U.S. patent application number 14/689194 was filed with the patent office on 2015-10-01 for protein tyrosine phosphatase mutations in cancers.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Kenneth W. Kinzler, Victor Velculescu, Bert Vogelstein, Zhenghe Wang.
Application Number | 20150275315 14/689194 |
Document ID | / |
Family ID | 35428947 |
Filed Date | 2015-10-01 |
United States Patent
Application |
20150275315 |
Kind Code |
A1 |
Wang; Zhenghe ; et
al. |
October 1, 2015 |
Protein Tyrosine Phosphatase Mutations in Cancers
Abstract
Tyrosine phosphorylation, regulated by protein tyrosine
phosphatases (PTPs) and kinases (PTKs), is important in signaling
pathways underlying tumorigenesis. A mutational analysis of the
tyrosine phosphatase gene superfamily in human cancers identified
83 somatic mutations in six PTPs (PTPRF, PTPRG, PTPRT, PTPN3,
PTPN13, PTPN14), affecting 26% of colorectal cancers and a smaller
fraction of lung, breast and gastric cancers. Fifteen mutations
were nonsense, frameshift or splice site alterations predicted to
result in truncated proteins lacking phosphatase activity. Five
missense mutations in the most commonly altered PTP (PTPRT) were
biochemically examined and found to reduce phosphatase activity.
Expression of wild-type but not a mutant PTPRT in human cancer
cells inhibited cell growth. These observations suggest that the
tyrosine phosphatase genes are tumor suppressor genes, regulating
cellular pathways that may be amenable to therapeutic
intervention.
Inventors: |
Wang; Zhenghe; (Baltimore,
MD) ; Velculescu; Victor; (Dayton, MD) ;
Kinzler; Kenneth W.; (Baltimore, MD) ; Vogelstein;
Bert; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Family ID: |
35428947 |
Appl. No.: |
14/689194 |
Filed: |
April 17, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13275958 |
Oct 18, 2011 |
9012145 |
|
|
14689194 |
|
|
|
|
11596349 |
Sep 27, 2007 |
8039210 |
|
|
PCT/US2005/017105 |
May 16, 2005 |
|
|
|
13275958 |
|
|
|
|
60571436 |
May 14, 2004 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/196; 435/21; 435/325; 435/455; 435/6.14; 506/2 |
Current CPC
Class: |
A61P 35/00 20180101;
C12Q 2600/112 20130101; C12Q 1/6886 20130101; C12Y 301/03048
20130101; C12Q 2600/136 20130101; C12Q 2600/158 20130101; C12N 9/16
20130101; G01N 33/5011 20130101; C12Q 2600/156 20130101; G01N
2333/916 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 9/16 20060101 C12N009/16; G01N 33/50 20060101
G01N033/50 |
Goverment Interests
[0001] This invention was made under contracts (CA43460 and
CA63934) with an agency (National Institutes of Health) of the
United States Government. The United States Government therefore
retains certain rights in the invention.
Claims
1. A method for identifying mutations involved in cancer,
comprising: determining nucleotide sequence differences in a human
nucleotide sequence in matched pairs of cancer cells and normal
cells, each pair being from a single individual, wherein the human
nucleotide sequence encodes a protein tyrosine phosphatase selected
from the group consisting of: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13,
and PTPN14.
2. The method of claim 1 wherein at least one of the nucleotide
sequence differences is in a region encoding a catalytic
domain.
3. The method of claim 1 wherein at least one of the nucleotide
sequence differences is a point mutation.
4. The method of claim 1 wherein at least one of the nucleotide
sequence differences is in a regulatory region.
5. The method of claim 1 further comprising determining whether a
nucleotide sequence difference is synonymous or non-synonymous.
6. The method of claim 1 further comprising determining whether a
nucleotide difference affects an evolutionarily conserved amino
acid residue.
7. The method of claim 1 wherein the protein tyrosine phosphatase
is PTPRT.
8. The method of claim 1 wherein the protein tyrosine phosphatase
is PTPRG.
9. The method of claim 1 wherein the cancer cells are selected from
the group consisting of bladder cancer, melanoma, breast cancer,
non-Hodgkin's lymphoma, colon and rectal cancer, pancreatic cancer,
endometrial cancer, prostate cancer, kidney cancer (renal cell),
skin (non-melanoma), leukemia, thyroid cancer, and lung cancer.
10. A method of screening test substances for use as anti-cancer
agents, comprising: contacting a test substance with a wild-type
form or a mutant form of a protein tyrosine phosphatase which is
mutated in cancer cells, wherein the protein tyrosine phosphatase
is selected from the group consisting of: PTPRF, PTPRG, PTPRT,
PTPN3, PTPN13, and PTPN14; testing activity of the form of the
protein tyrosine phosphatase, wherein a test substance which
increases the activity of the form of a protein tyrosine
phosphatase is a potential anti-cancer agent.
11. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is in a cell.
12. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is isolated from a cell.
13. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is in a cell of a cancer cell line.
14. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is in a cell which has been modified to express the
form of a protein tyrosine phosphatase.
15. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is PTPRF.
16. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is PTPRG.
17. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is PTPRT.
18. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is PTPN3.
19. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is wild-type.
20. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is PTPN13.
21. The method of claim 10 wherein the form of a protein tyrosine
phosphatase is PTPN14.
22. The method of claim 15 wherein the PTPRF has a mutation
selected from the group consisting of R218C, G645R, G1040V, R1333C,
V1390I, 387-468 substitution, and A381V.
23. The method of claim 16 wherein the PTPRG has a mutation
selected from the group consisting of T361M, A462V, T514M, R593W,
E955G, Y973C, R1312W, and I1326V.
