U.S. patent application number 12/449404 was filed with the patent office on 2011-05-19 for methods of diagnosing and prognosing lung cancer.
Invention is credited to Nabeel Bardeesy, Pasi Janne, Hongbin Ji, Bruce Johnson, Norman Sharpless, Kwok-Kin Wong.
Application Number | 20110119776 12/449404 |
Document ID | / |
Family ID | 39682366 |
Filed Date | 2011-05-19 |
United States Patent
Application |
20110119776 |
Kind Code |
A1 |
Wong; Kwok-Kin ; et
al. |
May 19, 2011 |
METHODS OF DIAGNOSING AND PROGNOSING LUNG CANCER
Abstract
The present invention provides methods of detecting cancer using
biomarkers.
Inventors: |
Wong; Kwok-Kin; (Arlington,
MA) ; Johnson; Bruce; (Arlington, MA) ; Janne;
Pasi; (Arlington, MA) ; Ji; Hongbin;
(Arlington, MA) ; Bardeesy; Nabeel; (Arlington,
MA) ; Sharpless; Norman; (Arlington, MA) |
Family ID: |
39682366 |
Appl. No.: |
12/449404 |
Filed: |
February 4, 2008 |
PCT Filed: |
February 4, 2008 |
PCT NO: |
PCT/US2008/052920 |
371 Date: |
January 31, 2011 |
Current U.S.
Class: |
800/10 ;
435/7.23; 506/16; 506/9 |
Current CPC
Class: |
G01N 2333/9121 20130101;
G01N 33/57423 20130101 |
Class at
Publication: |
800/10 ;
435/7.23; 435/6; 506/16; 506/9 |
International
Class: |
A01K 67/033 20060101
A01K067/033; G01N 33/574 20060101 G01N033/574; C12Q 1/68 20060101
C12Q001/68; C40B 40/06 20060101 C40B040/06; C40B 30/04 20060101
C40B030/04 |
Claims
1. A method for determining the aggressiveness of a lung cancer in
a mammal, said method comprising determining, in a test sample from
said mammal, the presence or absence of Lkb1 and correlating said
presence or absence with said aggressiveness.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said test sample is a tumor
biopsy.
4. The method of claim 1, wherein said absence of said Lkb1 or
decrease of the activity of said Lkb1 indicates said cancer is
aggressive.
5. The method of claim 1, wherein said presence of said intact Lkb1
indicates said cancer is not aggressive.
6. The method of claim 1, wherein said absence of said Lkb1 or
decrease of the activity of said Lkb1 indicates said cancer is
metastatic.
7. The method of claim 1, further comprising determining the
presence or absence of a mutation in K-ras, EGFR or BRAF.
8. A method of assessing the efficacy of a m-TOR inhibitor for
treating or inhibiting the growth of lung cancer in a patient,
comprising detecting inactivation of Lkb1 gene expression in a lung
cancer tumor from a subject, wherein said inactivation of Lkb1 gene
expression in the lung cancer tumor indicates treatment with an
m-TOR inhibitor is efficacious.
9. A method of diagnosing lung cancer or a predisposition to
developing lung cancer in a subject, comprising determining a level
of expression of an lung cancer-associated gene in a patient
derived tissue sample, wherein an increase of said level compared
to a normal control level of said gene indicates that said subject
suffers from or is at risk of developing lung cancer.
10. The method of claim 9, wherein said lung cancer-associated gene
is selected from the group consisting of LC 1-461, wherein an
increase in said level compared to a normal control level indicates
said subject suffers from or is at risk of developing lung
cancer.
11. The method of claim 9, wherein said increase is at least 10%
greater than said normal control level.
12. The method of claim 9, wherein said method further comprises
determining said level of expression of a plurality of lung
cancer-associated genes.
13. A lung cancer reference expression profile, comprising a
pattern of gene expression of two or more genes selected from the
group consisting of LC 1-461.
14. A kit comprising a detection reagent which binds to two or more
nucleic acid sequences selected from the group consisting of LC
1-461.
15. An array comprising a nucleic acid which binds to two or more
nucleic acid sequences selected from the group consisting of LC
1-461.
16. A transgenic animal whose genome comprises a mutant K-ras
oncogene and at least one Lkb1 null allele, wherein said transgenic
animal constitutively expresses a mutated K-ras protein in at least
one tissue and exhibits accelerated development of a lung
tumor.
17. The transgenic animal of claim 16 in which said animal is
homozygous null for Lkb1.
18. The transgenic animal of claim 16, wherein said K-ras oncogen
contains a G12D mutation.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
60/888,190 filed Feb. 5, 2007 and the contents of which are
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to generally to detecting cancer.
BACKGROUND OF THE INVENTION
[0003] Non-small cell lung cancer (NSCLC) is the most common and
lethal cancer world-wide. At least three major histologies of NSCLC
are described: squamous carcinoma (48%), large cell carcinoma (12%)
and adenocarcinoma (40%) The standard treatment for these patients
is systemic chemotherapy. However, systemic chemotherapy has modest
efficacy and has not greatly prolonged the median survival (8-12
months) or 5-year survival rates (2%) in these patients. Although
these sub-types differ markedly in histologic appearance and gene
expression, each is highly lethal, and until recently, little
clinical distinction has been made among these entities.
[0004] It is anticipated that a better understanding of the
molecular mechanisms involved in the initiation and progression
lung tumorigenesis--as well as the impact of environmental
exposures on lung carcinogenesis--would help guide the development
of better and more targeted lung cancer therapeutics as well as
potential prevention strategies. The recent identification of the
oncogenic kinase domain mutations in the epidermal growth factor
receptor (EGFR) in human lung adenocarcinomas and their association
with sensitivity to small molecule EGFR kinase inhibitors such as
gefitinib and erlotinib further support the that the molecular
understanding of the mechanisms involved in lung tumorigenesis will
lead to advances in patient screening, development of better
targeted therapeutics, and identification of patients who are best
suited for each type of targeted treatment. Activating K-RAS, EGFR,
and BRAF mutations comprise the most common oncogenic mutations in
human NSCLC. However, their interaction with other concurrent tumor
suppressor loss is not well understood.
SUMMARY OF THE INVENTION
[0005] The invention provides biological markers for monitoring,
diagnosing and prognosing lung cancer.
[0006] In one aspect the invention provides a method for
determining the aggressiveness of a lung cancer in a mammal, e.g.
human, by determining in a test sample from the mammal, the
presence or absence of Lkb1 expression or activity and correlating
the presence or absence with aggressiveness of the lung cancer. The
absence of Lkb1 expression or decrease of the activity of Lkb1
indicates the cancer is aggressive and/or metastatic. In contrast,
the presence of intact Lkb1, e.g. non-mutated indicates the cancer
is not aggressive. The test sample is for example a tumor
biopsy.
[0007] Optionally, the method includes determining the presence or
absence of an additional tumor biomarker such as determining the
presence or absence of a mutation in K-ras, EGFR or BRAF.
[0008] Also included in the invention is a method of assessing the
efficacy of an m-TOR inhibitor for treating or inhibiting the
growth of lung cancer in a patient, by detecting inactivation of
Lkb1 gene expression in a lung cancer tumor from a subject.
Inactivation of Lkb1 gene expression in the lung cancer tumor
indicates treatment with an m-TOR inhibitor is efficacious.
[0009] Lung cancer is diagnosed or a predisposition to developing
lung cancer in a subject by determining a level of expression of a
lung cancer-associated gene in a patient derived tissue sample. By
LC associated gene is meant a gene that is characterized by a level
of expression which differs in a cell obtained from a lung cancer
cell compared to a normal cell. A normal cell is one obtained from
lung tissue. A LC-associated gene includes for example LC 1-461. An
alteration, e.g., increase of the level of expression of the gene
compared to a normal control level of the gene indicates that the
subject suffers from or is at risk of developing lung cancer.
[0010] Alternatively, expression of a panel of LC-associated genes
in the sample is compared to a LC control level of the same panel
of genes. By LC control level is meant the expression profile of
the LC-associated genes found in a population suffering from lung
cancer.
[0011] Gene expression is increased or decreased 10%, 25%, 50%
compared to the control level. Alternately, gene expression is
increased or decreased 1, 2, 5 or more fold compared to the control
level. Expression is determined by detecting hybridization, e.g.,
on a chip, of a LC-associated gene probe to a gene transcript of
the patient-derived tissue sample.
[0012] The patient derived tissue sample is any tissue from a test
subject, e.g., a patient known to or suspected of having lung
cancer. For example, the tissue contains a sputum, blood, serum,
plasma or lung cell.
[0013] The invention also provides a LC reference expression
profile of a gene expression level of two or more of PRC 1-461.
[0014] The invention further provides a kit with a detection
reagent which binds to two or more LC nucleic acid sequences or
which binds to a gene product encoded by the nucleic acid
sequences. Also provided is an array of nucleic acids that binds to
two or more LC nucleic acids.
[0015] In another aspect, the invention includes a transgenic
animal whose genome contains a mutant K-ras oncogene, e.g. G12D
mutation and at least one Lkb1 null allele. The transgenic animal
constitutively expresses a mutated K-ras protein in at least one
tissue and exhibits accelerated development of a lung tumor.
Optionally, the animal is homozygous null for Lkb1.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A is a line graph showing the tumor-free survival of
mice treated with adeno-CRE. Cohort consists of K-ras (n=26), K-ras
Lkb1.sup.+/- (n=27), K-ras Lkb1.sup.L/L or L/- (n=56), and
Lkb1.sup.-/- (n=15). P<0.002 for pair-wise comparison between
K-ras and K-ras Lkb1.sup.+/-, and p<0.0001 for pair-wise
comparison between K-ras and K-ras Lkb1.sup.-/-.
[0019] FIG. 1B are photographs illustrating representative
histology of lesions in K-ras or K-ras Lkb.sup.L/L mice treated
with adeno-CRE at 2 weeks (top) or 4 weeks (bottom) after
treatment. Photographs are 100.times. original magnification.
[0020] FIG. 1C is a bar chart showing the quantification of early
lesions (<1 mm) found in K-ras or K-ras Lkb1.sup.L/L mice after
treatment with adeno-CRE. 2 week group consisted of K-ras (n=5) and
K-ras Lkb1.sup.L/L (n=6) mice, and 4 week groups consisted of K-ras
(n=4) and K-ras Lkb1.sup.L/L (n=5) mice. Error bars represent
.sup.+/- standard error of the mean (SEM).
[0021] FIG. 1D is a bar chart showing the quantification of tumors
of <3 mm in size from K-ras Lkb1.sup.L/L or L/- (n=12), K-ras
Lkb1.sup.+/- (n=8) and K-ras (n=10) mice 8 weeks after adeno-CRE
treatment. Error bars represent .sup.+/- SEM.
[0022] FIG. 1E is a photograph illustrating that mice lacking Lkb1
have increased metastasis. Representative photographs of lymph node
metastasis from K-ras Lkb1.sup.L/L or L/- mice. Dissection showed
that this was a lymph node separate from the lung itself. Note the
adenocarcinoma histology, which was found in all metastatic
lesions.
[0023] FIG. 2A are photographs showing K-ras Lkb1.sup.L/L or L/-
tumors have mixed histology. Representative tumors from K-ras
Lkb1.sup.L/L or L/- mice showing squamous histology (top), mixed
histology (middle) or large cell histology (bottom).
[0024] FIG. 2B are photographs showing immunohistochemical staining
of tumors from K-ras Lkb1.sup.L/L or L/- mice. Adenocarcinomas
(left) show high levels of prosurfactant protein C (SP-C), while
squamous tumors (right) show strong staining for pan-keratin and
p63. All pictures at 200.times. original magnification
[0025] FIG. 2C is a photograph of a Western blot analysis of Lkb1
and p63 expression in tumors from mice of indicated genotype.
Tubulin serves as a loading control.
[0026] FIG. 3 is a schematic illustration of microarray analysis of
K-ras-induced lung tumors. Two-way unsupervised hierarchical
clustering was performed on 6,781 unique and dynamic transcripts
(left). Excerpted gene clusters are shown at the right. Genes in
blue are overexpressed in human SCC and genes in red are known
regulators of metastasis. Lkb1 (Stk11) is indicated in orange.
[0027] FIG. 4A is a photograph of a blot showing MEFs increase
p16.sup.INK4a and Arf protein with passage. Cells were serially
passaged and assessed for Lkb1, p16.sup.INK4a, or Arf expression.
Actin serves as a loading control. Lysates represent a pool of two
independent lines. Lkb1.sup.-/- or Ink4a/Arf.sup.-/- MEFs serve as
negative controls (-).
[0028] FIG. 4B is a is a photograph of a blot showing Excision of
Lkb1 reduces accumulation of p16.sup.INK4a and Arf protein. Western
blot analysis of Lkb1, p16.sup.INK4a, and Arf protein levels at
various times after adenoviral treatment. Actin serves as a loading
control. * designates a non-specific background band. Arrow
designates Arf protein.
[0029] FIG. 4C is a bar chart showing excision of Lkb1 reduces
accumulation of p16.sup.INK4a and Arf mRNA MEFs. Taqman Real-Time
PCR analysis of p16.sup.INK4a or Arf levels in MEFs 16 days after
conditional excision of Lkb1 by adenoviral-CRE. Values represent
ratio of mRNA levels in Lkb1.sup.L/- (treated with adeno-empty) to
mRNA levels in Lkb1.sup.-/ (treated with Adeno-CRE). Data represent
4 independent experiments. Error bars represent .sup.+/- SEM.
[0030] FIG. 4D is a photograph of a blot showing forced expression
of Lkb1 in Lkb1-/-MEFs does not affect p16.sup.INK4a or Arf levels.
Late passage (P10) Lkb1 -/-MEFs were transduced with either pBABE
(V), pBABE-Lkb1 (WT), or kinase dead pBABE-Lkb1.sup.K78D (KD).
Levels of Lkb1, p16.sup.INK4a, Arf, and phosphorylated acetyl CoA
carboxylase-2 (p-ACC) were assessed 8 days after transduction.
Actin serves as a loading control.
[0031] FIG. 5A is a photograph of a blot showing expression of
exogenous LKB1 in A549 cells. A549 cells were stably transduced
with either pBABE-LKB1 (WT), or kinase dead pBABE-LKB1.sup.K78D
(KD) via retroviral infection and assessed for expression of LKB1
by western blotting. Tubulin serves as a loading control.
[0032] FIG. 5B is a photograph showing that LKB1 suppresses colony
formation in soft agar. A549 cells stably transduced with either
pBABE-LKB1 (WT), or kinase dead pBABE-LKB1.sup.K78D (KD) were
assessed for the ability to form colonies in soft agar.
Representative photographs of 4 independent experiments.
[0033] FIG. 5C is a bar chart showing that LKB1 suppresses colony
formation in soft agar. A549 cells stably transduced with either
pBABE-LKB1 (WT), or kinase dead pBABE-LKB1.sup.K78D (KD) were
assessed for the ability to form colonies in soft agar. Average
number of colonies per well from 4 independent experiments. Error
bars indicate .sup.+/- SEM.
[0034] FIG. 5D is a photograph illustrating that exogenous LKB1
suppresses metastasis in SCID mice. A549 cells were stably
transduced with either pBABE-LKB1 (WT), or kinase dead
pBABE-LKB1.sup.K78D (KD) and injected into the tail vein of SCID
mice. Representative photographs of lungs.
[0035] FIG. 6 is a schematic illustrating the tetracycline
bitransgenic regulatory system.
[0036] FIG. 7 is a schematic illustrating the Lkb1 signaling
pathways linking it to DNA damage response, growth control and cell
polarity
[0037] FIG. 8 are line graphs showing Power (red) and type 1 error
rates (green) for detecting mean differences of 0.58, 1, 1.32, and
1.58, plotted against the number of genes truly exhibiting a mean
difference of 0.58, 1, 1.32, and 1.58, assuming 15 samples for each
cohort.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The invention is based upon the discovery of biomarkers for
the detection and assessment of cancer. Specifically, it has been
shown that a somatic deficiency in the kinase, Lkb1 accelerates the
development of lung tumerigenesis. More specifically, Lkb1
deficiency in the setting of K-ras.sup.G12D mutation
(K-rasLkb1.sup.L/L) was associated with decreased tumor latency and
increased tumor aggressiveness including metastasis. Furthermore,
tumors from K-ras Lkb1.sup.L/L mice demonstrated mice exhibit the
full range of histologic subtypes (including squamous,
adenosquamous, and large-cell) that arise in humans, whereas
K-ras.sup.G12D mutation, Ink4a/Arf inactivation, or p53
inactivation alone or in combination result only in adenocarcinoma.
These findings suggest that, unexpectedly, Lkb1 influences cell
differentiation in addition to its roles in the suppression of cell
growth. Experiments in vitro demonstrate that LKB1 suppresses lung
tumorigenesis and progression through both p16.sup.INK4a-ARF-p53
dependent and independent mechanisms. These data indicate that LKB1
regulates lung tumor progression by controlling multiple aspects of
cell growth and differentiation.
[0039] Additionally, a comprehensive microarray analysis was
performed on K-ras induced lung tumors to identify commonly
over-expressed genes. Four hundred and sixty one genes were
up-regulated.
[0040] The genes whose expression levels are modulated (i.e.,
increased) in lung cancer are summarized in Table A and are
collectively referred to herein as "lung cancer-associated genes",
"LC nucleic acids" or "LC polynucleotides" and the corresponding
encoded polypeptides are referred to as "LC polypeptides" or "LC
proteins." Unless indicated otherwise, "LC" is meant to refer to
any of the sequences disclosed herein. (e.g., LC 1-461). The genes
have been previously described and are presented along with a
database accession number.
[0041] Accordingly, the invention provides methods of detecting and
evaluating the aggressiveness of lung cancer in a subject by
determining the presence or absence of Lkb1. In addition, the
differentially expressed genes identified herein are used for
diagnostic purposes and to develop gene targeted therapeutic
approaches to inhibiting lung cancer.
TABLE-US-00001 TABLE A LC Assignment CLID NAME 1 Tgfb2 Tgfb2
.parallel. transforming growth factor, beta 2 2 Zfp36l1 Zfp36l1
.parallel. zinc finger protein 36, C3H type-like 1 3 Fusip1 Fusip1
.parallel. FUS interacting protein (serine-arginine rich) 1 4 Abcc9
Abcc9 .parallel. ATP-binding cassette, sub-family C (CFTR/MRP),
member 9 5 Rnf130 Rnf130 .parallel. ring finger protein 130 6
Nfkbie Nfkbie .parallel. nuclear factor of kappa light polypeptide
gene enhancer in B-cells inhibitor, epsilon 7 Ppargc1b Ppargc1b
.parallel. peroxisome proliferative activated receptor, gamma,
coactivator 1 beta 8 Srrm2 Srrm2 .parallel. serine/arginine
repetitive matrix 2 9 Thrap3 Thrap3 .parallel. thyroid hormone
receptor associated protein 3 10 Smarca2 Smarca2 .parallel. SWI/SNF
related, matrix associated, actin dependent regulator of chromatin,
subfamily a, member 2 11 Tmcc3 Tmcc3 .parallel. transmembrane and
coiled coil domains 3 12 Fez2 Fez2 .parallel. fasciculation and
elongation protein zeta 2 (zygin II) 13 Suv39h1 Suv39h1 .parallel.
suppressor of variegation 3-9 homolog 1 (Drosophila) 14 Klf6 Klf6
.parallel. Kruppel-like factor 6 15 Col7a1 Col7a1 .parallel.
procollagen, type VII, alpha 1 16 Tspan17 Tspan17 .parallel.
tetraspanin 17 17 Gats Gats .parallel. opposite strand
transcription unit to Stag3 18 Tiparp Tiparp .parallel.
TCDD-inducible poly(ADP-ribose) polymerase 19 Sfrs8 Sfrs8
.parallel. splicing factor, arginine/serine-rich 8 20 Slc6a8 Slc6a8
.parallel. solute carrier family 6 (neurotransmitter transporter,
creatine), member 8 21 Trip12 Trip12 .parallel. thyroid hormone
receptor interactor 12 22 Wdsub1 Wdsub1 .parallel. WD repeat, SAM
and U-box domain containing 1 23 Vdr Vdr .parallel. vitamin D
receptor 24 Ece1 Ece1 .parallel. endothelin converting enzyme 1 25
Uxt Uxt .parallel. ubiquitously expressed transcript 26 Zdhhc4
Zdhhc4 .parallel. zinc finger, DHHC domain containing 4 27 Ndn Ndn
/// Pctk1 .parallel. necdin /// PCTAIRE-motif protein kinase 1 28
6330549H03Rik 6330549H03Rik .parallel. RIKEN cDNA 6330549H03 gene
29 Rab15 Rab15 .parallel. RAB15, member RAS oncogene family 30 Arf6
Arf6 .parallel. ADP-ribosylation factor 6 31 Dnase1l1 Dnase1l1
.parallel. deoxyribonuclease 1-like 1 32 1190002N15Rik
1190002N15Rik .parallel. RIKEN cDNA 1190002N15 gene 33 Clic4 Clic4
.parallel. chloride intracellular channel 4 (mitochondrial) 34
Txnip Txnip .parallel. thioredoxin interacting protein 35 Pim3 Pim3
.parallel. proviral integration site 3 36 Grin3b Grin3b .parallel.
glutamate receptor, ionotropic, NMDA3B 37 Pscd3 Pscd3 .parallel.
pleckstrin homology, Sec7 and coiled-coil domains 3 38 Macf1 Macf1
.parallel. microtubule-actin crosslinking factor 1 39 Pctk1 Pctk1
.parallel. PCTAIRE-motif protein kinase 1 40 Lyn Lyn /// LOC676654
.parallel. Yamaguchi sarcoma viral (v-yes-1) oncogene homolog ///
similar to Yamaguchi sarcoma viral (v-yes-1) oncogene homolog 41
Tspyl1 Tspyl1 .parallel. testis-specific protein, Y-encoded-like 1
42 Fbxw4 Fbxw4 .parallel. F-box and WD-40 domain protein 4 43
C80012 C80012 .parallel. expressed sequence C80012 44 Col16a1
Col16a1 .parallel. procollagen, type XVI, alpha 1 45 Sv2a Sv2a
.parallel. synaptic vesicle glycoprotein 2 a 46 Gm672 Gm672
.parallel. Gene model 672, (NCBI) 47 Centg2 Centg2 .parallel.
centaurin, gamma 2 48 Ahdc1 Ahdc1 .parallel. AT hook, DNA binding
motif, containing 1 49 Olfr65 Olfr65 .parallel. olfactory receptor
64 50 9130404D08Rik 9130404D08Rik .parallel. RIKEN cDNA 9130404D08
gene 51 Lgals8 Lgals8 .parallel. lectin, galactose binding, soluble
8 52 Slc6a6 Slc6a6 .parallel. solute carrier family 6
(neurotransmitter transporter, taurine), member 6 53 C79248 C79248
.parallel. expressed sequence C79248 54 Per3 Per3 .parallel. period
homolog 3 (Drosophila) 55 Nisch Nisch .parallel. nischarin 56 Mylip
Mylip .parallel. myosin regulatory light chain interacting protein
57 Abca1 Abca1 .parallel. ATP-binding cassette, sub-family A
(ABC1), member 1 58 Lfng Lfng .parallel. lunatic fringe gene
homolog (Drosophila) 59 Pitpnm1 Pitpnm1 .parallel.
phosphatidylinositol membrane-associated 1 60 Jak2 Jak2 .parallel.
