CA2754610C - Tumor-initiating cells and methods for using same - Google Patents

Abstract

Isolated and enriched tumor-initiating cell popula-tions, methods for preparing the same, and uses thereof.

Description

TUMOR-INITIATING CELLS AND METHODS FOR USING SAME
FIELD OF THE INVENTION
The present invention generally relates to highly tumorigenic cells, also called cancer stem cells or tumor-initiating cells, methods for isolating same and tumor-initiating cell markers for use in said methods. More particularly, the present invention relates to tumor-initiating cells having high levels of 5T4 expression (514high), optionally with no or low levels of CD24 expression (CD24-Aow) and with CD44 expression (CD444). The disclosed tumor-initiating cell populations are useful for identification of new drugs and targets for cancer therapy, and for testing the efficacy of existing cancer drugs.
BACKGROUND OF THE INVENTION
Signaling pathways that regulate setf-renewal and differentiation contribute to the cellular heterogeneity within tumors. The varying states of self-renewal and differentiation are evidenced by tumor subpapulations and individual tumor cells that exhibit disparate levels of in vivo tumorigenicity and in vitro clonogenicity. See Lobo et al., Annu. Rev. Cell Dev. Biol., 2007 23: 675-699;
Reya et al, Nature, 2001, 414: 105-111. The development of new tumor models has begun to enable the characterization of tumor heterogeneity at cellular levels. Implants of solid tumors in immunocompromised mice exhibit a rich architecture that reflects the histology of the original sample but that is not recapitulated in xenografts from cell lines cultured in serum. The culturing of cancer cells in defined serum-free media and/or three-dimensional matrices preserves the physiological characteristics of the cells more than culturing in media with serum (Lee et at, Cancer Cell, 2006, 9: 391-403). Fluorescence-activated cell sorting (FAGS) of cells from tumors, xenografts, and cell lines has facilitated the molecular characterization of specific tumor sub-populations.
In many tumors, cells defined by specific surface markers form tumors more efficiently than other cells in the same tumor. These cells are alternately referred to as multipotent tumor-initiating cells, cancer stem cells, tumor-initiating cells, and cancer-initiating cells. Tumor-initiating cells were first identified in the hematopoietic system (Bonnet & Dick, Nat. Med., 1997, 3(7): 730-737) and have since been identified in solid tumors, including tumors of the brain, breast, colon, head and neck, lung, melanoma, pancreas, and prostate. See Visvader &
Lindeman, Nat. Rev. Cancer, 2008, 8: 755-768 and reference cited therein. In a particular tumor type, the same set of cell surface markers can be used to isolate tumor-initiating cells from fresh tumor samples, xenografts, and cell lines.
See e.g,, Al-Hajj et al., Proc, Natl. Acad. Sci. USA, 2003, 1001 3983-3988;
Filmore &
Kuperwasser, Breast Cancer Res., 2008, 10: R25; Hermann et al, Cell Stern Cell, 2007, 11 313-323. Matsui et al., Blood, 2004, 103: 2332-2336, CD44, a marker of tumor-initiating cells in several tumor types, was recently shown to have a direct role in tumorigenesis and to be repressed by p53 (Godar et al., Cell, 2008, 134: 62-73), Tumor-initiating cells show resistance to standard therapies. For example.
tumor-initiating cells were highly enriched in samples from breast cancer patients that had received chemotherapy, suggesting an explanation for disease relapse following treatment (Yu et al., Cell, 2007, 131: 1109-1123). Similarly, CD133+

tumor-initiating cells in glioblastoma were resistant to irradiation that eradicated the more prevalent CD133- cells (Bao et al,, Cancer Res., 2006, 68: 6043-6048).
Thus, in the context of therapy, eliminating tumor-initiating cells might require targeting mechanisms other than those used to target the bulk of the tumor.
To develop treatments that significantly increase long-term patient survival in cancer, tumor-initiating cells responsible for tumor recurrence and metastasis represent an important therapeutic target for this disease. To meet this need, the present invention provides isolated and enriched populations of tumor-initiating cells that can be used to test the efficacy of new and existing cancer drugs.

- 2 -SUMMARY OF THE INVENTION
The present invention provides isolated and enriched tumor-initiating cell populations. In one aspect of the invention, an isolated tumor-initiating cell population is derived from a tumor cell population, the isolated tumor-initiating cell population comprising at least 90% tumor-initiating cells, wherein the tumor-initiating cells (i) express 514 at a level that is at least 2-fold higher than non-tumorigenic cells of the same origin, (ii) are tumorigenic, (iii) are capable of migration, (iv) are capable of self-renewal, and (v) generate tumors comprising non-tumorigenic cells. In another aspect of the invention, an enriched tumor-initiating cell population is provided, which is derived from a tumor cell population comprising tumor-initiating cells and non-tumorigenic cells, and wherein the tumor-initiating cells (i) express 514 at a level that is at least 2-fold higher than non-tumorigenic cells of the same origin, (ii) are tumorigenic, (iii) are capable of migration, (iv) are capable of self-renewal, (v) generate tumors comprising non-tumorigenic cells, and (vi) are enriched at least 2-fold compared to the tumor cell population. The isolated or enriched tumor-initiating populations may also express CD24 at a level that is at least 2-fold lower than non-tumorigenic cells of the same origin, and/or express CD44.
Also provided are methods of preparing isolated and enriched tumor-initiating cell populations. For example, a representative method of isolating or enriching a tumor-initiating cell population includes the steps of (a) providing dissociated tumor cells, wherein a majority of the cells express 514 at a low level and a minority of the cells express 514 at a high level; (b) contacting the dissociated tumor cells with an agent that specifically binds to 514; and (c) selecting cells that specifically bind to the agent of (b) to an extent that shows a high level of 514 expression that is at least about 2-fold greater than the low level; whereby a tumor-initiating cell population is isolated or enriched.
Optionally, the methods for preparing an isolated or enriched 514 expressing tunor initiating cell population include the additional steps of contacting the dissociated tumor cells with an agent that specifically binds to CD44; and selecting cells that specifically bind to the agent of to an extent that shows

- 3 expression of CD44 Optionally, the methods for preparing an isolated or enriched 5T4 expressing tuner initiating cell population may also include the steps of contacting the dissociated tumor cells with an agent that specifically binds to CD24; and selecting cells that specifically bind to the agent of to an extent that shows a low level of CD24 expression that is at least about 5-fold lower than non-tumorigenic cells of the same origin. Alternatively, the tumor initiating cell population can be enriched through culturing the primrary tumor cells in serum free conditions. In yet another representative method of the invention, isolating or enriching a 5T4 expressing tumor-initiating cell population can include contacting the dissociated tumor cells with an agent that specifically binds to CD24; and depleting cells that specifically bind to the agent of to an extent that shows a high level of CD24 expression that is at least about 5-fold greater than non-tumorigenic cells of the same origin.
Still further are provided methods of testing efficacy of a cancer drug or candidate cancer drug using the disclosed isolated or enriched tumor-initiating cell populations For example, such methods can include the steps of (a) providing an isolated or enriched tumor-initiating cell population: (b) contacting the tumor-initiating cells with a cancer drug or a candidate cancer drug; (c) observing a change in tumorigenic potential of the tumor-initiating cells following contacting the tumor-initiating cells with the cancer drug or candidate cancer drug.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1H show that the CD24-d0CD44+ cell phenotype marks tumor-initiating cells in the H460T non-small cell lung cancer line (NSCLC). CD24 1 wCD44+ cells are labeled "CD24"10 and CD24highCD44+ cells are labeled "CD24"h".
Figure 1A shows the results of flow cytometric analysis using expression of CD24 and CD44. Distinct populations of H460T were revealed by flow cytometry and labeling with anti-0O24 and anti-CD44 antibodies.

- 4 -Figure 1B shows mice that received subcutaneous implants of CD24-1mCD44+ cells or CD24h`ghCD44+ cells. Arrows, sites of implantation.
Figure 1C is a line graph showing a quantitative analysis results of the observation in Figure 16. Values indicate the average tumor measurement SEM (standard error of the mean).
Figure 1D is a line graph showing CD44 tumorigenesis based uon C044 expression in H460T cells. CD24-10wCD44hgh and CD24-n" CD441" cells were sorted and implanted subcutaneously into mice. Values indicate the average tumor measurement - SEM.
Figure 1E a line graph showing spheroid growth of sorted populations.
FACS-isolated CD2410" CD441- or CD24hIglICD44+ cells were cultured in suspension for 5 days to promote spheroid formation. Spheroids of 0.2mm diameter were transferred to individual wells of a 24-well plate and measured over a two-week time course. Values indicate the average spheroid volume SD
(standard deviation of the mean).
Figure IF a line graph showing the differential response of CD24-1"'CD4egh and CD24-10wCD4ew populations to mTOR inhibitor CC1-779.
Figure 1G is a bar graph showing the results of a transwell migration assay. CD2e0wCD44+ cells migrated efficiently in response to serum. The CD24-1mCD44+ value shows the average cell number normalized to CD24"hCD44+ for each experiment ( SD (n=4)).
Figure 1H shows micrographs depicting efficient migration on fibronectin of CD24-10wCD44+ spheroids 72 hours after spheroids were placed on fibronectin-coated slides.
Figures 2A-2E show the multipotency of CD2441 wCD44+ cells in H460T.
CD24-11"CD44+ cells are labeled "CD24-11 w" and CD24"hCD44+ cells are labeled "CD24h ".
Figure 2A shows the results of flow cytometric analysis of CD24-10"CD44+
cells and CD24h CD44+ following a three week culture. Distinct populations are revealed based upon CD24 and CD44 expression.

- 5 -Figure 2B shows the results of flow cytometric analysis of CD24 and CD44 expression in a representative tumor from sorted CD24-110wCD44+ cells.
Figure 2C shows the results of flow cytometric analysis of CO24 and C044 expression in clonal lines established from single sorted CD24-ImCD44+ or CD24"hCD44+ cells. The proportion of CD24 h cells in the transitioning CD24"
1" clones ranged from 10-70% depending on the clone. The CD24 distribution in each clone was steady over months in culture.
Figure 2D a line graph showing tumor growth from clonal lines presented in Figure 2C, CD24-1" cells were sorted from CD24-I" clones and CD24' cells were sorted from CO24hIgh clones, Values indicate the average tumor measurement 8 EM. Ti, transitioning clone; St, stable clone.
Figure 2E a line graph showing tumor growth of CD24-1 ' and CD2411 cells FACS-isolated from transitioning CD24-11cw clones. Values indicate the average tumor measurement 8 EM.
Figures 3A-3C show the multipotency of H46OT cells sorted based upon CD24 expression.
Figure 3A is a schematic drawing of the experimental design. Sorted cells were labeled with 2.5 pM CFSE (Invitrogen of Carlsbad, California, USA), washed extensively, and then plated with another sorted population in the ratio of the parent population (1 CD24-A"tD44+ to 3 CD24hIghCD44+), CFSE at 2,5 pM
had little or no effect on the growth of H460T cells over the time course of this experiment. After three days, cultures were analyzed for CD24, and the initial populations could be distinguished based on CFSE.
Figure 3B shows the results of flow cytometric analysis of labeled and -- unlabeled populations cultured individually or in combination.
Figure 3C is a histogram of replicate experiments shown in Figure 3B.
The transition of CD24410wCD44+ cells to CD2419hCD44+ was comparable when the cells were cultured alone or co-cultured with CO24"tD44+ or CD24-AawCD44+ cells, The stability of the CD24"hCD44+ phenotype was observed when CD244hCD444 cells were cultured alone or co-cultured with CD24-A"CD44+ or CD24"hCD44+ cells.

