ki-67 was used as a marker of cell
proliferation. The proliferation index (PI) was defined as the
percentage of MIB-1-positive tumor cells. Breast tumors in which
50% or greater of the cells assayed had a detected level of JAB1
protein were described as high JAB1 protein breast tumors, while
breast tumors in which less than 50% of the cells assayed had a
detectable level of JAB1 protein were described as low JAB1 protein
breast tumors.
[0240] Table 1 shows characteristics of the high JAB1 protein
breast tumors and of the low JAB1 protein breast tumors.
1 TABLE 1 Low JAB1 High JAB1 expression expression (n = 21) (n =
32) Tumor Characteristics Number % Number % P value Histologic Type
Ductal Carcinoma 18 of 47 38 29 of 47 62 0.7.sup.1 Lobular
carcinoma 2 of 3 67 1 of 3 33 Mixed 1 of 3 33 2 of 3 67 Grade 1 1
of 5 20 4 of 5 80 0.2.sup.1 2 10 of 29 34 19 of 29 66 3 10 of 29 53
9 of 19 47 Stage I 2 of 6 33 4 of 6 67 0.2.sup.1 II 9 of 28 32 19
of 28 68 III 7 of 13 54 6 of 13 46 IV 3 of 6 50 3 of 6 50 Lymph -
node metastasis Positive 16 of 34 47 18 of 34 53 0.15.sup.2
Negative 5 of 19 26 14 of 19 74 Estrogen Receptor Status Positive
14 of 33 42 19 of 33 58 0.3.sup.2 Negative 2 of 10 10 8 of 10 80
Progesterone Receptor Status Positive 10 of 27 37 17 of 27 63
>0.9.sup.1 Negative 6 of 16 38 10 of 16 62 .sup.1Chi-square test
.sup.2Fisher's exact test
[0241] JAB1 was detected in 43 (81%) of the 53 breast tumors. In 37
of the JAB1-positive tumors, JAB1 was predominantly found in the
nucleus, although weak cytoplasmic immunoreaction was also observed
in some cells. In the other 6 JAB1-positive tumors, JAB1 was
predominantly found in the cytoplasm, with weaker nuclear
immunoreactivity. The percentage of JAB1-positive tumor cells
ranged from 20% to 98%, with a mean of 65.6%+24.5 and a median of
70%. Thirty two (60%) of the 53 tumors showed high JAB1 protein
expression. JAB1 was not associated with the proliferation index.
Non-cancerous breast cells were also tested for the expression of
JAB1 protein. JAB1 was detected in about 10 to 30% of non-cancerous
breast cells, including ductal epithelial cells in proximate normal
and hyperplastic breast tissues.
[0242] p27 protein expression was examined in 49 of the breast
tumors included in this study. The percentage of cells assayed in
breast tumor cells with detected p27 protein ranged from 0.1% to
85%, with a mean.+-.s.d. of 33.5%.+-.24.5% and a median of 34.1%.
p27 protein was also expressed in the nuclei of nearby normal
epithelial, myoepithelial, and stromal cells. Breast tumors in
which 50% or greater of the cells assayed had a detected level of
p27 protein were described as high p27 protein breast tumors, while
breast tumors in which less than 50% of the cells assayed had a
detected level of p27 protein were described as low p27 protein
breast tumors. High p27 protein expression was found in 13 (27%) of
the 49 carcinomas.
[0243] Table 2 summarizes the relationship between JAB1 protein
levels and p27 protein levels in the 49 breast tumors. The
Mann-Whitney U test and Fisher's exact test were used in the
statistical analysis. An average of 27.2% of the breast tumor cells
examined in high JAB1 protein tumors had a detected level of p27,
while an average of 44.5% of the breast tumor cells examined in low
JAB1 protein tumors had a detected level of p27 (P=0.02,
Mann-Whitney U test). Of the 31 tumors that were high JAB1 protein
tumors, 26 of the tumors were low p27 protein tumors. Thus, it
appears that a high JAB1 protein level is correlated with a low p27
protein level in breast tumors.
2 TABLE 2 JAB1 level High Low P value p27 LI (mean % .+-. SD) 27.2
.+-. 23.2 44.5 .+-. 23.2 0.02.sup.1 p27 expression High 5 8
0.04.sup.2 Low 26 10 .sup.1Mann - Whitney U test .sup.2Fisher's
exact test
Example 2
Survival Rates
[0244] The available clinical data on the survival rate of the
female patients with breast carcinomas discussed above was examined
and is summarized in FIGS. 1A and 1B. FIG. 1B shows that a
significant difference in overall survival rates was found between
women with breast tumors that had a detected level of JAB1 protein
and women with breast tumors that did not have a detected level of
JAB1 protein. As defined herein, the "5-year overall survival rate"
is the percentage of surviving patients five years after the
patients' treatment or cancer diagnosis. The 5-year survival rate
includes patients that have experienced relapses or cancer
progression. After an average of 70 months, there was a 69% 5-year
overall survival rate among the women with breast tumors that had a
detected level of JAB1 protein, while there was a 100% 5-year
overall survival rate among the women with breast tumors that did
not have a detected level of JAB1 protein. In addition, there was
also a difference in progression-free survival rates between women
with breast tumors that had a detected level of JAB1 protein and
women with breast tumors that did not have a detected level of JAB1
protein.
[0245] "Progression-free survival rate" is the percentage of
survivors whose breast cancer had not progressed since the
patients' treatment or cancer diagnosis, which was about five years
earlier, in this case. There was an 80% 5-year progression-free
survival rate among the women with breast carcinomas that had a
detected level of JAB1 protein, while there was a 100% 5-year
progression-free survival rate among the women with breast tumors
that did not have a detected level of JAB1 protein. As all of the
women (that were studied over the five year period) that did not
have detected JAB1 protein in their breast tumors at the beginning
of the period survived and did not experience cancer progression,
while of the women that had detected JAB1 protein in their breast
tumors, 31% did not survive and 20% of the survivors experienced
breast cancer progression, testing a breast tumor sample for JAB1
protein expression provides a method of prognosticating a survival
rate after a specified period of time of a patient having breast
cancer. Furthermore, testing a breast tumor sample for JAB1 protein
provides a method of prognosticating a progression-free survival
rate after a specified period of time. The methods of
prognosticating survival rates may further include measuring or
estimating an amount of JAB1 protein in the sample.
Example 3
High JAB1 Expression is Directly Proportional to HER-2
Expression
[0246] JAB1 protein levels were examined in eight pairs of
non-cancerous and cancerous breast tissue samples from eight of the
patients of Example 1. The samples were obtained in a biopsy. The
samples were washed twice in cold 1.times.PBS that was diluted from
10.times.PBS, (Catalog #M6505, available from Fisher) and lysed at
4.degree. C. in lysis buffer (25 mM Hepes, pH 7.7, 400 mM NaCl,
0.5% Triton X-100, 1.5 mM MgCl.sub.2, 2 mM EDTA, 2 mM DTT, 0.1 mM
PMSF, protease inhibitors [including the following protease
inhibitors at the following final concentrations: leupeptin 10
.mu.g/ml, peptstatin 2 .mu.g/ml, antipain 50 .mu.g/ml, aprotinin 2
.mu.g/ml, chymostatin 20 .mu.g/ml, and benzamidine 2 .mu.g/ml] and
phosphatase inhibitors [including the following phosphatase
inhibitors at the following final concentrations: 50 mM NaF, 0.1 mM
Na.sub.3VO.sub.4, and 20 mM P-glycerophosphate]). Aliquots of cell
lysates containing about 70 mg of total protein were run on 10-12%
SDS-PAGE, transferred to polyvinylidene difluoride membranes
(Immobilon-P Transfer Membrane, Catalog #IPVH00010, available from
Millipore of Bedford, Mass.), and probed with primary polyclonal
antibodies to JAB1, available from Zymed, p27 antibodies available
from BD-Pharmingen, San Diego, Calif., and to HER-2, available from
Neomarkers of Fremont, Calif., all at 1:1000 dilutions. Goat
anti-mouse IgG (H&L)-HRP conjugate, Catalog #1706516, available
from Bio-Rad of Hercules, Calif. was used as the secondary antibody
for p27 and HER-2. HRP-protein A, Catalog #NA9120, from Amersham of
Piscataway, N.J. was used as the secondary antibody for JAB1. An
enhanced chemiluminescence (ECL) kit, Catalog #RPN.sub.2106,
available from Amersham Pharmacia, Piscataway, N.J. was used to
detect the proteins. Vinculin or .beta.-actin antibodies, available
from Sigma Chemical Co., St. Louis, Mo., served as internal
positive controls for the immunoblots.
[0247] The results of the western blots for the pairs of
non-cancerous and cancerous breast tissue samples revealed that
JAB1 protein levels were significantly higher in the cancerous
breast tissue samples than in the non-cancerous breast tissue
samples (FIG. 2A). It was also found that the level of JAB1 protein
in the cancerous breast tissue was directly proportional to the
amount of HER-2 protein in paired cancerous breast tissue samples
(FIG. 2B). HER-2 is a receptor tyrosine kinase that is often copy
number amplified and/or overexpressed in human cancers, including
breast cancer (see, Hung, et al., Gene 159:65-71 (1995), Slamon, et
al., Science 244:707-712 (1989), Berchuk, et al., Cancer Res.
50:4087-4091 (1990)). Overexpression of HER-2 has been associated
with tumor aggressiveness (Slamon, et al., Science 235:674-7
(1987)) and poor prognosis in breast cancer (Varley, et al.,
Oncogene 1:423-30 (1987)).
Example 4
Regulated Expression of JAB1 Affects Expression of p27
[0248] Four human breast cancer cell lines were used to examine the
effect of ectopic expression of JAB1 in breast cancer cells. The
following four breast cancer cell lines were used: BT-474,
MDA-MB-468, MDA-MB-231, and BT-549, all of which are available from
the ATCC. The BT-474 cells were cultured in Dulbeccos' minimal
Eagle medium supplemented with 10% fetal calf serum and 1%
penicillin-streptomycin, and the MDA-MB-468, MDA-MB-231, and BT-549
cells were cultured in RPMI-1640 supplemented with 10% fetal calf
serum and 1% penicillin-streptomycin. The cell lines were incubated
at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2.
[0249] The cell lines were transduced with a recombinant adenovirus
vector expressing a doxycycline-regulated (Tet-Off) form of JAB1.
The presence of doxycyline represses the expression of JAB1 by the
vector, while the absence of doxycycline allows the overexpression
of JAB1 by the vector. The recombinant vector was constructed using
the Adeno-X Tet-Off Expression System, Catalog #K1651-1, available
from Clontech, Palo Alto, Calif., according to the manufacturer's
recommendations. The full-length cDNA encoding for human JAB1, an
exemplary sequence which may be found at the National Center for
Biotechnology Information's GenBank database Accession number
U65928 (SEQ ID NO:8), was fused to a C-terminal Myc epitope tag by
introducing an Xba I site 1129 nucleotides downstream fro the JAB1
ATG and inserting the EcoR1/Xba I fragment of the JAB1 cDNA into an
EcoR1/Xbu I digested pcDNA3.1-Myc.His vector, Catalog #V800-20,
available from Invitrogen, Inc., Carlsbad, Calif. A BamH1I site and
an Afl II site were introduced at the ends of the JAB1-Myc
sequence, and the resulting BamHI/Afl II fragment was inserted into
the BamHI/Afl II restriction sites of the pTRE-shuttle vector to
generate a pTRE-JAB1-Myc construct.