Janus kinase 2 61 4732495E13Rik 4732495E13Rik .parallel. RIKEN cDNA
4732495E13 gene 62 Pscd1 Pscd1 .parallel. pleckstrin homology, Sec7
and coiled-coil domains 1 63 Tpp1 Tpp1 .parallel. tripeptidyl
peptidase I 64 Rbm7 Rbm7 .parallel. RNA binding motif protein 7 65
Drctnnb1a Drctnnb1a .parallel. down-regulated by Ctnnb1, a 66
Mospd1 Mospd1 .parallel. motile sperm domain containing 1 67 Huwe1
Huwe1 .parallel. HECT, UBA and WWE domain containing 1 68 Cfl2 Cfl2
.parallel. cofilin 2, muscle 69 Birc3 Birc3 .parallel. baculoviral
IAP repeat-containing 3 70 2410005O16Rik 2410005O16Rik .parallel.
RIKEN cDNA 2410005O16 gene 71 Zc3h12c Zc3h12c .parallel. zinc
finger CCCH-type containing 12C 72 Syk Syk .parallel. spleen
tyrosine kinase 73 Adamts15 Adamts15 .parallel. a disintegrin-like
and metallopeptidase (reprolysin type) with thrombospondin type 1
motif, 15 74 Npl Npl .parallel. N-acetylneuraminate pyruvate lyase
75 -- -- .parallel. Transcribed locus 76 Mef2d Mef2d .parallel.
myocyte enhancer factor 2D 77 Cdc42bpa Cdc42bpa .parallel. Cdc42
binding protein kinase alpha 78 Eln Eln .parallel. elastin 79
Serpina3n Serpina3n .parallel. serine (or cysteine) peptidase
inhibitor, clade A, member 3N 80 Lox Lox .parallel. lysyl oxidase
81 Cias1 Cias1 .parallel. cold autoinflammatory syndrome 1 homolog
(human) 82 Gdpd3 Gdpd3 .parallel. glycerophosphodiester
phosphodiesterase domain containing 3 83 H2-K1 H2-K1 .parallel.
Histocompatibility 2, K1, K region 84 Clasp2 Clasp2 .parallel. CLIP
associating protein 2 85 Cnnm3 Cnnm3 .parallel. cyclin M3 86 Nfia
Nfia .parallel. nuclear factor I/A 87 9430029L20Rik 9430029L20Rik
.parallel. RIKEN cDNA 9430029L20 gene 88 Pi4k2b Pi4k2b .parallel.
phosphatidylinositol 4-kinase type 2 beta 89 Ptger1 Ptger1
.parallel. Prostaglandin E receptor 1 (subtype EP1) 90 Leng8 Leng8
.parallel. leukocyte receptor cluster (LRC) member 8 91 Cables1
Cables1 /// LOC635753 .parallel. Cdk5 and Abl enzyme substrate 1
/// similar to Cdk5 and Abl enzyme substrate 1 92 Sorbs3 Sorbs3
.parallel. sorbin and SH3 domain containing 3 93 Adipor1 Adipor1
.parallel. adiponectin receptor 1 94 LOC547343 LOC547343 .parallel.
similar to H-2 class I histocompatibility antigen, L-D alpha chain
precursor 95 H2-D1 H2-D1 .parallel. histocompatibility 2, D region
locus 1 96 H2-D1 H2-D1 /// H2-L /// LOC547343 /// LOC636948
.parallel. histocompatibility 2, D region locus 1 ///
histocompatibility 2, D region /// similar to H-2 class I
histocompatibility antigen, L-D alpha chain precursor /// similar
to H-2 class I histocompatibility antigen, D-B alpha chain
precursor (H-2D(B)) 97 H2-L H2-L .parallel. histocompatibility 2, D
region 98 2310043N10Rik 2310043N10Rik .parallel. RIKEN cDNA
2310043N10 gene 99 Per1 Per1 .parallel. period homolog 1
(Drosophila) 100 Slc24a3 Slc24a3 .parallel. solute carrier family
24 (sodium/potassium/calcium exchanger), member 3 101 Slco3a1
Slco3a1 .parallel. solute carrier organic anion transporter family,
member 3a1 102 Epb4.1 Epb4.1 .parallel. erythrocyte protein band
4.1 103 Emilin1 Emilin1 .parallel. elastin microfibril interfacer 1
104 Usf2 Usf2 .parallel. upstream transcription factor 2 105 Trak1
Trak1 .parallel. trafficking protein, kinesin binding 1 106 Ctsb
Ctsb .parallel. cathepsin B 107 Man2b1 Man2b1 .parallel.
mannosidase 2, alpha B1 108 Adrbk1 Adrbk1 .parallel. adrenergic
receptor kinase, beta 1 109 BC018473 BC018473 .parallel. cDNA
sequence BC018473 110 Setdb1 Setdb1 .parallel. SET domain,
bifurcated 1 111 Zxdc Zxdc .parallel. ZXD family zinc finger C 112
Cetn1 Cetn1 .parallel. centrin 1 113 F2rl2 F2rl2 .parallel.
coagulation factor II (thrombin) receptor-like 2 114 Zfp346 Zfp346
.parallel. zinc finger protein 346 115 Eps15l1 Eps15l1 .parallel.
epidermal growth factor receptor pathway substrate 15-like 1 116
Xpo4 Xpo4 .parallel. exportin 4 117 Scly Scly .parallel.
selenocysteine lyase 118 Tlr5 Tlr5 .parallel. toll-like receptor 5
119 Chst12 Chst12 .parallel. carbohydrate sulfotransferase 12 120
Sipa1 Sipa1 .parallel. signal-induced proliferation associated gene
1 121 Samsn1 Samsn1 .parallel. SAM domain, SH3 domain and nuclear
localization signals, 1 122 Krt1-14 Krt1-14 /// Krt1-17 .parallel.
keratin complex 1, acidic, gene 14 /// keratin complex 1, acidic,
gene 17 123 Fgr Fgr .parallel. Gardner-Rasheed feline sarcoma viral
(Fgr) oncogene homolog 124 Scube1 Scube1 .parallel. signal peptide,
CUB domain, EGF-like 1 125 Unc5c Unc5c .parallel. unc-5 homolog C
(C. elegans) 126 Mmp24 Mmp24 .parallel. matrix metallopeptidase 24
127 Nxph3 Nxph3 .parallel. neurexophilin 3 128 Stau1 Stau1
.parallel. staufen (RNA binding protein) homolog 1 (Drosophila) 129
Ddi2 Ddi2 /// Rsc1a1 .parallel. DNA-damage inducible protein 2 ///
regulatory solute carrier protein, family 1, member 1 130 Pacs2
Pacs2 .parallel. phosphofurin acidic cluster sorting protein 2 131
Rac2 Rac2 .parallel. RAS-related C3 botulinum substrate 2 132
Impact Impact .parallel. imprinted and ancient 133 Trex1 Trex1
.parallel. three prime repair exonuclease 1 134 Sp4 Sp4 .parallel.
trans-acting transcription factor 4 135 2900002H16Rik 2900002H16Rik
.parallel. RIKEN cDNA 2900002H16 gene 136 D930015E06Rik
D930015E06Rik .parallel. RIKEN cDNA D930015E06 gene 137 Rbl2 Rbl2
/// LOC635075 .parallel. retinoblastoma-like 2 /// similar to
retinoblastoma- like 2 138 Parp8 Parp8 .parallel. poly (ADP-ribose)
polymerase family, member 8 139 Gnb4 Gnb4 .parallel. guanine
nucleotide binding protein, beta 4 140 Il1r1 Il1r1 .parallel.
interleukin 1 receptor, type I 141 Gfm1 Gfm1 .parallel. G
elongation factor, mitochondrial 1 142 Vps11 Vps11 .parallel.
vacuolar protein sorting 11 (yeast) 143 Epim Epim .parallel.
epimorphin 144 Cd37 Cd37 .parallel. CD37 antigen 145 Map3k1 Map3k1
.parallel. mitogen activated protein kinase kinase kinase 1 146
BC039093 BC039093 .parallel. cDNA sequence BC039093 147 Tug1 Tug1
.parallel. taurine upregulated gene 1 148 4631426J05Rik
4631426J05Rik .parallel. RIKEN cDNA 4631426J05 gene 149 Grn Grn
.parallel. granulin 150 Irf8 Irf8 .parallel. interferon regulatory
factor 8 151 Lycat Lycat .parallel. lysocardiolipin acyltransferase
152 Trps1 Trps1 .parallel. trichorhinophalangeal syndrome 1 (human)
153 Cysltr1 Cysltr1 .parallel. cysteinyl leukotriene receptor 1 154
T2bp T2bp .parallel. Traf2 binding protein 155 Tm6sf1 Tm6sf1
.parallel. transmembrane 6 superfamily member 1 156 Hdgfrp3 Hdgfrp3
/// Tm6sf1 .parallel. hepatoma-derived growth factor, related
protein 3 /// transmembrane 6 superfamily member 1 157 Il17ra
Il17ra .parallel. interleukin 17 receptor A 158 Hal Hal ///
LOC638196 .parallel. histidine ammonia lyase /// similar to
Histidine ammonia-lyase (Histidase) 159 Map3k8 Map3k8 .parallel.
mitogen activated protein kinase kinase kinase 8 160 Cd300lf
Cd300lf .parallel. CD300 antigen like family member F 161 Osbpl9
Osbpl9 .parallel. Oxysterol binding protein-like 9 162 BC013712
BC013712 .parallel. cDNA sequence BC013712 163 Igsf6 Igsf6
.parallel. immunoglobulin superfamily, member 6 164 LOC676654
LOC676654 .parallel. similar to Yamaguchi sarcoma viral (v-yes-1)
oncogene homolog 165 Prei4 Prei4 .parallel. preimplantation protein
4 166 Cebpb Cebpb .parallel. CCAAT/enhancer binding protein
(C/EBP), beta 167 Lst1 Lst1 .parallel. leukocyte specific
transcript 1 168 Siglecf Siglecf .parallel. sialic acid binding
Ig-like lectin F 169 Ccr1 Ccr1 .parallel. chemokine (C-C motif)
receptor 1 170 Rassf5 Rassf5 .parallel. Ras association
(RalGDS/AF-6) domain family 5 171 Vamp4 Vamp4 .parallel.
vesicle-associated membrane protein 4 172 Lgals7 Lgals7 .parallel.
lectin, galactose binding, soluble 7 173 Rcbtb2 Rcbtb2 .parallel.
regulator of chromosome condensation (RCC1) and BTB (POZ) domain
containing protein 2 174 Rhoq Rhoq .parallel. ras homolog gene
family, member Q 175 Dcamkl1 Dcamkl1 .parallel. double cortin and
calcium/calmodulin-dependent protein kinase-like 1 176 Apob48r
Apob48r .parallel. apolipoprotein B48 receptor 177 Slit2 Slit2
.parallel. slit homolog 2 (Drosophila) 178 Prkcb1 Prkcb1 .parallel.
protein kinase C, beta 1 179 Dmpk Dmpk .parallel. dystrophia
myotonica-protein kinase 180 Lamc1 Lamc1 .parallel. laminin, gamma
1
181 Rbpsuh Rbpsuh .parallel. recombining binding protein suppressor
of hairless (Drosophila) 182 2310016C16Rik 2310016C16Rik .parallel.
RIKEN cDNA 2310016C16 gene 183 Cd19 Cd19 .parallel. CD19 antigen
184 Src Src .parallel. Rous sarcoma oncogene 185 Cyp2j6 Cyp2j6
.parallel. cytochrome P450, family 2, subfamily j, polypeptide 6
186 Ikbkb Ikbkb .parallel. inhibitor of kappaB kinase beta 187
Pabpn1 Pabpn1 .parallel. poly(A) binding protein, nuclear 1 188
Mll1 Mll1 .parallel. myeloid/lymphoid or mixed-lineage leukemia 1
189 Wisp1 Wisp1 .parallel. WNT1 inducible signaling pathway protein
1 190 Ptplad2 Ptplad2 .parallel. protein tyrosine phosphatase-like
A domain containing 2 191 Dkk2 Dkk2 .parallel. dickkopf homolog 2
(Xenopus laevis) 192 Ankrd1 Ankrd1 .parallel. ankyrin repeat domain
1 (cardiac muscle) 193 Ltbp3 Ltbp3 .parallel. latent transforming
growth factor beta binding protein 3 194 Sspn Sspn .parallel.
sarcospan 195 Pfkfb3 Pfkfb3 .parallel.
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 196 Tuba1
Tuba1 .parallel. tubulin, alpha 1 197 Ptprs Ptprs .parallel.
protein tyrosine phosphatase, receptor type, S 198 Dync1i2 Dync1i2
.parallel. dynein cytoplasmic 1 intermediate chain 2 199 Lgals1
Lgals1 .parallel. lectin, galactose binding, soluble 1 200 Akr1b7
Akr1b7 .parallel. aldo-keto reductase family 1, member B7 201 Vim
Vim .parallel. vimentin 202 Pdgfa Pdgfa .parallel. platelet derived
growth factor, alpha 203 Postn Postn .parallel. periostin,
osteoblast specific factor 204 Itgb5 Itgb5 .parallel. integrin beta
5 205 Naalad2 Naalad2 .parallel. N-acetylated alpha-linked acidic
dipeptidase 2 206 Evl Evl .parallel. Ena-vasodilator stimulated
phosphoprotein 207 Syt11 Syt11 .parallel. synaptotagmin XI 208
Sparc Sparc .parallel. secreted acidic cysteine rich glycoprotein
209 Pkd1 Pkd1 .parallel. polycystic kidney disease 1 homolog 210
Mark1 Mark1 .parallel. MAP/microtubule affinity-regulating kinase 1
211 Chst2 Chst2 .parallel. carbohydrate sulfotransferase 2 212
Ptpre Ptpre .parallel. protein tyrosine phosphatase, receptor type,
E 213 Ncf4 Ncf4 .parallel. neutrophil cytosolic factor 4 214 Mnt
Mnt .parallel. max binding protein 215 4632428N05Rik 4632428N05Rik
.parallel. RIKEN cDNA 4632428N05 gene 216 Nid1 Nid1 .parallel.
nidogen 1 217 Apbb2 Apbb2 .parallel. amyloid beta (A4) precursor
protein-binding, family B, member 2 218 Stxbp1 Stxbp1 .parallel.
syntaxin binding protein 1 219 4732435N03Rik 4732435N03Rik
.parallel. RIKEN cDNA 4732435N03 gene 220 Pilrb Pilrb .parallel.
paired immunoglobin-like type 2 receptor beta 221 Ank Ank
.parallel. progressive ankylosis 222 Polb Polb .parallel.
polymerase (DNA directed), beta 223 Unc5b Unc5b .parallel. unc-5
homolog B (C. elegans) 224 Inhba Inhba .parallel. inhibin beta-A
225 Cxxc5 Cxxc5 .parallel. CXXC finger 5 226 Tnnt2 Tnnt2 .parallel.
troponin T2, cardiac 227 Adamts4 Adamts4 .parallel. a
disintegrin-like and metallopeptidase (reprolysin type) with
thrombospondin type 1 motif, 4 228 Mtap1b Mtap1b .parallel.
microtubule-associated protein 1 B 229 Ints6 Ints6 .parallel.
integrator complex subunit 6 230 Aplp1 Aplp1 .parallel. amyloid
beta (A4) precursor-like protein 1 231 Muc5ac Muc5ac .parallel.
mucin 5, subtypes A and C, tracheobronchial/gastric 232 Cdkn2b
Cdkn2b .parallel. cyclin-dependent kinase inhibitor 2B (p15,
inhibits CDK4) 233 Dbn1 Dbn1 .parallel. drebrin 1 234 Pyy Pyy
.parallel. peptide YY 235 Rnf38 Rnf38 .parallel. ring finger
protein 38 236 Bace1 Bace1 .parallel. beta-site APP cleaving enzyme
1 237 1200003C05Rik 1200003C05Rik .parallel. RIKEN cDNA 1200003C05
gene 238 Sh3glb1 Sh3glb1 .parallel. SH3-domain GRB2-like B1
(endophilin) 239 LOC637870 LOC637870 .parallel. similar to Nedd4 WW
binding protein 4 240 Tmepai Tmepai .parallel. transmembrane,
prostate androgen induced RNA 241 Skil Skil .parallel. SKI-like 242
LOC637870 LOC637870 /// LOC676013 .parallel. similar to Nedd4 WW
binding protein 4 /// similar to Nedd4 WW binding protein 4 243
Slc25a30 Slc25a30 .parallel. solute carrier family 25, member 30
244 Cpne1 Cpne1 .parallel. copine 1 245 Mfge8 Mfge8 .parallel. milk
fat globule-EGF factor 8 protein 246 Rnasel Rnasel .parallel.
ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent) 247
Mtap4 Mtap4 .parallel. microtubule-associated protein 4 248 Tcf4
Tcf4 .parallel. transcription factor 4 249 Raver1 Raver1 .parallel.
ribonucleoprotein, PTB-binding 1 250 Extl3 Extl3 .parallel.
exostoses (multiple)-like 3 251 Stx1a Stx1a .parallel. syntaxin 1A
(brain) 252 Gtf2ird1 Gtf2ird1 .parallel. general transcription
factor II I repeat domain-containing 1 253 5430405G24Rik
5430405G24Rik .parallel. RIKEN cDNA 5430405G24 gene 254 Fbln2 Fbln2
.parallel. fibulin 2 255 Col5a3 Col5a3 .parallel. procollagen, type
V, alpha 3 256 Ppp2r4 Ppp2r4 .parallel. protein phosphatase 2A,
regulatory subunit B (PR 53) 257 Tnip1 Tnip1 .parallel. TNFAIP3
interacting protein 1 258 Cic Cic .parallel. capicua homolog
(Drosophila) 259 C81521 C81521 .parallel. expressed sequence C81521
260 AI450540 AI450540 .parallel. expressed sequence AI450540 261
Rem2 Rem2 .parallel. rad and gem related GTP binding protein 2 262
Cplx2 Cplx2 .parallel. complexin 2 263 Igfbp4 Igfbp4 .parallel.
insulin-like growth factor binding protein 4 264 Vapb Vapb
.parallel. vesicle-associated membrane protein, associated protein
B and C 265 Slc6a6 Slc6a6 .parallel. Solute carrier family 6
(neurotransmitter transporter, taurine), member 6 266 BC023055
BC023055 .parallel. cDNA sequence BC023055 267 Sec61a2 Sec61a2
.parallel. Sec61, alpha subunit 2 (S. cerevisiae) 268 Ext2 Ext2
.parallel. exostoses (multiple) 2 269 Crtap Crtap .parallel.
cartilage associated protein 270 Pla1a Pla1a .parallel.
phospholipase A1 member A 271 Ncor2 Ncor2 .parallel. nuclear
receptor co-repressor 2 272 Suhw4 Suhw4 .parallel. suppressor of
hairy wing homolog 4 (Drosophila) 273 Ptn Ptn .parallel.
pleiotrophin 274 Ltbp4 Ltbp4 .parallel. latent transforming growth
factor beta binding protein 4 275 Chd7 Chd7 .parallel. Chromodomain
helicase DNA binding protein 7 276 Nkd2 Nkd2 .parallel. naked
cuticle 2 homolog (Drosophila) 277 Tgfb3 Tgfb3 .parallel.
transforming growth factor, beta 3 278 Ptpn1 Ptpn1 .parallel.
protein tyrosine phosphatase, non-receptor type 1 279 Nudt16 Nudt16
.parallel. nudix (nucleoside diphosphate linked moiety X)-type
motif 16 280 4631408O11Rik 4631408O11Rik .parallel. RIKEN cDNA
4631408011 gene 281 Gtf3c2 Gtf3c2 .parallel. general transcription
factor IIIC, polypeptide 2, beta 282 Arid1a Arid1a /// LOC675933
.parallel. AT rich interactive domain 1A (Swi1 like) /// similar to
AT rich interactive domain 1A isoform a 283 B3gnt1 B3gnt1
.parallel. UDP-GlcNAc:betaGal
beta-1,3-N-acetylglucosaminyltransferase 1 284 Stk11ip Stk11ip
.parallel. serine/threonine kinase 11 interacting protein 285
Rasal2 Rasal2 .parallel. RAS protein activator like 2 286 Myst4
Myst4 .parallel. MYST histone acetyltransferase monocytic leukemia
4 287 Tmlhe Tmlhe .parallel. trimethyllysine hydroxylase, epsilon
288 Camsap1 Camsap1 .parallel. calmodulin regulated
spectrin-associated protein 1 289 C80068 C80068 .parallel.
expressed sequence C80068 290 Plekhg2 Plekhg2 .parallel. pleckstrin
homology domain containing, family G (with RhoGef domain) member 2
291 Wipi1 Wipi1 .parallel. WD repeat domain, phosphoinositide
interacting 1 292 6820424L24Rik 6820424L24Rik .parallel. RIKEN cDNA
6820424L24 gene 293 Klf7 Klf7 .parallel. Kruppel-like factor 7
(ubiquitous) 294 Tm4sf1 Tm4sf1 .parallel. transmembrane 4
superfamily member 1 295 Mcpt8 Mcpt8 .parallel. mast cell protease
8 296 Has2 Has2 .parallel. hyaluronan synthase 2 297 Slc29a1
Slc29a1 .parallel. solute carrier family 29 (nucleoside
transporters), member 1 298 Cd44 Cd44 .parallel. CD44 antigen 299
Tnc Tnc .parallel. tenascin C 300 Pdgfc Pdgfc .parallel.
