U.S. patent application number 12/978286 was filed with the patent office on 2011-04-28 for use of id4 for diagnosis and treatment of cancer.
Invention is credited to Dave S.B. Hoon, Naoyuki Umetani.
Application Number | 20110098341 12/978286 |
Document ID | / |
Family ID | 42828725 |
Filed Date | 2011-04-28 |
United States Patent
Application |
20110098341 |
Kind Code |
A1 |
Hoon; Dave S.B. ; et
al. |
April 28, 2011 |
USE OF ID4 FOR DIAGNOSIS AND TREATMENT OF CANCER
Abstract
The invention relates to a method of determining whether a human
subject is suffering from or at risk for developing pancreatic
cancer by determining the methylation level of an ID4 gene promoter
or the expression level of an ID4 gene in a biological sample from
a human subject. Also disclosed are a method of analyzing the
methylation level of an ID4 gene promoter or the expression level
of an ID4 gene in a pancreatic cancer cell, and a method of
inhibiting the methylation of an ID4 gene promoter or enhancing the
expression of an ID4 gene by contacting a pancreatic cancer cell
with a compound that decreases the methylation level of an ID4 gene
promoter or increases the expression level of an ID4 gene in the
cell.
Inventors: |
Hoon; Dave S.B.; (Los
Angeles, CA) ; Umetani; Naoyuki; (Tokyo, JP) |
Family ID: |
42828725 |
Appl. No.: |
12/978286 |
Filed: |
December 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12417577 |
Apr 2, 2009 |
7888033 |
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12978286 |
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11345836 |
Feb 1, 2006 |
7588894 |
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12417577 |
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60649650 |
Feb 1, 2005 |
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Current U.S.
Class: |
514/44A ;
435/375; 514/43; 514/575 |
Current CPC
Class: |
C12Q 2600/136 20130101;
A61P 35/00 20180101; C12Q 1/6886 20130101; C12Q 2600/154
20130101 |
Class at
Publication: |
514/44.A ;
435/375; 514/43; 514/575 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C12N 5/09 20100101 C12N005/09; A61K 31/706 20060101
A61K031/706; A61K 31/16 20060101 A61K031/16; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method of inhibiting the methylation of an ID4 gene promoter
or enhancing the expression of an ID4 gene in a cell, comprising:
providing a pancreatic cancer cell; and contacting the cell with a
compound that decreases the methylation level of an ID4 gene
promoter or increases the expression level of an ID4 gene in the
cell.
2. The method of claim 1, wherein the pancreatic cancer cell is a
primary or metastatic pancreatic cancer cell.
3. The method of claim 1, wherein the compound is a demethylation
agent or a histone deacetylase (HDAC) inhibitor.
4. The method of claim 3, wherein the demethylation agent is
5-aza-cytidine or the HDAC inhibitor is Trichostatin.
5. The method of claim 1, wherein the compound is an ID4 protein, a
nucleic acid encoding an ID4 protein, or an agent that activates an
ID4 gene.
6. A method for treating pancreatic cancer, comprising
administering an effective amount of a compound that decreases the
methylation level of an ID4 gene promoter or increases the
expression level of an ID4 gene to a subject suffering from or at
risk for developing pancreatic cancer.
7. The method of claim 6, wherein the compound is a demethylation
agent.
8. The method of claim 7, wherein the demethylation agent is
5-aza-cytidine.
9. The method of claim 6, wherein the compound is a histone
deacetylase (HDAC) inhibitor.
10. The method of claim 9, wherein the HDAC inhibitor is
Trichostatin.
11. The method of claim 6, wherein the compound comprises
polynucleotides.
12. The method of claim 11, wherein the polynucleotides are
siRNAs.
13. The method of claim 6, wherein the compound is suspended in a
pharmaceutically-acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/417,577, filed Apr. 2, 2009, which is a continuation-in-part
of application of U.S. application Ser. No. 11/345,836, filed Feb.
1, 2006, which claims priority to U.S. Provisional Application Ser.
No. 60/649,650, filed Feb. 1, 2005. The contents of U.S.
Application Ser. Nos. 12/417,577, 11/345,836 and 60/649,650 are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates in general to inhibitors of
DNA binding proteins. More specifically, the invention relates to
the use of ID4 for diagnosis, prognosis, and treatment of
pancreatic cancer.
BACKGROUND OF THE INVENTION
[0003] Methylation of cytosines in CpG islands in the promoter
region affects promoter activity and can down-regulate gene
transcription. Because the promoter hypermethylation of genes in
cancer cells is as significant as deletions or mutations,
hypermethylation of key regulatory genes can play a significant
role in transformation and tumor progression. Progression of
transformed cells requires regulatory gene inactivation that
promotes growth, dedifferentiation, invasion, and/or
metastasis.