[0250] The PI-SceI and I-Ceu I digestion product of the
pTRE-JAB1-Myc shuttle vector was cloned into a pAdeno-X Viral DNA
vector via ligation. Recombinant infectious adenoviruses were then
produced by transfecting HEK (human embryonic kidney) 293 cells
with pAdeno-X-JAB1-Myc viral DNA. Successful transfection was
confirmed by detecting synthesis of a JAB1-Myc fusion protein by
immunoblotting with anti-Myc antibodies. Adenoviruses were
collected from the cells by centrifugation of the cell culture
medium from the plates at 1200 rpm for 10 minutes. Recipient cells
from the breast cancer cell lines BT-474, MDA-MD-468, MDA-MD 231,
and BT-549 that had been plated 12 to 24 hours before infection
were then co-transfected with a regulatory virus, adeno-X Tet-Off,
and the Ad-JAB1-Myc virus at a multiplicity of infection of 50 in
the presence or absence of 1 .mu.g/ml doxycycline, a tetracycline
analogue, in a tetracycline-free serum medium, Catalog #8630-1,
available from Clontech, Palo Alto, Calif. After 48 hours, cell
lysates from the transfected cells were prepared as described in
Example 3, and immunoblotting was performed as described in Example
3. Anti-Myc antibodies were used to detect the JAB1-Myc fusion
protein and anti-p27 antibodies were used to detect p27 protein.
.beta.-actin antibodies were used as a loading control. Protein
amounts were quantified by Phosphorlmager analysis. FIG. 3A shows
the western blot and FIG. 3B shows the quantitative results from a
western blot demonstrating that the breast cancer cell lines
transfected with the Ad-JAB1-Myc construct produced high levels of
Myc-JAB1 protein in the absence of doxycycline, and that p27
protein levels decreased when high levels of Myc-JAB1 protein were
produced. Thus, it appears that high levels of JAB1 in breast
cancer cells result in reduced levels of p27 protein in breast
cancer cells.
Example 5
Tumor Induction in Mice
[0251] The activity of the Ad-JAB1-Myc virus was also studied in
NIH3T3 cells transfected with the Ad-JAB1-Myc virus and in mice
that were injected with the transfected NIH3T3 cells. The NIH3T3
cells were transfected using the method described above for the
transfection of the HeLa cells, and western blots were performed on
the cell lysates of four transfected cell lines, JAB-myc clones
#1-4. FIG. 4A shows the results of the western blots. JAB-myc
clones #3 and #4 expressed a high level of Myc-JAB1 protein and a
low level of p27 protein.
[0252] In addition, it was found that the stable expression of
Myc-JAB1 in 3T3 cells increased cellular proliferation as measured
by [.sup.3H]-thymidine incorporation. As can be seen in FIG. 4B,
the increase in thymidine incorporation was directly proportional
to the expression of exogenous Myc-JAB1 in the various stable
clones. For thymidine incorporation, 1.times.10.sup.5 cells of each
Myc-JAB1 clone and the parental 3T3 cells were plated in six wells
of a 24-well plate. After 24 hours, the media was changed to
serum-free DMEM and incubated at 37.degree. C. for 24 hours. The
media was aspirated and replaced with DMEM containing serum and
lmCu/ml [.sup.3H]-thymidine (Amersham Biosciences, Piscataway,
N.J.), and incubated at 37.degree. C. for 1 hour. Cells were washed
twice sith PBS and solubilized in 200 mM NaOH. Counts per minute
were determined in a Liquid Scintillation Beta Analyzer (Packard
Instruments Co., Meridan, Conn.).
[0253] Additionally, morphology of parental NIH-3T3 cells and
NIH-3T3-JAB1C#4 were observed (FIG. 4C). NIH-3T3-JAB1C#4,
expression high levels of Myc-JAB1, exhibited morphologic
transformation compared to the control cells. The NIH-3T3-JABlC#4
cells were spindle-shaped and displayed highly refractile
morphology, with long protrusions and pseudopodia. In other
experiments, exogenous JAB1 expression was found to promote S-phase
cell cycle progression as measured by bromodeoxyuriding (BrdU)
incorporation and propidium iodide (PI) staining, the results of
which can be seen in FIG. 4D. The parental cells and stable clones
were serum starved and labeled with BrdU for 45 minutes. The cells
were then stained with fluorescent anti-BrdU antibodies and PI for
flow cytomotery analysis.
[0254] The JAB-myc clones #3 and #4 experienced the formation of a
large number of colonies after 1 or 2 weeks of growth on soft agar,
as shown in Table 3 and in FIG. 4E.
3TABLE 3 Average no. Soft- of colonies/ No. of cells Tumors >10
agar dish at 1 week; injected in mm in diameter Cells growth at 2
weeks mice by 42 days 3T3 Parental - 0 0 4 .times. 10.sup.6 0/5
(Control) 3T3 JAB1#C3 + 145 227 4 .times. 10.sup.6 3/5 3T3 JAB1#C4
+++ 165 336 4 .times. 10.sup.6 9/10
[0255] Approximately 4.times.10.sup.6 NIH3T3 cells, JAB-myc clone
#3 cells and JAB-myc clone #4 cells were separately injected into 5
nude mice each. The results are summarized in FIG. 5. None of the
mice injected with NIH3T3 cells had developed tumors by 40 days
after the injections; however, the mice injected with JAB-myc clone
#3 cells developed tumors averaging greater than 350 mm.sup.3 in
volume and the mice injected with JAB-myc clone #4 cells developed
tumors greater than 1000 mm.sup.2 in diameter. FIG. 4F demonstrates
that exogenous JAB1 expression induced tumorigenesis in nude mice.
Stable clones, #C3, C4 and control cells (NIH-3T3) were injected
s.c (6.times.10.sup.6 cells) into 6-week old female nude mice
(BALB/C). 5 mice were used for each cell line. After 35 days mice
developed tumors >10 mm only with clones C3 and C4 but not with
control injected clone. Pictures of each mice are shown at 35 and
42 days post-injection. FIG. 4G shows JAB1 expression promotes
tumor development in nude mice. Mice were injected as in FIG. 4F,
and tumor formation was scored weekly.
[0256] In addition, immunostaining for JAB1 and p27 was performed
in both normal and cancerous tissues in these mice. Mice-bearing
JAB1 tumors were isolated and paraffin-embedded tissue section sere
obtained and stained with monoclonal antibodies for JAB1 or p27 and
counterstained with hemotoxylin. In FIG. 4H, results showed low
JAB1 expression and high p27 expression in normal tissue, while the
inverse was seen in tumor tissues. FIG. 41 is a column chart
summarizing this data. 300 positive and negative cells were counted
in each of three fields for JAB1 and p27 in normal and four tumor
tissue samples, and the percent positive staining for each is
shown.
Example 6
JAB1 and p27 Levels in Normal, Hyperplastic-Benign and
Invasive-Neoplastic Lesions of Human Breast Tissue Samples
[0257] JAB1 levels increase with tumorigenicity, correlating with a
decrease in p27. In FIG. 6A, immunohistochemical staining of a
breast tumor progression array for JAB1 and p27. JAB1 levels are
low in normal tissue and increase with tumorigenesis. In FIG. 6B,
percent of cells staining positive for either JAB1 or p27 were
quantified and graphed.
Example 7
JAB1 Expression in Anaplastic Large Cell Lvmphomas
[0258] Lymph tumor and reactive lymph node tissues samples were
obtained from a study group of 66 patients with systemic ALCL. The
diagnosis of ALCL was based on morphological and immunohistologic
criteria as specified by the WHO classification. Consecutive
sections were cut from each sample and processed for
immunohistochemical analysis as described below. The
clinicopathological features of the patients are described in
Rassidakis, et al., Am. J. Pathol. 159:527-535 (2001), which is
incorporated by reference herein.
[0259] Consecutive sections were cut from each tumor specimen and
processed for immunohistochemical analysis with the LSAB+kit
available from DAKO as described below. The following monoclonal
antibodies were used in the immunohistochemical analysis of the
tumor sections: JAB1 antibody from clone 4D11D8, available from
Zymed, San Francisco, Calif., at a dilution of 1:400; p27 antibody
from DAKO clone SX53K8, available from DAKO, Carpinteria, Calif.,
at a dilution of 1:200; and a Ki-67 antibody, MIB-1, available from
Immunotech, Westbrook, Me., at a dilution of 1:120. The specificity
of the JAB1 antibody was tested in a competition study by using a
specific JAB1 peptide at a concentration of 100 .mu.M and an
unrelated peptide to stain full tissue sections of two normal
tonsils and two reactive lymph nodes. The specific JAB1 peptide
used was identical to the one used for the production of the JAB1
monoclonal antibody. Competition of the specific JAB1 peptide with
the JAB1 antibody resulted in a lack of JAB1 immunostaining in
control slides.
[0260] The sections from the tumor specimens were fixed in buffered
formalin and embedded in paraffin. 5-.mu.m thick paraffin-embedded
sections were mounted on poly-L-lysine-coated slides, dewaxed in
xylene, and rehydrated in a graded series of ethanol. Sections were
placed in plastic Coplin jars containing preheated target retrieval
solution available from Dako, heated in a household vegetable
steamer (Model Sunbeam 4713/5710, 900 W, available from
Sunbeam-Oster) and allowed to cool at room temperature for at least
15 minutes. The following steps were performed on the sections
using the DAKO Autostainer at room temperature: blocking with 3%
hydrogen peroxide in PBS, pH 7.4 for 5 minutes; blocking using
protein blocking solution available from DAKO for 5 minutes;
incubation with the monoclonal antibodies for 1 hour; incubated
with the secondary biotinylated antibody, Dako Catalog #K0690, for
30 minutes at room temperature; and developed with the
streptavidin/horseradish peroxidase complex of the LSAB+kit for 20
minutes at room temperature. The sections were then incubated with
streptavidin-hyperoxidase complex. 3,3-diaminobenzidine
tetrahydro-chloride (DAB), available from Biogenex, San Ramon,
Calif., was used as the chromogen and hematoxylin was used as the
counterstain, according the LSAB+kit instructions.
[0261] Tissue sections from normal tonsil were used as external
positive controls for p27, JAB1, and MIB-1 immunostaining. Reactive
small lymphocytes in all tissue sections served as internal
positive controls for each antibody. Slides stained with normal
rabbit serum, available from DAKO, without primary antibody were
used as negative controls.
[0262] Expression of JAB1 and p27 was evaluated in at least 1,000
tumor cells. Cells were considered JAB1-positive when nuclear
staining of JAB1 protein was detected, and cells were considered
p27-positive when nuclear staining of p27 protein was detected. p27
was detected mostly in the mantle and marginal zones of reactive
lymphoid follicles. The highly proliferating germinal center cells
were almost all p27 negative.