- 6 -Figures 4A-4C show CD244" identifies tumor-initiating cells in cultured HCC2429 cells.
Figure 4A shows the results of flow cytometric analysis of HCC2429 based on CO24 expression.
Figure 4B is a line graph showing differential tumorigenicity of CD24-4"CD44+ or CD24hi9hCD44+ HCC2429 populations. Values indicate the average tumor measurement SEM.
Figure 4C shows the results of flow cytometric analysis of CD2440vCD44+
or CD24highCD44+ populations 2 weeks after sorting. Cultured CD24-11"CD44+
cells can transition to CD24hi9hCD444, but CD24hi9hCD44+ do not transition to CD2441"CD44+ cells.
Figure 5 is a bar graph showing CD24 mRNA levels on Affymetrix GENECHe oligonucleotide arrays hybridized with triplicate samples of mRNA
transcripts prepared from CD24410wCD44+ and CD24hi9hCD44+ cells.
Figures 6A-6B show that the oncofetal protein 5T4 (TPBG) is expressed in H460T multipotent tumor-initiating cells.
Figure 6A shows the results of immunoblot analysis to detect 5T4 expression in CD24-40CD44+ and CD24higlICD44+ cells grown in media or treated with vehicle or all-trans retinoic acid.
Figure 6B is a bar graph showing 5T4 (TPBG) mRNA levels on Affymetrix GENECIIPPII oligonucleotide arrays hybridized with mRNA transcripts prepared from CD24-1mCD44+ and CD24highCD44+ cells. Values indicate the average of triplicate samples SD.
Figures 7A-7G show gene expression profiles associated with undifferentiated and differientiated 87426 primary culture cells.
Figure 7A shows micrographs of the 87426 primary culture of NSCLC
under conditions that promote growth (left) and differentiation at the air-liquid interface (right). Scale bars, 200 pM.
Figure 78 shows the results of immunoblot analysis to detect 5T4 expression at the indicated time points during differentiation.

- 7 -Figure 7C shows mRNA levels on Affymetrix GENECHIP oligonucleotide arrays hybridized with mRNA transcripts prepared from primary culture lung cancer cells under conditions of growth or differentiation. Values represent averages SD. FN1, fibronectin; VIM, vimentin.
Figure 70 shows the results of gene profiling experiments to compare gene expression in the cell line culture (H460T) and primary culture (87426A1) tumor models, See Example 3. The expression difference for genes that are above noise level in the H460T data set were compared. Statistical analysis yielded the False Discovery Rate of 0.0015.
Figure 7E is a bar graph showing mRNA levels of CO24 and C044 in serum-free primary culture of 87426 cells at days 0, 12, and 24 of differentiation.
Cultures were maintained in BEBM growth medium or differentiated for 12 and 24 days in CnT-23 medium containing 50nM retinoic acid and 1mM CaCl2, without exposing cells to the air-liquid interface.
Figure 7F shows the distribution of CD24 and CD44 expression in duplicate samples from the experiment presented in in Figure 7D. After 12 days of differentiation as a monolayer culture, the levels of cell surface CD24 expression increased and were retained up to 24 days. Cell surface levels of CD44 expression were decreased by 12 days and declined further by 24 days of differentiation.
Figure 7G is a bar graph showing mRNA levels of angiogenesis factors in serum-free primary culture of 87426 cells at day 0, 12, and 24 of differentiation, The levels of mRNA were determined using gene expression profiling as described in Example 6.
Figures 8A-8B show heterogeneous 5T4 expression in NSCLC primary implant xenografts.
Figure 8A shows flow cytometric analysis of 514 expression in dissociated xenografts from the 37622 line (left panel), cultured cells established from xenografts in serum-free medium (middle panel; shown after five weeks in culture), and in xenografts of implanted cells of the serum-free culture (right panel).

- 8 -

9 Figure 8B is a table showing tumor incidence in animals that were implanted with 5T4"h or 5T410w cells from dissociated 37622 or 60257 xenografts (#1, #2 indicate replicate experiments).
Figures 9A-90 show that Sox-2 induces differentiation of CD2441m cells.
Figure 9A is a bar graph showing Sox-2 mRNA levels from Affymetrix GENECHIW' oligonucleotide arrays hybridized with transcripts prepared from CD24-110w clones.
Figure 9B shows the results of immunoblot analysis to detect Sox-2 and Sox-11 transcripts in stable CD24-11" clones transfected with Sox-2-Flag, Sox-Flag, empty vector, or no DNA. Cells were harvested 24 hours after transfection and subjected to immunoblot with anti-Flag (top) or anti-3-actin antibody.
Figure 9C shows the results of flow cytometric analysis of CO24 in stable CD24-10'" clones transfected with Sox-2-Flag, Sox-11-Flag, empty vector, or no DNA after three weeks in culture.
Figure 9D is a histogram of the data presented in Figure 9C showing the percentage of 24"h cells in each sample.
Figures 10A-10B show sensitivity of CD24-'CD44+ cells to an anti-5T4-calicheamicin conjugate. CD24-11"CD44+ cells are labeled "CD24-iE"" and CD24"hCD44+ cells are labeled "CD24.
Figure 10A is a line graph showing the results of a four-day MTS assay.
Crosshairs indicate the sensitivity to free calicheamicin.
Figure 10B is a line graph showing the results of a clonogenic assay.
Crosshairs indicate the sensitivity to free calicheamicin.
Figure 11 is a line graph that showstumor growth of 5T4high and 5Tew cells in H460T clonal line 24N-26, which showed higher expression than in the H460T parental cell line. Cells were sorted based upon 5T4 expression and implanted subcutaneously into mice.
Values indicate the average tumor measurement SEM.
Figures 12A-12C show tumor volume regression of primary implant xenografts treated with an anti-5T4 antibody-calicheamicin conjugate. Diamond, vehicle; square, anti-5T4-calicheamicin conjugate: circle, anti-CD33-calicheamicin conjugate; triangle, cisplatin; asterisk (*), p<0.05.
Figure 12A is a line graph showing tumor volume regression of 37622 xenografts following treatment with anti-5T4 antibody-calicheamicin conjugate.
Animals were administered anti-5T4 antibody-calicheamicin conjugate on days 1, 5, and 9 after staging. Values indicate average tumor volume SEM.
Figure 12B a line graph showing tumor volume regression of 60274 xenografts following treatment with anti-5T4 antibody-calicheamicin conjugate.

Animals were administered anti-5T4 antibody-calicheamicin conjugate on days 1, 5, and 9 after staging. Values indicate average tumor volume SEM.
Figure 12C a line graph showing tumor volume regression of 60274 xenografts following treatment with anti-5T4 antibody-calicheamicin conjugate.

Animals were administered anti-514 antibody-calicheamicin conjugate on days 1.

5, and 9 after staging Values indicate average tumor volume SEM.
Figure 13 is a bar graph showing 5T4 (TPBG) mRNA levels on an Affymetrix GENECHIP oligonucleotide arrays hybridized with transcripts from multiple tumors (Ti 12, T3) of NSCLC primary implant lines 37622, 60274, and 60257. The 37622 xenografts were from nude mice and the 60274 and 60257 xenografts were from nod-scid mice.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods for the prospective identification of tumor-initiating cells that express the oncofetal antigen 5T4 and optionally, also express CD44 and low levels of CD24, These cells are highly tumorigenic in vitro and in viva are self-renewing, are capable of migration, and have the ability to differentiate. The disclosed tumor-initiating cell populations may also show apoptosis resistance and contribute to cancer relapse and metastasis. Also provided are methods for isolating tumor-initiating cell populations and for enriching tumor-initiating cells within a population. Still further are provided novel tumor-initiating cell markers.

-10-The tumor-initiating cell populations disclosed herein are useful for studying the effects of therapeutic agents on tumor growth, relapse, and metastasis.
Isolated tumor-initiating cells can be used to identify unique therapeutic targets, which can be used to generate antibodies that target tumor-initiating cells. The isolated tumor-initiating cells can also be used in screening assays to improve the probability that drugs selected based upon in vitro activity, or based upon cytotoxicity of tumor populations that include non-turnorigenic cells, will successfully eradicate disease and prevent relapse in vivo. Tumor-initiating cells isolated from patients may also be used to predict disease outcome and/or sensitivity to known therapies.
I. Tumor-Initiating Cells A tumor-initiating cell is known in the art to mean a cell (1) that is capable of generating one or more kinds of progeny with reduced proliferative or developmental potential (e.g., differentiated cells); (2) that has extensive proliferative capacity; and (3) that is capable of self-renewal or self-maintenance.
See e.g., Patten et al., Development, 1990, 110; 1001-1020. Thus, tumor-initiating cells share properties of stem cells found in adult tissues, (including cells of the blood, gut, breast ductal system, and skin) that constantly replenish cells lost during normal tissue functions.
The best-known example of adult cell renewal by the differentiation of stem cells is the hematopoietic system. Developmentally immature precursors such as hem atopoietic stem cells and progenitor cells respond to molecular signals gradually forming the varied blood and lymphoid cell types. Stem cells are also found in other tissues, including epithelial tissues (Slack. Science, 2000, 287: 1431-1433) and mesenchymal tissues (U.S. Patent No. 5.942,225). Cancer stem cells may arise from any of these cell types, for example, because of genetic damage in normal stem cells or by the dysregulated proliferation of stem cells and/or differentiated cells.
Tumor-initiating cells of the present invention may be derived from any cancer comprising tumorigenic stem cells, i.e., multipotent cells having an ability