[0004] Transcription factors containing a basic helix-loop-helix
(bHLH) motif regulate the expression of certain tissue-specific
genes and have important roles in cell differentiation and
embryonic developmental processes. DNA-binding activity of the bHLH
proteins is dependent on formation of homo- and/or hetero-dimers.
ID family proteins, which are distinct members of the
helix-loop-helix (HLH) protein family, contain the HLH-dimerization
domain but lack the DNA-binding basic domain. Consequently, ID
proteins dominantly inhibit binding to DNA and transcriptional
transactivation by forming heterodimers with bHLH proteins and
modulate various key developmental processes. Currently, four known
human ID proteins have been identified. Expression studies have
shown that ID proteins play critical roles in early embryonic
development. They are also involved in angiogenesis, lymphocyte
development, cell cycle control, and cellular senescence. The
involvement of ID proteins in neoplastic processes has been
suggested. Increased ID1 and ID2 expression has been reported in
various tumor types, including adenocarcinomas arising from the
colon and pancreas. Transgene expression of ID1 and ID2 in mice has
resulted in tumor formation in the intestinal epithelium and
lymphoid organs, respectively. Expression of ID3 has been more
variable; studies report both up-regulation and down-regulation in
different tumor types.
SUMMARY OF THE INVENTION
[0005] This invention relates to methods for diagnosis, prognosis,
and treatment of pancreatic cancer using ID4.
[0006] In one aspect, the invention features a method of
determining whether a human subject is suffering from or at risk
for developing pancreatic cancer. The method comprises obtaining a
biological sample from a human subject, and determining the
methylation level of an inhibitor of DNA binding 4 (ID4) gene
promoter or the expression level of an ID4 gene in the sample. If
the methylation level of the ID4 gene promoter in the sample is
higher than a control methylation level or the expression level of
the ID4 gene in the sample is lower than a control expression
level, it indicates that the human subject is likely to be
suffering from or at risk for developing pancreatic cancer.
[0007] In another aspect, the invention features a method of
analyzing the methylation level of an ID4 gene promoter or the
expression level of an ID4 gene. The method comprises providing a
pancreatic cancer cell, and determining the methylation level of an
ID4 gene promoter or the expression level of an ID4 gene in the
cell.
[0008] The method may further comprises analyzing the angiogenesis
or chemotaxis of the cell. If the methylation level of the ID4 gene
promoter in the cell is higher than a control methylation level or
the expression level of the ID4 gene in the cell is lower than a
control expression level, it indicates an increase in the
angiogenesis or chemotaxis of the cell.
[0009] The invention also provides a method of inhibiting the
methylation of an ID4 gene promoter or enhancing the expression of
an ID4 gene in a cell. The method comprises providing a pancreatic
cancer cell, and contacting the cell with a compound that decreases
the methylation level of an ID4 gene promoter or increases the
expression level of an ID4 gene in the cell.
[0010] For example, the compound may be a demethylation agent such
as 5-aza-cytidine or a histone deacetylase (HDAC) inhibitor such as
Trichostatin. Alternatively, the compound may be an ID4 protein, a
nucleic acid encoding an ID4 protein, or an agent that activates an
ID4 gene.
[0011] In the methods described above, the pancreatic cancer may be
primary or metastatic. The methylation level of the ID4 gene
promoter may be determined by quantitative methylation-specific
polymerase chain reaction (MSP) or bisulfite sequencing. The
expression level of the ID4 gene may be determined at the mRNA
level (e.g., by quantitative polymerase chain reaction (PCR)) or
the protein level (e.g., by immunohistochemistry (IHC)).
[0012] The above-mentioned and other features of this invention and
the manner of obtaining and using them will become more apparent,
and will be best understood, by reference to the following
description, taken in conjunction with the accompanying drawings.
These drawings depict only typical embodiments of the invention and
do not therefore limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. Structure of the promoter region of ID4 gene and the
primer design for methylation-specific PCR and bisulfite
sequencing. CpG sites in the annealing site of methylation-specific
PCR primers are indicated with "*". (TSS, transcription start
site)
[0014] FIG. 2. ID4 expression in pancreatic cancer cell lines.
[0015] FIG. 3. ID4 siRNA in CFPAC-1.
[0016] FIG. 4. ID4 siRNA in PANC-1.
[0017] FIG. 5. IHC for ID4 siRNA--CFPAC-1.
[0018] FIG. 6. Migration Assay--CFPAC-1.