[0263] In ALCLs, the percentage of tumor cells having detected p27
protein in the nucleus varied from 0 to 82.6% with a mean.+-.SD of
9.3.+-.19.6% and a median of 0.8%. p27 was localized principally in
the nucleus of tumor cells with variable staining intensity. 12
(82%) of the tumors were p27-positive, i.e., tumors that had
detected p27 protein in greater than 10% of the tumor cells
examined, while 44 (81.8%) of the tumors were p27-negative, i.e.,
tumors that had detected p27 protein in 10% or less of the tumor
cells examined. Of the 12 p27-positive ALCLs, 5 were anaplastic
lymphoma kinase (ALK)-positive, and 7 were ALK negative (P=0.7,
Fisher's exact test). ALK overexpression is often found in ALCLs,
and is associated with a favorable clinical outcome. p27 expression
was not statistically associated with clinical and laboratory
features. In a subset of 20 ALCLs (8 ALK-positive and 12
ALK-negative), 3 were p27-positive, and 17 were p27-negative.
[0264] As almost 89% of the tumors had a high detected level of
JAB1 protein, it is believed that testing a lymph tissue sample for
the presence and amount of JAB1 protein, as described above with
respect to breast tissue, may provide a method of diagnosing or
prognosticating the development of lymphoma.
[0265] JAB1 was detected in a large number of germinal center cells
as well as a number of lymphocytes in interfollicular areas. JAB1
immunoreactivity was predominantly localized in the nucleus of
lymphocytes, but a weaker cytoplasmic reaction was also observed in
a variable number of lymphocytes.
[0266] The percentage of tumor cells having detected JAB1 protein
in the nucleus ranged from 0 to 100% with a mean of 70.8.+-.29.7%
and a median of 85%. 47 of 53 (88.7%) tumors were JAB1-positive,
i.e., tumors that had detected JAB1 protein in greater than 10% of
the tumor cells examined, while 6 (11.3%) of the tumors were
JAB1-negative, i.e., tumors that had detected JAB1 protein in 10%
or less of the tumor cells examined. Of the 47 JAB1-positive
tumors, 15 were ALK-positive, and 32 were ALK-negative (P>0.9,
Fisher's exact test). JAB1 expression (>10% positive tumor
cells) was inversely associated with p27 expression. More
specifically, 40 of 47 (85.1%) JAB1-positive tumors were p27
negative, and 4 of 6 (66.7%) JAB1-negative tumors were p27 positive
(P=0.01 by Fisher's exact test).
[0267] p27 protein expression in the tumors was significantly
correlated with a lower 5-year survival rate and a lower 5-year
progression-free survival rate. The 5-year survival rate of
patients having p27-positive tumors was 45.7%, while the 5-year
survival rate of patients have p27-negative tumors was 90.1%. The
5-year progression-free survival rate of patients having
p27-positive tumors was 39.5%, while the 5-year progression-free
survival rate of patients having p27-negative tumors was 66.8%.
[0268] p27 protein expression in the tumors was inversely related
to JAB1 protein expression in the tumors. 85.1% of the
JAB1-positive tumors were p27-negative tumors and 66.7% of the
JAB1-negative tumors were p27 positive.
[0269] The relationship between p27 and JAB1 protein expression in
ALCL was further examined by performing western blots on 5 ALCL
cell lines. Two of the cell lines had a detected level of p27
protein and little or no detected JAB1 protein, where two other
cell lines had a detected level of JAB1 protein and little or no
detected p27 protein. One other cell line expressed a low level of
p27 protein and a high level of JAB1 protein. The results of the
western blots are further described in Example 7.
Example 8
Western Blots of Anaplastic Large Cell Lymphoma Cells
[0270] Five ALK-positive ALCL cell lines were used to examine the
amount of JAB1 protein and p27 protein in ALCL cells. The following
cell lines were used: Karpas 299, which was obtained from Dr. M.
Kadin, Boston, Mass., SR-786 and SU-SHL-1, which were obtained from
DSMZ, Braunschweig, Germany, JB-6, and TS-G.sub.1, which was
obtained from Dr. D. Jones, Houston, Tex. The cell lines are also
available from the ATCC. The cells were cultured in RPMI-1640
supplemented with 1% non-essential amino acids, 10% fetal calf
serum from Invitrogen, Corp., Grand Island, N.Y., and 1%
penicillin-streptomycin. The cell lines were incubated at
37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2.
[0271] Cell lysates from the ALCL cell lines were prepared and
western blots were performed as described in Example 3. The results
of the western blots showed that SR-786 and JB-6 cells had a
detected level of JAB1 protein and little or no detected p27
protein (FIG. 7). SU-DHL-1 and TG-S1 cells had a detected level of
p27 protein and little or no detected JAB1 protein. Karpas 299
cells had a relatively low detected level of p27 protein and a
relatively high detected level of JAB1 protein.
Example 9
JAB1 and p27 expression in a Variety of Cancers
[0272] In FIGS. 8 and 9, JAB1 and p27 expression is determined in
tissue array for ovarian cancer and colon carcinoma, respectively.
In FIG. 8, there is immunohistochemical staining with JAB1 and p27
in ovarian cancer. JAB1 is negative in normal case compare to
tumor. In FIG. 9, JAB1 expression intensifies with
adenoma.fwdarw.carcinoma.fwdarw.metastatic cancer, which indicates
that it is a useful marker for identifying stage of disease and
progression of cancer.
Example 10
Quantification by ELISA or FISH
[0273] While JAB1 protein may be detected and/or quantitated by
immunochemical staining and/or western blots, as described herein,
JAB1 protein may be detected and/or quantified by other methods,
such as enzyme-linked immunosorbent assays (ELISA) (see, for
example, FIG. 16) or fluorescence in situ hybridization (FISH).
[0274] Elisa
[0275] JAB1 protein was detected using an exemplary ELISA protocol
where 100 .mu.L of JAB1 monoclonal antibody at a concentration of 5
.mu.g/.mu.L in sodium bicarbonate buffer (50 mM NaHCO.sub.3 pH 9.6)
was placed in wells of a 96-well plate (Nunc F96 Maxisorp Immuno
plate, Catalog # 442404 Batch 059317-3) and was incubated at room
temperature for 2 hours. The JAB1 monoclonal antibodies used were
Catalog #4D11D7 and #3F10B10, both available from Zymed. The wells
were aspirated, and about 200 .mu.l of PBS-T (10 mM PBS with 0.05%
Tween-20) was added to each well to wash the wells. The plate was
shaken for about 5-10 minutes. The plate was then inverted and
shaken to remove the wash buffer. The PBS-T wash protocol was
repeated twice more. About 250 .mu.L of SuperBlock Dry Blend
Blocking Buffer, Pierce Catalog #37545, diluted with water and
having 0.05% Tween-20 was added to each well. The plate was
incubated on a shaker for 2 hours at room temperature or at
4.degree. C. overnight. The wells were then washed twice with
PBS-T.
[0276] 100 .mu.L of an antigen, such as GST-JAB1, GST, or serum,
was added to the wells. One set of wells received 50 ng/well of
antigen, and another set of wells received 400 ng/well of antigen.
The plate was then shaken at room temperature for 2 hours. The
wells were then washed three times with PBS-T. Biotinylated JAB1
monoclonal antibody, prepared using the Biotintag Micro
Biotinylation Kit, Sigma Catalog #S26-36 Lot 91K4876, was diluted
to 1:1000 in PBS-T. 100 .mu.L of the biotinylated JAB1 monoclonal
antibody was added to each well. The biotinylated JAB1 monoclonal
antibodies used were biotinylated Catalog #3F10B10 and #4D11D7,
which were used with wells coated with non-biotinylated #4D11D7 and
#3F10B10, respectively. The plate was shaken at room temperature
for 1 hour. The wells were then washed three times with PBS-T.
[0277] Streptavidin Peroxidase Conjugate was then diluted to
1:1000, and 100 .mu.L was added to each well. The plate was shaken
at room temperature for 1 hour. The wells were then washed three
times with PBS-T. 200 .mu.L of TMB Substrate
(3,3',5,5'-Tetramethylbenzidine), Sigma-Aldrich Catalog #T-8665,
was then added to the wells. The plate was incubated at room
temperature for 30 minutes. The plate was then scanned at 650 nm.
Visually, wells containing 50 ng/well of JAB1 antigen had
detectable staining, while wells containing 400 ng/well of JAB1 had
significantly darker staining. Wells without antigen or with GST
antigen had little or no detectable staining.
[0278] Fish
[0279] JAB1 copy number was studied in healthy human breast tissue
by performing FISH on the samples. It is believed that determining
JAB1 copy number in cells may provide an estimate of the level of
JAB1 protein in cells, as the presence of excess copies of a gene
may be correlated with overexpression of the protein encoded by the
gene. JAB1 probes were made by nick translation of DNA from BAC
clone RP11-92M10, which is publicly available. The DNA was purified
from the BAC clones with a Qiagen Maxi Kit, phenol chloroform
extracted, precipitated, and resuspended in water. The BRL BioNick
Labeling System, Catalog # 8247SA was used to perform the nick
translation. 10.times.A4 solution containing 200 .mu.M dATP, 200
.mu.M dGTP, 200 .mu.M dCTP, 500 mM Tris, pH 7.2, 200 mM MgCl.sub.2,
100 mM mercaptoethanol, 100 .mu.g/mL BSA, and water was prepared.
Then, a reaction mixture of 1 .mu.g of the purified BAC DNA, 5
.mu.L of 10.times. enzyme mix containing Dnase and Pol 1,5 .mu.L of
A4 solution, 1 .mu.L of cy3-dUTP, FITC-dUTP, or alexa-488 dUTP, and
distilled water to 50 .mu.L was prepared, mixed, and incubated at
15.degree. C. for about 90 minutes. The reaction was stopped by
heating at 75.degree. C. for 15 minutes. A sample of the reaction
was run on an agarose gel to confirm that probe fragments of about
300-800 base pairs were generated in the reaction.
[0280] The probes were used on 5-.mu.m thick paraffin-embedded
sections that were mounted on slides, as described above. The
slides were baked at 55-60.degree. C. for 1 to 2 hours. The
sections were dewaxed in xylene at room temperature for 3 ten
minute incubations. The sections were then washed twice for 10
minutes at room temperature in 100% ethanol. The slides were air
dried, and then incubated in preheated fresh 1 M NaSCN at
80.degree. C. for 10 minutes. The slides were then washed twice for
5 minutes in distilled water. Next, the slides were incubated in
prewarmed pepsin (1 mg/mL in 0.2 N HCl) for 10 minutes at
37.degree. C., and then washed twice for 5 minutes in distilled
water. The slides were then dehydrated at room temperature in 70%,
85%, and 100% ethanol for 3 minutes each. The slides were air
dried. The slides were then incubated in prewarmed denaturing
solution (70% formamide in 2.times.SSC) at 74.degree. C. for 5
minutes. The slides were then dehydrated at room temperature in
70%, 85%, and 100% ethanol for 3 minutes each. The slides were air
dried.