-11-to proliferate extensively or indefinitely, and which give rise to the majority of cancer cells, Within an established tumor, most cells have lost the ability to proliferate extensively and form new tumors, and a small subset of tumor-initiating cells proliferate to thereby regenerate the tumor-initiating cells as well as give rise to tumor cells lacking tumorigenic potential. Tumor-initiating cells may divide asymmetrically and symmetrically and may show variable rates of proliferation.
In contrast to tumor-initiating cells, non-tumorigenic tumor cells fail to form a palpable tumor upon transplantation into an immunocompromised host, wherein if the same number of non-fractionated, dissociated cancer cells were transplanted under the same circumstances, the tumor-initiating cells would form a palpable tumor in the same period. A palpable tumor is known to those in the medical arts as a tumor that is capable of being handled, touched, or felt, Non-tumorigenic cells also show decreased migration as compared to tumor-initiating cells, an inability to generate tumor-initiating cells, and increased expression of differentiation markers.
Representative cancers from which tumor-initiating cells may be isolated include cancers characterized by solid tumors, including 5T4-expressing tumors such as lung, ovarian, colorectal, and gastric tumors. Additional representative cancers from which tumor-initiating cells may be isolated include acoustic neuroma, acute lymphoblastic leukemia, acute myelogenous leukemia, acute myeloid leukemia, adenocarcinoma, adenosqaumous carcinoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, angiosarcoma, astrocytoma, B
cell lymphomas and leukemias, basal cell carcinoma, basaloid carcinoma, bile duct cancer, bile duct carcinoma, bladder cancer, bladder carcinoma, brain tumor, breast cancer, bronchogenic carcinoma, bulky disease NHL and VValdenstrom's Macroglobulinemia, carcinosarcom a, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, clear cell carcinoma, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, cystadenocarcinoma,

-12-embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's sarcoma, Ewing's tumor, fibrosarcoma, gallbladder cancer, gestational trophoblastic tumor, giant cell carcinoma, glioma, hairy cell leukemia, hemangioblastoma, hemangiomas of infancy and childhood, hematopoietic malignancies, hematopoietic malignancies including acute lymphoblastic leukemia, hepatoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL.
Hodgkin's lymphoma, hypopharyngeal cancer, including but not limited to low grade/follicular non-Hodgkin's lymphoma (NHL), intermediate grade diffuse NHL, intermediate grade/follicular NHL, islet cell carcinoma. Kaposi's sarcoma, kidney cancer, large cell carcinoma with rhabdoid phenotype, large cell lung carcinoma, laryngeal cancer, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcorria, lymphoblastic leukemia, lymphocytic leukemia, lymphoepitheliomalike carcinoma, malignant melanoma, malignant mesothelioma, malignant thymoma, medullary carcinoma, medulloblastoma melanoma, meningioma, mesothelioma, monocytic leukemia, multiple myeloma, mycosis funoides, myelogenous leukemia, myxosarcoma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung carcinoma, oligodendroglioma, oral cancer, oropharyngeal cancer.
osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic carcinoma, papillary adenocarcinomas, papillary carcinoma, parathyroid cancer, penile cancer, pinealoma, pituitary tumor, promyelocytic leukemia, prostate cancer, prostate cancer, pulmonary blastoma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, small cell lung carcinoma, small intestine cancer, small lymphocytic (SL) NHL, soft tissue sarcoma, squamous cell carcinoma, squamous cell lung carcinoma, stomach cancer. sweat gland carcinoma, synovioma, testicular cancer, thyroid cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilm's tumor.

- 13-Tumor-initiating cells may also be derived from cells associated with a proliferative disease, i.e., a class of diverse disorders and diseases characterized by a lack of control or poorly controlled cell division or proliferation.
Proliferative diseases include disorders associated with an overgrowth of connective tissues, such as various fibrotic conditions, including scleroderma, arthritis, juvenile arthritis, gouty arthritis, and liver cirrhosis, and conditions such as restenosis, arteriosclerosis, and proliferative diabetic retinopathy.
1,A. Tumor-Initiating Cell Markers Tumor-initiating cells may be selected using positive and negative molecular markers. A reagent that binds to a tumor-initiating cell positive marker (i.e., a marker expressed by tumor-initiating cells at elevated levels compared to non-tumongenic or differentiated cells) can be used for the selection of tumor-initiating cells. Positive markers for tumor-initiating cells may also be present on non-tumorigenic cancer cells, i.e., cancer cells other than tumor-initiating cells, but at reduced levels. Markers that are widely expressed may show a measurable change in expression level in tumor-initiating cells and/or may provide for resolution of tumor-initiating cells when used in combination with additional positive or negative markers. A reagent that binds to a tumor-initiating cell negative marker (i.e., a marker not expressed or expressed at measurably reduced levels by tumor-initiating cells can be used for the elimination of those tumor cells in the population that are not tumor-initiating cells For selection using positive and negative molecular markers, useful markers include those that are expressed on the cell surface such that live cells are amenable to sorting and tracking.
When assessing expression levels using techniques such as immunoblot, tumor-initiating cell positive markers are typically expressed at a level that is at least about 2-fold greater than differentiated cells of the same origin or non-tumorigenic cells, for example, at least about 4-fold greater, or at least about 5-fold greater, or at least about 8-fold greater, or at least about 10-fold greater, or at least about 15-fold greater, or at least about 20-fold greater, or at least about

- 14-50-fold greater, or at least about 100-fold greater. When assessing expression levels using flow cytometry, tumor-initiating cell positive markers are typically expressed at a level that is at least about 0.5 log greater than differentiated cells of the same origin or non-tumorigenic cells, for example, at least about 1 log greater. at least about 1.5 logs greater, at least about 2 logs greater, or at least about 3 logs greater. Conversely, when assessing expression levels using techniques such as immunoblot, tumor-initiating cell negative markers are typically expressed at a level that is at least about 2-fold less than differentiated cells of the same origin or non-tumorigenic cells, for example, at least about fold less, or at least about 8-fold less, or at least about 10-fold less, or at least about 15-fold less, or at least about 20-fold less, or at least about 50-fold less, or at least about 100-fold less. When assessing expression levels using flow cytometry, tumor-initiating cell negative markers are typically expressed at a level that is at least about 0.5 log less than differentiated cells of the same origin or non-tumorigenic cells, for example, at lease about 1 log less, at least about 1.5 logs less, at least about 2 logs less, or at least about 3 logs less.
Disclosed herein are 514, CD44, and CD24 markers that can be used alone or in combination for the prospective identification and isolation of tumor-initiating cells from lung. Expression of 5T4 and CD44 are positive markers.
whereas expression of CD24 is a negative marker. Thus, tumor-initiating cells of the invention include those expressing high levels of 5T4 (514m), moderate to high levels of C044 (CD44), and/or little or no expression of CO24 (CD24-11 ).

514 oncofetal antigen is a 72 kDa highly glycosylated transmembrane glycoprotein comprising a 42 kDa non-glycosylated core (Hole et al., Br. J.
Cancer, 1988, 571 239-46; Hole et al., Int. J. Cancer, 1990, 45: 179-84; PCT
International Publication No. W089/07947: U.S. Patent No. 5,869,053). Human 514 is expressed in numerous cancer types, including carcinomas of the bladder, breast, cervix, endometrium, lung, esophagus, ovary, pancreas, stomach, and testes, and is substantially absent from normal tissues, except for syncytiotrophoblast in placenta (see, e.g,, Southall et alõ Br. J. Cancer, 1990, 61:
89-95 (immunohistological distribution of 514 antigen in normal and malignant

-15-tissues); Mieke et al., Clin. Cancer Res., 1997, 3: 1923-1930 (low intercellular adhesion molecule 1 and high 5T4 expression on tumor cells correlate with reduced disease-free survival in colorectal carcinoma patients); Starzynska et al.
Br. J. Cancer, 1994, 69: 899-902 (prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma); Starzynska et al,. Br. J Cancer, 1992, 66: 867-869 (expression of 5T4 antigen in colorectal and gastric carcinoma); Jones et al., Br. J. Cancer, 1990, 61: 96-100 (expression of 5T4 antigen in cervical cancer); Connor & Stern, Int. J. Cancer, 1990, 46: 1029-(loss of MHC class-I expression in cervical carcinomas); Ali et al., Oral Oncology, 2001, 37: 57-64 (pattern of expression of the 514 oncofetal antigen on normal, dysplastic and malignant oral mucosa): PCT International Publication No.
W089/07947; US. Patent No, 5,869,053). For example, tissues reported to have no expression of 514 include the liver, skin, spleen, thymus, central nervous system (CNS), adrenal gland, and ovary. Tissues reported to have focal or low expression of 5T4 include the liver, skin, spleen; lymph node, tonsil, thyroid, prostate, and seminal vesicles. Weak-moderate diffuse expression of 514 has been reported in the kidney, lung, pancreas, pharynx, and gastro-intestinal tract. The only tissue reported to have high expression of 5T4 is syncytiotrophoblast. and 514 was also absent from normal serum or the serum of pregnant women (i.e., levels < 10 ng/rn1). Overexpression of 5T4 in tumors has been correlated with disease progression, and assessment of 514 expression has been suggested as a useful approach for identifying patients with poor prognosis. See, e.g., Mulder et al., Din. Cancer Res., 1997, 3: 1923-1930;
Naganuma et al., Anticancer Res,. 200, 22: 1033-1038: Starzynska et al., Br.
J.
Cancer, 1994, 69: 899-902; Starzynska et al., Eur. J. Gastroenterol. Hepatol., 1998, 10: 479-484; Wrigley et al., Int. J. Gynecol. Cancer, 1995, 5: 269-274.
C044 is a transmembrane glycoprotein that participates in cancer metastasis by modulating cell adhesiveness, motility, matrix degradation, proliferation, and/or cell survival. See e.g., Marhaba & Zoller, J. Mot Histol., 2004, 35(3): 211-231. CO24 antigen is a cell surface glycoprotein marker of differentiation that is used as a negative marker, i.e., tumor-initiating cells show

- 16-little or no CD24 expression (CD2e0w).
Alone or in combination, cells expressing CD44 and CD24-10w have been used to identify tumor-initiating cells in many tumor types, including tumors of the breast, colon, head and neck, and pancreas. See e.g., Al-Hajj et al., Proc. Natl. Acad. Sci, USA, 2003, 100:

3988; Yu et al., Cell, 2007, 131: 1109-1123; Dalerba et al.; Proc. Natl. Acad.
Sc!.
USA, 2007, 104: 10158-10163; Prince et al., Proc. Natl. Acad, Sc!, USA, 2007;
104: 973-978; and Li et al., Cancer Res., 2007, 67: 1030-1037, In addition to the 514 and CD44+ markers described herein, other potential tumor-initiating cell positive markers in lung cancer include SLUG;
fibronectin (FN1), and vimentin (VIM) (see Figure 7C); vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B);
vascular endothelial growth factor C (VEGF-C); platelet derived growth factor (PDGF), and insulin-like growth factor-I (PIGF) (see Figure 7F); CD133 (see Eramo et al., Cell Death Differ., 2008, 15: 504-514); and C0117 (Donnenberg et al., J. Control Release, 2007; 122(3): 385-391). Representative additional potential positive tumor-initiating cell markers in lung cancer include transforming growth factor p type III receptor (TGF13R111), netrin receptor UNC5D (Unc5D), patatin-like phospholipase domain-containing protein 4 (PNPLA4), inward rectifier potassium channel 2 (KCN,12), gamma-aminobutyric acid (GABA) A
receptor, beta 3 (GABRB3), dihydropyrimidine dehydrogenase (DPYD), sperm associated antigen 1 (SPAG1), intestinal cell (MAK-like) kinase (ICK), stanniocalcin 2 (STC2); defensin131 (Deff31), and FLJ38736. See Example 5.
Still additional positive tumor-initiating cell markers include CD133 (Bao et al.. Nature, 2006, 444: 756-760; O'Brien et al., Nature, 2007, 445: 106-110;
Ricci-Vitiani et al., Nature, 2007, 445: 111-115; and Hermann et al.; Cell Stem Cell, 2007, 1: 313-323), ALDH1 (Yu et al., Cell 2007, 131: 1109-1123), EpCAMhgh (Dalerba et al., Proc. Natl. Acad. Sc!. USA, 2007, 104: 10158-10163);

epithelial-specific antigen (ESA, Li et al., Cancer Res., 2007, 67: 1030-1037);
C090 (Li et al., Cancer Res., 2007, 67: 1030-1037); ABCG5 (Schatton et al., Nature, 2008, 451: 345-349); ABCG2 (Patrawala et al., Cancer Res., 2005, 65(14): 6207-6219; Kondo et al., Proc. Natl. Acad. Sc!. U.S.A., 2004, 101(3):

-17-781-786): VEGF receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, and platelet derived growth factor (PDGF) (see Figure 7F and Andersen et al., I. Thorac.
Oncol., 2009, [Epub ahead of print]); neuron-specific enolase (NSE), cytokeratin 19 fragment (CYFRA), Carcinoembryonic antigen (CEA), squatrious cell carcinoma antigen (SCC), CA 125, CA 15.3 and TAG-72.3 (see Molina et al., Tumour Biol., 2008, 29(6): 371-380); VLA-2, Tweak (INF-like weak inducer of apoptosis), EphB2, EphB3, human Sca-1 (BIG1), CD34, 31 integrin (CD29), CD150, CXCR4, and members of gene sets that are inversely correlated with differentiated primary culture as set forh in Tables 1 and 2. See Examples 3 and 5.
Markers that may be used for selection of tumor-initiating cells based upon low or negative expression include any gene expressed in differentiated or non-tumorigientic cells. Numerous such molecules are known in the art. In addition to the CD244t(m marker described herein, additional lung cancer tumor-initiating cell negative markers include MUCliCD227 and cytokeratin 4 (KRT4) (see Figure 7C and Kuemmel et al., Lung Cancer, 2009, 63(1): 98-105). Still additional makers useful for isolation or enrichment of lung cancer tumor-initiating cells include CEA, SLX, CYFRA, SCC, pro-gastrin-releasing peptide (ProGRP).
See Komagata & Yondea, Gan To Kagaku Ryoho, 2004, 31(10): 1609-1613.
Additional representative markers that may be used for selection of tumor-initiating cells based upon low or negative expression include members of gene sets that are correlated with differentiated primary culture as set forh in Tables 1 and 2. See Examples 3 and 5.
The above-noted markers can also be use for identification of tumor-initiating cells in cancers other than lung cancer.
In the case of colon or colorectal cancer or other cancers, additional positive markers that may be useful for identification of tumor-initiating cells include prostaglandin F2 receptor regulatory protein (PTGFRN), CD166 (or activated leukocyte adhesion molecule, ALCAM), CD164, CD82, transforming growth factor beta receptor 1 (TGFBR1), MET, ephrin-B2 (EFNB2), integrin alpha 6 (ITGA6; CD49f), teratocarcinoma-derived growth factor 1 (TDGFI), heparin-

- 18 -binding EGF-like growth factor (HBEGF), ABC family transporter ABCC4, ABC
family transporter ABC03, tumor-differentially-expressed gene 2 (TDE2), integrin beta 1 (ITGB1), tumor necrosis factor receptor superfamily 21 (TNFRSF21), C081 and CD9 (members of the transmembrane-4 superfamily (TM4SF or tetraspanins)), KIAA1324, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), FZD6 and FZD7 (VVnt receptors), BMPR1A, JAG1, integrin alpha V (ITGAV), NOTCH2, SOX4, HES1, HES6, atonal homolog 1 (ATOH1), E-cadherin (CDH1), Eph receptor 82 (EPHB2), v-myb myeloblastosis viral oncogene homolog (MYB), MYC, SOX9, PCGF1, PCGF4, PCGF5, ALDH1A1, and STRAP. An additional negative marker useful for identification of tumor-initiating cells in colon or colorectal cancer is T cell factor 4 (TCF4). See e.g., PCT International Publication No. WO 07/053648.
In a particular aspect of the invention, isolated tumor-initiating cell population comprises a majority of cells expressing 514 at a level that is at least about 2-fold higher than non-tumorigenic cells of the same origin. Tumor-initiating cells may also express 5T4 at a level that is at least about 4-fold higher than non-tumorigenic cells of the same origin, for example, at least about 5-fold higher, or at least about 8-fold higher, or at least about 10-fold higher, or at least about 15-fold higher, or at least about 20-fold higher, or at least about 50-fold higher, or at least about 100-fold higher. When 5T4 expression is assessed using flow cytometry, tumor-initiating cells may also express 5T4 at a level that is at least about 0,5 log higher than non-tumorigenic cells of the same origin, for example, at least about 1 log higher, or at least about 1.5 logs higher, or at least about 2 logs higher, or at least about 3 logs higher.
In another aspect of the invention, the tumor-initiating cell population comprises a majority of cells that express CD24 at a level that is at least about 2-fold lower than CD24 + non-tumorigenic cells of the same origin. Tumor-initiating cells may also express CD24 at a level that is at least about 4-fold lower than non-tumorigenic cells of the same origin, for example, at least about 5-fold lower, or at least about 8-fold lower, or at least about 10-fold lower, or at least about 15-fold lower, or at least about 20-fold lower, or at least about 50-fold lower, or at

-19-least about 100-fold lower. When CD24 expression is assessed using flow cytometry, tumor-initiating cells may also express CD24 at a level that is at least about 0.5 log lower than CD24 4' non-tumorigenic cells of the same origin, for example, at least about 1 log lower, or at least about 1.5 logs lower, or at least about 2 logs lower, or at least about 3 logs lower.
The tumor-initiating cell population can also comprise a majority of cells that express CD44 at a level that is at least about 2-fold higher than non-tumorigenic cells of the same origin. Tumor-initiating cells may also express CD44 at a level that is at least about 4-fold higher than non-tumorigenic cells of the same origin, for example, at least about 5-fold higher, or at least about 8-fold higher, or at least about 10-fold higher, or at least about 15-fold higher, or at least about 20-fold higher, or at least about 50-fold higher, or at least about 100-fold higher. When CD44 expression is assessed using flow cytometry, tumor-initiating cells may also express CD44 at a level that is at least about 0.5 log higher than non-tumorigenic cells of the same origin, for example, at least about 1 log higher, or at least about 1.5 logs higher, or at least about 2 logs higher, or at least about 3 logs higher.
An isolated tumor-initiating cell population is removed from its natural environment (such as in a solid tumor) and is at least about 75% free of other cells with which it is naturally present, but which lack the marker based on which the cells were isolated. For example, isolated tumor-initiating cell populations as disclosed herein are at least about 90%, or at least about 95%, free of non-tumorigenic cells. When referring to a tumor-initiating cell population that is described as a percentage purity, or a percentage free of non-tumorigenic cells, the cell stem cell subpopulation and total cancer cell population are typically quantified as live cells.
An enriched population of cells can be defined based upon the increased number of cells having a particular marker in a fractionated tumor-initiating cell population as compared with the number of cells having the marker in the non-fractionated cancer cell population. It may also be defined based upon tumorigenic function as the minimum number of cells that form tumors at limiting

- 20 -dilution frequency. An enriched tumor-initiating cell population can be enriched about 2-fold in the number of stern cells as compared to the non-fractioned tumor cell population, or enriched about 5-fold or more, such as enriched about 10-fold or more, or enriched about 25-fold or more, or enriched about 50-fold or more, or enriched about 100-fold or more. Enrichment can be measured with using any one of the tumor-initiating cell properties noted herein above, e.g., levels of marker expression or tumorigenicity.
The present invention provides methods for isolation of the disclosed tumor-initiating cell populations. For example, the method can comprise (a) providing dissociated tumor cells; (b) contacting the dissociated tumor cells with an agent that specifically binds to 5T4; (c) selecting cells that specifically bind to the agent of (b) at a level that is at least about 5-fold greater than cells that either do not express 514 or express 514 at a low level. The method can also comprise further selection based upon any of the positive or negative tumor-initiating cell markers disclosed herein or otherwise known in the art. When performing selection using a negative marker, e.g_ excluding cells that express one or more negative markers, tumor-initiating cells may be identified as cells that show reduced expression of the marker as compared to differentiated cells or non-tumorigenic cells. Representative methods for isolation or enrichment of 5Tegh. CD24w, and/or CD444' tumor-initiating cell populations are described in Examples 1-4, Tumor-initiating cells can be isolated or enriched by any suitable means known in the art, including FACS using a fluorochrome conjugated marker-binding reagent and primary culture using serum free conditions. Any other suitable method including attachment to and disattachment from solid phase, is also within the scope of the invention. Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography and panning with antibody attached to a solid matrix, e.g., a plate or other convenient support. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light

-21 -scattering detecting channels, impedance channels, eta Dead cells may be eliminated by selection with dyes that bind dead cells (such as propidium iodide (PI), or 7-RAD). Any technique may be employed that is not unduly detrimental to the viability of the selected cells.
LB. Functional Properties of Tumor-Initiating Cells As described herein, tumor-initiating cells of the invention are tumorigenic in vitro and in vivo, have characteristics of tumorigenic cells such as clonogenicity, and a highly proliferative nature. Subpopulations of lung tumor cell lines were identified that express 5Tegh, CD44+, and/or CD241"1 and that are significantly enriched for colony formation and proliferation. See Examples 1-4.
The injection of tumor-initiating cells into a host animal consistently results in the successful establishment of tumors more than 75% of the time, such as more than 80% of the time, or more than 85%, or more than 90%, or more than 95% of the time, or 100% of the time.
Cancer stem cells of the invention give rise to tumors with the same differentiation state of the tumor of origin. For example, tumor-initiating cells isolated from poorly and moderately differentiated tumors give rise to poorly and moderately differentiated tumors in vivo, respectively. The molecular profile of the resultant tumors is also similar to the tumor of origin, notwithstanding the prior selection of tumor-initiating cells. Thus, the tumor-initiating cells show a capacity to differentiate or give rise to non-tumorigenic cells that make up the majority of mature cancer populations.
The tumor-initiating cells of the invention have a capacity for self-renewal, as demonstrated by the ability of 514high and/or CD24-1 wCD44+ cells but not 5Tew and/or CD2e"hCD44+ cells to form tumors consistently. This feature allows tumor-initiating cells to retain multipotency and high proliferative potential throughout repeated cell divisions.
The tumor-initiating cells of the invention have a capacity for migration, as demonstrated by the ability of 5Tegh and/or CD24-'10wCD44+ cells to migrate.
In a transwell assay, 5T4hi9h and/or CD24-11"`CD44+ cells migrated in a serum-