24. The method of claim 17 wherein the PTPRT has a mutation
selected from the group consisting of A209T, V1269M, A209T, F248S,
Y280H, Y412F, N510K, T605M, V648G, R632X, A707T, D927G, A707V,
R1021X, F74S, L708P, R975X, LOH, Q987K, A1118P, T1368M, N1128I,
R1212W, M1259L, I395V, Y1351F, T1368M, R453C, K218T, R1346L, and
R790I.
25. The method of claim 18 wherein the PTPN3 has a mutation
selected from the group consisting of V154I, A239V, E610X, S300A,
F307L, and R330Q.
26. The method of claim 20 wherein the PTPN13 has a mutation
selected from the group consisting of H2Q, E952X, G1476C, R380X,
M2443I, R402X, S443N, A529D, Q1691X, K2131N, D2154H, R2205W,
R2338X, Y2279X, M2307T, I2458V, and E2474D.
27. The method of claim 21 wherein the PTPN14 has a mutation
selected from the group consisting of L56M, R293Q, S314P, Q332R,
R491Q, P525L, H633Y, P528L, T657M, E883D, and T1068M.
28. An isolated, mutant form of protein tyrosine phosphatase
selected from the group consisting of: PTPRF, PTPRG, PTPRT, PTPN3,
PTPN13, and PTPN14, wherein enzymatic activity of the mutant form
is reduced compared to wild-type.
29. The isolated, mutant form of claim 28 which is PTPRF.
30. The isolated, mutant form of claim 28 which is PTPRG.
31. The isolated, mutant form of claim 28 which is PTPRT.
32. The isolated, mutant form of claim 28 which is PTPN3.
33. The isolated, mutant form of claim 28 which is PTPN13.
34. The isolated, mutant form of claim 28 which is PTPN14.
35. The isolated, mutant form of claim 29 wherein the PTPRF has a
mutation selected from the group consisting of R218C, G645R,
G1040V, R1333C, V1390I, 387-468 substitution, and A381V.
36. The isolated, mutant form of claim 30 wherein the PTPRG has a
mutation selected from the group consisting of T361M, A462V, T514M,
R593W, E955G, Y973C, R1312W, and I1326V.
37. The isolated, mutant form of claim 31 wherein the PTPRT has a
mutation selected from the group consisting of A209T, V1269M,
A209T, F248S, Y280H, Y412F, N510K, T605M, V648G, R632X, A707T,
D927G, A707V, R1021X, F74S, L708P, R975X, LOH, Q987K, A1118P,
T1368M, N1128I, R1212W, M1259L, I395V, Y1351F, T1368M, R453C,
K218T, R1346L, and R790I.
38. The isolated, mutant form of claim 32 wherein the PTPN3 has a
mutation selected from the group consisting of V154I, A239V, E610X,
S300A, F307L, and R330Q.
39. The isolated, mutant form of claim 33 wherein the mutant PTPN13
has a mutation selected from the group consisting of H2Q, E952X,
G1476C, R380X, M2443I, R402X, S443N, A529D, Q1691X, K2131N, D2154H,
R2205W, R2338X, Y2279X, M2307T, I2458V, and E2474D.
40. The isolated, mutant form of claim 34 wherein the mutant PTPN14
has a mutation selected from the group consisting of L56M, R293Q,
S314P, Q332R, R491Q, P525L, H633Y, P528L, T657M, E883D, and
T1068M.
41. An isolated polynucleotide which encodes a mutant form of
protein tyrosine phosphatase selected from the group consisting of:
PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14, wherein enzymatic
activity of the mutant form is reduced compared to wild-type.
42. A host cell comprising the polynucleotide of claim 41.
43. A method of categorizing cancers, comprising: determining the
coding sequence for or the amino acid sequence of one or more
protein tyrosine phosphatase family members selected from the group
consisting of PTPRF, PTPRG, PTPN3, PTPN13, and PTPN14 in a sample
of a cancer tissue; identifying a somatic mutation of said one or
more protein tyrosine phosphatase family members in the cancer
tissue; assigning the cancer tissue to a group based on the
presence or absence of the somatic mutation.
44. The method of claim 43 wherein the mutation is one which
truncates the protein tyrosine phosphatase family member.
45. The method of claim 43 wherein the mutation is
non-synonymous.
46. The method of claim 43 wherein the mutation is somatic.
47. The method of claim 43 wherein the mutation is a point
mutation.
48. The method of claim 43 wherein the protein kinase family member
is PTPRF.
49. The method of claim 43 wherein the protein kinase family member
is PTPRG.
50. The method of claim 43 wherein the protein kinase family member
is PTPN3.
51. The method of claim 43 wherein the protein kinase family member
is PTPN13.
52. The method of claim 43 wherein the protein kinase family member
is PTPN14.
53. The method of claim 43 wherein the group is used to analyze or
design clinical trials.
54. The method of claim 43 wherein the group is used to correlate
with prognostic data.
55. The method of claim 43 wherein the group is used to correlate
with recurrence data.
56. The method of claim 43 wherein the group is used to select an
appropriate therapeutic agent.
57. The method of claim 43 wherein the group is used to identify
cancer.
58. The method of claim 43 wherein the cancer tissue is
colorectal.
59. The method of claim 43 wherein the cancer tissue is breast.
60. The method of claim 43 wherein the cancer tissue is lung.
61. The method of claim 43 wherein the cancer tissue is
gastric.
62. A method of inhibiting growth of cancer cells, comprising:
administering to cancer cells a polynucleotide encoding a wild-type
protein tyrosine phosphatase selected from the group consisting of
PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14, whereby growth of
the cancer cells is inhibited.
63. The method of claim 62 wherein the cancer cells are
colorectal.