[0281] The slide preparation conditions described above provide
only one example of reagent concentrations and processing
conditions that may be used. For example, pepsin can be used at a
concentration of 100 .mu.g/mL to 4 mg/mL for a time of 5 minutes to
15 minutes. NaSCN may be used at a concentration of 0.1M to 1M for
5 minutes to 30 minutes. The denaturation period can be 2 minutes
to 10 minutes.
[0282] One hundred ng of a Cy3-labeled locus-specific BAC probe,
100 ng of a FITC-labeled locus specific BAC probe, or 10 .mu.g of
human Cot 1 DNA was dissolved in 3 .mu.L of water. 7 .mu.L of
Master mix #1 (5 mL formamide, 1 .mu.m dextran sulfate, 1 mL
20.times.SSC) was added to the 3 .mu.L, and the resulting solution
was mixed, denatured at 70-74.degree. C. for 20 minutes, and
reannealed at 37.degree. C. for 30 minutes. The solution was added
to one of the prepared slides for examining a 20.times.20 mm area
on the slide. A solution containing the chromosome 8-specific
probe, CEP 8 DNA probe, Catalog #30-16008 or 32-132008, Vysis Inc.,
Downers Grove, Ill., was also prepared and added to the slide. The
slide was then covered with a plastic coverslip and put in a 50 mL
Falcon tube with 100 .mu.L of 50% formamide in 2.times.SSC. The
tube was capped and incubated horizontally with the slide flat in
the tube at 37.degree. C. overnight for up to 3 days.
[0283] After the incubation, the slides were washed in 3.times.SSC
at 74.degree. C. for 5 minutes. The slides were then washed twice
in 4.times.SSC, 0.1% Triton at 37.degree. C. for 10 minutes. The
slides were then washed in 3.times.SSC at room temperature for 10
minutes. DAPI was added to the slides, and the slides were covered
with coverslips and examined using fluorescence microscopy.
[0284] JAB1 was successfully visualized on chromosome 8 (FIG. 15).
One copy of JAB1 on chromosome 8 was detected in healthy human
breast tissue, and multiple copies were detected on Karpas 299
T-cell lymphoma and breast carcinoma cells (MDA-MD 231). The CEP8
centromere probe for chromosome 8 (8p11.1-q11.1) was utilized as a
control.
Example 11
Alteration of JAB1 Expression
[0285] In view of the high level of JAB1 protein expression in the
various, different tumors and cancer cell lines described above,
altering the expression of JAB1 in cancer cells thus provides a
method for treating some types of cancer. Details for two methods
of altering expression are presented in this Example.
[0286] JAB1 siRNAs were constructed to study the effect of JAB1
protein down regulation on p27 protein levels. A JAB1 primer pair
5'-TTCAACATGCAGGAAGCTCAG-3' (SEQ ID NO:1) and
5'-TTCTGAGCTTCCTGCATGTTG-3' (SEQ ID NO: 2) starting at nucleotide
50 downstream from the JAB1 ATG was used with the Silencer.TM.
siRNA Construction Kit (Catalog #1620), available from Ambion of
Austin, Tex. according to the manufacturer's instructions to form
the double stranded RNA (dsRNA) shown in FIG. 10. HeLa cells that
were 30-50% confluent were transfected with 100 nM of JAB1 siRNA or
100 nM of a control siRNA using Oligofectamine.TM. Reagent, Catalog
#12252-011, available from Invitrogen, Carlsbad, Calif., according
to the manufacturer's instructions. The control siRNA was made
using in the Silencer.TM. siRNA Construction Kit. The siRNA were
diluted in Opti-MEM I media, available from Gibco, before the
addition of Oligofectamine.TM. Reagent. Before the transfection,
serum-free and antibiotic-free Dulbeccos' minimal Eagle medium
(DMEM) was added to the cells. The siRNA in Oligofectamine.TM.
Reagent was then overlayed on the cells. The cells were incubated
for 5 hours at 37.degree. C. in a CO.sub.2 incubator. DMEM with 10%
serum was added to the cells on top of the siRNA mixture. The
transfected cells were harvested 48 hours post transfection and
analyzed by western blotting, as described elsewhere herein.
[0287] FIG. 11A shows the results of the western blot. Cell lysates
were prepared and subjected to western blotting analysis using
anti-JAB1, anti-p27, anti-cyclin, anti-pRb and anti-actin
antibodies. For the kinase assay (last panel), cyclin A was
immunoprecipitated from cell lysates and analyzed for
cyclinA/Cdk-2-associated activity using Histone 1B as a
substrate.
[0288] FIG. 11B reveals that the knockdown of endogenous JAB1
expression by an JAB1 sequence-related siRNA decreases the S-phase
progression in the cell cycle and increases the number of cells in
G1 phase. HeLa cells were transfected with JAB1 siRNA and Control
siRNA, and progression through the S-phase was measured with
anti-BrdU fluorescent antibodies and propidium iodide (PI) staining
for flow cytometry analysis.
[0289] An antisense JAB1 construct was made to further study the
effect of JAB1 protein down-regulation on p27 protein levels. The
antisense construct was made by subcloning a 200 base pair fragment
of human JAB1 cDNA in the antisense orientation into the Hind III
site of the vector EC1214A, which is described in Hu, S. X. et al.,
Cancer Research 57: 3339-3343 (1997), and Hu, H. J. et al.,
Oncogene 15: 2589-2596 (1997). EC1214A is a
tetracycline/doxycyline-regulted vector that represses the
expression of inserted DNA fragments in the presence of
tetracycline or doxycycline, and allows the expression of inserted
DNA fragments in the absence of tetracycline or doxycycline. The
antisense construct was named Tet.AS JAB1. The 200 base pair
antisense fragment (SEQ ID NO: 5) includes bases -173 to +73 of the
human JAB1 cDNA and was generated by PCR amplification of a
JAB1-Myc-His construct. The primers
5'-CACACAAAGCTTGAATTCCCAAGAGTCTAGG-3' (SEQ ID NO: 6) and
5'-CACACAAAGCTTTACTCTGAGCTTCTTGCAT-3' (SEQ ID NO: 7) were used in
the PCR amplification. HeLa cells were transfected with the
antisense JAB1 construct in the presence or absence of 1 .mu.g/ml
doxycycline. The transfected cells were harvested 36-48 hours post
transfection and analyzed by western blotting as described in
Example 3. The results of the western blot are shown in FIG. 11A,
and these are quantitated in FIG. 1B. In the absence of
doxycycline, the antisense JAB1 construct significantly
down-regulated the amount of JAB1 protein, while the level of p27
protein increased in the absence of doxycycline.
[0290] It is believed that contacting a tumor cell with the siRNA
or antisense constructs described herein provides methods of
treating cancer by reducing the expression of JAB1 protein in the
cell. It is recognized that other methods may be used to reduce the
expression of JAB1 proteins in cells, such as cancer cells, and
such methods are considered to be within the scope of this
invention.
Example 12
Knockdown of JAB1 Expression with Antisense or siRNA
Compositions
[0291] FIGS. 11A-11C show that depletion of endogenous JAB1 with
either antisense (AS) or siRNA but not control siRNA, promotes
p27-increased stability and leads to G1-arrest. In FIG. 11A,
expression of antisense JAB1 increased the endogenous level of p27.
HeLa cells were transfected with a tetracycline-inducible (Tet-Off
system) antisense JAB1. Cell lysates were immunoblotted with JAB1
and p27 antibodies Quantification of the immunoblots is shown on
the right. In FIG. 11B, there is depletion of JAB1 by siRNA oligos
in HeLa cells. Cells were transfected with siRNA targeting JAB1
(JAB1 siRNA) or a scrambled sequence (Control siRNA). Forty-eight
hours after transfection, cell lysates were prepared and were
subjected to western blotting analysis using anti-JAB1, anti-p27,
anti.Cyclin A, anti-pRb and anti-actin antibodies. For kinase assay
(last panel), Cyclin A was immunoprecipated from cell lysates and
analyzed for cyclinA/Cdk2-associated activity using Histone 1B as a
substrate. In FIG. 11C, knockdown of endogenous JAB1 expression
decreases the S-phase progression in cell cycle and increases 5 G1
cells. Hela cells transfected with JAB1 siRNA and Control siRNA.
Progression through S-phase was measured with anti-Brdu fluorescent
antibodies and propidium idodide (PI) staining for Flow cytometery
analysis.
[0292] FIG. 12 shows silencing with adenoviral vector expressing
JAB1 siRNA (Ad-JAB1siRNA). In FIG. 12A, there is a schematic of
pSIREN Adeno strategy (Adeno-X viral DNA, BD-Pharmingen). In FIG.
12B, inhibition of endogenous JAB1 with Ad-JAB1siRNA but not with
control Ad-LUCsiRNA, increases p27 expression levels. HeLa cells
were transduced (MOI 50) with either Luciferase-RNAi pSIREN Shuttle
vector or JAB1-RNAi pSIREN Shuttle vector. Cells were harvested 48
hours post-transfection and analyzed by western blotting analysis
using both anti-JAB1 and anti-p27 antibodies.
[0293] FIGS. 13A-13B demonstrate that depletion of JAB1 by siRNA
adenovirus causes accumulation of p27kip1 and induces G1 arrest in
MDA-MB 231 breast carcinoma cells. In FIG. 13A, MDA-MB 231 cells
were transduced with adenoviruses driven JAB1 siRNA, or Luciferase
siRNA as a control, at MOI 50. Forty eight hours after, protein
lysates were prepared and immunobloted with an anti-JAB1, anti-p27
and anti-Cyclin A antibodies. Anti-.beta. actin was used as a
loading control. SiRNA ablation of JAB1 increases the steady-state
level of p27Kip1 protein and decreased cyclin A levels. In FIG.
13B, siRNA ablation of JAB1 induces G1 arrest. Cells were treated
same as in FIG. 13A, and cell cycle profile was determined by
propidium iodine staining and FACS.
[0294] FIG. 14 demonstrates that siRNA ablation of JAB1 causes
p27kip1 accumulation and prevents S-phase re-entry in Karpas 299
T-cells lymphoma. In FIG. 14A, knockdown of JAB1 protein levels by
siRNA increases the steady-state level of p27 protein, decreases
cyclin A and phopho-Rb levels. Karpas 299 cells were transfected
with p-Siren JAB1 siRNA or luciferase siRNA as a control (5 .mu.g
each). Lysates were immunoblotted 48 h after with the indicated
antibodies. In FIG. 14B, siRNA ablation prevents S-phase re-entry.
Karpas 299 cells were treated as in FIG. 14A, and progression
through S-phase was measured with anti-BrdU fluorescent antibodies
and FACS 48 hr after. Forty-six % of control siRNA-treated cells
were in S-phase compared to 15% with siRNA JAB1.
Example 13
Delineation of the JAB1-JUN and JAB1-p27 Interaction Domains
[0295] In order to map the interaction domains for p27 and JUN in
JAB1, sequential N-terminal and C-terminal deletion constructs were
made and tested. In vitro expression analysis of JAB1 full length,
C- and N-terminal deletion mutants was performed using a
TnT-coupled reticulocyte lysate system (Promega, Madison, Wis.).