platelet-derived growth factor, C polypeptide 301 Tpcn1 Tpcn1
.parallel. two pore channel 1 302 Tmeff1 Tmeff1 .parallel.
transmembrane protein with EGF-like and two follistatin-like
domains 1 303 Cdyl Cdyl .parallel. chromodomain protein, Y
chromosome-like 304 Gpr35 Gpr35 .parallel. G protein-coupled
receptor 35 305 Col6a2 Col6a2 .parallel. procollagen, type VI,
alpha 2 306 Stab1 Stab1 .parallel. stabilin 1 307 Axin2 Axin2
.parallel. axin2 308 AI428795 AI428795 .parallel. expressed
sequence AI428795 309 Igf2 Igf2 .parallel. insulin-like growth
factor 2 310 Snai1 Snai1 .parallel. snail homolog 1 (Drosophila)
311 Mgl1 Mgl1 .parallel. macrophage galactose
N-acetyl-galactosamine specific lectin 1 312 Spp1 Spp1 .parallel.
secreted phosphoprotein 1 313 Pdgfrb Pdgfrb .parallel. platelet
derived growth factor receptor, beta polypeptide 314 Zfhx1a Zfhx1a
.parallel. zinc finger homeobox 1a 315 Pdzrn3 Pdzrn3 .parallel. PDZ
domain containing RING finger 3 316 Olfml3 Olfml3 .parallel.
olfactomedin-like 3 317 Cdh11 Cdh11 .parallel. cadherin 11 318
9030425E11Rik 9030425E11Rik .parallel. RIKEN cDNA 9030425E11 gene
319 Gpr124 Gpr124 .parallel. G protein-coupled receptor 124 320
Fstl1 Fstl1 .parallel. follistatin-like 1 321 Prrx1 Prrx1
.parallel. paired related homeobox 1 322 Timp1 Timp1 .parallel.
tissue inhibitor of metalloproteinase 1 323 Fn1 Fn1 .parallel.
fibronectin 1 324 Col1a1 Col1a1 .parallel. procollagen, type I,
alpha 1 325 Fbn1 Fbn1 .parallel. fibrillin 1 326 Col1a2 Col1a2
.parallel. procollagen, type I, alpha 2 327 Col5a2 Col5a2
.parallel. procollagen, type V, alpha 2 328 Col5a1 Col5a1
.parallel. Procollagen, type V, alpha 1 329 Col5a1 Col5a1
.parallel. procollagen, type V, alpha 1 330 Col3a1 Col3a1
.parallel. procollagen, type III, alpha 1 331 Spon1 Spon1
.parallel. spondin 1, (f-spondin) extracellular matrix protein 332
Cxcl14 Cxcl14 .parallel. chemokine (C-X-C motif) ligand 14 333 Dkk3
Dkk3 .parallel. dickkopf homolog 3 (Xenopus laevis) 334 Loxl1 Loxl1
.parallel. lysyl oxidase-like 1 335 Adamts5 Adamts5 .parallel. a
disintegrin-like and metallopeptidase (reprolysin type) with
thrombospondin type 1 motif, 5 (aggrecanase-2) 336 Sox17 Sox17
.parallel. SRY-box containing gene 17 337 Nedd9 Nedd9 .parallel.
neural precursor cell expressed, developmentally down-regulated
gene 9 338 Vegfc Vegfc .parallel. vascular endothelial growth
factor C 339 Slc16a2 Slc16a2 .parallel. solute carrier family 16
(monocarboxylic acid transporters), member 2 340 38968 38968
.parallel. septin 8 341 C2 C2 .parallel. complement component 2
(within H-2S) 342 Cfh Cfh .parallel. complement component factor h
343 Cpxm1 Cpxm1 .parallel. carboxypeptidase X 1 (M14 family) 344
Lum Lum .parallel. lumican 345 Mmp2 Mmp2 .parallel. matrix
metallopeptidase 2 346 Saa3 Saa3 .parallel. serum amyloid A 3 347
Sfrp1 Sfrp1 .parallel. secreted frizzled-related sequence protein 1
348 Masp1 Masp1 .parallel. mannan-binding lectin serine peptidase 1
349 Hspg2 Hspg2 .parallel. perlecan (heparan sulfate proteoglycan
2) 350 Itgbl1 Itgbl1 .parallel. integrin, beta-like 1 351 Lrrc35
Lrrc35 .parallel. leucine rich repeat containing 35 352 Abca8a
Abca8a .parallel. ATP-binding cassette, sub-family A (ABC1), member
8a 353 Dnajb5 Dnajb5 .parallel. DnaJ (Hsp40) homolog, subfamily B,
member 5 354 Mgp Mgp .parallel. matrix Gla protein 355 Serping1
Serping1 .parallel. serine (or cysteine) peptidase inhibitor, clade
G, member 1 356 Dnm3os Dnm3os .parallel. dynamin 3, opposite strand
357 Ptgis Ptgis .parallel. prostaglandin I2 (prostacyclin) synthase
358 Slc43a3 Slc43a3 .parallel. solute carrier family 43, member 3
359 Cyp2d22 Cyp2d22 .parallel. cytochrome P450, family 2, subfamily
d, polypeptide 22 360 Hoxa3 Hoxa3 .parallel. homeo box A3 361 Vldlr
Vldlr .parallel. very low density lipoprotein receptor 362 Col6a1
Col6a1 .parallel. procollagen, type VI, alpha 1 363 Ulk2 Ulk2
.parallel. Unc-51 like kinase 2 (C. elegans) 364 2900009I07Rik
2900009I07Rik .parallel. RIKEN cDNA 2900009I07 gene 365 Cd163 Cd163
.parallel. CD163 antigen 366 Paqr7 Paqr7 .parallel. progestin and
adipoQ receptor family member VII 367 Ube2r2 Ube2r2 .parallel.
ubiquitin-conjugating enzyme E2R 2 368 Bcl2l11 Bcl2l11 .parallel.
BCL2-like 11 (apoptosis facilitator) 369 Dpysl3 Dpysl3 .parallel.
dihydropyrimidinase-like 3 370 Stmn2 Stmn2 .parallel. stathmin-like
2 371 Leprel2 Leprel2 .parallel. leprecan-like 2 372 Kcnj8 Kcnj8
.parallel. potassium inwardly-rectifying channel, subfamily J,
member 8 373 Cd1d1 Cd1d1 .parallel. CD1d1 antigen 374 Mthfr Mthfr
.parallel. 5,10-methylenetetrahydrofolate reductase 375 Aebp1 Aebp1
.parallel. AE binding protein 1 376 Prg4 Prg4 .parallel.
proteoglycan 4 (megakaryocyte stimulating factor, articular
superficial zone protein) 377 Ccdc80 Ccdc80 .parallel. coiled-coil
domain containing 80 378 Cd248 Cd248 .parallel. CD248 antigen,
endosialin
379 Htra3 Htra3 .parallel. HtrA serine peptidase 3 380 Cygb Cygb
.parallel. cytoglobin 381 Loxl3 Loxl3 .parallel. lysyl oxidase-like
3 382 A930004K21Rik A930004K21Rik .parallel. RIKEN cDNA A930004K21
gene 383 Col11a1 Col11a1 .parallel. procollagen, type XI, alpha 1
384 Adpn Adpn .parallel. adiponutrin 385 Islr Islr .parallel.
immunoglobulin superfamily containing leucine-rich repeat 386 Lepr
Lepr .parallel. leptin receptor 387 Fndc1 Fndc1 .parallel.
fibronectin type III domain containing 1 388 Thbs2 Thbs2 .parallel.
thrombospondin 2 389 Ror2 Ror2 .parallel. receptor tyrosine
kinase-like orphan receptor 2 390 Fgfr1 Fgfr1 .parallel. fibroblast
growth factor receptor 1 391 Fap Fap .parallel. fibroblast
activation protein 392 Ptger3 Ptger3 .parallel. prostaglandin E
receptor 3 (subtype EP3) 393 Sox11 Sox11 .parallel. SRY-box
containing gene 11 394 Mmp7 Mmp7 .parallel. matrix metallopeptidase
7 395 Penk1 Penk1 .parallel. preproenkephalin 1 396 Stc2 Stc2
.parallel. stanniocalcin 2 397 Slc2a2 Slc2a2 .parallel. solute
carrier family 2 (facilitated glucose transporter), member 2 398
Asz1 Asz1 .parallel. ankyrin repeat, SAM and basic leucine zipper
domain containing 1 399 Ihh Ihh .parallel. Indian hedgehog 400 Lgr5
Lgr5 .parallel. leucine rich repeat containing G protein coupled
receptor 5 401 Col10a1 Col10a1 .parallel. procollagen, type X,
alpha 1 402 D18Ertd232e D18Ertd232e .parallel. DNA segment, Chr 18,
ERATO Doi 232, expressed 403 Ambp Ambp .parallel. alpha 1
microglobulin/bikunin 404 Pap Pap .parallel.
pancreatitis-associated protein 405 Tnfrsf19 Tnfrsf19 .parallel.
tumor necrosis factor receptor superfamily, member 19 406
B130052G07Rik B130052G07Rik .parallel. RIKEN cDNA B130052G07 gene
407 Ncam1 Ncam1 .parallel. neural cell adhesion molecule 1 408
Slit3 Slit3 .parallel. slit homolog 3 (Drosophila) 409 Muc3 Muc3
/// LOC666339 /// LOC677034 .parallel. mucin 3, intestinal ///
similar to mucin 17 /// similar to mucin 17 410 Col8a1 Col8a1
.parallel. procollagen, type VIII, alpha 1 411 Aldh1a2 Aldh1a2
.parallel. aldehyde dehydrogenase family 1, subfamily A2 412 Pdlim4
Pdlim4 .parallel. PDZ and LIM domain 4 413 AW822216 AW822216
.parallel. expressed sequence AW822216 414 Spsb1 Spsb1 .parallel.
splA/ryanodine receptor domain and SOCS box containing 1 415 F13a1
F13a1 .parallel. coagulation factor XIII, A1 subunit 416 Fstl3
Fstl3 .parallel. follistatin-like 3 417 Wnt2 Wnt2 .parallel.
wingless-related MMTV integration site 2 418 Cyp26a1 Cyp26a1
.parallel. cytochrome P450, family 26, subfamily a, polypeptide 1
419 Scamp5 Scamp5 .parallel. secretory carrier membrane protein 5
420 Nppa Nppa .parallel. natriuretic peptide precursor type A 421
C1qtnf6 C1qtnf6 .parallel. C1q and rumor necrosis factor related
protein 6 422 Tmem119 Tmem119 .parallel. transmembrane protein 119
423 Zmym3 Zmym3 .parallel. zinc finger, MYM-type 3 424 Sdc3 Sdc3
.parallel. syndecan 3 425 Hoxb2 Hoxb2 .parallel. homeo box B2 426
D530037H12Rik D530037H12Rik .parallel. RIKEN cDNA D530037H12 gene
427 Falz Falz .parallel. fetal Alzheimer antigen 428 Itsn1 Itsn1
.parallel. intersectin 1 (SH3 domain protein 1A) 429 Sema6d Sema6d
.parallel. sema domain, transmembrane domain (TM), and cytoplasmic
domain, (semaphorin) 6D 430 Atp11c Atp11c .parallel. Atpase, class
VI, type 11C 431 H2-Ea H2-Ea .parallel. histocompatibility 2, class
II antigen E alpha 432 Farp1 Farp1 .parallel. FERM, RhoGEF (Arhgef)
and pleckstrin domain protein 1 (chondrocyte-derived) 433 Smoc2
Smoc2 .parallel. SPARC related modular calcium binding 2 434 Sash1
Sash1 .parallel. SAM and SH3 domain containing 1 435 Cd47 Cd47
.parallel. CD47 antigen (Rh-related antigen, integrin-associated
signal transducer) 436 Large Large .parallel.
like-glycosyltransferase 437 F2r F2r .parallel. coagulation factor
II (thrombin) receptor 438 Gfpt2 Gfpt2 .parallel. glutamine
fructose-6-phosphate transaminase 2 439 C1qtnf1 C1qtnf1 .parallel.
C1q and tumor necrosis factor related protein 1 440 Mcpt2 Mcpt2
.parallel. mast cell protease 2 441 Ptprt Ptprt .parallel. protein
tyrosine phosphatase, receptor type, T 442 D1Bwg1363e D1Bwg1363e
.parallel. DNA segment, Chr 1, Brigham & Women's Genetics 1363
expressed 443 Rab2b Rab2b .parallel. RAB2B, member RAS oncogene
family 444 Rffl Rffl .parallel. ring finger and FYVE like domain
containing protein 445 Serpinb1a Serpinb1a .parallel. serine (or
cysteine) peptidase inhibitor, clade B, member 1a 446 Zfyve26
Zfyve26 .parallel. zinc finger, FYVE domain containing 26 447
Slamf6 Slamf6 .parallel. SLAM family member 6 448 Gdap10 Gdap10
.parallel. ganglioside-induced differentiation-associated-protein
10 449 Mafb Mafb .parallel. v-maf musculoaponeurotic fibrosarcoma
oncogene family, protein B (avian) 450 Tcf7l2 Tcf7l2 .parallel.
transcription factor 7-like 2, T-cell specific, HMG-box 451 Itga5
Itga5 .parallel. integrin alpha 5 (fibronectin receptor alpha) 452
Dock6 Dock6 /// LOC670024 .parallel. dedicator of cytokinesis 6 ///
similar to Dedicator of cytokinesis protein 6 453 Tesc Tesc
.parallel. tescalcin 454 Tspan5 Tspan5 .parallel. tetraspanin 5 455
Dopey2 Dopey2 .parallel. dopey family member 2 456 Coil Coil
.parallel. coilin 457 2810055F11Rik 2810055F11Rik .parallel. RIKEN
cDNA 2810055F11 gene 458 C1s C1s .parallel. complement component 1,
s subcomponent 459 Dmd Dmd .parallel. dystrophin, muscular
dystrophy 460 Dcn Dcn .parallel. decorin 461 1110018M03Rik
1110018M03Rik .parallel. RIKEN cDNA 1110018M03 gene
[0042] Inherited mutation in LKB1 results in the Peutz-Jeghers
syndrome (PJS), characterized by intestinal hamartomas and an
increased frequency of gastrointestinal and breast cancer.sup.36.
Somatic inactivation of LKB1 occurs in human lung
adenocarcinoma.sup.9,10,37, but its tumor suppressor role in this
tissue is uncertain. Although activation of many kinases (e.g.
BRAF, EGFR, etc.) is oncogenic, LKB1 appears unusual in cancer
biology in that its kinase activity conveys tumor suppressor
activity.sup.38. LKB1 can phosphorylate multiple cellular
substrates and has been implicated in playing important roles in a
myriad of cellular metabolic functions including protein synthesis,
gluconeogensis, adipogenesis, and steroidogenesis as well as cell
polarity. All the substrates of LKB1 that are relevant to tumor
suppression are not known, but AMPK, the kinase crucially involved
in regulating the mTOR pathway, has been shown to be one definite
LKB substrate.sup.39-41.
[0043] LKB1 phosphorylates and activates AMP Kinase (AMPK) in
settings of high AMP levels (i.e., low energy states). AMPK, in
turn, phosphorylates Tuberin, but unlike phosphorylation by Akt,
this activates Tuberin's GAP activity leading to inhibition of mTOR
activity. However, in LKB1-deficient cells, AMPK cannot be
activated, and mTOR remains constitutively active under conditions
of energy stress. Thus, mTOR is regulated by growth factor receptor
signaling, nutrient availability, and the energy status of the
cell. This ensures that a cell normally commits to growth upon
appropriate growth stimuli and environmental cues. Transformed and
cancer cells, however, bypass one or more of these control
mechanisms to grow without restraint. The mTOR pathway is
frequently deregulated in many different types of cancer.
[0044] In addition, recent studies suggest that LKB1 may play an
important role in mediating the cellular responses to DNA damage.
ATM, a kinase involved in DNA damage checkpoint and p53-dependent
apoptosis, phosphorylates LKB1 on Thr366 after radiation-induced
DNA damage.sup.43,44. This region of LKB1 had been shown to be
necessary for its growth suppression function.sup.43,44. Thus, the
loss of LKB1 might render the affected cells more prone to DNA
damage and increased genetic alterations. Lastly, reactive lipid
species, such as cyclopentenone, often generated in the setting of
chronic inflammation and oxidative stress, can inactivate the
functional activity of LKB1 through the formation of a covalent
adduct at Cys210, an important amino acid in LKB1's activation
loop.sup.45.
[0045] A human Lkb1 nucleic acid and polypeptide are shown in Table
1 and 2 respectively (SEQ ID NO:1 and SEQ ID NO:2).
TABLE-US-00002 TABLE 1 (SEQ ID NO: 1) Lkb1 Nucleic Acid Sequence
GCGTGTCGGGCGCGGAAGGGGGAGGCGGCCCGGGGCGCCCGCGAGTGAG
GCGCGGGGCGGCGAAGGGAGCGCGGGTGGCGGCACTTGCTGCCGCGGCC
TTGGATGGGCTGGGCCCCCCTCGCCGCTCCGCCTCCTCCACACGCGCGG
CGGCCGCGGCGAGGGGGACGCGCCGCCCGGGGCCCGGCACCTTCGGGAA
CCCCCCGGCCCGGAGCCTGCGGCCTGCGCCGCCTCGGCCGCCGGGAGCC
CCGTGGAGCCCCCGCCGCCGCGCCGCCCCGCGGACCGGACGCTGAGGGC
ACTCGGGGCGGGGCGCGCGCTCGGGCAGACGTTTGCGGGGAGGGGGGCG
CCTGCCGGGCCCCGGCGACCACCTTGGGGGTCGCGGGCCGGCTCGGGGG
GCGCCCAGTGCGGGCCCTCGCGGGCGCCGGGCAGCGACCAGCCCTGAGC
GGAGCTGTTGGCCGCGGCGGGAGGCCTCCCGGACGCCCCCAGCCCCCCG
AACGCTCGCCCGGGCCGGCGGGAGTCGGCGCCCCCCGGGAGGTCCGCTC
GGTCGTCCGCGGCGGAGCGTTTGCTCCTGGGACAGGCGGTGGGACCGGG
GCGTCGCCGGAGACGCCCCCAGCGAAGTTGGGCTCTCCAGGTGTGGGGG
TCCCGGGGGGTAGCGACGTCGCGGACCCGGCCTGTGGGATGGGCGGCCC
GGAGAAGACTGCGCTCGGCCGTGTTCATACTTGTCCGTGGGCCTGAGGT
CCCCGGAGGATGACCTAGCACTGAAAAGCCCCGGCCGGCCTCCCCAGGG
TCCCCGAGGACGAAGTTGACCCTGACCGGGCCGTCTCCCAGTTCTGAGG
CCCGGGTCCCACTGGAACTCGCGTCTGAGCCGCCGTCCCGGACCCCCGG
TGCCCGCCGGTCCGCAGACCCTGCACCGGGCTTGGACTCGCAGCCGGGA
CTGACGTGTAGAACAATCGTTTCTGTTGGAAGAAGGGTTTTTCCCTTCC
TTTTGGGGTTTTTGTTGCCTTTTTTTTTTCTTTTTTCTTTGTAAAATTT
TGGAGAAGGGAAGTCGGAACACAAGGAAGGACCGCTCACCCGCGGACTC
AGGGCTGGCGGCGGGACTCCAGGACCCTGGGTCCAGCATGGAGGTGGTG
GACCCGCAGCAGCTGGGCATGTTCACGGAGGGCGAGCTGATGTCGGTGG
GTATGGACACGTTCATCCACCGCATCGACTCCACCGAGGTCATCTACCA
GCCGCGCCGCAAGCGGGCCAAGCTCATCGGCAAGTACCTGATGGGGGAC
CTGCTGGGGGAAGGCTCTTACGGCAAGGTGAAGGAGGTGCTGGACTCGG
AGACGCTGTGCAGGAGGGCCGTCAAGATCCTCAAGAAGAAGAAGTTGCG
AAGGATCCCCAACGGGGAGGCCAACGTGAAGAAGGAAATTCAACTACTG
AGGAGGTTACGGCACAAAAATGTCATCCAGCTGGTGGATGTGTTATACA
ACGAAGAGAAGCAGAAAATGTATATGGTGATGGAGTACTGCGTGTGTGG
CATGCAGGAAATGCTGGACAGCGTGCCGGAGAAGCGTTTCCCAGTGTGC
CAGGCCCACGGGTACTTCTGTCAGCTGATTGACGGCCTGGAGTACCTGC
ATAGCCAGGGCATTGTGCACAAGGACATCAAGCCGGGGAACCTGCTGCT
CACCACCGGTGGCACCCTCAAAATCTCCGACCTGGGCGTGGCCGAGGCA
CTGCACCCGTTCGCGGCGGACGACACCTGCCGGACCAGCCAGGGCTCCC
CGGCTTTCCAGCCGCCCGAGATTGCCAACGGCCTGGACACCTTCTCCGG
CTTCAAGGTGGACATCTGGTCGGCTGGGGTCACCCTCTACAACATCACC
ACGGGTCTGTACCCCTTCGAAGGGGACAACATCTACAAGTTGTTTGAGA
ACATCGGGAAGGGGAGCTACGCCATCCCGGGCGACTGTGGCCCCCCGCT
CTCTGACCTGCTGAAAGGGATGCTTGAGTACGAACCGGCCAAGAGGTTC
TCCATCCGGCAGATCCGGCAGCACAGCTGGTTCCGGAAGAAACATCCTC
CGGCTGAAGCACCAGTGCCCATCCCACCGAGCCCAGACACCAAGGACCG
GTGGCGCAGCATGACTGTGGTGCCGTACTTGGAGGACCTGCACGGCGCG
GACGAGGACGAGGACCTCTTCGACATCGAGGATGACATCATCTACACTC
AGGACTTCACGGTGCCCGGACAGGTCCCAGAAGAGGAGGCCAGTCACAA
TGGACAGCGCCGGGGCCTCCCCAAGGCCGTGTGTATGAACGGCACAGAG
GCGGCGCAGCTGAGCACCAAATCCAGGGCGGAGGGCCGGGCCCCCAACC
CTGCCCGCAAGGCCTGCTCCGCCAGCAGCAAGATCCGCCGGCTGTCGGC
CTGCAAGCAGCAGTGAGGCTGGCCGCCTGCAGCCCGTGTCCAGGAGCCC
CGCCAGGTGCCCGCGCCAGGCCCTCAGTCTTCCTGCCGGTTCCGCCCGC
CCTCCCGGAGAGGTGGCCGCCATGCTTCTGTGCCGACCACGCCCCAGGA
CCTCCGGAGCGCCCTGCAGGGCCGGGCAGGGGGACAGCAGGGACCGGGC
GCAGCCCTCCCCCCTCGGCCGCCCGGCAGTGCACGCGGCTTGTTGACTT
CGCAGCCCCGGGCGGAGCCTTCCCGGGCGGGCGTGGGAGGAGGGAGGCG
GCCTCCATGCACTTTATGTGGAGACTACTGGCCCCGCCCGTGGCCTCGT
GCTCCGCAGGGCGCCCAGCGCCGTCCGGCGGCCCCGCCGCAGACCAGCT
GGCGGGTGTGGAGACCAGGCTCCTGACCCCGCCATGCATGCAGCGCCAC
CTGGAAGCCGCGCGGCCGCTTTGGTTTTTTGTTTGGTTGGTTCCATTTT
CTTTTTTTCTTTTTTTTTTTAAGAAAAAATAAAAGGTGGATTTGAGCTG
TGGCTGTGAGGGGTGTTTGGGAGCTGCTGGGTGGCAGGGGGGCTGTGGG
GTCGGGCTCACGTCGCGGCCGCCTTTGCGCTCTCGGGTCACCCTGCTTT
GGCGGCCCGGCCGGAGGGCAGGACCCTCACCTCTCCCCCAAGGCCACTG
CGCTCTTGGGACCCCAGAGAAAACCCGGAGCAAGCAGGAGTGTGCGGTC
AATATTTATATCATCCAGAAAAGAAAAACACGAGAAACGCCATCGCGGG
ATGGTGCAGACGCGGCGGGGACTCGGAGGGTGCCGTGCGGGCGAGGCCG
CCCAAATTTGGCAATAAATAAAGCTTGGGAAGCTTGGACCTGAAAAAAA AAA
TABLE-US-00003 TABLE 2 (SEQ ID NO: 2) Lkb1 Polypeptide Sequence
MEVVDPQQLGMFTEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKY
LMGDLLGEGSYGKVKEVLDSETLCRRAVKILKKKKLRRIPNGEANVKKE
IQLLRRLRHKNVIQLVDVLYNEEKQKMYMVMEYCVCGMQEMLDSVPEKR
FPVCQAHGYFCQLIDGLEYLHSQGIVHKDIKPGNLLLTTGGTLKISDLG
VAEALHPFAADDTCRTSQGSPAFQPPEIANGLDTFSGFKVDIWSAGVTL
YNITTGLYPFEGDNIYKLFENIGKGSYAIPGDCGPPLSDLLKGMLEYEP
AKRFSIRQIRQHSWFRKKHPPAEAPVPIPPSPDTKDRWRSMTVVPYLED
LHGADEDEDLFDIEDDIIYTQDFTVPGQVPEEEASHNGQRRGLPKAVCM
NGTEAAQLSTKSRAEGRAPNPARKACSASSKIRRLSACKQQ
Diagnostic and Prognostic Methods
[0046] The aggressiveness of lung cancers is determined by
examining the presence (e.g, expression) or absence of a Lkb1
nucleic acid, polypeptide or activity in a test sample (i.e., a
patient derived sample). Preferably, the test sample is a tumor
biopsy. A change in the level of the Lkb1 nucleic acid, polypeptide
or activity compared to a control sample is indicative of the
aggressiveness of the lung cancer in the subject. The absence of
the Lkb1 nucleic acid or polypeptide or a decrease in the activity
of Lkb1 in the test sample indicates that the cancer is aggressive,
thus a less favorable prognosis for the patient. An aggressive
tumor is metastatic, thus the absence of the Lkb1 nucleic acid or
polypeptide or a decrease in the activity of Lkb1 in the test
sample indicates that the tumor is metastatic. In contrast, the
presence of the Lkb1 nucleic acid or polypeptide or activity of
Lkb1 indicates that the lung cancer is not aggressive, thus a more
favorable prognosis for the patient.