[0019] FIG. 7. Angiogenesis Assay--CFPAC-1.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The invention is based at least in part upon the unexpected
discovery that the expression of transcription factor ID4 results
in the inhibition of pancreatic cancer cell chemotaxis and
endothelial cell angiogenesis, and ID4 expression is regulated by
hypermethylation of the CpG islands in the promoter region of
ID4.
[0021] Accordingly, the invention provides a method of analyzing
the methylation level of an ID4 gene promoter or the expression
level of an ID4 gene in a pancreatic cancer cell. The pancreatic
cancer cell may be obtained from a cell culture or a biological
sample from a subject having pancreatic cancer. It can be either a
primary pancreatic cancer cell or a metastatic pancreatic cancer
cell. The methylation level of an ID4 gene promoter or the
expression level of an ID4 gene in the cell is then determined.
[0022] "Subject," as used herein, refers to a human or animal,
including all vertebrates, e.g., mammals, such as primates
(particularly higher primates), sheep, dog, rodents (e.g., mouse or
rat), guinea pig, goat, pig, cat, rabbit, cow; and non-mammals,
such as chicken, amphibians, reptiles, etc. In a preferred
embodiment, the subject is a human. In another embodiment, the
subject is an experimental animal or animal suitable as a disease
model.
[0023] A biological sample from a subject can be a tissue sample
(e.g., a biopsy specimen sample, a normal or benign tissue sample,
a freshly prepared tumor sample, a frozen tumor tissue sample, a
paraffin-embedded tumor sample, a primary tumor sample, or a
metastasis sample) or a body fluid sample (e.g., any body fluid in
which cancer cells or acellular DNA may be present, including,
without limitation, blood, bone marrow, cerebral spinal fluid,
peritoneal fluid, pleural fluid, lymph fluid, ascites, serous
fluid, sputum, lacrimal fluid, stool, or urine). These tissues and
body fluids can be collected using any of the methods well known in
the art.
[0024] The ID4 gene is known in the art. For example, the human ID4
gene is reported to be located at Entrez Gene cytogenetic band
6p22-p21, Ensemble cytogenetic band 6p22.3, and HGNC cytogenetic
band 6p22.3. The DNA sequence around the human ID4 promoter region
is shown below.
TABLE-US-00001 (SEQ ID NO: 1) [Upstream 1,000 bp]
cattaatggcctaaattaagttacaggtatgaattttacataaaacagat
taatattatatgtcataatggaattttaaatattccgtgtccatgcattt
ttaatctttacgtgctctaattgaatgcgcaaggcaactgcatttcttga
gcccacttttgcatttagatggggtaaaagaaccccccccacgtttttgt
tttatttatattcccttcaccaaaaacctgcattcgattcggcattcttt
tccttctttttttttctacttttgctaagctttagcattttttaaaaaga
aaacggaaaggctacacattccattccatcattatggtttcggcaaatgt
gaaaaggcgaataatgaaacggaggagggaaatatagaacagaatgaacg
tgccttcttgaacagcgcgtctttcttaaggcactggaatcccacggatg
gagtgatgggtggcggagggtccctgggcgccgtgctattaggagtggca
gggtatccgcgagcagggcccaggcgctccctcagcagcctagtcgggat
aaggggggcggtggagagtgaattccggccgcacattcccgcagttcttc
gcaggaacttcgctctctcttttcccctcccttgggcacacatcagcctg
gcccgactcccactcagctctcttttctcagaaccccgacccacagcgtt
gacggaatggagtgcccttcccattggcccgagcgtcattccccgaggtg
gcactgcccgcctgattggctggccactccagaccccccgcccactcctc
cactcgggtagccggactccccgccccccagcaccgcccggagcccccgc
cctcgcctctccctccgcgcccccgcccgcgcgcccagcgggctccgctc
ggctcgcgctgcgacccggcccgcgcgctggtcccgcccccggggcgcac
ggctctataaatacagctgcgcggcgggccgggcgagagcgtagtggagg [Transcriptional
Start Site] AGGCGCGGTTGTGAGTAGTACCGGGAGTGGGGTGATCCCGGGCTAGGGGA
GCGCGGCGGCCGCGATCGGGCTTAGTCGGAGCTCCGAAGGGAGTGACTAG
GACACCCGGGTGGGCTACTTTTCTTCCGGTGCTTTTGCTTTTTTTTTCCT
TTGGGCTCGGGCTGAGTGTCGCCCACTGAGCAAAGATTCCCTCGTAAAAC
CCAGAGCGACCCTCCCGTCAATTGTTGGGCTCGGGAGTGTCGCGGTGCCC
CGAGCGCGCCGGGCGCGGAGGCAAAGGGAGCGGAGCCGGCCGCGGACGGG
GCCCGGAGCTTGCCTGCCTCCCTCGCTCGCCCCAGCGGGTTCGCTCGCGT
AGAGCGCAGGGCGCGCGCGATGAAGGCGGTGAGCCCGGTGCGCCCCTCGG
GCCGCAAGGCGCCGTCGGGCTGCGGCGGCGGGGAGCTGGCGCTGCGCTGC
CTGGCCGAGCACGGCCACAGCCTGGGTGGCTCCGCAGCCGCGGCGGCGGC
[0025] A "promoter" is a region of DNA extending 150-300 by
upstream from the transcription start site that contains binding
sites for RNA polymerase and a number of proteins that regulate the
rate of transcription of the adjacent gene. The promoter region of
the ID4 gene is known in the art. For example, the minimal promoter
region of human ID4 is located at -48-+32 (FIG. 1).