Lysates from the full length and deletion mutants were translated
in vitro and labeled with [.sup.35S]-methionine. Ten percent of the
labeled products were separated on SDS-PAGE (FIG. 17B). The gel was
then fixed and dried, and autoradiography was performed. Next, an
in vitro binding assay was performed by incubating the translation
products from the full length, C- and N-terminal deletion mutants
with either glutathione-S-transferase (GST) alone or a GST-p27
fusion protein, each of which being immobilized on glutathione
agarose. The results of the binding assay (summarized in FIG. 17A)
showed that all N-terminal deletion mutants bound p27, but no
C-terminal mutant bound p27. Such a result indicates that p27 finds
to JAB1 at a position of about amino acid 299 to amino acid 334
(FIG. 17C).
[0296] Similarly, analysis was performed for determining the
interaction domain for c-Jun. Again, full length, C- and N-terminal
deletion mutants with either glutathione-S-transferase (GST) alone
or a GST-c-Jun fusion protein (amino acids 1-79), each of which was
immobilized on glutathione agarose (FIG. 17D). The results of the
binding assay (summarized in FIG. 17A) showed that all C-terminal
mutants bound c-Jun, but none of the N-terminal mutants bound p27,
indicating that c-Jun binds to JAB1 at the JAB1 N-terminus between
amino acid 49 and amino acid 96. The binding motifs delineated with
these studies are the p27 binding domain on JAB1 (aa
298-334)-LAKATRDSCKTTIEAIHGLMSQVIKDKLFNQINIS (SEQ ID NO:11) and the
c-Jun docking domain on JAB1 (aa 49-92):
HHYFKYCKISALALLKMVMHARSGGNLEVMGL- MLGKVDGETMIIM (SEQ ID NO:12). In
specific embodiments, these binding domain sequences are delineated
further with similar or analogous studies (see below).
[0297] FIGS. 18A-18B show a small region of JAB1 is sufficient for
interaction with p27. Recombinant proteins were bacterially
expressed and purified as Glutathione-S-transferase (GST) alone or
fused to p27 (GST-p27). Results of a GST (lane 1) or GST-p27 (lanes
3-7) pull-down experiments with in vitro .sup.35S-methionine
labeled JAB1 (full length) is shown (FIG. 18A). The effect of
increasing concentrations (0, 0.08, 0.4, 2, 10 and 20 .mu.g/ml) of
synthetic JAB1-peptide #1 corresponding to JAB1-p27 binding-domain
(LAKATRDSCKTTIEAIHG; SEQ ID NO:13; FIG. 18B) (Bottom panel, lanes
2-7) or to a peptide with scrambled sequence (control) was examined
(Top panel, lanes 2-7). Reactions were incubated 1 hr at room
temperature and washed 5 times and bound proteins to
glutathione-Sepharose beads were loaded onto an SDS-PAGE.
Autoradiogram is shown after .sup.35S exposure.
Example 14
Sensitization of Herceptin Therapy by JAB1
[0298] Recently, the receptor HER2 (erbB2/neu) was found to be
overexpressed in approximately 20%-30% of breast cancers and is an
indicator of aggressiveness, poor prognosis, and poor response to
tamoxifen (Slamon et al., 1987; Ross and Fletcher, 1998). Herceptin
(trastuzumab), a humanized antibody to the receptor HER2, was
developed (Carter et al., 1992) to block HER2 signaling and was
tested in clinical trials in women with HER2 overexpressing tumors
(Carter et al., 1992; Baselga et al., 1999; Arteaga, 2003; Slamon
et al., 2001; Vogel et al., 2002). Treatment with Herceptin in
HER2-overexpressing tumors is advantageous over standard
chemotherapy because of less adverse side effects. However, the
response ranges only from 12%-34%, and many HER2 positive tumors
remain non-receptive to Herceptin treatment. In order to obtain the
full benefit of this new drug, we need to understand the mechanism
of drug resistance in these tumors.
[0299] An interesting link between drug resistance and p27, a
potent inhibitor of cell division, offers one mechanism by which
breast tumors evade Herceptin treatment. Cytoplasmic
mislocalization of p27 and subsequent inactivation (Tomoda et al.,
1999) was seen in .about.40% of tumors of various types of human
cancers, and its sequestration were significantly higher in the
tumors than in normal tissues (Loda et al., 1997; Guo et al., 1997;
Masciullo et al., 1999; Singh et al., 1998; Ciaparrone et al.,
1998). Decreased protein level of p27 is an important clinical
marker that correlates with poor prognosis in breast and colorectal
cancers (Loda et al., 1997; Catzavelos et al., 1997) as well as
lung, colon, ovarian, skin, lymphoma, gastric, pituitary adenoma,
and prostate adenocarcinoma (Guo et al., 1997; Esposito et al.,
1997; Tsihlias et al., 1999; Sgambato et al., 1997). Indeed, a link
between HER2/neu signaling and JAB1 regulation affecting the
turnover rate of p27 may exist. HER2 signaling leads to an increase
in p27 levels and induces G1 cell cycle arrest and tumor growth
inhibition (Yang et al., 2000). A recent report by Yang et al.,
indicated that in breast cancer cells, overexpression and
activation of HER2/neu proto-oncogene leads to mislocalization of
p27 to the cytoplasm, thereby facilitating p27 degradation (Yang et
al., 2001). Thus, in specific embodiments, a JAB1-overexpressing
tumor provides a protective barrier against Herceptin-mediated
upregulation of p27. Further, that inhibition of JAB1 would
increase the efficiency of Herceptin treatment. This mechanism of
drug resistance presents useful targets for therapeutics
intervention.
[0300] Therefore, the role of JAB1 in resistance to Herceptin
treatment was characterized. Herceptin treatment leads to an
increase in cellular p27 levels and G1 arrest. Using an adeno-JAB1
in herceptin sensitive SKBr3 and BT-474 cells (both cell lines are
HER-2 overexpressing cells) it is determined whether JAB1 inhibits
the effect of Herceptin through degradation of p27. Further,
inhibition of JAB1 through siRNA technology provides a novel
strategy to sensitize tumors to Herceptin-induced tumor growth
arrest and apoptosis, such as is demonstrated in a
herceptin-resistant model.
[0301] To determine whether overexpression of JAB1 is involved in a
Herceptin pathway, HER2-overexpressing breast cancer cells, SKBR3
and BT474, are utilized. These cells express low levels of JAB1 and
are transduced with a doxycyclin-regulated (Tet-Off system)
adenovirus (Ad-JAB1) at 50MOI. Further treatment with Herceptin
(101g/mL) in the absence (-) or presence (+) of doxycycline (1
.mu.g/mL) for 48 h, followed by western blotting and flow cytometry
analysis, is performed. Western blotting of total cell lysates and
also nuclear and cytoplasmic fractions will demonstrate the effect
of JAB1 levels on p27 export into the cytoplasm and p27 protein
degradation. It has been reported that Herceptin is unable to
induce apoptosis in BT474 cells in cell culture, but does result in
cell arrest. Additionally, Herceptin is able to induce apoptosis,
not cell arrest, in SKBR3 cells. Therefore, the biological effects
of Herceptin treatment in the presence or absence of exogenous JAB1
is assessed on cell cycle and apoptosis by flow cytometry
analysis.
[0302] Next, it is determined whether inhibition of JAB1 increases
the efficiency of Herceptin treatment. siJAB1 effectively lowers
endogenous JAB1 levels and restabilizes p27 level. BT474 and SKBR3
cells are treated with Herceptin in the presence and absence of
siRNA JAB1. Through western blotting the amount of p27 is
determined in the cytoplasm versus the nucleus. In specific
embodiments, siRNA reduces JAB1 levels inhibiting the transport of
p27 into the nucleus and p27-degradation. Further, the ability of
Herceptin to induce G1 arrest in BT474 cells and apoptosis in SKBR3
cells in the presence of siRNA JAB1 is analyzed, such as by flow
cytometry. In particular aspects of the invention, there is a
correlation between JAB1 levels and effective Herceptin treatment.
Previous findings have shown that Herceptin increases p27 at the
protein level and JAB1 is known to be an inhibitor of p27. In
specific embodiments, inhibition of JAB1 leads tumor cells into
cell cycle arrest.
[0303] FIG. 24 shows that JAB1 bypass Herceptin-mediated G1 arrest
in breast cancer cells. SKBR3 and BT474 express very low levels of
JAB1 to undectable and were plated (2.times.105 cells/well) 24 hr
prior treatment. Then, cells were transduced with a
doxycyclin-regulated (Tet-Off system) adenovirus (Ad-JAB1) at 20
MOI in presence (+) or absence (-) of Doxycyclin (a tetracycline
analog) and were exposed or not to Herceptin treatment (10
.mu.g/ml). 48 hr later all cells (suspension and adherent) were
collected, stained with propidium iodine and analyzed by flow
cytometry. Herceptin treatment causes an increase in G1 which was
override upon Ad-JAB1 expression (-Dox) in these cells. Expression
of JAB1 in these cells decreased the G1 by 11% and increase in S
phase by 16%. A skilled artisan recognizes that Herceptin.RTM.
(Genentech, South San Francisco, Calif.) is also referred to as
trastuzumab, or humanized monoclonal IgG1.
[0304] Therefore, JAB1 siRNA and/or therapy targeted to inhibited
JAB1 function can be utilized for improving herceptin therapy.
Thus, in specific embodiments, JAB1-associated therapeutics are
used in conjunction with herceptin or similar compositions.
Example 15
Clinicopathological Features of JAB1-Associated Cancer
[0305] Table 4 provides considerable data concerning
clinocopathological features, JAB1 expression, p27 status,
proliferative activity, and ploidy status for representative breast
cancer patients. In specific embodiments, similar data is obtained
by analogous methods for any other type of cancer.