[0047] Additionally, the presence or absence of a mutation the Lkb1
nucleic acid is indicative of the aggressiveness of the lung cancer
in the subject. For example, the presence of a mutation in the the
Lkb1 nucleic acid in the test sample indicates that the cancer is
aggressive, thus a less favorable prognosis for the patient.
Whereas the absence a mutation in the Lkb1 nucleic acid indicates
that the lung cancer is not aggressive, thus a more favorable
prognosis for the patient.
[0048] By aggressiveness it is meant that that the tumor is quick
growing and spreading (i.e, metastasizing.
[0049] The amount of the Lkb1 nucleic acid, polypeptide or activity
is determined in the test sample and compared to the expression of
the normal control level. By normal control level is meant the
expression level of a Lkb1 nucleic acid, polypeptide or activity
typically found in a subject not suffering from lung cancer.
[0050] The alteration in the amount of the Lkb1 nucleic acid,
polypeptide or activity is statistically significant. By
statistically significant is meant that the alteration is greater
than what might be expected to happen by change alone. Statistical
significance is determined by method known in the art. For example
statistical significance is determined by p-value. The p-values is
a measure of probability that a difference between groups during an
experiment happened by chance. (P(z.gtoreq.z.sub.observed)). For
example, a p-value of 0.01 means that there is a 1 in 100 chance
the result occurred by chance. The lower the p-value, the more
likely it is that the difference between groups was caused by
treatment. An alteration is statistically significant if the
p-value is at least 0.05. Preferably, the p-value is 0.04, 0.03,
0.02, 0.01, 0.005, 0.001 or less.
[0051] The "diagnostic accuracy" of a test, assay, or method
concerns the ability of the test, assay, or method to distinguish
between patients having aggressive lung cancer and non-aggressive
lung cancer is based on whether the patients have a "clinically
significant presence or absence" of a Lkb1 nucleic acid,
polypeptide or activity. By "clinically significant presence" is
meant that the presence of the Lkb1 nucleic acid, polypeptide or
activity in the patient (typically in a sample from the patient) is
higher or lower than the predetermined cut-off point (or threshold
value) for Lkb1 nucleic acid, polypeptide or activity and therefore
indicates that the patient has cancer for which the sufficiently
high presence of that protein is a marker.
[0052] The terms "high degree of diagnostic accuracy" and "very
high degree of diagnostic accuracy" refer to the test or assay for
that Lkb1 nucleic acid, polypeptide or activity with the
predetermined cut-off point correctly (accurately) indicating the
presence or absence of the cancer. A perfect test would have
perfect accuracy. Thus, for individuals who have aggressive lung
cancer, the test would indicate only positive test results and
would not report any of those individuals as being "negative"
(there would be no "false negatives"). In other words, the
"sensitivity" of the test (the true positive rate) would be 100%.
On the other hand, for individuals who did not have aggressive lung
cancer, the test would indicate only negative test results and
would not report any of those individuals as being "positive"
(there would be no "false positives"). In other words, the
"specificity" (the true negative rate) would be 100%. See, e.g.,
O'Marcaigh A S, Jacobson R M, "Estimating The Predictive Value Of A
Diagnostic Test, How To Prevent Misleading Or Confusing Results,"
Clin. Ped. 1993, 32(8): 485-491, which discusses specificity,
sensitivity, and positive and negative predictive values of a test,
e.g., a clinical diagnostic test.
[0053] Changing the cut point or threshold value of a test (or
assay) usually changes the sensitivity and specificity but in a
qualitatively inverse relationship. For example, if the cut point
is lowered, more individuals in the population tested will
typically have test results over the cut point or threshold value.
Because individuals who have test results above the cut point are
reported as having the disease, condition, or syndrome for which
the test is being run, lowering the cut point will cause more
individuals to be reported as having positive results (i.e., that
they have cancer). Thus, a higher proportion of those who have
cancer will be indicated by the test to have it. Accordingly, the
sensitivity (true positive rate) of the test will be increased.
However, at the same time, there will be more false positives
because more people who do not have the disease, condition, or
syndrome (i.e., people who are truly "negative") will be indicated
by the test to have Lkb1 nucleic acid, polypeptide or activity
values above the cut point and therefore to be reported as positive
rather than being correctly indicated by the test to be negative.
Accordingly, the specificity (true negative rate) of the test will
be decreased. Similarly, raising the cut point will tend to
decrease the sensitivity and increase the specificity. Therefore,
in assessing the accuracy and usefulness of a proposed medical
test, assay, or method for assessing a patient's condition, one
should always take both sensitivity and specificity into account
and be mindful of what the cut point is at which the sensitivity
and specificity are being reported because sensitivity and
specificity may vary significantly over the range of cut
points.
[0054] There is, however, an indicator that allows representation
of the sensitivity and specificity of a test, assay, or method over
the entire range of cut points with just a single value. That
indicator is derived from a Receiver Operating Characteristics
("ROC") curve for the test, assay, or method in question. See,
e.g., Shultz, "Clinical Interpretation Of Laboratory Procedures,"
chapter 14 in Teitz, Fundamentals of Clinical Chemistry, Burtis and
Ashwood (eds.), 4th edition 1996, W.B. Saunders Company, pages
192-199; and Zweig et al., "ROC Curve Analysis: An Example Showing
The Relationships Among Serum Lipid And Apolipoprotein
Concentrations In Identifying Patients With Coronary Artery
Disease," Clin. Chem., 1992, 38(8): 1425-1428.
[0055] An ROC curve is an x-y plot of sensitivity on the y-axis, on
a scale of zero to one (i.e., 100%), against a value equal to one
minus specificity on the x-axis, on a scale of zero to one (i.e.,
100%). In other words, it is a plot of the true positive rate
against the false positive rate for that test, assay, or method. To
construct the ROC curve for the test, assay, or method in question,
patients are assessed using a perfectly accurate or "gold standard"
method that is independent of the test, assay, or method in
question to determine whether the patients are truly positive or
negative for the disease, condition, or syndrome (for example,
coronary angiography is a gold standard test for the presence of
coronary atherosclerosis). The patients are also tested using the
test, assay, or method in question, and for varying cut points, the
patients are reported as being positive or negative according to
the test, assay, or method. The sensitivity (true positive rate)
and the value equal to one minus the specificity (which value
equals the false positive rate) are determined for each cut point,
and each pair of x-y values is plotted as a single point on the x-y
diagram. The "curve" connecting those points is the ROC curve.
[0056] The area under the curve ("AUC") is the indicator that
allows representation of the sensitivity and specificity of a test,
assay, or method over the entire range of cut points with just a
single value. The maximum AUC is one (a perfect test) and the
minimum area is one half. The closer the AUC is to one, the better
is the accuracy of the test.
[0057] By a "high degree of diagnostic accuracy" is meant a test or
assay (such as the test of the invention for determining the
clinically significant presence of Lkb1 nucleic acid, polypeptide
or activity, in which the AUC (area under the ROC curve for the
test or assay) is at least 0.70, desirably at least 0.75, more
desirably at least 0.80, preferably at least 0.85, more preferably
at least 0.90, and most preferably at least 0.95.
[0058] By a "very high degree of diagnostic accuracy" is meant a
test or assay in which the AUC (area under the ROC curve for the
test or assay) is at least 0.875, desirably at least 0.90, more
desirably at least 0.925, preferably at least 0.95, more preferably
at least 0.975, and most preferably at least 0.98.
[0059] Optionally, the subject is tested for carrying other
indicators of susceptibility of developing cancer. For example, the
presence or absence of a mutation in K-ras, EGFR or BRAF is
determined in the test sample.
[0060] The Lkb1 nucleic acid, polypeptide or activity are detected
in any suitable manner, but is typically detected by contacting a
sample from the patient with an antibody which binds the Lkb1
nucleic acid, polypeptide and then detecting the presence or
absence of a reaction product. The antibody may be monoclonal,
polyclonal, chimeric, or a fragment of the foregoing, as discussed
in detail above, and the step of detecting the reaction product may
be carried out with any suitable immunoassay. The sample from the
subject is typically a tumor biopsy as described above.
[0061] Lung cancer is also diagnosed by examining the expression of
one or more LC nucleic acid or polypeptide sequences from a test
population of cells, (i.e., a patient derived tissue sample) that
contain or suspected to contain a non-small cell lung cancer cell.
Preferably, the test cell population comprises a lung cell, e.g., a
cell obtained from the lung. Gene expression is also measured from
blood or other bodily fluids such as sputum.
[0062] Expression of one or more of a lung cancer-associated gene,
e.g., LC 1-461 is determined in the test cell and compared to the
expression of the normal control level. By normal control level is
meant the expression profile of the lung cancer-associated genes
typically found in a population not suffering from lung cancer. An
increase of the level of expression in the patient derived tissue
sample of the lung cancer associated genes indicates that the
subject is suffering from or is at risk of developing lung
cancer.
[0063] Also provided is a method of assessing the prognosis of a
subject with lung cancer by comparing the expression of one or more
LC sequences in a test cell population to the expression of the
sequences in a reference cell population derived from patients over
a spectrum of disease stages. By comparing gene expression of one
or more LC sequences in the test cell population and the reference
cell population(s), or by comparing the pattern of gene expression
over time in test cell populations derived from the subject, the
prognosis of the subject can be assessed. For example, an increase
in expression of one or more of the sequences LC 1-461 compared to
a normal control indicates less favorable prognosis.
[0064] The differentially LC sequences identified herein also allow
for the course of treatment of lung cancer to be monitored. In this
method, a test cell population is provided from a subject
undergoing treatment for lung cancer. If desired, test cell
populations are obtained from the subject at various time points
before, during, or after treatment. Expression of one or more of
the LC sequences, in the cell population is then determined and
compared to a reference cell population which includes cells whose
lung cancer state is known. The reference cells have not been
exposed to the treatment.
[0065] If the reference cell population contains no lung cancer
cells, a similarity in expression between LC sequences in the test
cell population and the reference cell population indicates that
the treatment is efficacious. However, a difference in expression
between LC sequences in the test population and this reference cell
population indicates the a less favorable clinical outcome or
prognosis.
[0066] By "efficacious" is meant that the treatment leads to a
reduction in expression of a pathologically upregulated gene,
increase in expression of a pathologically down-regulated gene or a
decrease in size, prevalence, or metastatic potential of lung
cancer in a subject. When treatment is applied prophylactically,
"efficacious" means that the treatment retards or prevents lung
cancer from forming. Assessment of lung cancer is made using
standard clinical protocols.
[0067] Efficaciousness is determined in association with any known
method for diagnosing or treating non-small cell lung cancer. Lung
cancer is diagnosed for example, by identifying symptomatic
anomalies, e.g., chronic cough, hoarseness, coughing up blood,
weight loss, loss of appetite, shortness of breath, wheezing,
repeated bouts of bronchitis or pneumonia, and chest pain or
histopathologically.
[0068] Expression of Lkb1 also allows the identification of
patients who will be responsive to systemic therapy, e.g. m-TOR
inhibitors. In this method, a tumor sample is provided from a
subject and Lkb1 expression is determined. Identification of
inactivation of the Lkb1 gene indicate that the therapy will be
efficacious. In contrast a the patient will likely not be
responsive to systemic treatment with an M-TOR inhibitor if the
Lkb1 gene is active. By inactivation is meant a decrease in Lkb1
expression or activity. For example the Lkb1 gene is inactivated
due to a mutation.
[0069] The subject is preferably a mammal. The mammal is, e.g., a
human, non-human primate, mouse, rat, dog, cat, horse, or cow.
Subjects are typically human females or human males.
[0070] The subject has been previously diagnosed as carrying a
cancer, and possibly has already undergone treatment for the
cancer. Alternatively, the subject has not been previously
diagnosis as carrying a cancer. The present invention is useful
with all patients at risk for a cancer. Although each type of
cancer has their own set of risk factors, the risk of developing
cancer increases as with aged, gender, race and personal and family
medical history. Other risk factors are largely related to
lifestyle choices, while certain infections, occupational exposures
and some environmental factors can also be related to developing
cancer. Lung cancer risk factors include personal and family
history of lung cancer, smoking, exposure to asbestos, radon or
other carcinogens, air pollution, Vitamin A deficiency, or
reoccurring inflammation.
[0071] Diagnosis of cancer is typically made through the
identification of a mass on an examination, though it may also be
through other means such as a radiological diagnosis, or
ultrasound. Treatment is typically through cytoreductive surgery.
In addition, many patients will require radiation therapy.
[0072] Expression of the Lkb1, a LC nuclic acid or polypeptide or
other cancer biomarkers (e.g., K-ras, EGFR or BRAf) is determined
at the protein or nucleic acid level using any method known in the
art. For example, Northern hybridization analysis using probes
which specifically recognize one or more of these sequences can be
used to determine gene expression. Alternatively, expression is
measured using reverse-transcription-based PCR assays, e.g., using
primers specific for the differentially expressed sequence of
genes. Expression is also determined at the protein level, i.e., by
measuring the levels of peptides encoded by the gene products
described herein, or activities thereof. Such methods are well
known in the art and include, e.g., immunoassays based on
antibodies to proteins encoded by the genes. Any biological
material can be used for the detection/quantification of the
protein or it's activity. Alternatively, a suitable method can be
selected to determine the activity of proteins encoded by the
marker genes according to the activity of each protein
analyzed.
[0073] Immunoassays carried out in accordance with the present
invention may be homogeneous assays or heterogeneous assays. In a
homogeneous assay the immunological reaction usually involves the
specific antibody (e.g., fibrinogen .alpha.C domain peptide or
hemoglobin polypeptide), a labeled analyte, and the sample of
interest. The signal arising from the label is modified, directly
or indirectly, upon the binding of the antibody to the labeled
analyte. Both the immunological reaction and detection of the
extent thereof are carried out in a homogeneous solution.
Immunochemical labels which may be employed include free radicals,
radioisotopes, fluorescent dyes, enzymes, bacteriophages, or
coenzymes.
[0074] In a heterogeneous assay approach, the reagents are usually
the sample, the antibody, and means for producing a detectable
signal. Samples as described above may be used. The antibody is
generally immobilized on a support, such as a bead, plate or slide,
and contacted with the specimen suspected of containing the antigen
in a liquid phase. The support is then separated from the liquid
phase and either the support phase or the liquid phase is examined
for a detectable signal employing means for producing such signal.
The signal is related to the presence of the analyte in the sample.
Means for producing a detectable signal include the use of
radioactive labels, fluorescent labels, or enzyme labels. For
example, if the antigen to be detected contains a second binding
site, an antibody which binds to that site can be conjugated to a
detectable group and added to the liquid phase reaction solution
before the separation step. The presence of the detectable group on
the solid support indicates the presence of the antigen in the test
sample. Examples of suitable immunoassays are radioimmunoassays,
immunofluorescence methods, or enzyme-linked immunoassays.
[0075] Those skilled in the art will be familiar with numerous
specific immunoassay formats and variations thereof, which may be
useful for carrying out the method disclosed herein. See generally
E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton,
Fla.); see also U.S. Pat. No. 4,727,022 to Skold et al. titled
"Methods for Modulating Ligand-Receptor Interactions and their
Application," U.S. Pat. No. 4,659,678 to Forrest et al. titled
"Immunoassay of Antigens," U.S. Pat. No. 4,376,110 to David et al.,
titled "Immunometric Assays Using Monoclonal Antibodies," U.S. Pat.
No. 4,275,149 to Litman et al., titled "Macromolecular Environment
Control in Specific Receptor Assays," U.S. Pat. No. 4,233,402 to
Maggio et al., titled "Reagents and Method Employing Channeling,"
and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled
"Heterogeneous Specific Binding Assay Employing a Coenzyme as
Label."
[0076] Antibodies are conjugated to a solid support suitable for a
diagnostic assay (e.g., beads, plates, slides or wells formed from
materials such as latex or polystyrene) in accordance with known
techniques, such as precipitation. An antibody or antibody fragment
that binds to CA-125 or CEA may optionally be conjugated to the
same support, as discussed above. Antibodies as described herein
may likewise be conjugated to detectable groups such as radiolabels
(e.g., 35 S, 125 I, 131 I), enzyme labels (e.g., horseradish
peroxidase, alkaline phosphatase), and fluorescent labels (e.g.,
fluorescein) in accordance with known techniques.
[0077] Screening Methods
[0078] An agent that inhibits the expression or activity of a lung
cancer-associated gene is identified by contacting a test cell
population expressing a lung cancer associated upregulated gene
with a test agent and determining the expression level of the lung
cancer associated gene. A decrease in expression compared to the
normal control level indicates the agent is an inhibitor of a lung
cancer associated upregulated gene and useful to inhibit non-small
cell lung cancer.
[0079] The differentially expressed sequences disclosed herein can
also be used to identify candidate therapeutic agents for treating
lung cancer. The method is based on screening a candidate
therapeutic agent to determine if it converts an expression profile
of LC 1-461 sequences characteristic of a lung cancer state to a
pattern indicative of a non lung cancer state.
[0080] In the method, a cell is exposed to a test agent or a
combination of test agents (sequentially or consequentially) and
the expression of one or more LC 1-461 sequences in the cell is
measured. The expression profile of the LC sequences in the test
population is compared to expression level of the LC sequences in a
reference cell population that is not exposed to the test
agent.
[0081] Alternatively, the screening of the present invention may
comprise the steps described below. A protein required for the
screening can be obtained as a recombinant protein by using the
nucleotide sequence of the marker gene. Based on the information on
the marker gene, one skilled in the art can select the biological
activity of a protein as an index of screening and a measurement
method for the activity.
[0082] (1) the step of contacting a candidate agent with the
protein encoded by a marker gene; and
[0083] (2) the step of selecting a candidate agent that alters the
activity of the protein as compared with that in a control.