- 22 -dependent manner more efficiently than 5Tew and/or CD24"IghCD44+ cells. In another assay, spheroids of 5T4high and/or CD24-1mCD44+ cells migrated across fibronectin coated slides, but no little or migration of cells in 5Tew and/or CD24+mghC044+ cells spheroids was observed after twenty-four hours.
IL Applications The tumor-initiating cell populations disclosed herein are useful for studying the effects of therapeutic agents on tumor growth, relapse, and metastasis. When isolated from a cancer patient, the efficacy of particular therapies can be tested and/or predicted based upon the unique genetic and molecular profile of the isolated population. Thus, the disclosed tumor-initiating cell populations provide means for developing personalized cancer therapies.
In one aspect of the invention, the genetic and molecular features of tumor-initiating cells are described to identify target molecules and/or signaling pathways. Accordingly, the present invention also provides arrays or microarrays containing a solid phase, e.g., a surface, to which are bound, either directly or indirectly, tumor-initiating cells (enriched populations of or isolated), polynucleotides extracted from tumor-initiating cells, or proteins extracted from the tumor-initiating cells. Monoclonal and polyclonal antibodies that are raised against the disclosed tumor-initiating cell populations may be generated using standard techniques. The identification of tumor-initiating cell target molecules, and agents that specifically bind tumor-initiating cells, will complement and improve current strategies that target the majority non-tumorigenic cells.
Microarrays of genomic DNA from tumor-initiating cells can also be probed for single nucleotide polymorphisms (SNP) to localize the sites of genetic mutations that cause cells to become precancerous or tumorigenic. The genetic and/or molecular profile of tumor-initiating cells may also be used in patient prognosis. See e.g., Glinsky et al., Jelin. Invest., 2005, 115(6): 1503-1521, which describes a death-from-cancer signature predictive of therapy failure.
in another aspect of the invention, the efficacy of cancer drugs or candidate cancer drugs can be tested by contacting isolated tumor-initiating cells

- 23 -with a test compound and then assaying for a change in tumor-initiating cell properties as described herein. For example, therapeutic compositions can be applied to tumor-initiating cells in culture at varying dosages, and the response of these cells is monitored for various time periods. Physical characteristics of these cells can be analyzed by observing cells by microscopy Induced or otherwise altered expression of nucleic acids and proteins can be assessed as is known in the art, for example, using hybridization techniques and Polymerase Chain Reaction (PCR) amplification to assay levels of nucleic acids, immunohistochemistry, enzymatic assays, receptor binding assays, enzyme-linked immunosorbant assays (ELISA), electrophoretic analysis, analysis with high performance liquid chromatography (HPLC), Western blots, radioimmunoassays (RIA), fluorescence activated cell sorting (FACs), etc.
The ability of therapeutic compounds to inhibit or decrease the tumorigenic potential of tumor-initiating cells can be tested by contacting turnor-initiating cells and a test compound, allowing a sufficient temporal period for response, and then assessing tumor-initiating cell growth in vitro. Following exposure to the test compound, the tumor-initiating cells can alternatively be transplanted into a host animal (i.e., preparation of a xenograft model, which is then monitored for tumor growth, cancer cell apoptosis, animal survival, etc.). In yet another screening format, test compounds are administered to a xenograft host animal (i.e., an animal bearing tumor-initiating cells and/or a resultant tumor). Additional phenotypes that may be assayed include cell viability, proliferation rate, regenerative capacity, and cell cycle distribution of tumor-initiating cells or resultant non-tumorigenic cancer cells, or any other phenotype relevant to therapeutic outcome.
Test compounds include known drugs and candidate drugs, for example, viruses, proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, antibodies, prodrugs, small molecules (e.g., chemical compounds), or any other substance that may have an effect on tumor cells whether such effect is harmful, beneficial, or otherwise. Test compounds include but are not limited to 2 2 2"-trichlorotriethylamine, 2-ethylhydrazide, 2 -pyrrolino-doxorubicin, 5-FU (5-

-24 -fluorouracii), 6-azauridine, 6-diazo-5-oxo-L-norieucine, 6-mercaptopurine, 6-thioguanine, a camptothecin, a sarcodictyin, ABRAXANEO. ABT-510 (Abbott Labs), aceglatone, acetogenins, aclacinomysins, actinomycin, ADRIAMYCINO, AG1478, AG1571 (SU 5271, Sugen), aidophosphamide glycoside, altretamine, -- aminoglutethimide, aminolevulinic acid, aminopterin, am sacrine, ancitabine, androgens, Angiostatin (EntreMed), Angizyme (AstraZeneca), anguidine, anti-metabolites, arabinoside (Ara-C"), authramycin, azacitidine, azaserine, aziridines, benzodopa, bestrabucil, bevacizumab, bevacizumab (AVASTINO
Genentech), bexarotene, bisantrene, bleornycin, BMS-275291 (Bristol Myers Squib), Bortezomib (VELCADE Millenium Pharm.), bryostatin, busulfan, cactinomycin, callystatin, calusterone, capecitabine, carabicin, carboplatin, carboquone, carrninomycin, carmofur, carmustine, carzinophilin, CC-1065, chlorambucii, chioranbucil, chlornaphazine, chiorozotocin, cholophosphamide, chromomycinis, cisplatin, Combrestatin (Oxigene), CPT-11, cryptophycins, -- cyanomorpholino-doxorubicin, cyclooxygenase-2 (COX-2) inhibitors exisulind, cyclophosphamide, cyclosphospharnide, cytarabine, CYTOXAN , dacarbazine, dactinomycin, daunomycin, daunorubicin, defofamine, demecolcine, deoxydoxorubicin, detorubicih, diaziquone, dideoxyuridine, difluorometlhylomithine (DM FO), docetaxel, dolastatin, doxifiuridine, doxorubicin.
-- clromostanolone propionate, duocarmycin, edatraxate, edatrexate, eleutherobin, elformithine, elliptinium acetate, enediyne antibiotics, eniluracil, enocitabine, epirubicin, epitiostanol, eriotinib (tarceva), Erlotinib (TARCEVA
Genentech/OSI
Pharm.), esorubicin, estramustine, ethylenimines, etoglucid, etoposide, etoposide (VP-16), floxuridine, fludarabine, folic acid analogues such as denopterin.
folic acid replenisher such as frolinic acid, fotemustine; France), Fulvestrant (FASLODEXO AstraZeneca), gacytosine, gallium nitrate, Gefitinib (IRESSAO
AstraZeneca), GEMZAR (gemcitabine), hydroxyurea, ibandronate, idarubicin, ifosfamide, Imatinib mesyiate (GLEEVEC Novartis), improsulfan and piposuifan, irinotecan, Lapatinib (GSK572016, lentinan, Letrozole (FEMARAO
-- Novartis), Leucovorin, lonnustine, Lonafamib (SC H 66336), lonidainine, losoxantrone, mannomustine, marcellomycin, marimastate (British Biotech),

- 25 -maytansinoids such as maytansine and ansamitocins, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan mepitiostane, mercaptopurine, rnethotrexate, methotrexate and 5-fluorouracil (5-FU), rnethylamelamines, meturedopa, mitobronitol, mitoguazone, mitolactol, mitomycin C, mitornycins, mitotane, mitoxantrone, mopidanmol, morpholino-doxorubicin, mycophenolic acid, MYLOTARGq (gemtuzumab ozogamicin, Wyeth), NAVELBINEO
(vinorelbine), Neovastat (Aeterna Zentaris), nimustine, nitraerine, nitrogen mustards, nogalamycin, novantrone; novembichin, olivomycins, or vinorelbine, ELOXATIN (Oxaliplatin Sanofi), paclitaxel, pancratistatin, pemetrexed disodium (ALIMTAO, pentostatin, peplomycin, phenamet, phenesterine, pipobroman, pirarubicin, podophyllinic acid, potfiromycin, prednimustine, procarbazine;
proteasome inhibitors, pteropterin, PTK787/ZK 222584 (Novartis), puromycin, quelamycin, ranimnustine, Rapamycin (Sirolimus, RAPAMUNEO, Wyeth), razoxane, retinoic acid, retinoids, rhizoxin, rodorubicin, roridin A, sizofuran, Sorafenib (6AY43 9006, Bayer), spirogermanium, spongistatin, streptonigrin, streptozocin. SU5416, SU6668 (Sugen), Sunitinib (Pfizer), SUTENTO (SU11248 Pfizer), T-2 toxin, TAXOL (paclitaxel; Bristol-Myers Squibb), TAXOTERE
(doxetaxel; Rhone-Poulenc Rorer), ternsirolimus (TORISELS, Wyeth), teniposide, tenuazonic acid, testolactone: anti-adrenals, thalidomide (Celgene), thiamiprine, thioguanine, thiotepa, topoisomerase inhibitor RFS 2000, topotecan, triaziquone, trichothecenes, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trilostane, trimethylomelamine, trimetrexate, trofosfamide, tubercidin, ubenimex, uracil mustard, uredopa, urethane, vaccines.
VEGF-Trap (Regeneron Pharm), verracurin A, vinblastine, vincristine, vindesine, vinorelbine, Vitaxin II (Medimmune) and Cilengitide (Merck KgaA), xeloda, ZD6474 (ZACTIMA AstraZeneca), zinostatin, zorubicin and pharmaceutically acceptable salts, acids derivatives and antibody conjugates of any of the above.
For use in any of the above-noted applications, or other applications, tumor-initiating cells of the invention may be cryopreserved until needed for use.
For example, the cells can be suspended in an isotonic solution, preferably a cell culture medium, containing a particular cryopreservant. Such cryopreservants

-26 -include dimethyl sulfoxide (DMSO), glycerol and the like. These cryopresewants are used at a concentration of 5-15%, such as 8-10% Cells are frozen gradually to a temperature of -10 C to -150 C, such as -20 C to -100 C, or at -150 C.
EXAMPLES
The following examples have been included to illustrate modes of the invention. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work well in the practice of the invention. These examples illustrate standard laboratory practices of the co-inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the invention, Isolation of CD24-1"CD44+ Tumor Initiating Cells Using Non Small Cell Lung Cancer Cell Lines H460 cells were obtained from the American Type Culture Collection (ATCC) in Manassas, Virginia, United States of America. The H460 cell line was derived from the pleural fluid of a patient with large cell cancer of the lung (Gazdar et al., Science, 1989, 246: 491494). HCC2429 cells were obtained from J. Minna. See Haruki et al., J. Med. Genet., 2005. 42(7)'558-64. All experiments with H460T cells were performed with cells between passage numbers 37-51, because these cells were observed to have more robust phenotypes than lower passage cells. Higher-passage cells are referred to as H460T to distinguish them from the low-passage H460 that were originally obtained from ATCC. All cells were incubated at 37 Celsius with 5.0% carbon dioxide (CO2). H460T cells were cultured in RPMI-1640 (GIBCO , available from Invitrogen of Carlsbad, California, USA), See Moore et al, JAMA, 1967, 199: 519-524, 10% fetal bovine serum (FBS, GIBCO , available from Invitrogen