[0026] Methods for extracting DNA from biological samples and
determining the methylation level of a gene promoter are well known
in the art. Commonly, DNA isolation procedures comprise lysis of
cells using detergents. After cell lysis, proteins are removed from
DNA using various proteases. DNA is then extracted with phenol,
precipitated in alcohol, and dissolved in an aqueous solution.
[0027] The methylation level of a gene promoter can be determined,
for example, by methylation-specific PCR, bisulfite sequencing
(COBRA), pyrosequencing, or methylation-sensitive restriction
enzymes.
[0028] More specifically, a method for determining the methylation
state of nucleic acids is described in U.S. Pat. No. 6,017,704,
which is incorporated herein in its entirety. Methylation-specific
PCR (MSP) is a technique whereby DNA is amplified by PCR dependent
upon the methylation state of the DNA. Determining the methylation
state of a nucleic acid includes amplifying the nucleic acid by
means of oligonucleotide primers that distinguishes between
methylated and unmethylated nucleic acids. MSP can rapidly assess
the methylation status of virtually any group of CpG sites within a
CpG island, independent of the use of methylation-sensitive
restriction enzymes. This assay entails initial modification of DNA
by sodium bisulfite, converting all unmethylated, but not
methylated, cytosines to uracil, and subsequent amplification with
primers specific for methylated versus unmethylated DNA. MSP
requires only small quantities of DNA, is sensitive to 0.1%
methylated alleles of a given CpG island locus, and can be
performed on DNA extracted from paraffin-embedded samples. MSP
eliminates the false positive results inherent to previous
PCR-based approaches which relied on differential restriction
enzyme cleavage to distinguish methylated from unmethylated DNA.
This method is very simple and can be used on small amounts of
tissue or few cells and fresh, frozen, or paraffin-embedded
sections. MSP product can be detected by gel electrophoresis,
capillary array electrophoresis, or real-time quantitative PCR.
[0029] Bisulfite sequencing is widely used to detect
5-methylcytosine (5-MeC) in DNA. It provides a reliable way of
detecting any methylated cytosine at single-molecule resolution in
any sequence context. The process of bisulfite treatment exploits
the different sensitivity of cytosine and 5-MeC to deamination by
bisulfite under acidic conditions, in which cytosine undergoes
conversion to uracil while 5-MeC remains unreactive. Assessment of
the paraffin-embedded specimen can be performed directly on a
tissue section (5-12 microns thick) on the slide using bisulfite
modification following extraction of DNA and MSP analysis.
[0030] Gene expression can be detected and quantified at mRNA or
protein level using a number of means well known in the art. To
measure mRNA levels, cells in biological samples (e.g., cultured
cells, tissues, and body fluids) can be lysed and the mRNA levels
in the lysates or in RNA purified or semi-purified from the lysates
determined by any of a variety of methods familiar to those in the
art. Such methods include, without limitation, hybridization assays
using detectably labeled gene-specific DNA or RNA probes and
quantitative or semi-quantitative RT-PCR (e.g., real-time PCR)
methodologies using appropriate gene-specific oligonucleotide
primers. Alternatively, quantitative or semi-quantitative in situ
hybridization assays can be carried out using, for example, unlysed
tissues or cell suspensions, and detectably (e.g., fluorescently or
enzyme-) labeled DNA or RNA probes. Additional methods for
quantifying mRNA levels include RNA protection assay (RPA), cDNA
and oligonucleotide microarrays, and colorimetric probe based
assays.
[0031] Methods of measuring protein levels in biological samples
are also known in the art. Many such methods employ antibodies
(e.g., monoclonal or polyclonal antibodies) that bind specifically
to target proteins. In such assays, an antibody itself or a
secondary antibody that binds to it can be detectably labeled.