[0084] Alternatively, the screening of the present invention may
comprise the steps described below. A reporter construct required
for the screening can be prepared by using the transcriptional
regulatory region of a marker gene. When the transcriptional
regulatory region of a marker gene has been known to those skilled
in the art, a reporter construct can be prepared by using the
previous sequence information. When the transcriptional regulatory
region of a marker gene remains unidentified, a nucleotide segment
containing the transcriptional regulatory region can be isolated
from a genome library based on the nucleotide sequence information
of the marker gene.
[0085] (1) the step of preparing a reporter construct that ensures
the expression of the reporter gene under control of the
transcriptional regulatory region of the marker gene;
[0086] (2) the step of contacting a candidate agent with host cells
containing and capable of expressing the above-mentioned reporter
construct; and
[0087] (3) the step of measuring the expression level of the
reporter gene, and selecting a candidate agent that has an activity
of altering the expression level when compared with that in a
control.
[0088] There is no limitation on the type of candidate agent in the
screening of the present invention. The candidates of the present
invention can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam
(1997)Anticancer Drug Des. 12:145).
[0089] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem.
37:1233.
[0090] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Bio Techniques 13:412), or on beads (Lam (1991)
Nature 354:82), chips (Fodor (1993) Nature 364:555), bacteria (U.S.
Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;
and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci.
USA 89:1865) or phage (Scott and Smith (1990) Science 249:386;
Devlin (1990) Science 249:404; Cwirla et al. (1990) Proc. Natl.
Acad. Sci. USA 87:6378; and Felici (1991) J. Mol. Biol.
222:301).(United States Patent Application 20020103360)
[0091] An agent effective i in suppressing expression of
overexpressed genes is deemed to lead to a clinical benefit such
compounds are further tested for the ability to prevent cancer cell
growth.
[0092] Differences in the genetic makeup of individuals can result
in differences in their relative abilities to metabolize various
drugs. An agent that is metabolized in a subject to act as an
anti-cell lung cancer agent can manifest itself by inducing a
change in gene expression pattern in the subject's cells from that
characteristic of a cancerous state to a gene expression pattern
characteristic of a non-cancerous state. Accordingly, the
differentially expressed LC sequences disclosed herein allow for a
putative therapeutic or prophylactic anti-lung cancer agent to be
tested in a test cell population from a selected subject in order
to determine if the agent is a suitable ant lung cancer agent in
the subject.
[0093] To identify an anti-lung cancer agent, that is appropriate
for a specific subject, a test cell population from the subject is
exposed to a therapeutic agent, and the expression of one or more
of LC 1-461 sequences is determined. For example a test cell
population is incubated in the presence of a candidate agent and
the pattern of gene expression of the test sample is measured and
compared to one or more reference profiles, e.g., a non-lung cancer
reference expression profile or an lung cancer reference expression
profile.
[0094] A decrease in expression of one or more of the sequences LC
1-461 oin a test cell population relative to a reference cell
population containing non-lung cancer is indicative that the agent
is therapeutic.
[0095] The test cell population is any cell expressing the lung
cancer-associated genes. For example, the test cell population
contains an epithelial cell. For example, the test cell is
immortalized cell line derived from a lung cancer cell.
[0096] The test agent can be any compound or composition.
[0097] Kits
[0098] The invention also includes an Lkb1 or LC-detection reagent,
e.g., a nucleic acid that specifically binds to or identifies one
or more NSC nucleic acids such as oligonucleotide sequences, which
are complementary to a portion of a Lkb1 or LC nucleic acid or
antibodies which bind to proteins encoded by a Lkb1 or LC nucleic
acid. The reagents are packaged together in the form of a kit. The
reagents are packaged in separate containers, e.g., a nucleic acid
or antibody (either bound to a solid matrix or packaged separately
with reagents for binding them to the matrix), a control reagent
(positive and/or negative), and/or a detectable label. Instructions
(e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay
are included in the kit. The assay format of the kit is a Northern
hybridization or a sandwich ELISA known in the art.
[0099] For example, Lkb1 or LC detection reagent, is immobilized on
a solid matrix such as a porous strip to form at least one Lkb1 or
LC detection site. The measurement or detection region of the
porous strip may include a plurality of sites containing a nucleic
acid. A test strip may also contain sites for negative and/or
positive controls. Alternatively, control sites are located on a
separate strip from the test strip. Optionally, the different
detection sites may contain different amounts of immobilized
nucleic acids, i.e., a higher amount in the first detection site
and lesser amounts in subsequent sites. Upon the addition of test
sample, the number of sites displaying a detectable signal provides
a quantitative indication of the amount of Lkb1 or LC present in
the sample. The detection sites may be configured in any suitably
detectable shape and are typically in the shape of a bar or dot
spanning the width of a teststrip.
[0100] Alternatively, the kit contains a nucleic acid substrate
array comprising one or more nucleic acid sequences. The nucleic
acids on the array specifically identify one or more nucleic acid
sequences represented by LKB1 ore LC 1-461. The expression of 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the sequences
represented by LC 1-461 are identified by virtue if the level of
binding to an array test strip or chip. The substrate array can be
on, e.g., a solid substrate, e.g., a "chip" as described in U.S.
Pat. No.5,744,305.
[0101] Arrays and Pluralities
[0102] The invention also includes a nucleic acid substrate array
comprising one or more nucleic acid sequences. The nucleic acids on
the array specifically corresponds to one or more nucleic acid
sequences represented by LC 1-461. The level expression of 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50 or more of the sequences
represented by LC 1-461 are identified by detecting nucleic acid
binding to the array.
[0103] The invention also includes an isolated plurality (i.e., a
mixture if two or more nucleic acids) of nucleic acid sequences.
The nucleic acid sequence are in a liquid phase or a solid phase,
e.g., immobilized on a solid support such as a nitrocellulose
membrane. The plurality includes one or more of the nucleic acid
sequences represented by LC 1-461. In various embodiments, the
plurality includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40 or 50
or more of the sequences represented by LC 1-461
[0104] Chips
[0105] The DNA chip is a device that is convenient to compare
expression levels of a number of genes at the same time. DNA
chip-based expression profiling can be carried out, for example, by
the method as disclosed in "Microarray Biochip Technology " (Mark
Schena, Eaton Publishing, 2000), etc.
[0106] A DNA chip comprises immobilized high-density probes to
detect a number of genes. Thus, expression levels of many genes can
be estimated at the same time by a single-round analysis. Namely,
the expression profile of a specimen can be determined with a DNA
chip. The DNA chip-based method of the present invention comprises
the following steps of:
[0107] (1) synthesizing cRNAs or cDNAs corresponding to the marker
genes;
[0108] (2) hybridizing the cRNAs or cDNAs with probes for marker
genes; and
[0109] (3) detecting the cRNA or cDNA hybridizing with the probes
and quantifying the amount of mRNA thereof.
[0110] The cRNA refers to RNA transcribed from a template cDNA with
RNA polymerase. A cRNA transcription kit for DNA chip-based
expression profiling is commercially available. With such a kit,
cRNA can be synthesized from T7 promoter-attached cDNA as a
template by using T7 RNA polymerase. On the other hand, by PCR
using random primer, cDNA can be amplified using as a template a
cDNA synthesized from mRNA.
[0111] On the other hand, the DNA chip comprises probes, which have
been spotted thereon, to detect the marker genes of the present
invention. There is no limitation on the number of marker genes
spotted on the DNA chip. For example, it is allowed to select 5% or
more, preferably 20% or more, more preferably 50% or more, still
more preferably 70% or more of the marker genes of the present
invention. Any other genes as well as the marker genes can be
spotted on the DNA chip. For example, a probe for a gene whose
expression level is hardly altered may be spotted on the DNA chip.
Such a gene can be used to normalize assay results when assay
results are intended to be compared between multiple chips or
between different assays.
[0112] A probe is designed for each marker gene selected, and
spotted on a DNA chip. Such a probe may be, for example, an
oligonucleotide comprising 5-50 nucleotide residues. A method for
synthesizing such oligonucleotides on a DNA chip is known to those
skilled in the art. Longer DNAs can be synthesized by PCR or
chemically. A method for spotting long DNA, which is synthesized by
PCR or the like, onto a glass slide is also known to those skilled
in the art. A DNA chip that is obtained by the method as described
above can be used for diagnosing a disease X according to the
present invention.
[0113] The prepared DNA chip is contacted with cRNA, followed by
the detection of hybridization between the probe and cRNA. The cRNA
can be previously labeled with a fluorescent dye. A fluorescent dye
such as Cy3(red) and Cy5 (blue) can be used to label a cRNA. cRNAs
from a subject and a control are labeled with different fluorescent
dyes, respectively. The difference in the expression level between
the two can be estimated based on a difference in the signal
intensity. The signal of fluorescent dye on the DNA chip can be
detected by a scanner and analyzed by using a special program. For
example, the Suite from Affymetrix is a software package for DNA
chip analysis.
[0114] Transgenic Animals
[0115] In another aspect, the present invention includes transgenic
animals containing a heterologous (or exogenous) gene construct.
Specifically, the invention provided a transgenic animal whose
genome contains a mutant K-ras oncogene and at least one Lkb1 null
allele. Optionally, animal is homozygous null for Lkb1. The animal
exhibits accelerated development of a lung tumor. Preferably the
K-ras mutation is a G12 D mutation.
[0116] The preparation of a transgenic mammal requires introducing
a nucleic acid construct that will be used to express a nucleic
acid encoding a light-generating fusion protein into an
undifferentiated cell type, e.g., an embryonic stem (ES) cell. The
ES cell is then injected into a mammalian embryo, where it will
integrate into the developing embryo. The embryo is then implanted
into a foster mother for the duration of gestation.
[0117] Embryonic stem cells are typically selected for their
ability to integrate into and become part of the germ line of a
developing embryo so as to create germ line transmission of the
heterologous gene construct. Thus, any ES cell line that has this
capability is suitable for use herein. One mouse strain that is
typically used for production of ES cells is the 129J strain. A
preferred ES cell line is murine cell line D3 (American Type
Culture Collection catalog no. CRL 1934). The cells are cultured
and prepared for DNA insertion using methods well known in the art,
such as those set forth by Robertson (Robertson, In:
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed., IRL Press, Washington, D.C., 1987.). Insertion
of the nucleic acid construct into the ES cells can be accomplished
using a variety of methods well known in the art including for
example, electroporation, microinjection, and calcium phosphate
treatment.
[0118] The term "transgene" is used herein to describe genetic
material that has been or is about to be artificially inserted into
the genome of a mammalian cell, particularly a mammalian cell of a
living animal. The transgene is used to transform a cell, meaning
that a permanent or transient genetic change, preferably a
permanent genetic change, is induced in a cell following
incorporation of an heterologous nucleic acid, such as DNA. A
permanent genetic change is generally achieved by introduction of
the DNA into the genome of the cell. Vectors for stable integration
include plasmids, retroviruses and other animal viruses, YACs, and
the like. Of interest are transgenic mammals, e.g. cows, pigs,
goats, horses, etc., and particularly rodents, e.g., rats, mice,
etc. Preferably, the transgenic animals are mice.
[0119] Transgenic animals comprise an heterologous nucleic acid
sequence present as an extrachromosomal element or stably
integrated in all or a portion of its cells, especially in germ
cells. Unless otherwise indicated, it will be assumed that a
transgenic animal comprises stable changes to the germline
sequence. During the initial construction of the animal, "chimeras"
or "chimeric animals" are generated, in which only a subset of
cells have the altered genome. Chimeras are primarily used for
breeding purposes in order to generate the desired transgenic
animal. Animals having a heterozygous alteration are generated by
breeding of chimeras. Male and female heterozygotes are typically
bred to generate homozygous animals.
[0120] The heterologous gene is usually either from a different
species than the animal host, or is otherwise altered in its coding
or non-coding sequence. The introduced gene may be a wild-type
gene, naturally occurring polymorphism, or a genetically
manipulated sequence, for example having deletions, substitutions
or insertions in the coding or non-coding regions. Where the
introduced gene is a coding sequence, it is usually operably linked
to a promoter, which may be constitutive or inducible, and other
regulatory sequences required for expression in the host animal. By
"operably linked" is meant that a DNA sequence and a regulatory
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules, e.g., transcriptional
activator proteins, are bound to the regulatory sequence(s). The
transgenic animals of the invention can comprise other genetic
alterations in addition to the presence of the heterologous gene.
For example, the host's genome may be altered to affect the
function of endogenous genes, contain marker genes, or other
genetic alterations such as are described in the Examples.
[0121] Although not necessary to the operability of the invention,
the transgenic animals described herein may comprise alterations to
endogenous genes in addition to (the genetic alterations described
above. For example, the host animals may be either "knockouts"
and/or "knockins" for a target gene(s) as is consistent with the
goals of the invention (e.g., the host animal's endogenous
HIF1.alpha. may be "knocked out" and/or the endogenous
bioluminescent fusion protein "knocked in". Knockouts have a
partial or complete loss of function in one or both alleles of an
endogenous gene of interest. Knockins have an introduced transgene
with altered genetic sequence and/or function from the endogenous
gene. The two may be combined, for example, such that the naturally
occurring gene is disabled, and an altered form introduced.
[0122] In a knockout, preferably the target gene expression is
undetectable or insignificant. For example, a knock-out of an gene
means that function of the gene has been substantially decreased so
that expression is not detectable or only present at insignificant
levels. This may be achieved by a variety of mechanisms, including
introduction of a disruption of the coding sequence, e.g.,
insertion of one or more stop codons, insertion of a DNA fragment,
etc., deletion of coding sequence, substitution of stop codons for
coding sequence, etc. In some cases the exogenous transgene
sequences are ultimately deleted from the genome, leaving a net
change to the native sequence. Different approaches may be used to
achieve the "knock-out". A chromosomal deletion of all or part of
the native gene may be induced, including deletions of the
non-coding regions, particularly the promoter region, 3' regulatory
sequences, enhancers, or deletions of gene that activate expression
of genes. A functional knock-out may also be achieved by the
introduction of an anti-sense construct that blocks expression of
the native genes (See, e.g., Li and Cohen (1996) Cell 85:319-329).
"Knock-outs" also include conditional knock-outs, for example where
alteration of the target gene occurs upon exposure of the animal to
a substance that promotes target gene alteration, introduction of
an enzyme that promotes recombination at the target gene site (e.g.
Cre in the Cre-lox system), or other method for directing the
target gene alteration postnatally.
[0123] A "knockin" of a target gene means an alteration in a host
cell genome that results in altered expression or function of a
native target gene. Increased (including ectopic) or decreased
expression may be achieved by introduction of an additional copy of
the target gene, or by operatively inserting a regulatory sequence
that provides for enhanced expression of an endogenous copy of the
target gene. These changes may be constitutive or conditional, i.e.
dependent on the presence of an activator or represser. The use of
knockin technology may be combined with production of exogenous
sequences to produce the transgenic animals of the invention.
[0124] A selection marker can be any nucleic acid sequence that is
detectable and/or assayable. Examples of selection markers include
positive selection markers and negative selection markers. Positive
selection markers include drug resistance genes; e.g., neomycin
resistance genes or hygromycin resistance genes, or
beta-galactosidase genes. Negative selection markers, e.g.,
thymidine kinase gene, diphtheria toxin gene and ganciclovir are
useful in the heterologous gene construct in order to eliminate
embryonic stem (ES) cells that do not undergo homologous
recombination. The selection marker gene is usually operably linked
to its own promoter or to another strong promoter from any source
that will be active or can easily be activated in the cell into
which it is inserted; however, the marker gene need not have its
own promoter attached as it may be transcribed using the promoter
of the light-generating fusion protein gene to be suppressed. In
addition, the marker gene will normally have a polyA sequence
attached to the 3'
[0125] "Enhancer elements" include nucleic acid sequences that are
bound by polypeptides associated with transcription, and are
usually in cis with the nucleic acid encoding a light-generating
fusion protein. Examples of enhancer elements include cyclic AMP
response elements (CRE), serum response elements (SRE), nuclear
factor B (NF-.kappa.B), activator protein 1 (AP-1), serum response
factor (SRF), and p53 binding sites. These enhancer elements may
further include a TATA box.
[0126] The heterologous gene construct may be constitutively
expressed in the transgenic mammal. The gene construct may
expressed in specific tissues, e.g., the construct is under the
control of a tissue-specific promoter.
EXAMPLE 1
General Methods
Mouse Colony and Tumor Analysis:
[0127] All mice were housed and treated in accordance with
protocols approved by the institutional care and use committees for
animal research at the Dana-Farber Cancer Institute and the
University of North Carolina. The LSL-K-ras.sup.G12D mice (K-ras)
were provided in a mixed genetic background by Dr. Tyler Jacks. All
cohorts in Table I were of a similar, mixed genetic background
(.about.75% C57B1/6, .about.25% FVB/n and 129SvEv). Over 500 mice
were analyzed in a standard manner for Table I, but CRE-treated
littermates of less informative genotypes (e.g. compound
heterozygotes, etc.) and animals treated with empty adenovirus are
not shown in the interest of brevity. In all cases, heterozygote
mice showed tumor-prone phenotypes intermediate to the wild-type
and homozygous mutant animals. For CRE-expression, 5.times.10.sup.6
pfu adenoviral-CRE (purchased from University of Iowa adenoviral
core) was administered intra-nasally as previously
described.sup.19,23.
TABLE-US-00004 TABLE I Comparison of lung cancer cohorts: Median
Squamous # survival Tumor or Mixed Genotype treated (wks)#
Mult.& Histology Metastasis Comments KRas 26 24 Med. 0 of 16 0
of 19 See also.sup.19,23-25. Lkb1.sup.L/- or .sup.L/L 15 >40 NA
NA NA No tumors observed p53.sup.L/L 16 >40 NA 0 of 1 NA See
also.sup.28. p16.sup.INK4a-/- 15 29 Low 0 of 5 NA High frequency of
fatal p53.sup.L/L pulmonary hemorrhage. KRas 19 24 Med. 0 of 12 3
of 15 p16.sup.INK4a-/- (20%) KRas 17 14 High 0 of 9 4 of 9 Compare
with.sup.26. See also.sup.24,37. p53.sup.L/L (44%) KRas 26 22 High
0 of 11 0 of 11 Compare with.sup.26. Ink4a/Arf-/- KRas 27 19 High 0
of 18 7 of 22 Lkb1.sup.L/+ or +/- (32%) KRas Lkb1.sup.L/L or L/- 56
9 High 15 of 27 27 of 44 2 of 27 mice also (56%) (61%) demonstrated
Large Cell histology #Median Latency shown is after Adeno-CRE
treatment at 5-6 weeks of age, estimated by Kaplan Meier analysis.
&Tumor multiplicity: Low < 3 per lung section, Medium = 3-10
per lung section, High > 10 per lung section
Histology and Immunohistochemistry
[0128] Mice were sacrificed and the left lungs were dissected. The
right lung and mediastinal structures were inflated with neutral
buffered 10% formalin for 10 minutes and fixed in 10% formalin
overnight at room temperature. Fixed tissues were embedded in
paraffin, sectioned at 5 m, and Hematoxylin and eosin (H&E)
stained (Department of Pathology in Brigham and Women's Hospital).
Immunohistochemical analyses were performed as described.sup.36,37.
The antibodies used were: CCSP (sc-9772, Santa Cruz), SPC (AB3786,
Chemicon), pan-Keratin (Z0622, Dako), p63 (ab3239, Abcam), p-AMPK
(2535, Cell signaling), Phospho-Acetyl-CoA Carboxylase (Ser79)
(3661, Cell Signaling), and VEGFc (2712, Cell signaling).
In Vitro Analyses:
[0129] Murine Embryo Fibroblasts (MEFs) cultured from day 13.5
embryos were serially passaged in DMEM (GIBCO)+10% Fetal Bovine
Serum (Sigma), 50M -mercaptoethanol (Sigma), and pen/strep
antibiotic (Invitrogen) on a 3T9 protocol at 21% O.sub.2. For Lkb1
replacement, late passage (P18) Lkb1.sup.-/- MEFs or A549 cells
(ATCC) were transduced with either pBABE-puro, or pBABE-Lkb1
(wild-type), or pBABE-Lkb1.sup.K78I, selected with 1 g/ml puromycin
for 4 days, and then harvested for RNA and protein after 4 days
without selection. For conditional excision of Lkb1, Lkb1.sup.L/-
MEFs were treated with .sup.10.sup.10 PFU/mL of Adenoviral-CRE or
Adenoviral-Empty for 24 hours. Cells were then passaged according
to 3T9 protocol for an additional 15 days.
Western Blotting and mRNA Analysis:
[0130] Western blot assays were performed as previously
described.sup.38 with antibodies against p16.sup.INK4a (M-156,
Santa Cruz), Arf (ab-80, Abcam), Actin (C-1, Santa Cruz), Lkb1
(Rabbit polyclonal antibody, 1:5000 dilution), tubulin (clone DM
1A, Sigma-Aldrich Co), p63 (4892, Cell signaling). Expression of
mRNA was analyzed by quantitative TaqMan real-time PCR as
previously described with some modifications.sup.39. Reactions were
carried out using cDNA equivalent to 80 ng RNA and performed in
triplicate for each sample. 18S rRNA was used as a loading control
for all reactions. Primer set for 18S (Hs99999901_s1) was purchased
from Applied Biosystems; p16.sup.INK4a and Arf primers were
generated as previously described.sup.39.
Soft Agar Assay
[0131] Parental A549 cell and A549 stable cell lines with
expression of wt LKB1 and LKB1 K78I cells EGFR-expressing NIH-3T3
cells were suspended in a top layer of RPMI1640 containing 10% FBS
and 0.4% Select agar (Gibco/Invitrogen) at 5,000 cells per well in
triplicate in 6-well plates and plated on a bottom layer of
RPMI1640 containing 10% FBS and 1% Select agar. After 2-wk culture,
cells were stained with 0.5 ml of Crystal Violet for 1 hour. The
colonies were then counted in triplicate wells from ten fields
photographed with a 10.times. objective.
In Vivo Lung Seeding Assay of NSCLC Cell Lines
[0132] Parental A549 cell and A549 stable cell lines with
expression of wt LKB1 and LKB1 K78I cells were injected into SCID
mice intravenously via tail veins. After 8-week of inoculation, the
mice were sacrificed and the lungs were dissected for both gross
inspection and histology analysis.