- 27 -of Carlsbad, California, USA), 2mM additional glutamine, 1001U/mipenicillin, pg/ml streptomycin, 1 mM sodium pyruvate, 0.1% sodium bicarbonate, 0,45%
additional glucose, and 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). HCC2429 cells were cultured in RPMI-1640, 10% FBS, 2mM
additional glutamine, 100 ILPm! penicillin, 100 pg/ml streptomycin.
Karyotyping and short tandem repeat (STR) analysis of H460T confirmed its H460 origin, however the Y chromosome was absent in H460T cells and present in most H460 cells.
For flow cytometry analysis, cells were harvested with GIBCOO
TRYPLEim, washed in Hanks Balanced Salt Solution without calcium and magnesium (HBSS) with 3% heat-inactivated calf serum (HICS), incubated with 100 pg/ml DNase, 5 mM MgCl2 and 50 pg/ml human immunoglobulin (IgG), incubated with antibodies or isotype controls, washed, and resuspended in HBSS

with 3% HICS, 25 pg/m I DNase, 1 mM MgCl2, and 25 mM HEPES. Xenografts were minced to a paste-like consistency, incubated in Collagenase/Hyaluronidase (Stem Cell Technologies of Vancouver, British Columbia, Canada) for 1 hour with frequent mixing in a 37 C water bath, and filtered through a 40-micron filter. The cell suspension was treated with Red Blood Cell Lysis Buffer (Roche Diagnostics Corporation of Indianapolis, Indiana, USA) followed by ACCU-PREP (Axis of Oslo, Norway), both according to manufacturer's instructions. Anti-human CO24 (#555428) and CD44 (#559942) monoclonal antibodies were obtained from BD Biosciences of San Jose, California, USA). Anti-5T4 antibody clone H8 (Hole & Stern, Br, J. Cancer, 1988, 57(3): 239-46) was obtained from Oxford Biomedica of Oxford, United Kingdom.
Labeling the H460T NSCLC cell line with anti-CD24 and anti-CD44 antibodies revealed distinct populations with a stable distribution over long periods in culture (Figure 1A).When these populations were separated by fluorescence-activated cell sorting and implanted subcutaneously into immunocompromised nu/nu mice, the CD24-1 ''CD44+ cells formed large tumors rapidly, whereas the CD24highCD44+ cells slowly formed small tumors (p <0.001 Figures 1B-1C). CD24-11mCD44 cells also formed larger tumors than the third

- 28 -population, CO24410wCD4e1 (p < 0.02: Figure 1D). These studies demonstrated that CD2441 w and CD44+ enrich for tumorigenic potential in H460T.
The CD24-10wCD44+ cells were phenotypically distinct from the CD24hI9hCD4e cells in several additional assays. First, the CD24410wCD44+
cells grew more rapidly than the CD244hCD44+ cells in three-dimensional culture as spheroids (p <0001; Figure 1E). No difference in proliferation rate, cell cycle profile, or cell size was detected between the populations in two-dimensional culture.
Second, the populations exhibited a differential response to the mTOR
inhibitor CCI-779, a rapamycin analog that was recently approved for treatment of advanced renal cell carcinoma (Faivre et al., Nat, Rev. Drug Discov., 2006, 5:
671-688), The CD2ewCD44+ cells were 5- to 10-fold more resistant to CCI-779 than CD2eghCD444' cells (Figure 1F). In contrast, the populations responded equally to camptothecin, 5-fluoruracil, and ionizing radiation, indicating that the differential response to CCI-779 was specific.
Third, CD24-1"CD44+ cells migrated more efficiently than CD24h`ghCD44+
cells, as shown using a transwell migration assay and a spheroid growth assay.

To perform a transwell migration assay, sorted cells that were cultured overnight in growth medium and then serum-starved for 24 hours. 500,000 cells per well were plated in serum-free media in 8.0-micron pore 24mm-diameter transwells.
Media with or without serum was added to the outer chamber and cells were incubated for 16-18 hours. Cells were fixed with formaldehyde and stained with crystal violet. Cells were carefully scraped from the inner chamber with wet and dry Q-tips such that cells that had migrated to the outer chamber could be counted under the microscope. Eight to ten fields per well were counted. Using this assay, CD24-10wCD44+ cells migrated 2.5-fold more efficiently than CD2eghCD44+ cells in a serum-dependent manner (n=4: Figure 1G), To perform a spheroid growth assay, 100,000 sorted cells in 5m1 of culture medium were seeded on 60mm polystyrene cell culture dishes previously coated with 5m1 of tissue culture grade agar (0.7%) in culture medium. The dishes were incubated for 5 days at 37 C. Spheroids with a diameter of 0.2mm were selected

- 29 -and placed on fibronectin-coated slides (BD Biosciences). Migration of cells in CD24-10'CD44+ spheroids across the fibronectin-coated slides was evident after 24 hours and at subsequent timepoints, but little or no migration of cells in CD24h19hCD4e spheroids was observed (Figure 1 H). There was no difference in growth rate of the spheroids over the 3-day period of this experiment.
To determine whether the tumor-initiating population of H460T also possessed stem cell-like characteristics, sorted cells were maintained in culture and monitored regularly by flow cytometry. CD24-110WCD44+ cells always gave rise to a significant population of CD24hghCD44+ cells evident as early as three days after the sort (Figure 2A). In contrast, CD24"hCD44+ cells remained CD24"11CD44+ through the latest time point of two months post-sort (Figure 2A).
These results indicated a multipotency phenotype of the tumor-initiating cells. To determine whether the observed transition occurs in the context of the parental line, labeled and unlabeled populations were co-cultured. When co-cultured with CD24"tD44+ cells, CD24-1"CD444- cells were also multipotent (Figures 3A-3C), which implied that the mutlipotency phenotype exists in culture of parental H460T. Multipotency was also observed in vivo: xenografts grown from CD24-10wC044+ cells typically contained -50% CD24"'l cells (Figure 28).
Clonal analysis was performed to verify that the multipotency could be followed at the single cell level Colonies from single CD24-bwC044+ or CD24"hCD44+ cells were expanded into clonal lines. Most (23/31) of the CD24-II"CD44+ derived clonal lines contained >10% CD24h]gh cells ("transitioning clones"), but some (8/31) contained <1% CD24"h cells (stable clones"). All (6/6) CD241mghCD44+ derived clonal lines contained 100% CD24"h cells (Figure 20). Consistent with the above results from the sorted parental line, sorted CD24-11"CD44+ cells from all tested CD24-n"CD44+ clonal lines were highly tumorigenic, while sorted CD24"hC044+ cells from all tested CD24"hCD44+
clonal lines formed small or no tumors (Figure 20).
To test whether CD24m9hCD444' cells show reduced tumorigenic potential as compared to CD24-1mC044+ cells. transitioning CD24-10wCD44 clonal lines were sorted into CD24-1 wCD44+ and CD24highCD44+ cells, and the sorted cells

- 30 -were implanted into animals. In three clonal lines, the CD2441"CD44+ cells formed larger tumors than the CD24highCD44+ cells (p <0.005 in clone 24N-4; p = 0.01 in clone 24N-10; p < 0.05 in clone 24N-25; Figure 2E). No significant difference was observed in three other clonal lines. Thus CD24410wCD44+ cells from H460T can give rise to less tumorigenic, functionally distinct CD24tughCD44+
cells. These results demonstrate the existence of multipotent tumor-initiating cells in H460T.
Other NSCLC cell lines were assessed for heterogeneity with respect to CD24 and C044. The HCC2429 line (Dang et al, J. Natl. Cancer Inst., 2000, 92, 1355-1357) contained two distinct CD24 populations, CD24" and CD24high (Figures 4A). FACS-isolated CD2440w cells formed significantly larger tumors than CD24hi9h cells (p < 0.05; Figure 4B). In addition, CD24" cells gave rise to CD24 high cells in culture, whereas CD24hi9h cells remained CD24 high (Figure 4C).

Identification of 5T4+ Tumor Initiating Cells In Non Small Cell Lung Cancer Cell Lines To identify genes that might underlie the phenotypic differences between CD24410wCD44+ and CD24h CD44+ cells, gene expression profiles were generated from triplicate samples of FACS-isolated populations. As expected, the mRNA levels of CO24 were consistently high in CD24hignCD44+ cells and low in CD24410wCD44. cells (Figure 5). Levels of 514 (also known as TPBG) were 4.5-fold higher in CD244I0wCD44+ cells compared to CD24highCD44+ cells (Figure 6B).
To determine 5T4 expression under conditions of growth and differentiation, cells were harvested at several time points, and protein extracts were subjected to immunoblot analysis. Cells were washed with PBS and lysed in 0.5% %/iv NP40 in 25m1µ.4 Tris-buffered saline pH 7.4 (TBS). After protein estimation (M1CROBCArm Protein Assay Kit, Pierce of Rockford, Illinois, USA), the lysates were mixed with non-reducing Laemmli sample buffer (Biorad of Hercules, California, USA) and 10pg samples were loaded in each well of a non-

-31 -reducing 4-20% polyacrylamide gradient gel (NOVEXO, available from Invitrogen of Carlsbad, California, USA). The samples were run for two to three hours at 125 volts and transferred to a PVDF (polyvinylidene fluoride) membrane by means of a Novex electrophoresis transfer system. The membrane was blocked overnight with 5% milk in Tris-buffered saline Tween-20 (TBST) with 1%
goat serum, probed with anti-5T4 antibody H8 at 1pg/m1 in 5% milk in TBST, washed and probed with HRP conjugated goat anti-mulgG at 1:5,000 dilution.
The ECL detection system was used (Amersham of Burlington, Massachusetts, USA). lmmunoblot analysis with anti-5T4 antibody showed expression of 5T4 protein in CD24410/CD44+ but not CD24h1 CD44+ cells (Figure 6A).
Immunofluorescence of parental H460T with anti-5T4 and anti-CD24 antibodies demonstrated that 5T4 and CD24 stainings were exclusive and that nearly all of the CD24"110wCD44+ cells also expressed 5T4, 5T4 was expressed in all of the CD2441m/CD44+ clonal lines described in Example 1.
To further assess 514 expression in tumor cell lines, sorted cells were treated with all-trans retinoic acid to induce differentiation and then subjected to immunoblot analysis. Sorted cells were obtained by FACS and 2.3x105 cells of each immunophenotype were plated in 6-well dishes in complete growth medium. After 24 hours, medium was removed, cells were washed 2 times with PBS and re-fed with 0.5% FBS growth medium. Medium was removed 24 hours later and replaced with 0.5% FBS growth medium supplemented with vehicle control or 1 OpM all-trans retinoic acid (Sigma of St. Louis, Missouri, USA).
Cells were cultured for 72 hours, washed in PBS and lysed directly in ix Laemmli buffer (Bio-Rad of Hercules, California, USA) for anti-5T4 western blot analysis.
514 expression was dramatically reduced in the treated CD24-ficmCD444. cells (Figure 6A), which indicated that 5T4 was associated with the undifferentiated state in cancer cells.