4TABLE 4 SUMMARY OF CLINICOPATHOLOGICAL FEATURES, JAB1 EXPRESSION,
P27 STATUS, PROLIFERATIVE ACTIVITY, AND PLOIDY STATUS T. % nuclear
Localiz. SAMPLE AGE HISTOLOGY SIZE GRADE LN STAGE ER PR JAB1 of
JAB1 p27(LI) MIB1(PI) 2 75 MIXED 3 2 positive II negative positive
95 N 6% 18% 5 65 IDC 2.4 3 negative II negative positive 85 N 12%
5% 6 77 IDC 2.2 2 negative II positive positive 50 C <0.5% 9% 7
73 IDC 1 2 positive II ND ND 98 N 55% 8% 8 52 IDC 2.6 3 positive II
positive positive 0 55% 26% 10 51 IDC 5 2 positive III positive
positive 40 N 24% 10% 11 67 IDC 7.8 2 positive II positive positive
76 N 6% 7% 12 48 IDC + DCIS 3.8 1 Negative II positive negative 65
C <0.5% ND 13 42 IDC 2.8 3 Negative II negative positive 0
<0.5% 11% 14 66 IDC 7 2 Positive III positive negative 68 N 42%
33% 15 68 IDC 4.2 2 Positive III negative negative 75 N <0.5% ND
16 61 IDC 5 2 Positive III positive positive 45 N 34% 20% 17 35 IDC
2 3 Positive II negative negative 90 C <0.5% 42% 18 64 IDC 2 3
Positive II positive positive 75 N 40% 17% 19 71 MIXED 1.8 2
Positive II ND ND 5 N 35% 16% 20 48 IDC 5 2 Positive III positive
positive 90 N 46% 15% 21 58 IDC 2.5 3 Negative III negative
negative 95 N 39% 72% 22 67 IDC 3 2 Negative II positive positive
50 N 3% 24% 23 73 ILC 2.6 2 Negative II positive positive 80 N 4%
14% 24 39 IDC 2.2 3 Positive II positive negative 30 N 21% 83% 25
70 IDC 4.8 2 Positive III positive positive 5 N 69% 14% 26 51 IDC 4
3 Positive II positive positive 30 N 76% 14% 27 67 IDC + DCIS 2 2
Negative II positive positive 80 N 3% 13% 28 62 IDC 3 2 Positive II
positive positive 95 N 33% 19% 29 72 IDC 2 1 Positive II positive
positive 95 N 85% 16% 30 76 IDC 0.9 2 Negative I negative positive
70 N 32% 32% 31 54 ILC 4.5 2 Positive IV ND ND 30 N ND ND 33 64 IDC
5 3 Positive III negative negative 1 N 85% 28% 35 81 IDC 2 1
Negative I positive positive 70 C 12% ND 36 67 IDC 5.2 3 Positive
IV positive negative 30 N 34% 41% 37 75 IDC 10 2 Positive IV
positive positive 80 N 63% 16% 38 66 IDC 5 3 Positive II positive
positive 75 N ND 7% 39 69 IDC 3 2 Negative II ND ND 5 N 15% ND 40
65 IDC 5 3 Positive III positive negative 5 N 78% 16% 41 60 IDC 1.5
2 Negative I ND ND 40 N 40% 24% 42 88 IDC 7.5 3 Positive IV ND ND
65 C <0.5% ND 43 37 IDC + DCIS 11.2 3 Negative II positive
negative 0 ND 31% 44 52 IDC 3.6 3 Negative II positive negative 97
N 31% ND 45 90 IDC 4.2 2 Negative II ND ND 95 N 42% 9% 48 65 IDC 3
3 Positive III ND ND 0 28% 29% 49 67 ILC 7.3 2 Positive III
positive positive 20 N 58% 11% 50 64 IDC 3 2 Positive II ND ND 90 N
<0.5% 12% 51 66 IDC 4.4 3 Positive III positive positive 55 N
35% 21% 52 48 IDC 2.5 1 Positive II positive negative 30 N 53% 9%
53 39 IDC 8.5 2 Positive III positive positive 0 ND 14% 55 60 IDC +
DCIS 2 1 Positive I ND ND 60 N 21% 22% 56 54 IDC 6 2 Negative II
negative negative 60 N 43% 10% 57 53 IDC 9 3 Positive III negative
negative 55 C 14% 13% 58 46 IDC 9.5 2 Positive II positive positive
25 N 54% 17% 60 74 IDC 1.8 2 Negative I positive negative 70 N 20%
10% 61 88 IDC 7 2 Negative II positive positive 90 N 42% 12% 62 81
IDC 2 2 Negative I ND ND 20 N 41% 15%
[0306] A variety of conclusions may be obtained from this data. For
example, it can be seen that there is a correlation between high
levels of JAB1 expression in the nucleus and low levels of p27
expression. This can be further associated with later stages of
breast cancer (FIG. 19).
[0307] By extracting data from Table 4, the value corresponding to
the ratio of nuclear vs. cytoplasmic localization of JAB1 among all
JAB1 positive tumors having more than .gtoreq.50% staining was
determined. This shows that there is a direct correlation between
the presence of JAB1-positive staining in the nucleus with stage
progression in breast cancer. In specific embodiment, the ratio for
Stage I JAB1 nuclear vs. cytoplasmic expression is about 3.0; the
ratio for Stage II JAB1 nuclear vs. cytoplasmic expression is about
4.66; and the ratio for Stage III JAB1 nuclear vs. cytoplasmic
expression is about 5. Thus, by determining JAB1 levels and its
subcellular localization (nuclear versus cytoplasmic, for example),
this is an indicator of tumor progression.
Example 16
Characterization of JAB1 Promoter Region and Transcriptional Start
Site
[0308] FIGS. 20A-20C demonstrate characterization of the JAB1
promoter region and its transcriptional start site. In FIG. 20A, 1,
2 and 3 kb upstream of the mRNA start site have been amplified by
PCR, and the JAB1 promoter regions were predicted by using Proscan
V1.7. Primers were designed to amplify 1, 2, and 3 kb upstream of
the ATG. In FIG. 20B, PCR amplification products of the predicted
regions are identified on the agarose gel. In FIG. 20C, there is
the transcriptional start site of the Jab1 gene. Primer extension
from 20 .mu.g of total RNA from human cells using a JAB1 specific
primer and the Promega provided control is shown. The primer
extension product (+P) marks the start of transcription. Sequencing
using the same primer is shown (G,A,T,C). Seventy-seven nucleotides
in the G lane corresponds to the band in the primer extension
lane.
[0309] FIG. 21 provides the reverse complement of the JAB1 promoter
sequence and the corresponding transcription factor binding sites,
as well as the transcription start site at +1. This sequence is
provided as SEQ ID NO:17 and is included at a GenBank Gene ID No.
10987. SEQ ID NO:18 provides a reverse complement sequence upstream
of JAB1 comprising about 3 kb 5' of the ATG start of Jab 1.
[0310] Thus, in specific embodiments, the promoter region of JAB1
is utilized for methods and/or compositions suitable for the
invention. Exemplary therapeutics may target inhibition of these
regulatory regions by interfering with a regulator (such as a
transcription factor) that binds to these specific cis-regulatory
DNA regions. Specific-DNA competitions aimed to inhibit these
bindings could be utilized as well. Thus, using the promoter region
to screen for one or more drug/small inhibitors that may inhibit
JAB1 mRNA expression, and therefore protein production, is within
the scope of the invention.
Example 15
Jab1/p27 Expression in Normal and Neoplastic Pancreas
[0311] JAB1/p27 expression was assayed in normal vs. neoplastic
pancreatic tissue (FIG. 22). JAB1 and p27 immunostainings are shown
in brown. Three different type of cells (ductal, acinar and islet
cells) comprise the pancreas. Normal ducts are negative for JAB1
and positive for p27 stainings (Left panel). Neoplastic carcinoma
arise from the duct cells that are positive for JAB1 and negative
for p27 (Right panel). Brown staining illustrates specific
immunostainings. (-): negative; (+): Positive stainings. Thus, in
specific embodiments of the present invention, the scope of the
invention comprises pancreatic cancer.
Example 16
Jab 1/p27 Expression in Normal and Neoplastic Pancreas
[0312] FIG. 23 shows p27 and JAB1 Expression in lymphoma types are
demonstrated, including the following exemplary types: Hodgkin's
Lymphoma; high grade non-Hodgkin's lymphomas (ALCL, DLBCL and
Burkitt); intermediate grade non-Hodgkin's lymphomas (MCL and
follicular lymphoma); and low grade non-Hodgkin's lymphomas
(CLL/SLL). Thus, in specific embodiments of the present invention,
the scope of the invention comprises lymphoma, such as Hodgkins'
lymphoma or non-Hodgkin's lymphoma.
[0313] It is to be understood that this invention is not limited to
the particular methodology, protocols, formulations and reagents
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs.
REFERENCES
[0314] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing, for
example, the methods and compositions that are described in the
publications which might be used in connection with the presently
described invention. The publications discussed above and
throughout the text are provided solely for their disclosure prior
to the filing date of the present application. Nothing herein is to
be construed as an admission that the inventors are not entitled to
antedate such disclosure by virtue of prior invention.
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[0368] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the invention as defined by the appended claims. Moreover, the
scope of the present application is not intended to be limited to
the particular embodiments of the process, machine, manufacture,
composition of matter, means, methods and steps described in the
specification. As one will readily appreciate from the disclosure,
processes, machines, manufacture, compositions of matter, means,
methods, or steps, presently existing or later to be developed that
perform substantially the same function or achieve substantially
the same result as the corresponding embodiments described herein
may be utilized. Accordingly, the appended claims are intended to
include within their scope such processes, machines, manufacture,
compositions of matter, means, methods, or steps.