Statistical Analysis
[0133] Tumor-free survival and comparisons of tumor numbers and
colonies in soft agar were analyzed using Graphpad Prism4.
Statistical analysis were performed using nonparametric
Mann-Whitney test. Comparisons of mRNA levels were made using the
unpaired student t-test. All error bars indicate .sup.+/- standard
error of the mean (SEM).
Microarray Analysis
[0134] Total RNA was extracted, amplified and labeled as described
(Giovanni et al) and hybridized to Mouse430A2 GeneChip Arrays
(Affymetrix, Santa Clara, Calif.) representing 22690 unique
transcripts. Probe level intensity CEL files were preprocessed
using the Robust Multichip Average.sup.40-42 as implemented in
Bioconductor (http://www.bioconductor.org/). Gene expression data
were filtered using low stringency, pre-defined criteria: probe set
intensity (>32 in all samples) and dynamic variation (more than
2-fold over the entire sample set). After filtering, 9239 probe
sets remained upon which unsupervised 2-way hierarchical clustering
was performed (We can put a reference here if we have the space:
Eisen M B, Spellman P T, Brown P O, Botstein D: Cluster analysis
and display of genome-wide expression patterns. Proc Natl Acad Sci
USA 1998, 95(25):14863-14868). Multiple probe sets that presented
the same genes were collapsed by taking the median value for that
gene per array yielding 6871 unique genes, upon which 2-way
hierarchical clustering was performed (FIG. 3). Excerpted clusters
are shown in FIG. 3. 1).
EXAMPLE 2
Analysis of Tumor Suppressor Function of LKB1 in Context with
Activation of K-RAS in an In Vivo Lung Cancer Mice Model
[0135] To discern the relationship among tumor suppressor lesions
in lung carcinogenesis, a conditionally activatable Lox-Stop-Lox
K-ras.sup.G12D (hereafter K-ras) allele.sup.19 and four conditional
(L/L) or germline null (-/-) alleles: Lkb1.sup.L/L (Ref.),
p53.sup.L/L (Ref..sup.20), Ink4a/Arf -/- (Ref..sup.21), and
p16.sup.INK4a-/- (Ref..sup.22) were intercrossed. Somatic,
lung-specific K-ras activation and/or tumor suppressor inactivation
was accomplished through inoculation of young adult mice (5-6 weeks
of age) with adenoviral-CRE by inhalation as reported.sup.19,23.
This method transduced a small percentage of pulmonary cells,
predominantly of the medium airways. Animals were sacrificed at
scheduled time points or for morbidity, and comprehensive autopsies
performed to determine tumor number, histology, invasion and
metastasis (See, Table I). Control animals of each genotype were
treated with empty adenovirus, but no tumors were noted in any
cohort in the absence of transient CRE expression.
[0136] Isolated K-ras activation led to tumors with high
multiplicity but relatively long latency and low aggressiveness. In
contrast, concomitant p16.sup.INK4a and p53 inactivation in animals
lacking the activatable K-ras allele produced infrequent, but
highly lethal, hemorrhagic tumors; suggesting that K-ras mutation
initiates tumorigenesis while p16.sup.INK4a and p53 constrain tumor
progression (See, Table I). Potent cooperation was noted between
somatic K-ras activation and somatic loss of p53. In contrast to
the overexpression setting, however, only modest cooperation was
noted between single copy K-ras mutation and p16.sup.INK4a
inactivation alone or in combination with Arf (Ink4a/Arf-/-)
inactivation (See, Table I). These data demonstrate that p16 and
p53 combine to suppress pulmonary tumorigenesis, but highlight a
more prominent tumor suppressor role for p53 than p16.sup.INK4a or
Arf in response to single-copy K-ras mutation in the lung.
[0137] Surprisingly, however, the strongest genetic interaction
between any two alleles was that seen when K-ras mutation was
combined with homozygous Lkb1 inactivation (See, Table I and FIG.
1a). The median survival for K-ras Lkb1.sup.L/L or L/- mice was 9
weeks after Cre-inoculation compared with a 14 week median survival
seen in K-ras p53.sup.L/L mice, the next most tumor-prone cohort.
Significant cooperation was also noted between K-ras activation and
heterozygous Lkb1 mutation; although loss of the wild-type allele
was not seen in the tumors of heterozygous mice Inactivation of
Lkb1 alone was not sufficient for pulmonary neoplasia, as
Lkb1.sup.L/L or L/- mice did not develop tumors after Cre-treatment
in the absence of K-ras activation (See, Table I and FIG. 1a). In
accord with their more rapid clinical progression, mice harboring
simultaneous K-ras mutation and Lkb1 loss showed an increased
frequency of metastasis. Additionally, tumors from these mice
showed an enhanced spectrum of tumor histology relative to the
other genotypes analyzed (See, Table I). These data demonstrate
potent in vivo cooperation between K-ras activation and Lkb1 loss
in lung tumorigenesis.
EXAMPLE 3
Evaluation of the Cooperation Between K-RAS Activation and LKB1
Loss in Lung Tumorigenesis
[0138] Time course experiments were performed to better delineate
the cooperation between K-ras activation and Lkb1 loss in lung
tumorigenesis. (FIGS. 1b-d). Two weeks after treatment with
Adeno-CRE, there were few discernable lesions in the lungs of K-ras
mice, while K-ras Lkb1.sup.L/L or L/- mice harbored a significantly
increased tumor burden, which was pronounced by 4 weeks (FIGS. 1b,
c). In addition to facilitating the formation of pulmonary lesions
after K-ras activation, Lkb1 loss clearly enhanced progression: at
eight weeks post-CRE treatment, large tumors (>3 mm) were seen
with high frequency in K-ras Lkb.sup.L/L or L/- or K-ras
Lkb1.sup.+/- mice, but not in animals harboring K-ras activation
alone (FIG. 1d). Even after 28 weeks of observation, tumors greater
than 3 mm were not seen in K-ras mice. These results indicate that
Lkb1 efficiently constrains lung tumor initiation within days of
somatic K-ras activation, as well as tumor progression at later
time points.
[0139] Local invasion or metastasis was not observed in in K-ras
mice. In contrast, lung tumors from K-ras Lkb1.sup.L/L or L/- mice
displayed local invasion into the pleura as well as metastases to
lymph nodes and bone. Regional lymph node metastasis was observed
in approximately one fourth of K-ras Lkb.sup.+/- or L/+ mice, and
in the majority of K-ras Lkb.sup.L/L or L/- mice (FIG. 1e, Table
1). Metastasis to the axial skeleton of four K-ras Lkb1.sup.L/L or
L//- mice and one K-ras Lkb1.sup.+/mouse (Table 1) were noted.
These results suggest reduced Lkb1 gene dosage facilitates
metastasis in K-ras-induced lung cancers.
[0140] Analysis of tumors from K-ras Lkb1.sup.L/L or L/- mice
revealed distinct differences in tumor histopathology compared to
tumors in the other cohorts listed in Table I. Consistent with
prior reports.sup.19,24-28, all tumors from K-ras mice with or
without Ink4a/Arf or p53 inactivation were of the characteristic
adenocarcinoma subtype, as were tumors from
p16.sup.INK4a-/-p53.sup.L/L mice. In contrast, the lungs 17 of 27
Adeno-CRE treated K-ras Lkb1.sup.L/L or L/- mice demonstrated a
histology other than pure adenocarcinoma: 15 of 27 lungs harbored
squamous cell carcinoma (SCC) or adenosquamous tumors (mixed) and 2
of 27 lungs showed large cell carcinoma (LCC) (FIG. 2a).
Importantly, even though K-ras Lkb1.sup.L/L or L/- (or +/-)mice
demonstrated decreased survival and increased metastasis (Table I),
the metastatic tumors did not demonstrate squamous histology. These
data indicate that Lkb1 modulates lung tumor differentiation.
EXAMPLE 4
LKB1 Modulates Lung Tumor Differentiation
[0141] Analysis of tumors from K-ras Lkb1.sup.L/L or L/- mice
revealed distinct differences in tumor histopathology compared to
tumors in the other cohorts listed in Table I. Consistent with
prior reports.sup.19, 24-28, all tumors from K-ras mice with or
without Ink4a/Arf or p53 inactivation were of the characteristic
adenocarcinoma subtype, as were tumors from
p16.sup.INK4a-/-p53.sup.L/L mice. In contrast, the lungs 17 of 27
Adeno-CRE treated K-ras Lkb1.sup.L/L or L/- mice demonstrated a
histology other than pure adenocarcinoma: 15 of 27 lungs harbored
squamous cell carcinoma (SCC) or adenosquamous tumors (mixed) and 2
of 27 lungs showed large cell carcinoma (LCC) (FIG. 2a).
Importantly, even though K-ras Lkb1.sup.L/L or L/- (or +/-)mice
demonstrated decreased survival and increased metastasis (Table I),
the metastatic tumors did not demonstrate squamous histology. These
data indicate that Lkb1 modulates lung tumor differentiation.
EXAMPLE 5
Protein Expression Analysis
[0142] Protein expression analyses confirmed these differences in
tumor histology. Squamous tumors from K-ras Lkb1.sup.L/L or L/-
mice did not stain for pro-surfactant protein C (SP-C), a marker of
type II pneumocytes expressed in lung adenocarcinoma, but did
demonstrate strong staining for pan-keratin and p63, markers of SCC
(FIG. 2b). In contrast, expression of pan-keratin and p63 was low
or absent in adenocarcinoma. Western blot analysis of tumors from
K-ras Lkb1 mice showed that p63 was only expressed in squamous
tumors lacking Lkb1 expression (FIG. 2c). While some
adenocarcinomas from K-ras Lkb1.sup.L/L or L/- mice demonstrated
very low expression of Lkb1, immunostaining suggested that this was
stromally derived in at least some of these tumors (data not
shown). Therefore, complete absence of Lkb1 expression appeared
consistent with each of the four observed histologies:
adenocarcinoma, squamous carcinoma, large cell carcinoma, and mixed
tumors, whereas retained Lkb1 expression was only noted in
adenocarcinomas.
EXAMPLE 6
RNA Expression Profiling
[0143] To study the mechanism whereby loss of Lkb1 modifies the
histological versatility and malignancy of lung cancer, RNA
expression profiling was performed of lung tumors with or without
Lkb1 loss (FIG. 3). Thirteen tumors from 10 K-ras mice of the
indicated histologies and Lkb1 genotypes were analyzed using
Affymetrix arrays. Expression data were normalized, filtered, and
collapsed as described in the methods, and unsupervised
hierarchical clustering was performed using 6,871 unique and
dynamic transcripts. This unbiased analysis revealed three discrete
groups of tumors with regard to gene expression. The most distinct
group (E-G) was comprised of squamous or adenosquamous (mixed)
tumors from K-ras Lkb1.sup.L/L or L/-mice. These tumors showed a
marked increase in the expression of genes (e.g. p63, Krt5,
desmoplakin, PTH-like peptide, Sox2; Cluster A) known to be
overexpressed in human squamous lung cancer compared to
adenocarcinoma.sup.29-31. The full 135 gene list of the squamous
cluster (r>0.85) appears in Supp. Data File 2. These tumors also
demonstrated a sharply reduced expression of the Lkb1 (Stk11)
transcript (Cluster B). Therefore, loss of Lkb1 expression is
associated with tumors harboring a transcriptional profile that is
highly similar to that of human squamous lung cancers.
[0144] Based on gene expression, the adenocarcinomas further
clustered into two groups that were specified by Lkb1 expression.
Tumors A-C from K-ras Lkb1.sup.+/+ mice showed high expression of
Lkb1 and several other transcripts associated with carbohydrate
metabolism (e.g. Acetyl-CoA acyltransferase and lactate
dehydrogenase; Cluster B). DAVID analysis.sup.32 suggested that the
235 transcripts that strongly correlated (r>0.86,) with Lkb1
expression were significantly enriched for genes involved in ATP
synthesis as well as metabolism of fatty acids and carbohydrates,
consistent with the known role of Lkb1 in regulating the
nutrient-sensing AMPK pathway.sup.33. Correspondingly, these tumors
harbored increased expression of the active, phosphorylated forms
of AMPK and an AMPK target (Acetyl Co-A carboxylase or ACC)
compared to tumors lacking Lkb1. The other group of
adenocarcinomas, from K-ras Lkb1.sup.L/+ or L/-mice (tumors D, H
and I), were characterized by reduced, but not necessarily absent,
expression of Lkb1. In accord with the increased frequency
metastasis of this group (Table I), these tumors also demonstrated
increased expression of several genes associated with angiogenesis
and/or metastasis. For example, the 461 gene cluster (r>0.65,
Supp. Data File 4) surrounding Cluster C contained Nedd9, Vegf-c,
lysl oxidases (Lox, Loxl1 and Loxl3), Pdgf's (A, B and C), Pdgf
receptor, and MMP2. Increased Vegf-c expression was confirmed by
immunohistochemical analysis (Supp. FIG. 2). In particular, several
of the pro-metastasis transcripts in Cluster C are targets of
hypoxia-inducible factors (HIF), consistent with the hypothesis
that LKB1 loss activates HIF signaling.sup.34. Moreover, as
previously reported.sup.27, these metastatic adenocarcinomas
appeared more fibrotic by trichrome staining, consistent with
increased expression of extracellular matrix proteins (e.g.
Fibronectin and Vimentin) in this cluster. Therefore, Lkb1 appears
to activate metabolic regulators and repress metastasis genes in
K-ras induced lung adenocarcinomas.
EXAMPLE 7
In Vitro Analysis
[0145] In an effort to further delineate these disparate
anti-cancer functions of Lkb1, in vitro analyses was performed.
Previous reports have suggested that loss of Lkb1 and p16.sup.INK4a
are mutually exclusive in human lung adenocarcinoma.sup.3, and that
loss of Lkb1 attenuates expression of p16.sup.INK4a and Arf protein
in response to oncogenic Ras in murine embryo fibroblasts.sup.15.
Cultured murine embryo fibroblasts (MEFs) was used to examine the
relationship between Lkb1 and expression of the Ink4a/Arf tumor
suppressor locus. Upon culture, wild-type MEFs demonstrated a rapid
increase in Lkb1 protein expression, which preceded the
culture-induced expression of p16.sup.INK4a and Arf (FIG. 4a).
Inactivation of Lkb1 expression through CRE expression in
Lkb1.sup.L/- MEFs substantially reduced the passage-dependent
increase in p16.sup.INK4a and Arf protein (FIG. 4b) and mRNA (FIG.
4c). This effect was likely due to changes in transcription, as no
difference in mRNA decay rate was seen for the Arf transcript
between Lkb1.sup.L/- and Lkb1.sup./-MEFs (not shown).
Re-introduction of wild-type Lkb1 or Lkb1.sup.K78I, a kinase-dead
mutant, into wild-type or Lkb1.sup.-/- MEFs did not, however,
result in a further increased p16.sup.INK4a or Arf expression (FIG.
4d). Therefore, Lkb1 activity cooperates with culture-induced
stresses to potentiate Ink4a/Arf transcription in MEFs, but Lkb1
appears unable to enhance Ink4a/Arf expression after
culture-induced activation of the locus. As K-ras Ink4a/Arf-/- mice
show enhanced tumor progression compared to K-ras mice (Table I),
transcriptional activation of Ink4a/Arf expression may in part
explain the anti-progression effects of Lkb1 in mice with somatic
K-ras activation. As K-ras Ink4a/Arf-/-and K-ras p53.sup.L/L mice
are less tumor-prone than K-ras Lkb1.sup.L/L or L/- mice and do not
develop squamous tumors, however, clearly some effects of Lkb1 on
tumor progression and differentiation are independent of
p16.sup.INK4a and Arf-p53.
[0146] To investigate the INK4a/ARF-independent effects of LKB1 on
metastasis, A549 cells, a human lung carcinoma cell line, that
harbor a K-RAS activating mutation was and lack INK4a/ARF and LKB1
expression was used. A549 cells stably expressing equivalent LKB1
or LKB1.sup.K78I were established (FIG. 5a) through transduction
with amphotropic retrovirus. Although the in vitro proliferation
was not dramatically different among the lines regardless of LKB1
expression (data not shown), the A549-wt LKB1 cells demonstrated a
profound inability to form colonies in soft agar (FIG. 5b, c) or
metastasize to the lung after tail-vein injection in SCID mice
(FIG. 5d). The suppression of metastasis by LKB1 required its
kinase activity, as A549-LKB1.sup.K78I cells demonstrated soft agar
growth and metastasis comparable to the parental A549 line (FIG.
5b-d). These results, are consistent with the genetic data (Table
I) as well as a prior report.sup.13, suggest that LKB1 kinase
activity represses lung cancer metastasis independent of INK4a/ARF
function.
EXAMPLE 8
Genetical Dissection and Comparison of the Role of LKB1 in the
Initiation and Progression of K-RAS, EGFR, and BRAF Mutant-Driven
Lung Tumorigenesis
[0147] Multiple cytogenetic and molecular studies have shown that
there are many genetic changes in human lung carcinomas resulting
in the inactivation of tumor suppressor genes and
mutation/activation of oncogenes.sup.61,62. Thus, it is virtually
certain that in lung carcinomas (or other tumor types) that K-ras,
EGFR, or BRAF mutants are not the sole mutation that occurs.
Furthermore, these additional genetic alterations likely play
important roles in the initiation and progression of tumorigenesis
as well as sensitivities of the tumors to treatments. LKB1
mutations appear to be relatively common (.about.25%) in NSCLC and
thus, are likely to occur concurrently with K-ras, EGFR and BRAF
mutations. A recent study suggested that LKB1 mutations are
commonly found with K-RAS mutations in human lung cancers. It has
been recently shown that Lkb1 cooperates with K-ras mutant to
shorten the latency of lung tumorigenesis in the lox-stop-lox K-ras
knock-in model (See, examples above). These preliminary findings
will be confirmed in the tet-op K-ras G12D mouse model. This is
important, as the lox-stop-lox K-ras model has a different level
(endogenous level) of K-ras expression than the tet-op K-ras model
(overexpression), and this might have a differential effect on
tumorigenesis. Furthermore, the K-RAS locus is commonly amplified
in human NSCLCs, thus suggesting K-RAS is over-expressed in this
tumor type. Similarly, it will be determined whether Lkb1
deficiency will synergize with EGFR and BRAF mutations in lung
tumorigenesis and if the tet-op-K-ras cohorts can serve as a
positive control.
[0148] The inventors have generated and characterized the
doxycycline inducible tet-op-K-ras, tet-op-EGFR, tet-op-BRAF mutant
alleles Upon induced expression of K-ras, EGFR or BRAF mutant in
the lung epithelial cells, these mice develop lung adenocarcinomas
with a latency of 12 to 16 weeks (See. Examples above) To
specifically delete the Lkb1 conditional alleles in the same lung
cells that express mutant K-ras, EGFR, or BRAF, a recently
generated SPC-Cre-ER.sup.T2 allele will be employed. The SPC
promoter will direct the expression of the Cre-recombinase/estrogen
receptor (Cre-ER.sup.T2) mutant fusion protein specifically in the
lung type II pneumocytes epithelial cells (in the same cell
compartment that are expressing the K-ras/EGFR/BRAF mutants. The
administration of tamoxifen will activate the Cre recombinase
activity. Using these unique alleles, the following 12 cohorts of
mice (30 mice in each cohort) will be generated:
[0149] 1. Lkb1 L/L, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(no doxy and no tamoxifen: control)
[0150] 2. Lkb1 L/L, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(no doxy and tamoxifen: control)
[0151] 3. Lkb1 L/L, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(doxy and no tamoxifen: control)
[0152] 4. Lkb1 L/L, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(doxy and tamoxifen: experimental cohort)
[0153] 1a. Lkb1 L/L, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (no doxy and no tamoxifen: control)
[0154] 2a. Lkb1 L/L, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (no doxy and tamoxifen: control)
[0155] 3a. Lkb1 L/L, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and no tamoxifen: control)
[0156] 4a. Lkb1 L/L, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and tamoxifen: experimental cohort)
[0157] 1b. Lkb1 L/L tet-op-BRAF V600E, CCSP-rtTA, SPC-Cre-ER.sup.T2
(no doxy and no tamoxifen: control)
[0158] 2b. Lkb1 L/L, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (no doxy and tamoxifen: control)
[0159] 3b. Lkb1 L/L, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and no tamoxifen: control)
[0160] 4b. Lkb1 L/L, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and tamoxifen: experimental cohort)
[0161] At 3 weeks of age, mice from cohorts 2, 4, 2a, 4a, 2b, and
4b will be injected intraperitionally with 4 mg of tamoxifen over a
5-day period to activate the Cre-recombinase activity in the type
II pneumocytes and delete the Lkb1 alleles as described above.
[0162] Mice from cohorts 3, 3a, 3b, 4, 4a, and 4b will be
administered doxycycline continuously through their drinking water
to activate the expression of mutant K-ras, EGFR, and BRAF. During
these experiments, mice will be inspected daily Monday through
Friday. Any mouse that show evidence of respiratory distress or
illness will be euthanized (along with age-matched controls from
the other cohorts) and subjected to analysis. To determine if the
loss of Lkb1 affects overall survival of these mice, 10 mice from
each cohort will be followed until it is time for them to be
euthanized and a Kaplan-Meier survival curve will be generated. Of
note, the person deciding which mice should be euthanized will be
blinded regarding their specific genotypes. In addition to
measuring effects on survival, we will also determine if loss of
Lkb1 affects K-ras, EGFR, and BRAF mutant-induced tumor burden. For
these studies, three mice from each cohort will be sacrificed by
CO.sub.2 inhalation at 3, 6, 9, 12, 15, and 18 weeks after
continuous administration for the lung tumor burden analyses and
assessment of Lkb1 signaling. At the time of sacrifice, each organ
from the mouse, including the heart, bones, liver, spleen, brain,
kidney, and adrenal glands will be microscopically inspected for
visible signs of metastases. Additionally, these organs will be
fixed in formalin for subsequent detailed histological analyses.