64. The method of claim 62 wherein the cancer cells are breast.
65. The method of claim 62 wherein the cancer cells are lung.
66. The method of claim 62 wherein the cancer cells are
gastric.
67. The method of claim 62 wherein the cancer cells are in
culture.
68. The method of claim 62 wherein the cancer cells are in a human
body.
69. The method of claim 62 wherein the polynucleotide is
administered by intratumoral injection.
70. The method of claim 62 wherein the cancer cells comprise one or
two mutant alleles of a protein tyrosine phosphatase selected from
the group consisting of PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and
PTPN14.
71. A method of identifying cancer cells in a sample collected from
a human, comprising: determining the coding sequence for or the
amino acid sequence of one or more protein tyrosine phosphatase
family members selected from the group consisting of PTPRF, PTPRG,
PTPN3, PTPN13, and PTPN14 in a sample collected from the human,
wherein the sample is selected from the group consisting of a
suspected cancer tissue, blood, serum, plasma, and stool;
identifying a somatic mutation of said one or more protein tyrosine
phosphatase family members in the cancer tissue; identifying the
sample as containing cancer cells if a somatic mutation is
identified.
72. The method of claim 71 wherein the protein tyrosine phosphatase
family member is PTPRF.
73. The method of claim 71 wherein the protein tyrosine phosphatase
family member is PTPRG.
74. The method of claim 71 wherein the protein tyrosine phosphatase
family member is PTPN3.
75. The method of claim 71 wherein the protein tyrosine phosphatase
family member is PTPN13.
76. The method of claim 71 wherein the protein tyrosine phosphatase
family member is PTPN14.
77. The method of claim 1 wherein the protein tyrosine phosphatase
is PTPRF.
78. The method of claim 1 wherein the protein tyrosine phosphatase
is PTPN3.
79. The method of claim 1 wherein the protein tyrosine phosphatase
is PTPN13
80. The method of claim 1 wherein the protein tyrosine phosphatase
is PTPN14.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of cancer. In
particular, it relates to diagnosis, prognosis, treatment, drug
discovery, target discovery, clinical testing for cancer.
BACKGROUND OF THE INVENTION
[0003] Phosphorylation of tyrosine residues is a central feature of
most cellular signaling pathways, including those affecting growth,
differentiation, cell cycle regulation, apoptosis and invasion (1,
2). This phosphorylation is coordinately controlled by protein
tyrosine kinases (PTKs) and phosphatases (PTPs). Although a variety
of PTK genes have been directly linked to tumorigenesis through
somatic activating mutations (3-6) only a few PTP genes have been
implicated in cancer (7-10). Moreover, it is not known how many or
how frequently members of the PTP gene family are altered in any
particular cancer type.
[0004] The PTP gene superfamily is composed of three main families:
(i) the classical PTPs, including the receptor PTPs (RPTPs) and the
non-receptor PTPs (NRPTPs); (ii) the dual specificity phosphatases
(DSPs), which can dephosphorylate serine and threonine in addition
to tyrosine residues; and (iii) the low molecular weight
phosphatases (LMPs) (1).
[0005] There is a continuing need in the art to identify new
therapeutic targets, identify new drugs, improve diagnosis,
prognosis, and therapy of cancers.
SUMMARY OF THE INVENTION
[0006] One aspect of the invention is a method for identifying
mutations involved in cancer. Nucleotide sequence differences are
determined in a human nucleotide sequence between matched pairs of
cancer cells and normal cells. Each matched pair of cells is
isolated from a single individual. The human nucleotide sequence
encodes a protein tyrosine phosphatase selected from the group
consisting of: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14.
[0007] Another aspect of the invention is a method of screening
test substances for use as anti-cancer agents. A test substance is
contacted with a wild-type form of a protein tyrosine phosphatase
or a mutant form of a protein tyrosine phosphatase which is mutated
in cancer cells. Activity of the form of the protein tyrosine
phosphatase is tested. A test substance which increases the
activity of the form of a protein tyrosine phosphatase is a
potential anti-cancer agent. The protein tyrosine phosphatase is
selected from the group consisting of: PTPRF, PTPRG, PTPRT, PTPN3,
PTPN13, and PTPN14.
[0008] One embodiment of the invention provides an isolated, mutant
form of a protein tyrosine phosphatase. The phosphatase is selected
from the group consisting of: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13,
and PTPN14. Enzymatic activity of the mutant form is reduced
compared to wild-type.
[0009] Another embodiment of the invention provides an isolated
polynucleotide which encodes a mutant form of protein tyrosine
phosphatase. The phosphatase is selected from the group consisting
of: PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14. Enzymatic
activity of the mutant form is reduced compared to wild-type.
[0010] Still another aspect of the invention is a method of
categorizing cancers. The coding sequence for or the amino acid
sequence of one or more protein tyrosine phosphatase family members
in a sample of a cancer tissue is determined. The family member is
selected from the group consisting of PTPRF, PTPRG, PTPRT, PTPN3,
PTPN13, and PTPN14.
[0011] A somatic mutation of the one or more protein tyrosine
phosphatase family members is identified in the cancer tissue. The
cancer tissue is assigned to a group based on the presence or
absence of the somatic mutation.
[0012] According to another aspect of the invention a method of
inhibiting growth of cancer cells is provided. A polynucleotide
encoding a wild-type protein tyrosine phosphatase is administered
to cancer cells. The phosphatase is selected from the group
consisting of PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14,
Growth of the cancer cells is thereby inhibited.