-32-A Differentiation Model for Tumor Initiating Cells In Non Small Cell Lung Cancer Primary Cultures Primary serum-free cultures were established from freshly resected NSCLC samples, The cells were cultured under conditions to promote self-renewal or induced to differentiate by exposure to the air-liquid interface in the presence of retinoic acid. The air-liquid interface is considered to be a physiological environment for lung cells and has been used to study fetal lung development (Vaughan et al. Differentiation, 2006, 741 141-148). To induce differentiation using this model, cultures were prepared and treated as follows.
Millicell 11.AM PET hanging cell culture inserts (Millipore of Billerica, Massachusetts. USA) were placed inside 6-well dishes. Membranes were pre-wet with phosphate buffered saline (PBS). 25x105 primary cells obtained from 87426A1 tumor tissue were plated onto each insert and filled with BEBM
medium. After 1-2 days, medium was removed from upper and lower chambers, rinsed with PBS and CnT-23 medium containing 50nM retinoic acid and 1mM
CaCl2 (Millipore of Billerica, Massachusetts, USA) was added back to lower chamber leaving cells in upper chamber exposed to the air. Lifted cultures were fed every 2 days with fresh medium or harvested at indicated time points in Buffer RLT (QIAGEN of Valencia, California, USA) for RNA isolation or TBS
(Tris-buffered saline)/0.5%NP40 (Tergitol-type NP-40, Sigma-Aldrich of St, Louis, Missouri, USA) for anti-5T4 Western blot analysis. For gene expression profiling, replicate growth samples were analyzed together, and due to limited sample, differentiation samples from days 8, 16, and 24 were pooled and analyzed together. Live cell imaging revealed that monolayer cultures efficiently formed 3D-stratified epithelium upon exposure to the air-liquid interface and 50 nM
retinoic acid for 18 days (Figure 7A).
To determine 5T4 expression under conditions of growth and differentiation, cells were harvested at several timepoints, and protein extracts were subjected to immunoblot analysis as described in Example 2. 5T4 levels

- 33 -were high under growth conditions and decreased quickly and dramatically upon differentiation (Figure 78).
To obtain a global view of this differentiation model, the experiment was repeated and gene expression profiles were generated from cells under conditions of growth and differentiation. See Examples 5 and 6. Consistent with the above results, 514 expression decreased and CD24 expression increased during differentiation (Figure 70). The gene expression profiles of the primary culture in growth and differentiation were also compared to those of the H460T

CD24-1mCD44+ and CD24highCD44+ populations (see Example 5). A significant fraction of the genes that were expressed at higher levels during differentiation of the primary culture were also expressed at higher levels in the CD24"11CD44+
cells (FDR = 0.0015). For statistical comparison of the H4601 and 87426 data sets, the top 250 upregulated genes in the differentiated 87426 culture were compared in the H460T populations. Figure 7D shows the expression difference for genes that are above noise level in the H4601 data set. Statistical analysis yielded the False Discovery Rate of 0.0015. This analysis indicates that these very different experimental systems are physiological models of the differentiation hierarchy in NSCLC. The microarray data were confirmed by flow cytometry (Figures 7E-7F).
The expression profiles also revealed striking patterns of genes involved in epithelial-mesenchymal transition and angiogenesis. The epithelial-mesenchymal transition markers vimentin, fibronectin, Slug, and Twist were expressed at high levels under growth conditions compared to the differentiated state (Figure 70). In contrast, the epithelial markers mucin and several cytokeratins were expressed at high levels during differentiation compared to growth conditions (Figure 7C). The angiogenesis factors VEGF-A, -B, C. PDGF-A and -C, and PIGF, were expressed at significantly higher levels under growth conditions compared to differentiation (Figure 7G).
An unbiased meta-analysis of the expression data revealed several gene signatures with significant expression changes during differentiation (Table 1).
Gene sets with higher expression in undifferentiated cells included signatures of

-34 -poor clinical prognosis, stem cells, oncogenic signaling, and developmental signaling. Gene sets with higher expression in differentiated cells included signatures of better clinical prognosis, differentiated tumors, and differentiated cells. Information from the Broad Institute's Molecular Signatures Database used in this analysis is presented in Table 2, including a list of genes (members) for each of the gene sets identified in Table 1. All NTk's in Table 1 have a false discovery rate (FDR) <= 0.01. NTk > 0 indicates direct correspondence with differentiated primary culture. NTk < 0 indicates inverse correspondence with differentiated primary culture.

Gene Sets With Significant Changes During Differentiation of a Primary Serum-Free Culture of NSCLC

Additional notes NTk2 HCC SURVIVAL GOOD VS POOR UP 3.816 Up in HCC with good survival LIZUKA_G1_GR_G2 3.844 Enriched in well- vs. moderately differentiated HCC

_ _ _ 4.515 Up during differentiation of 3T3-L1 into adipocytes ADIP DIFF UP 4.384 Up during differentiation of 3T3-L1 into adipocytes TISSUE_DEVELOPMENT 4.182 Gene Ontology Term EPIDERMIS DEVELOPMENT 7.495 Gene Ontology Term MORPHOGENESIS_OF_AN_EPITHELIUM 4.153 Gene Ontology Term

- 35 -KERATINOCYTE DIFFERENTIATION 11.11 Gene Ontology Term ECTODERM_DEVELOPMENT 6,949 Gene Ontology Term EPITHELIAL CELL DIFFERENTIATION 6,661 Gene Ontology Term VANTVEER_BREAST_OUTCOME_GOOD_VS_POOR_DN -5.368 Poor prognosis marker genes BRCA_PROGNOSIS_NEG -4,922 Higher expression associated with poor prognosis HCC_SURVIVAL_GOOD_VS_POOR_DN -8.651 Expressed in HCC with poor survival LI FETAL VS WT KIDNEY DN -7.394 Down in fetal kidney vs Wilms tumor HSA05222 SMALL CELL LUNG CANCER -5.451 Genes involved in SCLC
FLOTHO CASP8AP2 MRD DIFF -4.529 Associated with minimal residual disease in ALL
CANCER NEOPLASTIC META UP
-4.754 Meta-analysis: tumor relative to normal tissue CANCER_UNDIFFERENTIATED_META_UP -7.089 Meta-analysis: upregulated in undifferentiated cancer STEMCELL COMMON UP -4.524 Enriched in mouse ESC, HSC, NSC
BHATTACHARYA ESC UP -4,198 Upregulated in undifferentiated hESC
BROWN MYELOID PROLIF AND SELF RENEWAL -4,33 IDX TSA ON CLUSTER2 -5.681 _ Down during 3T3-L1 differentiation

-36 -ADIPOGENESIS_HMSC_CLASS8_DN -4.235 Down during differentiation of hMSC into adipocytes CTNNBl_oncogenic_signature -4.174 Cells expressing activated beta-catenin WNT_TARGETS -4.321 From literature TGFBETA_C4_UP -7.326 Up by TGFb treatment of fibroblasts TGFBETA_EARLY_UP -4.085 Up in skin fibroblasts after TGFb (early) TGFBETA_ALL_UP -6.334 Up by TGFb treatment of fibroblasts, any tirnepoint RAS ONCOGENIC SIGNATURE -8.879 Cells expressing H-ras SRC ONCOGENIC SIGNATURE -3.904 Cells expressing c-Sic =
SCHUMACHER MYC UP _4.02 Upregulated by myc in P493-6 B cell IGF1 NIH3T3 UP -4.25 Up after IGF1 treatment of NIH3T3-1GF1R
SERUM FIBROBLAST CELLCYCLE -5.821 From a variety of human fibroblast lines SERUM FIBROBLAST CORE UP -8.071 Up in serum in variety of human fibroblast lines CHANG SERUM RESPONSE UP -7.01 OLDAGE_DN -4.00 Down in fibroblasts from old relative to young P21253_ANY_DN -6.411 Ectopic p21 expression; p53 dependent changes; any timepoint

- 37 -P21 P53 EARLY DN -4.149 Ectopic p21 expression; p53 dependent changes; early timepoint P21 P53 MIDDLE DN -4.743 Ectopic p21 expression; p53 dependent changes; middle timepoint H3A04512 ECM RECEPTOR INTERACTION -8.649 MENSE HYPDXIA UP -8.337 Hypoxia-induced in HeLa and astrocytes =
POSITIVE REGULATION OF CELL PROLIFERATION -5.054 Gene Ontology Term REGULATION OF CELL MIGRATION -4.391 Gene Ontology Term Standard Name in Molecular Signatures Database of the Broad Institute.
2 NTk, normalized t statistic.

- 38 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME
ORGANISM
NAME
HCC_SURVIVAL_ msigclb V2.5 24-Mar-08 c2:786 Human GOOD_VS_POOR
UP
LIZUKA_Gl_GR_ msigdb V2.5 24-Mar-08 c2:796 Human

- 39 -STANDARD
EXTERNAL DETAILS URL CHIP CATEGORY CODE
NAME
HCC_SURVIVAL_ SECLACCESSION c2 GOOD_VS_POOR
UP
LIZUKA_Gl_GR_ SEO,ACCESSION c2

-40 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
HCC_SURVIVAL_ Yujin Hoshida Broad institute Genes highly expressed in GOOD_VS_POOR hepatocellular carcinoma with UP good survival.
LIZUKA_Gl_GR_ Yujin Hoshida Broad Institute Genes highly expressed in well G2 differentiated vs.
moderately differentiated hepatoceliular carcinoma

-41 -STANDARD
FULL DESCRIPTION
NAME
HCC SURVIVAL
GOOD_VS_POOR
UP
LIZUKA_Gl_GR_ AB - Using high-density oligonucleotide array we comprehensively analyzed expression levels 32 of 12600 genes in 50 hepatocellular carcinoma (HCC) samples with positive hepatitis C virus (HCV) serology (well (G1) moderately (G2) and poorly (G3) differentiated tumors) and 11 non-tumorous livers (L1 and LO) with and without HCV infection. We searched for discriminatory genes of transition (LO vs. L1 Li vs. G1 G1 vs. G2 G2 vs. G3) with a supervised learning method and then arranged the samples by self-organizing map (SOM) with the discriminatory gene sets. The SOM arranged the five clusters on a unique sigmoidal curve in the order LO L1 G1 G2 and G3. The sample arrangement reproduced development-related features of HCC such as p53 abnormality. Strikingly G2 tumors without venous invasion were located closer to the G1 cluster and most G2 tumors with venous invasion were located closer to the G3 cluster (P,--0.001 by Fisher's exact test). Our present profiling data will serve as a framework to understand the relation between the development and dedifferentiation of HCC.