Sequence CWU 1
1
18 1 21 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 1 ttcaacatgc aggaagctca g 21 2 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 2
ttctgagctt cctgcatgtt g 21 3 21 RNA Artificial Sequence Description
of Artificial Sequence Synthetic Primer 3 aacaacaugc aggaagcuca g
21 4 21 RNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 4 aacugagcuu ccugcauguu g 21 5 199 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 5
tactctgagc ttcctgcatg ttgttggcca gttcccaggt tttctgggcc ataccgctcc
60 cggacgccgc catcgccgag gaagcggaga agttgtcgtc tctacaacca
agacgcaact 120 ttacctcgct aggtttccgg gtgtgggcct gaccctccgc
accacgggaa caaactctta 180 cctagactct tgggaattc 199 6 31 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 6 cacacaaagc ttgaattccc aagagtctag g 31 7 31 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 7
cacacaaagc tttactctga gcttcttgca t 31 8 1292 DNA Homo sapiens 8
gaattcccaa gagtctaggt aagagtttgt tcccgtggtg cggagggtca aggcccacac
60 ccggaaacct agcgaggtaa agttgcgtct tggttgtaga gacgacaact
tctccgcttc 120 ctcggcgatg gcggcgtccg ggagcggtat ggcccagaaa
acctgggaac tggccaacaa 180 catgcaggaa gctcagagta tcgatgaaat
ctacaaatac gacaagaaac agcagcaaga 240 aatcctggcg gcgaagccct
ggactaagga tcaccattac tttaagtact gcaaaatctc 300 agcattggct
ctgctgaaga tggtgatgca tgccagatcg ggaggcaact tggaagtgat 360
gggtctgatg ctaggaaagg tggatggtga aaccatgatc attatggaca gttttgcttt
420 gcctgtggag ggcactgaaa cccgagtaaa tgctcaggct gctgcatatg
aatacatggc 480 tgcatacata gaaaatgcaa aacaggtggg ccaccttgaa
aatgcaatcg ggtggtatca 540 tagccaccct ggctatggct gctggctttc
tgggattgat gttagtactc agatgctcaa 600 tcagcagttc caggaaccat
ttgtagcagt ggtgattgat ccaacaagaa caatatccgc 660 agggaaagtg
aatcttggcg cctttaggac atacccaaag ggctacaaac ctcctgatga 720
aggaccttct gagtaccaga ctattccact taataaaata gaagattttg gtgtacactg
780 caaacaatat tatgccttag aagtctcata tttcaaatcc tctttggatc
gcaaattgct 840 tgagctgttg tggaataaat actgggtgaa tacgttgagt
tcttctagct tgcttactaa 900 tgcagactat accactggtc aggtctttga
tttgtctgaa aagttagagc agtcagaagc 960 ccagctggga cgagggagtt
tcatgttggg tttagaaacg catgaccgaa aatcagaaga 1020 caaacttgcc
aaagctacaa gagacagctg taaaactacc atagaagcta tccatggatt 1080
gatgtctcag gttattaagg ataaactgtt taatcaaatt aacatctctt aaacagtctc
1140 tgagaagtac tttacctgaa agacagtatg agaaaaatat tcaagtacac
tttaaaacca 1200 gttacccaaa atctgattag aagtataagg tgctctgaag
tgtcctaaat attaatatcc 1260 tgtaataaag ctctttaaaa tgaaaaaaaa aa 1292
9 1510 DNA Homo sapiens 9 gactatacca ctcccatacc ctataacttt
gtttgttcta tttcacacat ataattttcc 60 gagacaagat gttctcattt
aagcaacaag aagattcgtc tctcgctatt actgtaactg 120 ctgtttatat
cgtcatgtcc cggaaaggtc cctgtcttcc ctgaatggtc tctaccaact 180
tcacctccgg ttctaggtgt catggctgcc ccaagagtct aggtaagagt ttgttcccgt
240 ggtgcggagg gtcaaggccc acacccggaa acctagcgag gtaaagttgc
gtcttggttg 300 tagagacgac aacttctccg cttcctcggc gatggcggcg
tccgggagcg gtatggccca 360 gaaaacctgg gaactggcca acaacatgca
ggaagctcag agtatcgatg aaatctacaa 420 atacgacaag aaacagcagc
aagaaatcct ggcggcgaag ccctggacta aggatcacca 480 ttactttaag
tactgcaaaa tctcagcatt ggctctgctg aagatggtga tgcatgccag 540
atcgggaggc aacttggaag tgatgggtct gatgctagga aaggtggatg gtgaaaccat
600 gatcattatg gacagttttg ctttgcctgt ggagggcact gaaacccgag
taaatgctca 660 ggctgctgca tatgaataca tggctgcata catagaaaat
gcaaaacagg ttggccgcct 720 tgaaaatgca atcgggtggt atcatagcca
ccctggctat ggctgctggc tttctgggat 780 tgatgttagt actcagatgc
tcaatcagca gttccaggaa ccatttgtag cagtggtgat 840 tgatccaaca
agaacaatat ccgcagggaa agtgaatctt ggcgccttta ggacataccc 900
aaagggctac aaacctcctg atgaaggacc ttctgagtac cagactattc cacttaataa
960 aatagaagat tttggtgtac actgcaaaca atattatgcc ttagaagtct
catatttcaa 1020 atcctctttg gatcgcaaat tgcttgagct gttgtggaat
aaatactggg tgaatacgtt 1080 gagttcttct agcttgctta ctaatgcaga
ctataccact ggtcaggtct ttgatttgtc 1140 tgaaaagtta gagcagtcag
aagcccagct gggacgaggg agtttcatgt tgggtttaga 1200 aacgcatgac
cgaaaatcag aagacaaact tgccaaagct acaagagaca gctgtaaaac 1260
taccatagaa gctatccatg gattgatgtc tcaggttatt aaggataaac tgtttaatca
1320 aattaacatc tcttaaacag tctctgagaa gtactttacc tgaaagacag
tatgagaaaa 1380 atattcaagt aacactttaa aaccagttac ccaaaatctg
attagaagta taaggtgctc 1440 tgaagtgtcc taaatattaa tatcctgtaa
taaagctctt taaaatgaaa aaaaaaaaaa 1500 aaaaaaaaaa 1510 10 334 PRT
Homo sapiens 10 Met Ala Ala Ser Gly Ser Gly Met Ala Gln Lys Thr Trp
Glu Leu Ala 1 5 10 15 Asn Asn Met Gln Glu Ala Gln Ser Ile Asp Glu
Ile Tyr Lys Tyr Asp 20 25 30 Lys Lys Gln Gln Gln Glu Ile Leu Ala
Ala Lys Pro Trp Thr Lys Asp 35 40 45 His His Tyr Phe Lys Tyr Cys
Lys Ile Ser Ala Leu Ala Leu Leu Lys 50 55 60 Met Val Met His Ala
Arg Ser Gly Gly Asn Leu Glu Val Met Gly Leu 65 70 75 80 Met Leu Gly
Lys Val Asp Gly Glu Thr Met Ile Ile Met Asp Ser Phe 85 90 95 Ala
Leu Pro Val Glu Gly Thr Glu Thr Arg Val Asn Ala Gln Ala Ala 100 105
110 Ala Tyr Glu Tyr Met Ala Ala Tyr Ile Glu Asn Ala Lys Gln Val Gly
115 120 125 Arg Leu Glu Asn Ala Ile Gly Trp Tyr His Ser His Pro Gly
Tyr Gly 130 135 140 Cys Trp Leu Ser Gly Ile Asp Val Ser Thr Gln Met
Leu Asn Gln Gln 145 150 155 160 Phe Gln Glu Pro Phe Val Ala Val Val
Ile Asp Pro Thr Arg Thr Ile 165 170 175 Ser Ala Gly Lys Val Asn Leu
Gly Ala Phe Arg Thr Tyr Pro Lys Gly 180 185 190 Tyr Lys Pro Pro Asp
Glu Gly Pro Ser Glu Tyr Gln Thr Ile Pro Leu 195 200 205 Asn Lys Ile
Glu Asp Phe Gly Val His Cys Lys Gln Tyr Tyr Ala Leu 210 215 220 Glu
Val Ser Tyr Phe Lys Ser Ser Leu Asp Arg Lys Leu Leu Glu Leu 225 230
235 240 Leu Trp Asn Lys Tyr Trp Val Asn Thr Leu Ser Ser Ser Ser Leu
Leu 245 250 255 Thr Asn Ala Asp Tyr Thr Thr Gly Gln Val Phe Asp Leu
Ser Glu Lys 260 265 270 Leu Glu Gln Ser Glu Ala Gln Leu Gly Arg Gly
Ser Phe Met Leu Gly 275 280 285 Leu Glu Thr His Asp Arg Lys Ser Glu
Asp Lys Leu Ala Lys Ala Thr 290 295 300 Arg Asp Ser Cys Lys Thr Thr
Ile Glu Ala Ile His Gly Leu Met Ser 305 310 315 320 Gln Val Ile Lys
Asp Lys Leu Phe Asn Gln Ile Asn Ile Ser 325 330 11 35 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 11 Leu Ala Lys Ala Thr Arg Asp Ser Cys Lys Thr Thr Ile Glu
Ala Ile 1 5 10 15 His Gly Leu Met Ser Gln Val Ile Lys Asp Lys Leu
Phe Asn Gln Ile 20 25 30 Asn Ile Ser 35 12 45 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 12
His His Tyr Phe Lys Tyr Cys Lys Ile Ser Ala Leu Ala Leu Leu Lys 1 5
10 15 Met Val Met His Ala Arg Ser Gly Gly Asn Leu Glu Val Met Gly
Leu 20 25 30 Met Leu Gly Lys Val Asp Gly Glu Thr Met Ile Ile Met 35
40 45 13 18 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 13 Leu Ala Lys Ala Thr Arg Asp Ser Cys
Lys Thr Thr Ile Glu Ala Ile 1 5 10 15 His Gly 14 1288 DNA Homo
sapiens 14 ctaggtaaga gtttgttccc gtggtgcgga gggtcaaggc ccacacccgg
aaacctagcg 60 aggtaaagtt gcgtcttggt tgtagagacg acaacttctc
cgcttcctcg gcgatggcgg 120 cgtccgggag cggtatggcc cagaaaacct
gggaactggc caacaacatg caggaagctc 180 agagtatcga tgaaatctac
aaatacgaca agaaacagca gcaagaaatc ctggcggcga 240 agccctggac
taaggatcac cattacttta agtactgcaa aatctcagca ttggctctgc 300
tgaagatggt gatgcatgcc agatcgggag gcaacttgga agtgatgggt ctgatgctag
360 gaaaggtgga tggtgaaacc atgatcatta tggacagttt tgctttgcct
gtggagggca 420 ctgaaacccg agtaaatgct caggctgctg catatgaata
catggctgca tacatagaaa 480 atgcaaaaca ggttggccgc cttgaaaatg
caatcgggtg gtatcatagc caccctggct 540 atggctgctg gctttctggg
attgatgtta gtactcagat gctcaatcag cagttccagg 600 aaccatttgt
agcagtggtg attgatccaa caagaacaat atccgcaggg aaagtgaatc 660
ttggcgcctt taggacatac ccaaagggct acaaacctcc tgatgaagga ccttctgagt
720 accagactat tccacttaat aaaatagaag attttggtgt acactgcaaa
caatattatg 780 ccttagaagt ctcatatttc aaatcctctt tggatcgcaa
attgcttgag ctgttgtgga 840 ataaatactg ggtgaatacg ttgagttctt
ctagcttgct tactaatgca gactatacca 900 ctggtcaggt ctttgatttg
tctgaaaagt tagagcagtc agaagcccag ctgggacgag 960 ggagtttcat
gttgggttta gaaacgcatg accgaaaatc agaagacaaa cttgccaaag 1020
ctacaagaga cagctgtaaa actaccatag aagctatcca tggattgatg tctcaggtta
1080 ttaaggataa actgtttaat caaattaaca tctcttaaac agtctctgag
aagtacttta 1140 cctgaaagac agtatgagaa aaatattcaa gtaacacttt
aaaaccagtt acccaaaatc 1200 tgattagaag tataaggtgc tctgaagtgt