[0013] Yet another aspect of the invention is a method of
identifying cancer cells in a sample collected from a human. The
coding sequence for or the amino acid sequence of one or more
protein tyrosine phosphatase family members in a sample collected
from the human is determined. The family member is selected from
the group consisting of PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and
PTPN14. The sample is selected from the group consisting of a
suspected cancer tissue, blood, serum, plasma, and stool. A somatic
mutation of said one or more protein tyrosine phosphatase family
members is identified in the cancer tissue. The sample is
identified as containing cancer cells if a somatic mutation is
identified.
[0014] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with reagents and methods for detection, diagnosis, therapy, and
drug screening pertaining to cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. Distribution of mutations in PTPRT, PTPN13, PTPN14,
PTPRG, PTPRF, and PTPN3. Black arrows indicate location of missense
mutations, red arrows indicate location of nonsense mutations or
frameshifts, and boxes represent functional domains (B41, band 41;
CA, carbonic anhydrase; FN3, fibronectin type III; IG,
immunoglobulin; MAM, meprin/A5/PTP.mu.; PDZ, postsynaptic density,
discs large, zonula occludans; PTPc, catalytic phosphatase domain).
Black stars indicate PTPRT mutants evaluated for phosphatase
activity (see results in FIG. 2), and red star indicates PTPRT
mutant evaluated for effects on cell proliferation (see results in
FIG. 3).
[0016] FIG. 2A-2B. Evaluation of phosphatase activity of mutant
PTPRT. (FIG. 2A) Saturation kinetics of wild-type and mutant PTPRT.
His-tagged versions of PTPRT protein segments comprising the two
catalytic domains containing wild-type (WT) and tumor-specific
mutant sequences were expressed in bacteria and purified using
nickel affinity chromatography. Equal amounts of WT and mutant
proteins were used to evaluate enzyme kinetics. The rate of
hydrolysis of substrate (DiFMUP) is plotted against increasing
substrate concentration. Data were fitted to the Michaelis-Menton
equation and the resulting kinetic parameters of WT and mutant
proteins are indicated (FIG. 2B).
[0017] FIG. 3A-3B. PTPRT overexpression suppresses growth of human
cancer cells. (FIG. 3A) HCT116 colorectal cancer cells were
transfected with wild-type (WT) PTPRT construct, truncated R632X
mutant PTPRT construct, or empty pCI-Neo vector. The photographs
show colonies stained with crystal violet after 14 days of
geneticin selection. (FIG. 3B) Number of resistant colonies (mean
of two 25 cm.sup.2 flasks) for WT PTPRT, mutant PTPRT, and empty
vector.
[0018] FIG. 4. Expression analysis of PTPRT. PTPRT expression was
evaluated by reverse transcription of total RNA followed by PCR
amplification using a forward primer from exon 31 and a reverse
primer from exon 32. Expression analysis of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as a
control. Tissues analyzed are indicated above. PBL represents
peripheral blood leukocytes; DiFi and Hct116 represent cancer cell
lines derived from the colon.
[0019] FIG. 5. (Table S1.) Mutations of the tyrosine phosphatome in
human cancers
[0020] FIG. 6. (Table S2.) Tyrosine phosphatome genes analyzed (SEQ
ID NO: 15-101; encoded amino acids SEQ ID NO: 102-187).
DETAILED DESCRIPTION OF THE INVENTION
[0021] The inventors have discovered that protein tyrosine
phosphatase genes are the targets of somatic mutations in cancers,
suggesting that these genes function as tumor suppressors in human
cells. 87 genes were identified as being members of the protein
tyrosine phosphatase superfamily. These include members of the (i)
the classical PTPs, (ii) the dual specificity phosphatases (DSPs),
and (iii) the low molecular weight phosphatases (LMPs). See FIG. 6.
Screening a collection of colorectal cancers identified six
different genes of the classical PTPs which are the object of
somatic mutations in the colorectal cancers. Some of these genes
are also the subject of somatic mutations in breast, lung, and/or
gastric cancers. Screening collections of other types of cancers
will undoubtedly uncover other sets of the superfamily which are
somatically mutated. Other types of cancers which can be screened
for mutations include: bladder, melanoma, breast, non-Hodgkin's
lymphoma, pancreatic, endometrial, prostate, kidney, skin,
leukemia, thyroid, and lung.
[0022] Phosphatases which can be screened can be chosen from those
shown in FIG. 6. Other phosphatases may be identified for
screening, for example, using different bioinformatics criteria as
described below. The primers identified below for amplification and
sequencing of the phosphatases can be used, or other primers can be
used as is convenient for the practitioner. For identification of
mutations, determined sequences in samples can be compared to known
sequences in the literature. For example, for each of the 87
phosphatases of FIG. 6, a GenBank and a Celera accession number are
provided. Sequences determined in samples can be compared to the
wild-type sequences provided in the databases and attached sequence
listing. However, a better indication of involvement in cancer is
provided by comparing a determined sequence to that in a normal
tissue of the same human. Such a comparison indicates that a change
is a somatic mutation. Sequences in databases refer to the
sequences that existed in the databases as of May 14, 2004. GenBank
stores and records sequences according to the dates on which they
were indicated as the most recent update. Thus the sequences
available on May 14, 2004 are maintained and publicly available.
See also the sequence listing. It is well recognized in the art
that there is variation in the human population of wild-type
protein and nucleic acid sequences. Such variation typically
maintains sequences within a 95% identity range, more typically
within a 97% identity range, and more typically within a 99%
identity range.
[0023] Matched pairs of cells for determining somatic mutations
ideally are cells from a single individual. Typically the cells are
of the same type, e.g., lung cancer cells and normal lung cells. If
a body sample such as blood or stool is being examined, then normal
cells can be selected from any body tissue as a comparator.