The left lung will be removed and snap-frozen in liquid nitrogen
for protein, DNA, and RNA analyses. When macroscopic tumors are
evident, they will be dissected out of the tumor to enable tumor
specific molecular studies. The right lung (with the left main
bronchus ligated with a suture) will then be inflated at 25 cm
H.sub.2O pressure with 10% buffered formalin for 10 minutes via of
an intratracheal catheter. The right lung will then be removed and
fixed in 10% buffered formalin for 24 hours before embedding in
paraffin. Serial mid-sagittal sections (5 .mu.M thickness) will be
obtained for histological analysis.
[0163] Data on the histological types, lung tumor multiplicity as
well as the grade of the tumor will be tabulated on each lung.
Paraffin-embedded lung is cut in to 4 um sections, stained with
H&E and tumor-quantified using Bioquant software; specifically,
the size and number of specific tumors are determined. Using this
software, lung sections are captured in a 2.times. field and a line
is drawn around the periphery of the lung. The software then
calculates the area of the tumor (mm.sup.2). The same tools are
used to determine the size of each tumor in the lung (total tumor
area/total lung area.times.100). These measurements will be
compared at different time points after Cre activation. Experiments
will be performed at all six time points (5 mice of each genotype
per time point).
EXAMPLE 9
Assessment of LKB1 Signaling in Lung Tumors
[0164] Lung tumors will be evaluated by western blot and IHC
analyses to probe the activity of the known LKB1 signaling pathway.
When tumors arise, they will be evaluated by IHC, using specific
antibodies against LKB1 to ensure the loss of LKB1 and antibodies
against P-S6 Kinase and P-S6 as readouts for activated mTOR
signaling. IHC will be performed Drs. Padera, using previously
described methods. Colored signal will be generated with a combined
secondary antibody-peroxidase kit (Envision+; DAKO, Carpinteria,
Calif.), according to the manufacturer's instructions. The slides
will be evaluated the pathologists using light microscopy to select
and analyze the 100.times.-magnification field with the highest
concentration of positive-staining tumor cells with a clear
background. Positive cases are defined by the presence of membrane,
cytoplasmic, or nuclear staining, depending on the antigen. The
grading of antigen expression is performed independently by at
least two of the pathologists. The presence of positive staining in
in situ lesions is noted. Scoring of invasive tumor is performed by
two methods as previously described.sup.69. In the first method,
scores of 0-3 are assigned according to the percentage of positive
tumor cells (0=0%; 1=<25%; 2=25-50%; 3=>50%) and the
intensity of staining (0=0; 1=1+; 2=2+; 3=3+). The two scores are
multiplied to give an overall score of 0-9, of which 0-2 is
considered negative, 3-6 moderate, and 7-9 strong staining. The
second method of scoring examines the intensity of staining of
tumor cells compared to adjacent normal cells. Here, the intensity
of staining in greater than 50% of the tumor is compared to the
intensity of staining in adjacent normal glands on the same slide
and scored on a scale of 1-3, where 1=tumor staining less than
normal, 2=tumor equivalent to normal and 3=tumor greater than
normal. A score of 3 is considered as overexpression. The choice of
the scoring method will depend on the antigen. Controls for the
specificity of an antibody used for IHC will include tumor derived
from xenografts that are known to express or not express a given
protein as determined by western blot analysis. Data will be
collected on the intensity and frequency of staining using
immunohistochemistry and a semi-quantitative scoring system will be
used as described above. Furthermore, tumors will be categorized as
positive or negative. For example, for the first scoring method
described above, scores 0-2 and 3-6 would be grouped together as
"negative," and scores 7-9 would be "positive." The most
appropriate groupings will be determined empirically for each
antigen. The list of antibodies suitable for use in this analyses
are listed in Table 2.
TABLE-US-00005 TABLE 2 Antibody epitope Company Cat
Phospho-AMPKalpha (Thr172) (40H9) Rabbit mAb pT172 Cell Signaling
2535S Phospho-Acetyl-CoA Carboxylase (Ser79) Rabbit pS79 Cell
Signaling 3661S polyclonal Ab Phospho-S6 Ribosomal Protein
(Ser235/236) pS235/236 Cell Signaling 2211S Rabbit polyclonal Ab
Phospho-Akt (Ser473) (736E11) Rabbit mAb pS473 Cell Signaling 3787
Phospho-p44/42 MAP Kinase (Thr202/Tyr204) pT202/pY204 Ceo Signaling
4376 (20G11) Rabbit mAb Phospho-EGF Receptor (Tyr1068) Rabbit
pY1068 Cell Signaling 2234 polyclonal Ab Akt1 isoform specific
(2H10) Mouse mAb NA Cell Signaling 4057 SPC amino acids 1-33 of
human Chemicon 07-647 Pro-Surfactant Protein C CC10 (T-18) epitope
near the C-terminus Santa Cruz Biotech sc-9772 of mouse CC10 Raf-B
(H-145) N/A Santa Cruz Biotech SC-9002 VEGF (Ab-7) Clone VG1 N/A
Neomarker/Labvision Ab-7 Anti-LKB1, clone 5c10 1-433 of human LKB1
Upstate/Millipore 05-832
[0165] Protein, DNA and RNA will be harvested from any found tumors
in the left lungs. With these reagents, the level of mutated kinase
expression and autophosphorylation will be analyzed as well as the
activation status of the various downstream pathways via western
blot and expression analyses. The loss of Lkb1 in the tumors from
the various cohorts with genotyping and western analyses will be
assessed. All tumors found will also be cultured in an attempt to
establish cell lines. These cell lines will greatly facilitate the
in vitro signal transduction and drug sensitivity studies.
[0166] To determine whether or not the temporal sequence of genetic
alterations play differential and distinct roles in the initiation
and progression of lung tumorigenesis, the same experiment as
outlined above with the same identical cohorts of mice above (30
mice in each cohort), but switch the sequence of Lkb1 inactivation
and K-ras/EGFR/BRAF mutant induction. First doxycycline will be
administered continuously through the drinking water to all the
mice in the cohort. After 8 weeks on doxycycline, two treated mice
from each cohort will be sacrificed and their lungs harvested for
immunohistochemical staining and western analyses to confirm the
expression of the K-ras, EGFR, or BRAF mutant expression and the
activation of the downstream pathways. After the confirmation, the
remaining 18 mice in each cohort will be injected intraperitionally
with 4 mg of tamoxifen over a 5-day period to activate the
Cre-recombinase activity in the nucleus to delete the Lkb1 alleles.
Three mice from each cohort will be sacrificed by CO.sub.2
inhalation at 3, 6. 9, 12, 15, and 18 weeks after tamoxifen
treatment for analysis. Identical analyses on these mice as
outlined for the initial reverse temporal sequence will be
performed.
[0167] The results from both sets of experiments will be compared
directly, in particular, the histological types, lung tumor
multiplicity as well as the grade of the tumor. [0168] Lastly, the
following mouse cohorts (30 mice per cohort) will be generated:
[0169] 1. Lkb1 L/+, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(no doxy and no tamoxifen: control)
[0170] 2. Lkb1 L/+, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(no doxy and tamoxifen: control)
[0171] 3. Lkb1 L/+, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(doxy and no tamoxifen: control)
[0172] 4. Lkb1 L/+, tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2
(doxy and yes tamoxifen: experimental cohort)
[0173] 1a. Lkb1 L/+, tet-op-EGFR L858R, CCSP-rtTA,SPC-Cre-ER.sup.T2
(no doxy and no tamoxifen: control)
[0174] 2a. Lkb1 L/+, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (no doxy and tamoxifen: control)
[0175] 3a. Lkb1 L/+, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and no tamoxifen: control)
[0176] 4a. Lkb1 L/+, tet-op-EGFR L858R, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and tamoxifen: experimental cohort)
[0177] 1b. Lkb1 L/+, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (no doxy and no tamoxifen: control)
[0178] 2b. Lkb1 L/+, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (no doxy and yes tamoxifen: control)
[0179] 3b. Lkb1 L/+, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and no tamoxifen: control)
[0180] 4b. Lkb1 L/+, tet-op-BRAF V600E, CCSP-rtTA,
SPC-Cre-ER.sup.T2 (doxy and yes tamoxifen: experimental cohort)
[0181] The same experiments as outlined above for the initial
cohorts to determine whether the loss of a single copy of Lkb1
(haploinsufficiency) impact on the phenotypes of the K-ras, EGFR,
and BRAF mutant-driven lung tumors.
EXAMPLE 10
Use Pharmacological and Genetic Approaches to Dissect the Role of
Dysregulated Pathway Caused by LKB1 Loss that are Involved in the
Initiation and Progression of K-RAS, EGFR, and BRAF Lung
Tumorigenesis
[0182] Lkb1 is implicated in various aspects of cellular metabolism
and polarity control. Lkb1 exerts its effects on diverse cellular
functions through phosphorylation of different cellular substrates.
In addition to the first identified physiological substrate of Lkb1
kinase, AMPK, 13 other AMPK-related serine/threonine kinases have
been subsequently identified as Lkb1 substrates. Of these different
pathways, the most characterized is the AMPK/TSC1/TSC2/mTOR
signaling pathways, as this pathway is frequently activated in
various types of cancers. In the preliminary studies, it was
revealed in K-ras-driven lung tumors with LKB1 loss had elevated
mTOR activity. The role of the activated AMPK/TSC1/TSC2/mTOR
pathway caused by the loss of Lkb1 function in worsening the
biological behavior of the K-ras LKB1 compound mutant lung tumors
will be determined
[0183] To determine if the activated mTOR pathway is the main cause
for the more aggressive phenotype seen in the K-ras/Lkb1 compound
mutant lung tumors 78 mice from each of the following cohorts will
be generated:
[0184] 1. Tet-op-K-ras G12D, CCSP-rtTA, SPC-Cre-ER.sup.T2 (control
cohort)
[0185] 2. Tet-op-K-ras G12D, CCSP-rtTA, Lkb1 L/L, SPC-Cre-ER.sup.T2
(experimental cohort)
[0186] Seventy-eight mice will be used for experiments in which
Lkb1 is deleted after K-ras-driven tumors are present, and the
other 78 mice will be used for experiments in which Lkb1 is deleted
before K-ras expression is induced. For the first set of
experiments, Lkb1 will be deleted after K-ras tumors are
formed.
[0187] In the experiment in which Lkb1 is deleted before K-ras
expression is induced, 78 mice at 3 weeks of age from the each
cohort will be injected intraperitionally with 4 mg of tamoxifen
over a 5 day period to activate the Cre recombinase activity in the
nucleus (deleting both alleles of LKB1 in pneumocytes in cohort 2).
These tamoxifen-treated animals will be then administered
doxycycline continuously through their diet to activate the
expression of the mutant K-ras. Concurrently, in each cohort, 26
mice will be treated once daily with 2 mg/kg of rapamycin
intraperitionally, 26 mice with 4 mg/kg of rapamycin
intraperitionally and 26 mice with placebo vehicle. These dosing
schedules are well established and have been shown to inhibit mTOR
activity in xenograft models. Eight mice from each of the
sub-cohorts will be followed until it is appropriate for them to be
euthanized, and a Kaplan-Meier survival curve will be generated. To
evaluate the effects of mTOR inhibition on the phenotype of the
K-ras LKB1 compound mutant lung tumors, three mice from each
sub-cohort will be sacrificed by CO.sub.2 inhalation following 3,
6, 9, 12, 15, and 18 weeks of doxycycline treatment. At the time of
sacrifice, tumor burden, tumor histology, and inhibition of the
mTOR signaling by methods outlined above will be evaluated. If
possible, the mice will be sacrificed 4 hours after the
administration of the last dose of the drug to ensure uniformity,
as serum from the mouse will be collected at the time of harvest to
assess rapamycin levels. In addition, any tumor nodules from these
tumor-bearing mice will be microdissected out. PCR and southern
analyses on DNA isolated from these microdissected tumors will be
utilized to verify if the Lkb1 recombination indeed occurs.
Furthermore, we will assess tumors via IHC and western blot
analysis to document loss of LKB1 protein expression. By comparing
the results from the different cohorts, it can be determined if
activated mTOR pathway plays a significant role in the altered
phenotype seen in the K-ras Lkb1 compound mutant mice.
[0188] The above experiments will allow for the determination of
the role of activated mTOR pathway in K-ras Lkb1 mutant lung tumor
initiation.
[0189] To evaluate the potential differing effects on the
initiation and progression of K-ras Lkb1 compound mutant-driven
lung tumorigenesis, the same experiments as outlined above will be
repeated with the same identical cohorts of mice (80 mice in each
cohort). Doxycycline will be administered continuously through
their diet to all mice in the cohort. After 8 weeks on doxycycline,
two treated mice from each cohort will be sacrificed and their
lungs harvested for immunohistochemical staining and western
analyses to confirm the expression of the mutant K-ras expression
and the activation of the downstream pathways. After the
confirmation, the remaining mice in each cohort will be injected
intraperitionally with 4 mg of tamoxifen over a 5-day period to
activate Cre-recombinase activity to delete the Lkb1 alleles. The
remaining 78 mice from each cohort will be divided into 3
sub-cohorts and be administered placebo, rapamycin at 2 mg/kg and
rapamycin at 4 mg/kg once daily as outlined above while remaining
on continuous doxycycline treatment. Eight mice from each
sub-cohort will be utilized to generate Kaplan-Meier survival
curves. As described above, three mice from each sub-cohort will be
sacrificed by CO.sub.2 inhalation following 3, 6, 9, 12, 15, and 18
weeks of doxycycline treatment. At the time of sacrifice, tumor
burden, tumor histology, and inhibition of the mTOR signaling will
be evaluated by methods outlined above.
[0190] As TSC1/TSC2 is directly downstream of AMPK but upstream of
mTOR, if the effect seen with loss of Lkb1 in the K-ras/Lkb1 tumors
is mainly due to the activated mTOR pathway, genetic inactivation
of the TSC1/TSC2 complex in the K-ras lung tumors should give a
similar phenotype as the K-ras Lkb1 mutant mice. A Tsc1 conditional
knockout allele into lox-stop-lox K-ras conditional mouse strain
(K-ras L/+) will be breed and the following colonies will be
generated:
[0191] 1. Tsc1 +/+, K-ras L/+
[0192] 2. Tsc1 L/L, K-ras L/+
[0193] 3. Tsc1 L/+, K-ras L/+
[0194] 4. Lkb1 +/+, K-ras L/+
[0195] 5. Lkb1 L/L, K-ras L/+
[0196] 6. Lkb1 L/+, K-ras L/+
[0197] Tumors will be induced in the K-ras L/+ mice by treating the
mice with a recombinant adenovirus expressing Cre recombinase
(adeno-Cre) via inhalation as has been done previously. Expression
of the Cre recombinase promotes a recombination event that induces
expression of the mutant K-ras. In brief, when the mice are
.about.7 weeks old, they will be anaesthetized with 0.013 ml/kg of
Avertin solution. 5 million colony-forming units of adenoviral Cre
(purchased from the University of Iowa) will be administered in a
solution of MEM and CaCl2. The solution will be applied to the
nares of the anesthetized mice. This procedure invariably induces
tumors in M. musculus and the mice usually become ill from
respiratory distress starting at about 8 weeks. During these
experiments, mice will be inspected daily, Monday through Friday.
Any mice that show evidence of respiratory distress or illness will
be euthanized (along with age-matched controls from the other
cohorts) and subjected to analysis. To determine if the loss of
Tsc1 affects overall survival of these mice and directly compare
the results to LKB1 loss, 10 mice from each cohort will be followed
until it is time for them to be euthanized and a Kaplan-Meier
survival curve will be generated. Of note, the person deciding
which mice should be euthanized will be blinded regarding their
specific genotypes. In addition to measuring effects on survival,
it will also be determined if loss of Tsc1 affects K-ras-induced
tumor burden. For these studies, five mice from each cohort will be
sacrificed by CO.sub.2 inhalation at 6, 8, 12, 18, and 21 weeks
after Cre administration for the lung tumor burden and
immunohistochemical analyses. At the time of sacrifice, each organ
from the mouse, including the heart, bones, liver, spleen, brain,
kidney, and adrenal glands we be inspected macroscopically for
visible signs of metastases. Additionally, these organs will be
fixed in formalin for subsequent detailed histological analyses.
The left lung will be removed and snap-frozen in liquid nitrogen
for protein, DNA, and RNA analyses. When macroscopic tumors are
evident, they will be dissected out of the tumor to enable tumor
specific molecular studies. The right lung (with the left main
bronchus ligated with a suture) will then be inflated at 25 cm H2O
pressure with 10% buffered formalin for 10 minutes via of an
intratracheal catheter. The right lungs will then be removed and
fixed in 10% buffered formalin for 24 hours before embedding in
paraffin. Serial mid-sagittal sections (5 .mu.M thickness) will be
obtained for histological analysis. Tumor genotyping will also be
performed to confirm recombination and deletion of the
Tsc1locus.
[0198] These experiments will determine if loss of Tsc1 effects
K-ras mutant-induced tumorigenesis in a similar manner as Lkb1
loss. It is anticipated that loss of Tsc1 will accelerate K-ras
tumorigenesis. However, these Lkb1 experiments are necessary
controls for direct comparison to the impact Tsc1 loss in K-ras
lung tumorigenesis.
[0199] To globally determine the effect of LKB1 in
K-ras/EGFR/BRAF-driven tumorigenesis expression profiling from the
RNA derived from the lung tumor nodules collected from the tumor
bearing cohorts (3, 4, 3a, 4a, 3b, 4b) described above will be
performed. All the tumors to be profiled will first be confirmed
for the presence or absence of Lkb1 by genotyping and
immunohistochemical analyses and for their precise histological
classification by the three pathologists. It is anticipated that in
the K-ras Lkb1 mutant cancers, there will be both adenocarcinomas
and squamous cell carcinomas. RNA for profiling from 15
adenocarcinomas from K-ras alone tumors (from cohort 3) and 15
adenocarcinomas from K-ras Lkb1 -/- compound mutant tumors (from
cohort 4) will be prepared. 15 lung squamous cell carcinomas from
the K-ras Lkb1 mutant mouse cohorts will also be collected. It is
not expected to have any lung squamous cell carcinomas from the
K-ras alone cohort. 15 paired EGFR, EGFR Lkb1 -/-, BRAF, BRAF Lkb1
-/- adenocarcinomas from the other cohorts will also be collected.
If tumors of other histological types emerge from these cohorts,
RNA from them will be prepared (up to 15 tumors from each
histological subtype) for expression profiling.
[0200] Briefly, RNA will be prepared from the mouse tumors
generated from the different cohorts using the standard trizol
method and digested with DNase I, and purified through the Qiagen
column. The purified RNA will be sent to the Dana-Farber Cancer
Institute Microarray Core Facility for expression profiling using
the Affymetrix Mouse Expression Array 430A2.0 (22,626 known
genes).
[0201] Data generated from each profiling will be downloaded as
"cel" image files. Low-level analyses will be performed using the
dChip software. These analyses will include image analysis (grid
alignment, target detection, intensity extraction, and local
background correction); normalization based on an invariant set and
subsequent median smoothing; and model-based expression indices
will be computed for each probe.
[0202] High-level analyses of the gene expression data will be
performed in the R statistical computing environment. A total of
approximately 105 samples (see above) will be expression-profiled
to determine the transcriptional level of 22,626 known genes. Gene
expression profiles between paired K-ras, EGFR, and BRAF cohorts
with and without Lkb1 function will also be compared. In addition,
the transcription signature of the K-ras alone driven tumors will
also be compared to those driven by EGFR or BRAF alone.
Specifically, we will compare gene expression profiles of: (1)
K-ras, Lkb1 +/+ vs. K-ras, Lkb1 -/-, (2) EGFR, Lkb1 +/+ vs. EGFR,
Lkb1 -/-, (3) BRAF, Lkb1 +/+ vs. BRAF, Lkb1 -/-, (4) K-ras, Lkb1
+/+ vs. EGFR, Lkb1 +/+ vs. BRAF, Lkb1 +/+, (5) K-ras, Lkb1 -/- vs.
EGFR, Lkb1 -/- vs. BRAF, Lkb1 -/-. Both individual gene analyses
and pathway analyses will be performed. For individual gene
analyses, individually differentially expressed genes will be
studied by performing the comparisons listed above using two-sample
t-tests tailored towards micro-array analysis, e.g., using the
software package SAM.sup.72,73. To control for the error rates of a
large number of hypothesis tests, the False Discovery Rates will be
calculated. Each cohort to be compared will have 15 tumors, and the
number of genes to be compared will depend on filtering of
unexpressed genes done in the low level analysis.
[0203] To assess expected power of our study, preliminary data
comparing gene expression profiles of K-ras Lkb1 +/+ tumors vs.
K-ras Lkb1 -/- tumors will be used to estimate the standard errors
and a reasonable range of effect sizes and performed power
calculations using the method of Tibshirani, which gives estimates
of false discovery rates and false negative rates and which is
particularly suitable, as it makes few assumptions regarding
correlation and variance of genes. Power in this situation is
defined as 1-FDR and depends on the mean difference between groups
of logged expression values, sample size, and the number of genes
that are truly significant for that effect size (for Tibshirani's
method this is set equal to the number of genes called
significant). Plots of FDR estimates for mean differences of 0.58,
1, 1.32, and 1.58 (corresponding to fold changes of 1.5, 2, 2.5,
and 3 for the raw unlogged data) are given below.
[0204] From these plots on FIG. 8 for a sample size of 15 per
cohort and a mean difference of 1 unit in the logged scale between
two cohorts, it is expected that greater than 80% power if the
hypothesized number of genes with a 2-fold change in expression
level is between 10 and 100. It is expected to be over 90% power if
the hypothesized number of genes with a 2-fold change in expression
level is greater than 100. For a mean difference of 0.58 (1.5-fold
change in a gene's unlogged expression), we expect greater than 60%
power if the true number of genes with a 1.5 fold change is greater
than 50. For a mean difference of 1.32 in the log-scale (2.5 fold
changes in the original scale), the estimated power is greater than
80% and becomes greater than 90% if the number of such genes is
greater than 100. For a mean difference of 1.58 (3-fold change in
the original scale) with 100 such genes, the estimated power is
greater than 90%. Preliminary data finds that the number of
significantly expressed genes is close to 1000 when comparing
K-ras, Lkb1 +/+ tumors vs. K-ras, Lkb1 -/- tumors. Hence it is
expected that the proposed sample sizes are likely to have
excellent power for comparing different cohorts. In each case
shown, the estimated false negative rate (type 1 error) is low.