Alternatively, the antibody can be conjugated with biotin, and
detectably labeled avidin (a polypeptide that binds to biotin) can
be used to detect the presence of the biotinylated antibody.
Combinations of these approaches (including "multi-layer sandwich"
assays) familiar to those in the art can be used to enhance the
sensitivity of the methodologies. Some of these protein-measuring
assays (e.g., ELISA or Western blot) can be applied to bodily
fluids or to lysates of test cells, and others (e.g.,
immunohistological methods or fluorescence flow cytometry) applied
to unlysed tissues or cell suspensions. Methods of measuring the
amount of a label depend on the nature of the label and are known
in the art. Appropriate labels include, without limitation,
radionuclides (e.g., .sup.125I, .sup.131I, .sup.35S, .sup.3H, or
.sup.32P), enzymes (e.g., alkaline phosphatase, horseradish
peroxidase, luciferase, or .beta.-glactosidase), fluorescent
moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin,
GFP, or BFP), or luminescent moieties (e.g., Qdot.TM. nanoparticles
supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Other
applicable assays include quantitative immunoprecipitation or
complement fixation assays.
[0032] The invention further provides methods for pancreatic cancer
diagnosis and prognosis. One method is used to determine whether a
subject is suffering from or at risk for developing pancreatic
cancer. In this method, a biological sample is obtained from a
subject, and the methylation level of an ID4 gene promoter or the
expression level of an ID4 gene in the sample is determined. If the
methylation level of the ID4 gene promoter in the sample is higher
than a control methylation level or the expression level of the ID4
gene in the sample is lower than a control expression level, the
subject is likely to be suffering from or at risk for developing
pancreatic cancer, e.g., primary or metastatic pancreatic cancer.
The control methylation level of the ID4 gene promoter and the
control expression level of the ID4 gene may be, for example, the
methylation level and the expression level detected in a biological
sample from a normal subject or a normal biological sample from the
test subject.
[0033] In another method, a pancreatic cancer cell is provided, and
the methylation level of an ID4 gene promoter or the expression
level of an ID4 gene in the cell is determined as described above.
The angiogenesis or chemotaxis of the cell is then analyzed. If the
methylation level of the ID4 gene promoter in the cell is higher
than a control methylation level or the expression level of the ID4
gene in the cell is lower than a control expression level, it
indicates an increase in the angiogenesis or chemotaxis of the
cell.
[0034] The angiogenesis of a cell may be analyzed using any of the
methods known in the art. For example, in vivo or model system may
be analyzed by induction of blood vessels, induction of
angiogenesis growth factors (VEGF), and chick embryo
chorioallantoic membrane (CAM) assay.
[0035] Methods for analyzing the chemotaxis of a cell are also
known in the art. Such methods include in vitro cell migration or
invasive assay using Boyden Chambers and cell culture wound healing
assay.
[0036] In addition, the invention provides a method for screening
drugs for treating pancreatic cancer. The method involves the steps
of providing a pancreatic cancer cell that contains an ID4 gene
promoter or expresses an ID4 gene, contacting the cell with a test
compound, and determining the methylation level of the ID4 gene
promoter or the expression level of the ID4 gene in the cell. If
the methylation level of the ID4 gene promoter in the cell is lower
than a control methylation level or the expression level of the ID4
gene in the cell is higher than a control expression level, the
test compound is identified as a candidate for treating pancreatic
cancer. The control methylation level and the control expression
level may be, for example, the methylation level and the expression
level detected in the pancreatic cancer cell prior to the
contacting step. The method may optionally include a step of
manufacturing the identified candidate compound.
[0037] The test compounds of the present invention can be obtained
using any of the numerous approaches (e.g., combinatorial library
methods) known in the art. See, e.g., U.S. Pat. No. 6,462,187. Such
libraries include, without limitation, peptide libraries, peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone that is resistant
to enzymatic degradation), spatially addressable parallel solid
phase or solution phase libraries, synthetic libraries obtained by
deconvolution or affinity chromatography selection, and the
"one-bead one-compound" libraries. Compounds in the last three
libraries can be peptides, non-peptide oligomers, or small
molecules. Examples of methods for synthesizing molecular libraries
can be found in the art. Libraries of compounds may be presented in
solution, or on beads, chips, bacteria, spores, plasmids, or
phages.
[0038] To identify a compound for treating pancreatic cancer, a
pancreatic cancer cell or a subject suffering from pancreatic
cancer is provided. The cell or the subject contains an ID4 gene
promoter or expresses an ID4 gene. It may be a cell or subject that
naturally contains an ID4 gene promoter or expresses an ID4 gene,
or alternatively, a cell or subject that contains a recombinant
form of an ID4 gene promoter or expresses a recombinant form of an
ID4 gene.