[0024] Mutations that are relevant to cancer can be in almost any
region of the phosphatases, because the relevant mutations are
loss-of-function mutations. Thus the mutations can be in the
catalytic domain or in other portions of the protein. Mutations can
also be in non-coding, regulatory regions of the gene.
Non-synonymous mutations change the encoded amino acids of a
protein. Thus such mutations are highly likely to be functionally
relevant to cancer. Mutations in residues that are evolutionarily
conserved among species are also highly likely to be functionally
relevant to cancer.
[0025] Since loss of protein tyrosine phosphatase activity appears
to be detrimental to cells, reacquisition of activity should have a
positive, therapeutic effect. Test substances can be tested for
their ability to enhance the activity of PTPs by contacting a
wild-type or mutant PTP with a test substance. The PTP can be
isolated from cells and contacted in a cell-free system, or the PTP
may be in cells, either genetically engineered host cells or native
cells which express the PTP. The cells can be tested in culture or
in a model non-human animal system. Typically the cells will be
somatic cells. The PTP can be any mutant or wild-type form,
especially one of the six PTPs identified as mutant in colorectal
cancers, but also may any of the 87 identified below. One of the
mutant forms identified in the present study can be used (see FIG.
5) or other mutant forms, for example those found in other types of
cancer. PTPs can be isolated from producing host cells or native
producer cells. One means of purifying a PTP is disclosed in
example 7, in which a His tag is added to the PTP by cloning and
then used to purify the PTP using nickel affinity beads. A
desirable test substance for becoming a candidate anti-cancer agent
will enhance the activity of a wild-type and/or mutant PTP or
enhance PTP activity of a cell with wild-type, mutant, or both
types of PTP.
[0026] Polynucleotides comprising coding sequences for PTPs, in
particular mutant PTPs found in cancer cells, can be naturally
occurring coding sequences or coding sequences which are
synthesized based on the genetic code and the amino acid sequence
of a mutant PTP. The coding sequences can be inserted in expression
vectors so that quantities of the mutant PTPs can be produced
efficiently and used in drug screening assays. Alternatively, host
cells which contain expression vectors encoding mutant PTPs can be
used for drug screening assays. The mutant PTPs may be reduced in
enzyme activity, for example with a higher K.sub.m or with a lower
K.sub.cat than wild-type. The mutant PTPs may alternatively have no
detectable enzymatic activity.
[0027] Isolated polynucleotides are polynucleotides which are
separated from the chromosome upon which they normally reside in
the human genome. They are typically separated from the genes which
flank them on a normal human chromosome. They may be in a vector
with an origin of replication, or they may simply be an isolated
linear piece of nucleic acid. The polynucleotides encoding PTPs may
or may not contain the introns which are present in the human
genome.
[0028] Cancer tissues can be categorized on the basis of which, if
any, phosphatase mutation(s) they contain. Any of the PTPs
demonstrated to harbor cancer-related mutations can be used for the
categorization. Somatic mutations are identified on the basis of a
difference between an affected tissue and a normal tissue of the
same individual. Categorization of the tissue can be used for
stratifying patients for clinical trials, for analyzing data from
clinical trials, for correlating with prognostic data (such as
recurrence, metastasis, and life expectancy), as well as for
selecting an appropriate course of treatment for a cancer patient.
The PTP categorization can be used in conjunction with other data,
for example, histopathological data, to identify a cancer.
Similarly, PTP somatic mutation analysis can be used in any tissue
or body sample to diagnose cancer. Presence of a mutant PTP or
coding sequence in a tissue or body sample indicates the presence
of cancer cells, either in the sample itself, or in a tissue which
drains into the sample. Thus, for example, detection of PTP mutants
in a fecal sample reflects the presence of colorectal cancer cells
in the human from whom the sample was taken. Body samples which can
be tested include without limitation suspected cancerous tissues,
stool, sputum, tears, saliva, blood, plasma, serum, urine, and
bronchoalveolar lavage.
[0029] The mutational data associating loss of PTP function with
cancers strongly suggests that PTPs are tumor suppressors.
Therefore wild-type PTP coding sequences can be used as therapeutic
agents for treating tumors. Wild-type PTP coding sequences are
shown in the sequence listing. These sequences or wild-type
sequences which are at least 95% identical, at least 96% identical,
at least 97% identical, at least 98% identical, or least 99%
identical, can be used to deliver wild-type PTP to tumor cells. The
coding sequences may or may not contain introns. Sequences for any
of the six PTPs identified as somatically mutated in colorectal
cancers may be used, as well as any of the other PTPs identified in
FIG. 6.
[0030] Viral or non-viral vectors may be used for delivery of
polynucleotides. For example, adenoviruses, adeno-associated
viruses, herpes viruses, and retroviruses can be used for delivery.
Non-viral vectors include liposomes, nanoparticles and other
polymeric particles. Any vectors or techniques known in the art may
be used for delivering genes to cells or humans. See, e.g., Gene
Therapy Protocols, Paul D. Robbins, ed., Human Press, Totowa, N.J.,
1997. Vectors may not be necessary according to some protocols, and
coding sequences can be administered without a means of
replication. Administration of gene therapy vectors can be by any
means known in the art, including but not limited to intravenous,
intramuscular, intratumoral, intranasal, intrabronchial, and
subcutaneous injections or administration. An effective amount of
polynucleotide is one which inhibits growth of cancer cells in a
measurable amount. Preferably the tumor regresses and shrinks, or
at least ceases to grow larger.
[0031] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
Identification of PTP Gene Superfamily Members
[0032] We employed a combination of Hidden Markov Models
representing catalytic domains of members of the PTP superfamily to
identify 53 classical PTPs (21 RPTPs and 32 NRPTPs), 33 DSPs, and
one LMP in the human genome (12). This analysis revealed a set of
genes representing all known human PTPs (13) as well as seven
putative PTPs.