[0205] Although individual gene analysis is useful, cellular
processes often affect sets of genes. Traditional analyses have
focused on individually highly ranked genes. However, this approach
suffers from several major limitations: (1) Long lists of
individually significant genes without a single encompassing theme
are difficult to interpret. (2) Single gene analyses miss important
pathway effects as cellular processes often affect sets of genes
and individually highly ranked genes are often downstream genes, so
moderate changes in many genes may give more insight into
biological mechanisms than dramatic change in a single gene. (3)
Individual highly ranked genes can be poorly annotated and are
often not reproducible from studies to studies. Knowledge-based
studies on gene sets, e.g. genetic pathways are more biologically
interpretable and reproducible. Biological knowledge based pathway
analysis will also be performed. Examples of candidate lung cancer
pathways we will consider include the mTOR and the angiogenesis
pathway as seen from our preliminary studies. Pathways will be
constructed based on: (1) genes demonstrated to be associated with
a particular gene ontology term; (2) genes demonstrated to be
associated with a particular KEGG pathway; (3) gene lists manually
assembled based on the previous literature.
[0206] Gene set enrichment analysis (GSEA), the principle component
analysis, the global testing approach and logistic version of the
kernel machine method can be used to test for modified regulation
of entire groups of genes between samples cohorts. GSEA is suitable
for comparative tests, while the others are suitable for
self-contained tests. Since this pathway analysis is
knowledge-based, self-contained tests are more suitable. The
Principle Components Analysis method essentially performs dimension
reduction by constructing weighted averages of genes comprising a
pathway. Weights are based on directions of greatest variability of
the genes in the pathway and can be found by finding the
eigenvectors corresponding to the largest eigenvalues of the
covariance matrix of the genes. Each weighted average can then be
considered as a super-gene and we will test for differences in
these super-genes between pathways via standard multivariate tests,
the Hotelling's T.sup.2-test in the two-group comparison case and
in the multiple groups setting, MANOVA. The principle component
analysis is particularly suitable when the number of subjects per
group is small. Both the global testing method and the kernel
machine method are based on logistic regression by regression
cohort status on gene expressions. The global testing method and
principal components method can also show which genes, and what
combination of genes, drive the difference between cohorts for each
pathway. For the global testing method, influence plots are
generated while for the principal components analysis method the
weights used to find the super-genes indicate the relative
importance of each gene in generating a difference. This can
potentially elucidate the mechanisms by which a pathway is
affected. The logistic analog of the kernel machine method further
allows interactions among genes within a pathway when comparing
different cohorts.
[0207] From the above expression profiling experiments and
analyses, it is anticipated that the expression profiling will
confirm that the mTOR pathways are hyper-activated in tumors
without Lkb1 function. More importantly, it is also anticipated
that many novel Lkb1 dependent genes and pathways that are involved
in lung cancer progression and metastases will be discovered. In
addition, there might be unique Lkb1 cancer relevant pathways that
are activated/inactivated depending on the initial oncogenic
stimuli. (For example, Lkb1 dependent pathway X is only activated
in EGFR mutant driven lung tumors while Lkb1 dependent pathway Y is
only activated in K-ras mutant driven lung tumors.) It is also
anticipated that the expression profiling to confirm that the mTOR
pathways are hyper-activated in tumors without Lkb1 function.
EXAMPLE: 11
Determination of the the Impact of Concentrated Ambient Particles
(CAPs), an Environmental Insult, on Inflammation, Proliferation,
Apoptosis, and LKB1 Function on K-RAS, EGFR, and BRAT Mutant
Mice
[0208] With the generation of different genetically engineered mice
that are prone to develop lung cancer, we now have the tools and
reagents to examine the environmental interaction with these
defined oncogenic stimuli and determine the role of environmental
insult on lung cancer initiation and progression. Concentrated air
particles (CAPs) system is a well-established and ideal model to
dissect the role of air pollution in lung cancer progression
through its effect on the primed lung epithelial compartment in our
genetically defined lung oncogenic mouse models. It is hypothesized
that prolonged CAPs exposure will cause chronic lung inflammation.
The chronic lung inflammatory environment will generate reactive
oxidative species leading to increased DNA damage, and genetic
inactivation of important lung tumor suppressors such as LKB1, and
accelerate lung tumorigenesis. The following series of experiments
will test this hypothesis.
[0209] The following 6 cohorts of mice (60 mice per cohort) will be
generated:
[0210] 1. Tet-op-K-ras G12D, CCSP-rtTA, (no doxy: control)
[0211] 2. Tet-op-K-ras G12D, CCSP-rtTA, (doxy: experimental
cohort)
[0212] 1a. Tet-op-EGFR L858R, CCSP-rtTA, (no doxy: control)
[0213] 2a. Tet-op-EGFR L858R, CCSP-rtTA (doxy: experimental
control)
[0214] 1 b. Tet-op-BRAF V600E, CCSP-rtTA, (no doxy: control)
[0215] 2b. Tet-op-BRAF V600E, CCSP-rtTA, (doxy: experimental)
[0216] At 10 weeks of age, mice from cohort 2, 2a, and 2b will be
put on continuous doxycycline diet. All the mice from each of the
cohorts will then be randomly divided into two equal groups (30
mice in each sub-cohort). One group of mice will be exposed The
Harvard/EPA Ambient Particle Concentrator (HAPC) generates
concentrated aerosols of outdoor air particles that can
subsequently be directly delivered to animals (described below) for
a total of 20 weeks. The other group will be exposed to filtered
air in an identical exposure chamber.
[0217] The Harvard/EPA Ambient Fine Particle Concentrator (HAPC)
will be used to expose animals to concentrated ambient fine
particles (0.1-2.5 .mu.m). Boston atmospheres typically consist of
particles generated by vehicle exhaust, power plant emissions, home
heating, and transported aerosols. Employment of this system allows
for the direct investigation of the potential harm of "real world"
particles in our genetically defined mouse models. Briefly, the
HAPC used for the animal inhalation studies consists of three
components: (1) a high-volume conventional impactor with a
2.5-.mu.m cutoff size, (2) a series of three virtual impactors with
a 0.1-.mu.m cutoff size (concentrator stages I, II, and III), and
(3) an animal exposure chamber. The first impactor is a high-volume
conventional impactor (Fractionating Sampler, Anderson, Inc.,
Atlanta, Ga.) and removes particles larger than 2.5 .mu.m operating
at 5000 L/min, while smaller particles escape collection. The
deflected flow of the conventional impactor is drawn through a
series of three virtual Impactors. Each virtual impactor
accelerates all airborne particles in a rectangular nozzle. These
particles cross the deflected air streamlines, and enter a
slit-shaped collection probe, while particles smaller than 0.1
.mu.m follow the deflected streamlines, or enter the collection
probe, but are unlikely to be concentrated. Particles in the size
range 0.1-2.5 .mu.m pass through the collection probe and are
referred to as the minor flow (20% of the total flow). The minor
flow of the third stage virtual impactor contains the concentrated
aerosol. The total flow rate into the third stage is 50 L/min, and
concentrated particles are supplied to the animal exposure chamber
at 40 L/min. The remaining 10 L/min is used for characterization of
the aerosol. The total concentration factor for the three virtual
impactor stages is about 30. The concentrated air from stage III of
the virtual impactor is supplied to the whole-body animal exposure
unit (described below) through a manifold connection port on top of
the chamber. Sham exposures are also performed in the exposure unit
at the same pressure and flow rate with the air passed through a
glass fiber filter (Gelman, Type A/E, Ann Arbor, Mich.) connected
at the inlet of the chamber to remove particles. The filter
efficiency is 99.6-99.9%. The use of the three virtual impactor
stages results in a pressure drop of about 10 in H2O. The aerosol
flow rate provides for a residence time in the exposure chamber of
about 3.5 minutes. This residence time results in minimal particle
losses on the chamber wall. The virtual impactor residence time is
only a few seconds. The portable chamber for rodent concentrated
air particle (CAPs) exposure has a volume of 142 L and is specially
designed to fit inside a larger 1000-L stainless steel and glass
outer chamber. Because the air pumping units are downstream from
the concentrator/inhalation chamber, both the external chamber and
the exposure chamber are operated under a negative pressure
(.about.10 in H2O). The chamber is designed to optimally utilize
the flow rates from the concentrator to deliver the aerosol to the
rodents. A manifold connection port on top of the chamber allows
for direct connection to the HAPC. The unit is rectangular in shape
with a grated floor to allow waste products to fall into
trapezoidal waste collection areas underneath; drain valves in the
bottom of the trapezoidal waste collection areas allow for removal
of animal waste during exposure. A second manifold on the back of
the unit is connected to a negative pressure system to maintain air
exchange in the system. Up to six stainless steel wire-mesh cages
(holding up to 50 animals each) can be placed into the unit at one
time.
[0218] CAPs characterization will be performed weekly to ensure
that there is no dramatic change in its composition during the
course of the experiments using gravimetric particle mass
determinations, liquid chromatography for sulfate, X-ray
fluorescence for elemental analysis and thermal and optical
reflectance method for elemental carbon analyses.
[0219] Mice from all the cohorts will be monitored closely for
clinical signs of stress or tumors. Any mouse that shows any
clinical signs of having stress or tumors will be sacrificed for
analyses into the causality and nature of pathology. In addition
four mice from each cohort will be sacrificed for analyses at weeks
2, 4, 8, 12, and 16 weeks post CAPs or filtered air exposure. The
remaining 10 mice from each cohort are used to generate Kaplan
Meier curves.
[0220] At the time of sacrifice, each organ from the mouse
including the heart, lung, pancreas, liver, spleen, kidney,
bladder, and intestines will be inspected macroscopically for signs
of abnormality and fix them in formalin for subsequent detailed
histological analyses Blood and serum from each mouse via cardiac
puncture will also be collected. For the lungs, prior to fixation,
bronchioalevolar lavage (BAL) will be performed to obtain fluids
and cells. As described above, ligated left lung will be harvested
and snap-frozen for subsequent DNA, RNA, and protein analyses. The
right lung will then be inflated at 25 mm with 3% gluteraldehyde in
0.1 M cacodylte buffer to 25 cm H.sub.2O (for 15 min), then ligated
and removed. This procedure would preserve the physiological air
space architecture. The lungs collected from the different cohorts
will be assessed for tumor burden. The tumors will also be analyzed
for loss of Lkb1 via IHC and western analyses as well as activated
mTOR pathway.
[0221] Bone marrow from the femurs will be harvested to generate
primary bone marrow culture suspension. Metaphase spreads from
these primary cells derived from the various cohorts will be
prepared to quantitatively determine the frequency of chromosomal
aberrations caused by chronic oxidative and mutagenic stress from
the CAPs. This series of experiments will determine the potency of
CAPs as a carcinogen and inducers of DNA double strand breaks.
[0222] Immuno-staining with Ki67 antibody will be performed on
histological sections of the lungs and the other organs from the
six cohorts harvested at the determined time points to assess the
steady state rate of proliferation in response to chronic CAPs
exposure in the various organs from the different cohorts.
Similarly, TUNEL analyses will be performed to determine the steady
state rate of apoptosis.
[0223] The steady state level of the lung and the circulating
pro-inflammatory cytokines including IL-1.beta., IL-2, IL-4, IL-5,
IL-6, IL-10, IL-12(p70), TNF.alpha., IFN.gamma. and GM-CSF will be
quantitatively determined using the Upstate
Beadlyte.RTM./Luminex.RTM. multiplex mouse multi-cytokine detection
system which allowed for sensitive and accurate measurement of all
the above cytokine simultaneously in a small volume of serum.
[0224] As the temporal relationship between environmental exposure
to CAPs and oncogene activation might be important to lung
tumorigenesis and progression. Thus, the experiments described
above will be repeated in a different temporal sequence. Using the
same cohorts of animals as described above, animals in the
different cohorts we be chronically exposed the either to CAPs or
filtered air starting at 3 weeks of age. After 20 weeks of
exposure, all the mice will be placed on filtered air, and the mice
in 2, 2a, and 2b will be put on continuous doxycycline diet. This
set of experiment will address the impact of existing chronic lung
inflammation caused by environmental insult (CAPs) on
oncogene-driven lung tumorigenesis.
[0225] Lastly, if there are major differences in the between the
CAPs exposed mouse cohorts and the control cohorts in either the
latency, aggressiveness or the histology of lung tumors, the tumors
from each of the cohorts will be isolated and similar expression
profiling analyses will be performed as detailed above to determine
the pathways that are de-regulated as a result of the CAPs
exposure.
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Other Embodiments
[0268] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
213286DNAHomo sapiens 1gcgtgtcggg cgcggaaggg ggaggcggcc cggggcgccc
gcgagtgagg cgcggggcgg 60cgaagggagc gcgggtggcg gcacttgctg ccgcggcctt
ggatgggctg ggcccccctc 120gccgctccgc ctcctccaca cgcgcggcgg
ccgcggcgag ggggacgcgc cgcccggggc 180ccggcacctt cgggaacccc
ccggcccgga gcctgcggcc tgcgccgcct cggccgccgg 240gagccccgtg
gagcccccgc cgccgcgccg ccccgcggac cggacgctga gggcactcgg
300ggcggggcgc gcgctcgggc agacgtttgc ggggaggggg gcgcctgccg
ggccccggcg 360accaccttgg gggtcgcggg ccggctcggg gggcgcccag
tgcgggccct cgcgggcgcc 420gggcagcgac cagccctgag cggagctgtt
ggccgcggcg ggaggcctcc cggacgcccc 480cagccccccg aacgctcgcc
cgggccggcg ggagtcggcg ccccccggga ggtccgctcg 540gtcgtccgcg
gcggagcgtt tgctcctggg acaggcggtg ggaccggggc gtcgccggag
600acgcccccag cgaagttggg ctctccaggt gtgggggtcc cggggggtag
cgacgtcgcg 660gacccggcct gtgggatggg cggcccggag aagactgcgc
tcggccgtgt tcatacttgt 720ccgtgggcct gaggtccccg gaggatgacc
tagcactgaa aagccccggc cggcctcccc 780agggtccccg aggacgaagt
tgaccctgac cgggccgtct cccagttctg aggcccgggt 840cccactggaa
ctcgcgtctg agccgccgtc ccggaccccc ggtgcccgcc ggtccgcaga
900ccctgcaccg ggcttggact cgcagccggg actgacgtgt agaacaatcg
tttctgttgg 960aagaagggtt tttcccttcc ttttggggtt tttgttgcct
tttttttttc ttttttcttt 1020gtaaaatttt ggagaaggga agtcggaaca
caaggaagga ccgctcaccc gcggactcag 1080ggctggcggc gggactccag
gaccctgggt ccagcatgga ggtggtggac ccgcagcagc 1140tgggcatgtt
cacggagggc gagctgatgt cggtgggtat ggacacgttc atccaccgca
1200tcgactccac cgaggtcatc taccagccgc gccgcaagcg ggccaagctc
atcggcaagt 1260acctgatggg ggacctgctg ggggaaggct cttacggcaa
ggtgaaggag gtgctggact 1320cggagacgct gtgcaggagg gccgtcaaga
tcctcaagaa gaagaagttg cgaaggatcc 1380ccaacgggga ggccaacgtg
aagaaggaaa ttcaactact gaggaggtta cggcacaaaa 1440atgtcatcca
gctggtggat gtgttataca acgaagagaa gcagaaaatg tatatggtga
1500tggagtactg cgtgtgtggc atgcaggaaa tgctggacag cgtgccggag
aagcgtttcc 1560cagtgtgcca ggcccacggg tacttctgtc agctgattga
cggcctggag tacctgcata 1620gccagggcat tgtgcacaag gacatcaagc
cggggaacct gctgctcacc accggtggca 1680ccctcaaaat ctccgacctg
ggcgtggccg aggcactgca cccgttcgcg gcggacgaca 1740cctgccggac
cagccagggc tccccggctt tccagccgcc cgagattgcc aacggcctgg
1800acaccttctc cggcttcaag gtggacatct ggtcggctgg ggtcaccctc
tacaacatca 1860ccacgggtct gtaccccttc gaaggggaca acatctacaa
gttgtttgag aacatcggga 1920aggggagcta cgccatcccg ggcgactgtg
gccccccgct ctctgacctg ctgaaaggga 1980tgcttgagta cgaaccggcc
aagaggttct ccatccggca gatccggcag cacagctggt 2040tccggaagaa
acatcctccg gctgaagcac cagtgcccat cccaccgagc ccagacacca
2100aggaccggtg gcgcagcatg actgtggtgc cgtacttgga ggacctgcac
ggcgcggacg 2160aggacgagga cctcttcgac atcgaggatg acatcatcta
cactcaggac ttcacggtgc 2220ccggacaggt cccagaagag gaggccagtc
acaatggaca gcgccggggc ctccccaagg 2280ccgtgtgtat gaacggcaca
gaggcggcgc agctgagcac caaatccagg gcggagggcc 2340gggcccccaa
ccctgcccgc aaggcctgct ccgccagcag caagatccgc cggctgtcgg
2400cctgcaagca gcagtgaggc tggccgcctg cagcccgtgt ccaggagccc
cgccaggtgc 2460ccgcgccagg ccctcagtct tcctgccggt tccgcccgcc
ctcccggaga ggtggccgcc 2520atgcttctgt gccgaccacg ccccaggacc
tccggagcgc cctgcagggc cgggcagggg 2580gacagcaggg accgggcgca
gccctccccc ctcggccgcc cggcagtgca cgcggcttgt 2640tgacttcgca
gccccgggcg gagccttccc gggcgggcgt gggaggaggg aggcggcctc
2700catgcacttt atgtggagac tactggcccc gcccgtggcc tcgtgctccg
cagggcgccc 2760agcgccgtcc ggcggccccg ccgcagacca gctggcgggt
gtggagacca ggctcctgac 2820cccgccatgc atgcagcgcc acctggaagc
cgcgcggccg ctttggtttt ttgtttggtt 2880ggttccattt tctttttttc
tttttttttt taagaaaaaa taaaaggtgg atttgagctg 2940tggctgtgag
gggtgtttgg gagctgctgg gtggcagggg ggctgtgggg tcgggctcac
3000gtcgcggccg cctttgcgct ctcgggtcac cctgctttgg cggcccggcc
ggagggcagg 3060accctcacct ctcccccaag gccactgcgc tcttgggacc
ccagagaaaa cccggagcaa 3120gcaggagtgt gcggtcaata tttatatcat
ccagaaaaga aaaacacgag aaacgccatc 3180gcgggatggt gcagacgcgg
cggggactcg gagggtgccg tgcgggcgag gccgcccaaa 3240tttggcaata
aataaagctt gggaagcttg gacctgaaaa aaaaaa 32862433PRTHomo sapiens
2Met Glu Val Val Asp Pro Gln Gln Leu Gly Met Phe Thr Glu Gly Glu1 5
10 15Leu Met Ser Val Gly Met Asp Thr Phe Ile His Arg Ile Asp Ser
Thr 20 25 30Glu Val Ile Tyr Gln Pro Arg Arg Lys Arg Ala Lys Leu Ile
Gly Lys 35 40 45Tyr Leu Met Gly Asp Leu Leu Gly Glu Gly Ser Tyr Gly
Lys Val Lys 50 55 60Glu Val Leu Asp Ser Glu Thr Leu Cys Arg Arg Ala
Val Lys Ile Leu65 70 75 80Lys Lys Lys Lys Leu Arg Arg Ile Pro Asn
Gly Glu Ala Asn Val Lys 85 90 95Lys Glu Ile Gln Leu Leu Arg Arg Leu
Arg His Lys Asn Val Ile Gln 100 105 110Leu Val Asp Val Leu Tyr Asn
Glu Glu Lys Gln Lys Met Tyr Met Val 115 120 125Met Glu Tyr Cys Val
Cys Gly Met Gln Glu Met Leu Asp Ser Val Pro 130 135 140Glu Lys Arg
Phe Pro Val Cys Gln Ala His Gly Tyr Phe Cys Gln Leu145 150 155
160Ile Asp Gly Leu Glu Tyr Leu His Ser Gln Gly Ile Val His Lys Asp
165 170 175Ile Lys Pro Gly Asn Leu Leu Leu Thr Thr Gly Gly Thr Leu
Lys Ile 180 185 190Ser Asp Leu Gly Val Ala Glu Ala Leu His Pro Phe
Ala Ala Asp Asp 195 200 205Thr Cys Arg Thr Ser Gln Gly Ser Pro Ala
Phe Gln Pro Pro Glu Ile 210 215 220Ala Asn Gly Leu Asp Thr Phe Ser
Gly Phe Lys Val Asp Ile Trp Ser225 230 235 240Ala Gly Val Thr Leu
Tyr Asn Ile Thr Thr Gly Leu Tyr Pro Phe Glu 245 250 255Gly Asp Asn
Ile Tyr Lys Leu Phe Glu Asn Ile Gly Lys Gly Ser Tyr 260 265 270Ala
Ile Pro Gly Asp Cys Gly Pro Pro Leu Ser Asp Leu Leu Lys Gly 275 280
285Met Leu Glu Tyr Glu Pro Ala Lys Arg Phe Ser Ile Arg Gln Ile Arg
290 295 300Gln His Ser Trp Phe Arg Lys Lys His Pro Pro Ala Glu Ala
Pro Val305 310 315 320Pro Ile Pro Pro Ser Pro Asp Thr Lys Asp Arg
Trp Arg Ser Met Thr 325 330 335Val Val Pro Tyr Leu Glu Asp Leu His
Gly Ala Asp Glu Asp Glu Asp 340 345 350Leu Phe Asp Ile Glu Asp Asp
Ile Ile Tyr Thr Gln Asp Phe Thr Val 355 360 365Pro Gly Gln Val Pro
Glu Glu Glu Ala Ser His Asn Gly Gln Arg Arg 370 375 380Gly Leu Pro
Lys Ala Val Cys Met Asn Gly Thr Glu Ala Ala Gln Leu385 390 395
400Ser Thr Lys Ser Arg Ala Glu Gly Arg Ala Pro Asn Pro Ala Arg Lys
405 410 415Ala Cys Ser Ala Ser Ser Lys Ile Arg Arg Leu Ser Ala Cys
Lys Gln 420 425 430Gln
* * * * *
References