cctaaatatt aatatcctgt aataaagctc 1260 tttaaaatga aaaaaaaaaa
aaaaaaaa 1288 15 1288 DNA Homo sapiens 15 gtaagagttt gttcccgtgg
tgcggagggt caaggcccac acccggaaac ctagcgaggt 60 aaagttgcgt
cttggttgta gagacgacaa cttctccgct tcctcggcga tggcggcgtc 120
cgggagcggt atggcccaga aaacctggga actggccaac aacatgcagg aagctcagag
180 tatcgatgaa atctacaaat acgacaagaa acagcagcaa gaaatcctgg
cggcgaagcc 240 ctggactaag gatcaccatt actttaagta ctgcaaaatc
tcagcattgg ctctgctgaa 300 gatggtgatg catgccagat cgggaggcaa
cttggaagtg atgggtctga tgctaggaaa 360 ggtggatggt gaaaccatga
tcattatgga cagttttgct ttgcctgtgg agggcactga 420 aacccgagta
aatgctcagg ctgctgcata tgaatacatg gctgcataca tagaaaatgc 480
aaaacaggtt ggccgccttg aaaatgcaat cgggtggtat catagccacc ctggctatgg
540 ctgctggctt tctgggattg atgttagtac tcagatgctc aatcagcagt
tccaggaacc 600 atttgtagca gtggtgattg atccaacaag aacaatatcc
gcagggaaag tgaatcttgg 660 cgcctttagg acatacccaa agggctacaa
acctcctgat gaaggacctt ctgagtacca 720 gactattcca cttaataaaa
tagaagattt tggtgtacac tgcaaacaat attatgcctt 780 agaagtctca
tatttcaaat cctctttgga tcgcaaattg cttgagctgt tgtggaataa 840
atactgggtg aatacgttga gttcttctag cttgcttact aatgcagact ataccactgg
900 tcaggtcttt gatttgtctg aaaagttaga gcagtcagaa gcccagctgg
gacgagggag 960 tttcatgttg ggtttagaaa cgcatgaccg aaaatcagaa
gacaaacttg ccaaagctac 1020 aagagacagc tgtaaaacta ccatagaagc
tatccatgga ttgatgtctc aggttattaa 1080 ggataaactg tttaatcaaa
ttaacatctc ttaaacagtc tctgagaagt actttacctg 1140 aaagacagta
tgagaaaaat attcaagtaa cactttaaaa ccagttaccc aaaatctgat 1200
tagaagtata aggtgctctg aagtgtccta aatattaata tcctgtaata aagctcttta
1260 aaatgaaaaa aaaaaaaaaa aaaaaaaa 1288 16 1309 DNA Homo sapiens
16 gtgtcatggc tgccccaaga gtctaggtaa gagtttgttc ccgtggtgcg
gagggtcaag 60 gcccacaccc ggaaacctag cgaggtaaag ttgcgtcttg
gttgtagaga cgacaacttc 120 tccgcttcct cggcgatggc ggcgtccggg
agcggtatgg cccagaaaac ctgggaactg 180 gccaacaaca tgcaggaagc
tcagagtatc gatgaaatct acaaatacga caagaaacag 240 cagcaagaaa
tcctggcggc gaagccctgg actaaggatc accattactt taagtactgc 300
aaaatctcag cattggctct gctgaagatg gtgatgcatg ccagatcggg aggcaacttg
360 gaagtgatgg gtctgatgct aggaaaggtg gatggtgaaa ccatgatcat
tatggacagt 420 tttgctttgc ctgtggaggg cactgaaacc cgagtaaatg
ctcaggctgc tgcatatgaa 480 tacatggctg catacataga aaatgcaaaa
caggttggcc gccttgaaaa tgcaatcggg 540 tggtatcata gccaccctgg
ctatggctgc tggctttctg ggattgatgt tagtactcag 600 atgctcaatc
agcagttcca ggaaccattt gtagcagtgg tgattgatcc aacaagaaca 660
atatccgcag ggaaagtgaa tcttggcgcc tttaggacat acccaaaggg ctacaaacct
720 cctgatgaag gaccttctga gtaccagact attccactta ataaaataga
agattttggt 780 gtacactgca aacaatatta tgccttagaa gtctcatatt
tcaaatcctc tttggatcgc 840 aaattgcttg agctgttgtg gaataaatac
tgggtgaata cgttgagttc ttctagcttg 900 cttactaatg cagactatac
cactggtcag gtctttgatt tgtctgaaaa gttagagcag 960 tcagaagccc
agctgggacg agggagtttc atgttgggtt tagaaacgca tgaccgaaaa 1020
tcagaagaca aacttgccaa agctacaaga gacagctgta aaactaccat agaagctatc
1080 catggattga tgtctcaggt tattaaggat aaactgttta atcaaattaa
catctcttaa 1140 acagtctctg agaagtactt tacctgaaag acagtatgag
aaaaatattc aagtaacact 1200 ttaaaaccag ttacccaaaa tctgattaga
agtataaggt gctctgaagt gtcctaaata 1260 ttaatatcct gtaataaagc
tctttaaaat gaaaaaaaaa aaaaaaaaa 1309 17 873 DNA Homo sapiens 17
taataaaaaa attaaacatt gtattaactg taggaagaaa acaaaccagt tacccagatt
60 aattacgctt tgggaacaaa tgcggttcac aggagtgcca aagatttaac
tgtaacctgt 120 tacgagaact gagtactgga cattttcaga tcagtaatgg
cacattatat atacgtagat 180 gttaactttt aaccaagcgc aaaaaagcaa
tcaaggagta cccactgcct cctcgcatcg 240 atagaaaaag cccatctgca
agtgaagtgc caagccacca cgagcccgtt atcttttacg 300 catgtacagt
gagtcatctt gaagtaaacg ggaatgccat gtttatcttc ctctcaacca 360
gcttccagaa cgacttttag ctcagttgta caacagacag ccttaccttt tagtctttca
420 acaaacttat ctcatttaag gtacctatac ccacacaaaa acactttccg
ccctccacat 480 cccgctctta aggctccagc tacctttaat atggcggagg
ccgagcctgc gcattagaag 540 cagagaaggc aaataccagt ttctggaata
acgttacatg cccttcttcc ggtttttccg 600 agacaagatg ttctcattta
agcaacaaga agattcgtct ctcgctatta ctgtaactgc 660 tgtttatatc
gtcatgtccc ggaaaggtcc ctgtcttccc tgaatggtct ctaccaactt 720
cacctccggt tctaggtgtc atggctgccc caagagtcta ggtaagagtt tgttcccgtg
780 gtgcggaggg tcaaggccca cacccggaaa cctagcgagg taaagttgcg
tcttggttgt 840 agagacgaca acttctccgc ttcctcggcg tac 873 18 3067 DNA
Homo sapiens 18 gacggagcag ggggggcctg gccgttgggg agagaaggcg
catgggaacc aaggaaccgc 60 tcgcccccgg gccctggagg tggctgacta
ggaagcgtcg tcaggcaacc agctatcaac 120 aacaccgcgg gcagcagcga
ccgcagtggc ccaggaatcc ggaccgtgta accagggaac 180 ctccccgacc
ccggcagctg caagacccct gctcacagaa ctcccgcccc caaccctcat 240
aactcgtagt caccgatgcc ggcctcacgg cggcgaaatc ccaactctcc acacccttaa
300 ctcgggcacc tcccccaccg cacccagccg ccacgagacc ccctgccgac
aagaaacccc 360 acccccaccc ccagattcca gagcctgtaa ctctggcacg
tcgccggcct agttgcctcg 420 agacctacgc cccgagggag ccactgcccc
tgcccccgct ccggggcagc gaatgcagcc 480 ccaggaccct ccaccctcaa
cctacaccac cccaggccac acctcgccac tcacgctccc 540 catccgcggc
tggctgactc ttactcaggg gagcgggctc gcgtccgggg agacacacag 600
tgctctagag gatcgtcgcg gaccgaagag gttacagcgg ccacctggag cgggaacagc
660 atgacagacc tccgggccgg ggctccgccc ctcaggccca gccctccgct
cgcccttcgc 720 caaccgccgg gtacggcccc gcccccactg caggcggccg
cgcggcaaga catcgccccc 780 tgctgtcctg gaggccgcat agctgcccgc
tgctctcggt tcgccagtac gctggccggg 840 gacttggtca actcgttctc
ctgctgtgcc caggggctat taaactgtgg gggccacttc 900 tcaggctaaa
tctattgcag cctctccagc ctcccatgca ccagccagct taggaacagt 960
gtaagagcct ggaaagaaaa atggggaggg gtggtagtga gaggaaaagt gttggcatac
1020 ctctcagggc attactccta gcccaggagt gtggtcaacg ttgggtcctg
ggctttcatt 1080 accattttca acatattatt cagttcaccc caatacaatc
gcatatggcc atatgctacc 1140 actagaaaag agacctctag tttcccagga
gagtggaact aagggtaggt attaatgtac 1200 tattttaccc taaatttctt
tgtaagatgg ttttaaagga aattttcttc aacggaaagt 1260 cagtgccaaa
acagcaagct gtggtgtgga ttcaataatt ctaatgtttt tgtcttaact 1320
gattattaca acgcattgta acagttatag aagcacctac ggagacttac agacataatg
1380 gaaggaatgg taattggagc gtcaagaaag agttcgtggt cgatctcact
ggactgcatt 1440 tttaaagatt aataggagtt cccaggcaga aaatttacga
aagagcgttt caagcaatgc 1500 tgaccacata tgcaaagcca aagtaaagat
atctgcattg ctagaatgaa aagtttcaag 1560 gggggactaa aaagagatga
agctggaaga ggtgcatagg ggccaggttg ttaacagctg 1620 tggtgctatc
ttttcttttt tcttcttttt tctttctttc tttctttttt tttttttttt 1680
tttttttgag actggctctg tctaacaggc tggagtggcg caatctcgga tcactgcaac
1740 ttccgcctcc tgggttcaaa cgattctcct gcctcagcct catgagtagc
cgggactaca 1800 ggcgcacatc atcacgcccg gctaattttt ttttgtattt
ttttagtaga gacggggttt 1860 caccatgttg gccaggctgg tcttgaactc
ccgacctcaa gtgatcagcc cgcctcggct 1920 gcccaaagtg ctgggattac
aggcgtgagc caccgcgcct ggcctgtcat gctatctttt 1980 caggaaataa
aagaaaaaaa aatcttgaac acctatgcta ggtgctagga gataaaacaa 2040
ggaacgcaga cctgctccct gtgtcttgaa acttaaaatc ttgcacggat ggaagaatga
2100 gatggagaaa aaagaaaacc taaaacacgt gaactaataa aaaaattaaa
cattgtatta 2160 actgtaggaa gaaaacaaac cagttaccca gattaattac
gctttgggaa caaatgcggt 2220 tcacaggagt gccaaagatt taactgtaac
ctgttacgag aactgagtac tggacatttt 2280 cagatcagta atggcacatt
atatatacgt agatgttaac ttttaccaaa gcgcaaaaaa 2340 gcaatcaagg
agtacccact gcctcctcgc atcgatagaa aaagcccatc tgcaagtgaa 2400
gtgccaagcc accacgagcc cgttagcttt tacgcatgta cagtgagtca tcttgaagta
2460 aacgggaatg ccatgtttat cttcctctca accagcttcc agaacgactt
ttagctcagt 2520 tgtacaacag acagccttac cttttagtct ttcaacaaac
ttatctcatt taaggtacct 2580 atacccacac aaaaacactt tccgccctcc
acatcccgct cttaaggctc cagctacctt 2640 taatatggcg gaggccgagc
ctgcgcatta gaagcagaga aggcaaatac cagtttctgg 2700 aataacgtta
catgcccttc ttccggtgcg gaagactata ccactcccat accctataac 2760
tttgtttgtt ctatttcaca catataattt tccgagacaa gatgttctca tttaagcaac
2820 aagaagattc gtctctcgct attactgtaa ctgctgttta tatcgtcatg
tcccggaaag 2880 gtccctgtct tccctgaatg gtctctacca acttcacctc
cggttctagg tgtcatggct 2940 gccccaagag tctaggtaag agtttgttcc
cgtggtgcgg agggtcaagg cccacacccg 3000 gaaacctagc gaggtaaagt
tgcgtcttgg ttgtagagac gacaacttct
ccgcttcctc 3060 ggcgtac 3067
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