Example 2
Identification of PTP Gene Superfamily Members with Mutations
[0033] As an initial screen to evaluate whether these phosphatases
are genetically altered in human cancer, we analyzed the coding
exons of all 87 members of this gene family in 18 colorectal
cancers. A total of 1375 exons from all annotated RPTPs, NRPTPs,
DSPs and LMPs were extracted from genomic databases (12). These
exons were PCR-amplified from cancer genomic DNA samples and
directly sequenced using dye terminator chemistry (12). Whenever a
presumptive mutation was identified, we attempted to determine
whether it was somatically acquired (i.e., tumor specific) by
examining the sequence of the gene in genomic DNA from normal
tissue of the relevant patient.
[0034] From the 3.3 Mb of sequence information obtained, we
identified six genes containing somatic mutations, including three
members of the RPTP subfamily (PTPRF, PTPRG, and PTPRT) and three
members of the NRPTP subfamily (PTPN3, PTPN13 and PTPN14). These
six genes were then further analyzed for mutations in another 157
colorectal cancers. Through this strategy we identified 77
mutations in the six genes, in aggregate affecting 26% of the
colorectal tumors analyzed (Table S1, FIG. 1). Examination of these
six genes in seven other tumor types identified PTPRT mutations in
two of 11 (18%) lung cancers and two of 12 gastric cancers (17%),
and PTPRF mutations in one of 11 (9%) lung cancers and one of 11
(9%) breast cancers. No mutations were identified in 12 pancreatic
cancers, 12 ovarian cancers, 12 medulloblastomas or 12
glioblastomas (FIG. 5 (Table S1), FIG. 1). In total, 83
nonsynonymous mutations were observed, all of which were somatic in
the cancers that could be assessed (12).
[0035] Fifteen of the 83 mutations were nonsense, frameshift or
splice site alterations, all of which were predicted to result in
aberrant or truncated proteins. In 16 tumors both alleles of the
phosphatase gene appeared to be mutated, a characteristic often
associated with tumor suppressor genes. The majority of tumors with
PTP gene mutations also contained mutations in KRAS or BRAF, and
nine tumors contained alterations in previously reported tyrosine
kinase genes (FIG. 5 (Table S1)). Thus the mutant phosphatases
identified in this study are likely to operate through cellular
pathways distinct from those associated with previously identified
mutant kinases.
Example 3
Analysis of Mutation Types
[0036] Analysis of mutations in tumors is complicated by the fact
that mutations can arise either as functional alterations affecting
key genes underlying the neoplastic process or as nonfunctional
"passenger" changes. The multiple waves of clonal expansion and
selection that occur throughout tumorigenesis lead to fixation of
any mutation that had previously occurred in any predecessor cell,
regardless of whether the mutation was actually responsible for the
clonal expansion. Two independent lines of evidence suggest that
the sequence alterations we observed are functional. First, the
ratio of nonsynonymous to synonymous mutations provides an
indication of selection, as synonymous alterations usually do not
exert a growth advantage. There were no somatic synonymous
mutations detected in the colorectal cancers analyzed, resulting in
a ratio of nonsynonymous to synonymous mutations of 77 to 0, much
higher than the expected 2:1 ratio for non-selected passenger
mutations (p<1.times.10-6). Second, the prevalence of mutations
in the coding regions of the analyzed genes was .about.19 per Mb of
tumor DNA, similar to the prevalence of functional somatic
alterations observed in other gene families (e.g., the tyrosine
kinome (6)) and significantly higher than the prevalence of
nonfunctional alterations previously observed in the cancer genomes
(.about.1 per Mb, p<0.01)(14). These data support the idea that
these mutations were the targets of selection during
tumorigenesis.
Example 4
Effect of Point Mutations on Enzymatic Activity
[0037] The great majority of the nonsense and frameshift mutations
(FIG. 1) would result in polypeptides devoid of C-terminal
phosphatase catalytic domains, thereby leading to inactivation of
the phosphatase. To evaluate whether tumor-specific point mutations
alter phosphatase activity, we biochemically tested mutant versions
of PTPRT, the most frequently mutated PTP in the superfamily.
Mutations in both intracellular PTP domains (D1 and D2) were
evaluated. His-tagged versions of the catalytic region of wild-type
PTPRT, two D1 mutants (Q987K and N11281), and three D2 mutants
(R1212W, R1346L and T1368M) were produced in bacteria and analyzed
for phosphatase activity using 6,8-difluoro-4-methylumbelliferyl
phosphate (DiFMUP) as a substrate (FIG. 2) (12). All D1 and D2
mutants had reduced phosphatase activity compared with the
wild-type protein (FIG. 2). Interestingly, the kinetic parameter
K.sub.cat was reduced in both D1 mutants, while K.sub.m was
increased in all three D2 mutants, suggesting that mutations in the
two domains may have different effects on enzymatic activity.
Although the D2 domain has been thought to usually be catalytically
inactive (1), these results are consistent with recent studies that
show that the D2 domain is important for phosphatase activity in
some receptor phosphatases (15). These biochemical data on missense
mutations, coupled with the large number of truncating mutations
noted above, suggest that PTPRT functions as a tumor suppressor
gene.
Example 5
Phosphatase Functions as Tumor Suppressor
[0038] To determine whether PTPRT inhibits tumor cell growth, we
transfected wild-type PTPRT into HCT116 colorectal cancer cells
(12). An identical expression vector containing an R632X mutant of
PTPRT was used for comparison. Wild-type PTPRT potently inhibited
cell growth in this assay, as seen by the substantial decrease in
the number of neomycin resistant colonies compared with the R632X
mutant or with vector alone (FIG. 3A, 3B). Similar results with
wild-type and mutant PTPRT were also observed in DLD1 colorectal
cancer cells.