[0039] The information obtained from the diagnostic and prognostic
methods and the screening assays may be used for optimizing the
design of a compound for treating pancreatic cancer, manufacturing
a candidate compound identified using the screening assays, or
treating pancreatic cancer with appropriate compounds.
[0040] The compounds of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include
the compounds and pharmaceutically acceptable carriers.
"Pharmaceutically acceptable carriers" include solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Other active compounds can also be
incorporated into the compositions.
[0041] A pharmaceutical composition is often formulated to be
compatible with its intended route of administration. See, e.g.,
U.S. Pat. No. 6,756,196. Examples of routes of administration
include parenteral, e.g., intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (topical), transmucosal, and
rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0042] In one embodiment, the compounds are prepared with carriers
that will protect the compounds against rapid elimination from the
body, such as a controlled release formulation, including implants
and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0043] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form," as used herein, refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0044] Moreover, the invention provides a method of inhibiting the
methylation of an ID4 gene promoter or enhancing the expression of
an ID4 gene in a cell in vivo and in vitro. The method involves the
steps of providing a pancreatic cancer cell, and contacting the
cell with a compound that decreases the methylation level of an ID4
gene promoter or increases the expression level of an ID4 gene in
the cell.
[0045] For example, the compound may be an ID4 protein or a nucleic
acid encoding an ID4 protein. Additionally, the compound may be an
agent that activates an ID4 gene, including transcription factor
proteins, serum growth factors, tissue inhibitor of matrix
metalloproteinase-1 (TIMP-1), and YY1 (a lineage-specific repressor
of transcriptional inhibitors).
[0046] The therapeutic compound can be a demethylating agent such
as 5-aza-cytidine or a compound capable of demethylating CpG
islands methylated in promoter regions. These compounds can reverse
gene silencing and activate gene expression. Other types of
compounds are histone deacetylase (HDAC) inhibitors such as
Trichostatin which can modify histones in chromatin regions and
activate genes silenced by methylation of CpG islands in promoter
regions. There are HDAC inhibitors available for in vitro and
clinical trials.
[0047] The invention also provides a method for treating pancreatic
cancer. The method involves the steps of identifying a subject
suffering from or at risk for developing pancreatic cancer, and
administering to the subject an effective amount of a compound that
decreases the methylation level of the ID4 gene promoter or
increases the expression level of the ID4 gene in the subject. A
subject to be treated may be identified in the judgment of the
subject or a health care professional, and can be subjective (e.g.,
opinion) or objective (e.g., measurable by a test or diagnostic
method such as those described above).
[0048] The term "treating" is defined as administration of a
substance to a subject with the purpose to cure, alleviate,
relieve, remedy, prevent, or ameliorate a disorder, symptoms of the
disorder, a disease state secondary to the disorder, or
predisposition toward the disorder. A subject to be treated may be
identified, e.g., using the diagnostic method described above.
[0049] An "effective amount" is an amount of a compound that is
capable of producing a medically desirable result in a treated
subject. The medically desirable result may be objective (i.e.,
measurable by some test or marker) or subjective (i.e., subject
gives an indication of or feels an effect). The treatment methods
can be performed alone or in conjunction with other drugs and/or
radiotherapy. See, e.g., U.S. Patent Application 20040224363.
[0050] In one in vivo approach, a therapeutic compound (e.g., a
compound that decreases the methylation level of an ID4 gene
promoter or increases the expression level of an ID4 gene in a
subject) itself is administered to a subject.
[0051] Generally, the compound will be suspended in a
pharmaceutically-acceptable carrier and administered orally or by
intravenous (i.v.) infusion, or injected or implanted
subcutaneously, intramuscularly, intrathecally, intraperitoneally,
intrarectally, intravaginally, intranasally, intragastrically,
intratracheally, or intrapulmonarily. For treatment of cancer, the
compound is preferably delivered directly to tumor cells, e.g., to
a tumor or a tumor bed following surgical excision of the tumor, in
order to kill any remaining tumor cells. For prevention of cancer
invasion and metastases, the compound can be administered to, for
example, a subject that has not yet developed detectable invasion
and metastases but is found to have an increased methylation level
of an ID4 gene promoter or a decreased expression level of an ID4
gene. The dosage required depends on the choice of the route of
administration, the nature of the formulation, the nature of the
subject's illness, the subject's size, weight, surface area, age,
and sex, other drugs being administered, and the judgment of the
attending physician. Suitable dosages are in the range of
0.01-100.0 mg/kg. Wide variations in the needed dosage are to be
expected in view of the variety of compounds available and the
different efficiencies of various routes of administration. For
example, oral administration would be expected to require higher
dosages than administration by i.v. injection. Variations in these
dosage levels can be adjusted using standard empirical routines for
optimization as is well understood in the art. Encapsulation of the
compound in a suitable delivery vehicle (e.g., polymeric
microparticles or implantable devices) may increase the efficiency
of delivery, particularly for oral delivery.