Example 6
Discussion
[0039] The combination of these genetic, biochemical, and cellular
data suggest that PTPRT and the other identified phosphatases are
likely to act as tumor suppressors. This is consistent with the
function of other phosphatases implicated in tumorigenesis
(7,8,16), and with the general role of phosphatases in inhibiting
various growth promoting signaling pathways (2). The absence of
biallelic mutations in a subset of the analyzed tumors suggests
that some alterations may act in a dominant negative fashion or may
affect gene dosage, mechanisms that have been previously involved
in inactivation of other tumor suppressor genes (17, 18).
[0040] Little is known about the functional role of the tyrosine
phosphatases discussed here. PTPN13 appears to be involved in
apoptosis (19) and may be partly responsible for the anti-tumor
effects of tamoxifen (20). Overexpression of PTPN3 inhibits growth
of NIH/3T3 cells, possibly through interaction with valosin
containing protein (VCP/p97) (21). PTPN14 and PTPRF are thought to
play a role in cell adhesion by regulating tyrosine phosphorylation
of adherens junction proteins (22, 23). As increased
phosphorylation of adherens junctions has been shown to increase
cell motility and migration (22, 24), mutational inactivation of
these genes may be an important step in cancer cell invasion and
metastasis. PTPRG maps to chromosome 3p14.2, a region frequently
lost in lung, renal and early stage breast tumors, and is thought
to be a target of the translocation at 3p14 in familial renal cell
carcinoma (25-27). However, no point mutations in PTPRG (28) or any
of the other genes identified here have been previously described
in any cancer. PTPRT is expressed in the developing central nervous
system and in the adult cerebellum (29) and had not been thought to
play a role in the growth or differentiation of other tissues. We
have found that PTPRT is expressed in a variety of human tissues,
including normal colon epithelium as well as cells derived from
colorectal cancers (FIG. 4).
Example 7
Materials and Methods
[0041] Identification of PTP genes. Protein tyrosine phosphatase
genes were identified by analysis of InterPro (IPR) phosphatase
domains present within the Celera draft human genome sequence.
IPR003595, IPR000340, IPR000751 and IPR002115 were used to search
all known and predicted genes for classical PTPs (RPTPs and
NRPTPs), DSPs, DSPs related to CDC25, and LMPs, respectively. This
resulted in identification of 91 tyrosine phosphatases, three of
which were pseudogenes and therefore not analyzed further. PTEN,
which has been determined to act primarily as a lipid phosphatase
was also not analyzed.
[0042] PCR, sequencing, and mutational analysis. Sequences for all
available annotated exons and adjacent intronic sequences of
identified PTP, DSP and LMP genes were extracted from Celera draft
human genome sequence (website: celera.com) or from GenBank
(website: genbank.nlm.nih.gov). Celera and public accession numbers
of all analyzed genes are available in FIG. 6 (Table S2).
[0043] Primers for PCR amplification and sequencing were designed
using the Primer 3 program (website:
genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), and were
synthesized by MWG (High Point, N.C.) or IDT (Coralville, Iowa).
PCR amplification and sequencing were performed on tumor DNA from
18 early passage cell lines as previously described (6) using a 384
capillary automated sequencing apparatus (Spectrumedix, State
College, Pa.). Sequence traces were assembled and analyzed to
identify potential genomic alterations using the Mutation Explorer
software package (SoftGenetics, State College, Pa.). Of the 1375
exons extracted, 92% were successfully analyzed, each in an average
of 17 tumor samples. All mutations listed in Table S1 were
determined to be somatic except in 10 cases in which no normal
tissue was available for comparison.
[0044] Construction of wild-type and mutant PTPRT proteins. The
region encoding two catalytic domains of PTPRT was cloned by PCR
using Platinum Hi-fidelity Taq polymerase (Invitrogen, Carlsbad,
Calif.) from human fetal brain cDNA with primers
GGAATTCCATATGGCCTTACCAGAGGGGCAGACAG (SEQ ID NO: 1) and
CGGGATCCCCCAGTTACTGCCATTCACA (SEQ ID NO: 2) and cloned in frame
fused to the 6X His tag of pET19b expression vector (Novagen,
Madison, Wis.). The Q987K, N1128I, R1212W, R1346L and T1368M
mutants were made using sexual PCR (Ref 30). The primers
CAAAAGTCCTTTACAGTCTCCTTCATCGGACCTTGAGTCGCAATG (SEQ ID NO: 3) and
AAGGAGACTGTAAAGGACTTTTGGAG (SEQ ID NO: 4) were used as mutagenic
primers for the Q987K mutant; the primers
CCATGCTTGACATGGCCGAGATTGAAGGGGTGGTGGACATCTTC (SEQ ID NO: 5) and
ATCTCGGCCATGTCAAGCATGG (SEQ ID NO: 6) were used as mutagenic
primers for the N11281 mutant; the primers
CAATGCTGCAGTCCTCGGGCCACACACGGGGTGTCACAATG (SEQ ID NO: 7) and
TGGCCCGAGGACTGCAGCATTG (SEQ ID NO: 8) were used as mutagenic
primers for the R1212W mutant; the primers
CTATACGATAACCATCCTGTGGCAGGGCCATGTTACAGATGCG (SEQ ID NO: 9) and
TGCCACAGGATGGTTATCGTATAG (SEQ ID NO: 10) were used as mutagenic
primers for the R1346L mutant; and the primers
GAGCGCTTGGAGGGGGGCATGTCCCGGTAGGCAGGCC (SEQ ID NO: 11) and
TGCCCCCCTCCAAGCGCTC (SEQ ID NO: 12) were used as mutagenic primers
for the T1368M mutant. For expression of recombinant proteins,
BL21-DE3 bacteria were grown to late log phase and induced with 1
mM IPTG for 3 hours at 37.degree. C. Bacterial lysates were made by
sonication in lysis buffer (1 mM Tris, 1 M NaCl, 10 mM imidazole
0.1% igepal, pH 8.0) and incubated with Ni-NTA beads for 45 min at
4.degree. C. The Ni-NTA beads were washed with 50 mM imidazole
buffer (40 mM Tris, 100 mM NaCl, 50 mM imidazole, pH8.0) and bound
protein was eluted with 500 mM imidazole.