[0052] In some embodiments, polynucleotides are administered to a
subject. Polynucleotides can be delivered to target cells by, for
example, the use of polymeric, biodegradable microparticle or
microcapsule devices known in the art. Another way to achieve
uptake of the nucleic acid is using liposomes, prepared by standard
methods. The polynucleotides can be incorporated alone into these
delivery vehicles or co-incorporated with tissue-specific or
tumor-specific antibodies. Alternatively, one can prepare a
molecular conjugate composed of a polynucleotide attached to
poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine
binds to a ligand that can bind to a receptor on target cells.
"Naked DNA" (i.e., without a delivery vehicle) can also be
delivered to an intramuscular, intradermal, or subcutaneous site. A
preferred dosage for administration of polynucleotide is from
approximately 10.sup.6 to 10.sup.12 copies of the polynucleotide
molecule.
[0053] The following examples are intended to illustrate, but not
to limit, the scope of the invention. While such examples are
typical of those that might be used, other procedures known to
those skilled in the art may alternatively be utilized. Indeed,
those of ordinary skill in the art can readily envision and produce
further embodiments, based on the teachings herein, without undue
experimentation.
EXAMPLE
DNA Methylation of Transcription Factor ID4 Regulates Chemotaxis
and Angiogenesis of Pancreatic Cancer
Introduction
[0054] Epigenetic modifications of transcription factor ID4, a
helix-loop-helix inhibitory transcription factor, appear to promote
cancer progression. Pancreatic cancer is often characterized by
epigenetic modifications of tumor suppressor genes. It was believed
that loss of ID4 expression may have a role in the progression of
pancreatic cancer, and that ID4 regulates the growth and invasion
of pancreatic cancer.
Methods
[0055] Established pancreatic cancer cell lines were screened for
ID4 expression by a quantitative real-time RT-PCR (qRT) assay. In
cell lines with absent ID4 expression, epigenetic modifications of
the ID4 promoter region CpG islands were assessed to determine the
role of DNA hypermethylation as the cause of absent ID4 expression.
A short-interfering RNA (siRNA) assay for ID4 was developed to
assess the specific contributions of ID4 gene expression to
pancreatic cancer cell proliferation, chemotaxis, and angiogenesis.
Abrogation of ID4 expression in pancreatic cancer cells lines by an
siRNA assay was measured by qRT and immunohistochemistry (IHC).
Tumor specimens from patients with pancreatic ductal adenocarcinoma
(PDAC) were assessed for DNA hypermethylation of ID4.
[0056] Pancreatic cancer lines assessed included PANC1, MiaPACA2,
CAPAN1, Hs766T, BxPC3, COLO357, AsPC1, and CFPAC-1. DNA from cell
lines was extracted using DNAzol. DNA from paraffin-embedded
specimens was extracted using a Qiagen kit. DNA was modified with
sodium bisulfite to assess methylation of specific CpG islands in
the promoter region of ID4. Methylation specific PCR was performed
for ID4 DNA with primers for methylated/unmethylated ID4. The
methylation status of ID4 was determined by CEQ analysis (Beckman
Coulter).
Results
Cell Lines
[0057] Screening of eight pancreatic cancer cell lines (AsPC-1, MIA
PaCa-2, PANC-1, CAPAN-1, Hs766T, CFPAC-1, BxPC-3, and COLO357) for
ID4 expression by qRT demonstrated that 5 of 8 (62%) cell lines
showed no ID4 expression (FIG. 2). Cell lines with absent ID4
expression were assessed for hypermethylation of the ID4 DNA
promoter region. Methylation specific PCR demonstrated complete
methylation of the CpG islands of the ID4 promoter region in these
cell lines.
Clinical Specimens
[0058] Specimens from 20 patients who underwent curative resection
for pancreatic cancer were accrued. Analysis of 20 PDAC (pancreatic
ductal adenocarcinoma) specimens demonstrated complete methylation
of the ID4 promoter region CpG islands in primary tumors in 17 of
20 (85%) patients.
Short Interfering RNA (siRNA) Assay
[0059] siRNA transfection was performed with Lipofectamine2000.
Positive (Lamin) and negative (nonsense) siRNA controls were
included. Transfection efficiency was assessed by qRT and IHC. The
efficiency of siRNA knockout on ID4 expression measured by qRT
showed 80%-90% decrease in ID4 expression with siRNA. This result
was corroborated by IHC. See FIGS. 3-5.
Cell Viability
[0060] Cells were assessed after transfection with ID4 siRNA.
Trypan blue exclusion test was performed. Abrogation of ID4
expression in pancreatic cancer cells resulted in an increase in
endothelial cell angiogenesis by an in vitro assay. Similarly,
there was an increase in cancer cell chemotaxis with a decrease in
ID4 expression. However, there was no significant change in
proliferation with ID4 siRNA. See FIGS. 6-7.
Conclusions
[0061] ID4 undergoes epigenetic silencing. It promotes growth and
abrogates migration. Expression of transcription factor ID4 appears
to result in the inhibition of pancreatic cancer cell chemotaxis
and endothelial cell angiogenesis, verifying its endogenous
inhibitory role. ID4 expression appears to be regulated by DNA
promoter region CpG island hypermethylation, which is a frequent
clinical finding. This study identifies a transcription factor that
suppresses pancreatic cancer progression. Potential future
treatment strategies should favor recapitulation of ID4
expression.
Discussion
[0062] ID4 is frequently silenced in pancreatic cancer, which may
enhance the migratory/invasive phenotype of pancreatic cancer.
Recapitulation of ID4 expression in human pancreatic cancer may
preferentially limit migration/invasion of pancreatic cancer.
[0063] All publications cited herein are incorporated by reference
in their entirety.
Sequence CWU 1
1
111500DNAHomo sapiensmisc_featureDNA sequence surrounding the human
ID4 promoter region 1cattaatggc ctaaattaag ttacaggtat gaattttaca
taaaacagat taatattata 60tgtcataatg gaattttaaa tattccgtgt ccatgcattt
ttaatcttta cgtgctctaa 120ttgaatgcgc aaggcaactg catttcttga
gcccactttt gcatttagat ggggtaaaag 180aacccccccc acgtttttgt
tttatttata ttcccttcac caaaaacctg cattcgattc 240ggcattcttt
tccttctttt tttttctact tttgctaagc tttagcattt tttaaaaaga
300aaacggaaag gctacacatt ccattccatc attatggttt cggcaaatgt
gaaaaggcga 360ataatgaaac ggaggaggga aatatagaac agaatgaacg
tgccttcttg aacagcgcgt 420ctttcttaag gcactggaat cccacggatg
gagtgatggg tggcggaggg tccctgggcg 480ccgtgctatt aggagtggca
gggtatccgc gagcagggcc caggcgctcc ctcagcagcc 540tagtcgggat
aaggggggcg gtggagagtg aattccggcc gcacattccc gcagttcttc
600gcaggaactt cgctctctct tttcccctcc cttgggcaca catcagcctg
gcccgactcc 660cactcagctc tcttttctca gaaccccgac ccacagcgtt
gacggaatgg agtgcccttc 720ccattggccc gagcgtcatt ccccgaggtg
gcactgcccg cctgattggc tggccactcc 780agaccccccg cccactcctc
cactcgggta gccggactcc ccgcccccca gcaccgcccg 840gagcccccgc
cctcgcctct ccctccgcgc ccccgcccgc gcgcccagcg ggctccgctc
900ggctcgcgct gcgacccggc ccgcgcgctg gtcccgcccc cggggcgcac
ggctctataa 960atacagctgc gcggcgggcc gggcgagagc gtagtggagg
aggcgcggtt gtgagtagta 1020ccgggagtgg ggtgatcccg ggctagggga
gcgcggcggc cgcgatcggg cttagtcgga 1080gctccgaagg gagtgactag
gacacccggg tgggctactt ttcttccggt gcttttgctt 1140tttttttcct
ttgggctcgg gctgagtgtc gcccactgag caaagattcc ctcgtaaaac
1200ccagagcgac cctcccgtca attgttgggc tcgggagtgt cgcggtgccc
cgagcgcgcc 1260gggcgcggag gcaaagggag cggagccggc cgcggacggg
gcccggagct tgcctgcctc 1320cctcgctcgc cccagcgggt tcgctcgcgt
agagcgcagg gcgcgcgcga tgaaggcggt 1380gagcccggtg cgcccctcgg
gccgcaaggc gccgtcgggc tgcggcggcg gggagctggc 1440gctgcgctgc
ctggccgagc acggccacag cctgggtggc tccgcagccg cggcggcggc 1500
* * * * *