[0045] Phosphatase kinetic analysis. Various concentrations of
6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP--Molecular
Probes D6567) were incubated with 800 ng of purified protein in 40
mM Tris-HCl pH8.0, 100 mM NaCl, 5 mM CaCl2, and 10 mM DTT in a 100
uL reaction. The reaction was incubated at 37.degree. C. for 30
minutes and fluorometric measurements were taken at an excitation
wavelength of 360 nm and an emission wavelength of 460 nm and
extrapolated to a standard curve of
6,8-difluoro-4-methylumbelliferone. The data were fitted to the
Michaelis-Menton equation using GraphPad Prism v. 3.02.
[0046] Cell proliferation assays. Full length wild-type or R632X
mutant PTPRT cDNA sequences were cloned into the pCI-Neo vector
(Promega, Madison, Wis.). Subconfluent HCT116 and DLD1 colorectal
cancer cells were transfected with equal amounts of the wild-type
PTPRT construct, R632X mutant PTPRT construct, or empty vector and
grown for 48 hours. Cells were then trypsinized and plated in T25
flasks with fresh media containing geneticin. Cells were grown for
2-3 weeks and stained with crystal violet. The expression level and
the mutational status of PTPRT are not known in either HCT116 or
DLD1 cells as no normal tissues from the same patients are
available as controls.
[0047] Expression analysis. Total RNAs from various human tissues
were purchased form BD Bioscience (San Jose, Calif.) and equal
amounts were reverse transcribed into cDNAs with random primers.
PTPRT expression was examined by PCR using primers
CCACATCGTGAAAACACTGC (SEQ ID NO: 13) and CAACAGGAGACCCCTCAGAA (SEQ
ID NO: 14) which are located in exons 31 and 32 and result in a 284
bp product.
REFERENCES
[0048] The disclosure of each reference cited is expressly
incorporated herein. [0049] 1. B. G. Neel, N. K. Tonks, Curr. Opin.
Cell Biol. 9, 193 (1997). [0050] 2. T. Hunter, Philos. Trans. R.
Soc. Lond. B. Biol. Sci. 353, 583 (1998). [0051] 3. P.
Blume-Jensen, T. Hunter, Nature 411, 355 (2001). [0052] 4. H.
Davies et al., Nature 417, 949 (2002). [0053] 5. H. Rajagopalan et
al., Nature 418, 934 (2002). [0054] 6. A. Bardelli et al., Science
300, 949 (2003). [0055] 7. J. Li et al., Science 275, 1943 (1997).
[0056] 8. P. A. Steck et al., Nat. Genet 15, 356 (1997). [0057] 9.
M. Tartaglia et al., Nat. Genet 34, 148 (2003). [0058] 10. S. Saha
et al., Science 294, 1343 (2001). [0059] 11. A. R. Forrest et al.,
Genome Res. 13, 1443 (2003). [0060] 12. Materials and Methods are
available as Supporting Online Material. [0061] 13. J. N. Andersen
et al., Mol. Cell. Biol. 21, 7117 (2001). [0062] 14. T. L. Wang et
al., Proc. Natl. Acad. Sci. USA 99, 3076 (2002). [0063] 15. A.
Organesian et al., Proc. Natl. Acad. Sci. USA 100, 7563 (2003)
[0064] 16. S. S. Wang et al., Science 282, 284 (1998). [0065] 17.
S. E. Kern et al., Science 256, 827 (1992). [0066] 18. R. Fodde, R.
Smits, Science 298, 761 (2002). [0067] 19. G. Bompard, C. Puech, C.
Prebois, F. Vignon, G. Freiss, J. Biol. Chem. 277, 47861 (2002).
[0068] 20. G. Freiss, C. Puech, F. Vignon, Mol. Endocrinol. 12, 568
(1998). [0069] 21. S. H. Zhang, J. Liu, R. Kobayashi, N. K. Tonks,
J. Biol. Chem. 274, 17806 (1999). [0070] 22. C. Wadham, J. R.
Gamble, M. A. Vadas, Y. Khew-Goodall, Mol. Biol. Cell. 14, 2520
(2003). [0071] 23. T. Muller, A. Choidas, E. Reichmann, A. Ullrich,
J. Biol. Chem. 274, 10173 (1999). [0072] 24. O. Ayalon, B. Geiger,
J. Cell. Sci. 110, 547 (1997). [0073] 25. S. LaForgia et al., Proc.
Natl. Acad. Sci. USA 88, 5036 (1991). [0074] 26. I. Panagopoulos et
al., Cancer Res. 56, 4871 (1996). [0075] 27. K. Kastury et al.,
Genomics 32, 225 (1996). [0076] 28. T. Druck et al., Cancer Res.
55, 5348 (1995). [0077] 29. P. E. McAndrew et al., J. Comp. Neurol.
391, 444 (1998). [0078] 30. R. Higuchi, B. Krummel, R. K. Saiki,
Nucleic Acids Res. 16, 7351 (1988).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150275315A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150275315A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References