-42 -STANDARD
MEMBERS
NAME
HCC_SURVIVAL_ 0101, EPHX1, KHK, FU14665, F138, SERPIND1, CES3, PRO0800, ACADS, WALL
GOOD_VS_POOR HADH2, MST1, CYP4F11, ABCA6, FU22578, PK1R, 5LC25A10, CABC1, DKFZp434F2, UP MGC35366, GLYAT, FU10851, RHBG, SLC27A5, INSR, VPREB1, MASP2, ALAS', CGR11, RNF29, L0C162427, AQP9, BDH, FH R5, C1QTN F4, MUCDHL, CC116, TIP120B, L0C339263, RODFI, ABCG5, ALS2CR19, RBP5, NDRG2, BAAT, L0C94431, FTHFD, A0X1, HAGH, DGAT2L1, MTSS1, FLJ22195, L0C149703, TTBK1, SELENBP1, CYP2,12, KLK3, MGC15419, AMFR, NF1, UGT1A6, WBSCR14, CRYL1, H PA, F12, MRPL46, AP0A5, KAA0977, HPD, STARD10, RAB-R, L0C91614, GJB1, PNUTL2, KIAA0888, L0C155066, E21G4) SERPINF1, C4BPB, ALDH4A1, FL110948, RGN, ECHS1, CUTL2, FU13941, LEAP-2, C20orf166, F10, CPN2, PC, L0C158402, LPIN1, L0051204, HA01, DSG1, NDUFS2, ZNF297, 7NF288, CPB2, APCS, ITPR2, ABHD6, AGL, FACL6, FACL2, NIG1, CD01, UGT2B15, L6R, M0C23940, SLC38A3, P8, NECAB2, 51C2A2, FU34658, PCK1, SLC22A1L, CES1, FU12331, FU14146, ABCG8, SERPING1, EHHADH, ANXA9, TRA53, SALL1, ITIH1, MSRA, ZP3, RPi89, DCXR, ASGR1, OTC, G6PC, APOC3, SERPINC1, LR8, WNW, ABCC2, PCCB, PRDX6, MAP4K1, ENPP5, CPT2, HOD, PR00195, C2, PRKWNK3, FU10035, ACF, IVD, PIPDX, SDR1, PACE4, PLEKHE1, HSA250839, PINK1, FU20581, UGT2B4, CYP2D6, NX17, ACOX2, SEC14L2, ALTE, CES2, SPINK2, CABIN1, SULT2A1, SRD5A1, MPDZ, CYP3A4, AMT, PAH, KNG, SGPL1, RDH5, FM03, SLC6Al2, NSlGl, USH3A, FACL1, PCYT2, WNT11, EGLN2, SLC35D1, FU30679, PPAP2A, AR, UNKL, MGC24039, MVK, CRAT, DPYS, AMACR
LIZUKA_G1_GR_ M87434, M12963, W28281, M97936, L07633, AA883502, AB007447, Z99129, 02 M97935, 050312, U07364, N625844, AF061258

-43-STANDARD
MEMBERS SYMBOLIZED
NAME
HCC_SURVIVAL_ 0101, EPHX1, KHK, FU14665, F138, SERPIND1, CES3, PRO0800, ACADS, HYAL1, GOOD_VS_POOR HADH2, MST1, CYP4F11, ABCA6, FU22578, PK1R, 5LC25A10, CABC1, DKFZp434F2, UP MGC35366, GLYAT, FU10851, RHBG, SLC27A5, INSR, VPREB1, MASP2, ALAS', CGR11, RNF29, L0C162427, AQP9, BDH, FH R5, C1QTNF4, MUCDHL, CC116, TIP120B, L0C339263, RODH, ABCG5, ALS2CR19, RBP5, NDRG2, BAAT, L0C94431, FTHFD, A0X1, HAGH, DGAT2L1, MTSS1, FU22195, L0C149703, TTBK1, SELENBP1, CYP2,12, KLK3, MGC15419, AMFR, NF1, UGT1A6, WBSCR14, CRYL1, H PA, F12, MRPL46, AP0A5, KÃAA0977, HPD, STARD10, RAB-R, L0C91614, GJB1, PNUTL2, KIAA0888, L0C155066, E21G4) SERPINF1, C4BPB, ALDH4A1, FU10948, RGN, ECHS1, CUTL2, FU13941, LEAP-2, C20orf166, F10, CPN2, PC, L0C158402, LPIN1, L0051204, HA01, DSG1, NDUFS2, ZNF297, ZNF288, CPB2, APCS, ITPR2, ABHD6, AGL, FACL6, FACL2, NLG1, CD01, UGT2B15, L6R, M0C23940, SLC38A3, P8, NECAB2, 51C2A2, FU34658, PCK1, SLC22A1L, CES1, FU12331, FU14146, ABCG8, SERPING1, EHHADH, ANXA9, TRA53, SALL1, ITIH1, MSRA, ZP3, RNB9, DCXR, ASGR1, OTC, G6PC, APOC3, SERPINC1, LR8, WNW, ABCC2, PCCB, PRDX6, MAP4K1, ENPP5, CPT2, HOD, PR00195, C2, PRKWNK3, FU10035, ACF, IVD, PIPDX, SDR1, PACE4, PLEKHE1, HSA250839, PINK1, FU20581, UGT2B4, CYP2D6, NX17, ACOX2, SEC14L2, ALTE, CES2, SPINK2, CABIN1, SULT2A1, SRD5A1, MPDZ, CYP3A4, AMT, PAH, KNG, SGPL1, RDH5, FM03, SLC6Al2, NSlGl, USH3A, FACL1, PCYT2, WNT11, EGLN2, SLC35D1, FU30679, PPAP2A, AR, UNKL, MGC24039, MVK, CRAT, DPYS, AMACR
LIZUKA_Gl_GR_ STAT1, UBE2L6, PDLIM5, KCNJ4, OAS2, PSME1, ADH1C, GABARAPL1, TRAFD1, 02 HSF2, L0C201229, KCNJ8

- 44 -STANDARD

NAME
HCC_SURVIVAL_ GOOD_VS_POOR
1.11) LIZUKA_Gl_GR_ 15710396 lizuka N, Oka M, Yamada-Okabe H, Mori N, 32 Tamesa T, Okada T, Takemoto N, Sakamoto K, Hamada K, lshitsuka H, Miyarnoto T, Uchimura 5, Hamamoto

-45 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
IDX JSA_UP_CL rnsigclb V2.5 '24-Mar-08 .c2:1507 Mouse

- 46 -STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
1DX_TSA_UP_CL GENE _SYMBOL c2

-47 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
IDX_TSA_UP_CL 121 John Newman Washington University Up-regulated at 48-96 hours USTER5 during differentiation of 3T34.1 fibroblasts into adipocytes with IDX (insulin, dexamethasone and isobutylxanthine), vs.
fibroblasts treated with IDX TSA
to prevent differentiation (duster 5)

-48 -STANDARD
FULL DESCRIPTION
NAME
IDX_TSA_UP_CL AB - During cellular differentiation and development it is recognized that many complex USTER5 molecular mechanisms as well as precise patterns of differentially expressed genes occur in directing precursor cells toward a given lineage. Using microarray-based technology we examined gene expression across the course of 3T3-L1 aclipocyte differentiation. Total cellular RNA was isolated at times 0 2 8 16 24 48 and 96 h following treatment with either standard hormonal inducers of differentiation insulin dexamethasone isobutylmethylxanthine (IDX) or IDX plus trichostatin A (TsA) a histone deacetylase inhibitor and potent adipogenic inhibitor. cRNA was synthesized from cellular RNA and hybridized to high density Affymetrix MG_U74Av2 microarray gene chips containing 12 488 cDNA/Expressed Sequence Tags (ESTs) probe sets. From the IDX-only treated cells all probe sets that were either unchanged or differentially expressed less than 2-fold throughout differentiation with respect to time 0 preadipocytes were excluded from further analyses.
This selection resulted in a net of 1686 transcripts 859 were increased in expression and 827 were decreased in expression at least 2-fold across differentiation. To focus in on genes that were more specific to differentiation the same analysis was performed on IDX
plus TsA-treated non-differentiating cells and all probe sets from the IDX-only group that exhibited similar expression profiles in the non-differentiating TsA-treated group were excluded leaving a total of 1016 transcripts that were regulated only under differentiating conditions.
Six hundred and thirty-six of these transcripts were elevated at least 2-fold and 380 exhibited a decrease in expression relative to time 0 preadipocytes. This group of genes was further analyzed using hierarchical clustering and self organizing maps and resulted in the identification of numerous genes not previously known to be regulated during adipocyte differentiation. Many of these genes may well represent novel aclipogenic mediators and markers of adipogenesis.

- 49 -STANDARD
MEMBERS
NAME
IDX_TSA_UP_CL MAPK6, TALD01, RREB1, RASD1, PHB, PEXI4, USMG5, BC12113, ACSL1, C200RF45, USTERS C180RF8, TYSND1, FAM82C, MKNK2, PTGES2, CHP, NDUFB10, C220RF13, GYS1, C2ORF7, PHB2, LOC642393, GPHN, L0C440567, ABHD5, MRPL34, COX6A1, COX4NB, IFNGR1, JAGN1, COQ% C60RF72, RPP14, C3, GK. PRPS1, HBLD1, SWA1, PIM3, G6PD, GPI, TBL2, DBI, AK2, XBP1, GNPAT, CBR3, TWM8A, SLC5A6, TXNDC14, TIMM17A, PSMA1, PPARG, MRPL12, LRRC59, COX17, PLA2G12A, UQCR, TOMM40, ESRRA, NDUFAB1, ETFB, ACO2, PRDX3, ACADM, IVD, SORBS1, CYCl, MRPS34, PDIA6, SDHD, PEXI1A, KIAA1161, ADAM12, MRPL18, MRPS2, MTX2, MRPL15, GRPEL1, NDUFA8, ALDOA, TUMM23, PPA1, 5RP44, ORMDL3, MGST3, TMEM97, MRP526, BCAP31, ARL61P2, TIMM9, ECHS1, ATP5G1, PSMA5, POR, PC, PDCD8, DNAH39, LPIN1, ZNRF2, CRAT, FAM73B, N101, PTRF, LMAN2, FDX1

- 50 -STANDARD
MEMBERS SYMBOLIZED
NAME
IDX_TSA_UP_CL MAPK6, TALD01, RREB1, RASD1, PHB, PEX14, USMG5, BCL2L13, ACSL1, C200RF45, USTERS C180RF8, TYSND1, FAM82C, MKNK2, PTGES2, CHP, NDUFB10, C220RF13, GYS1, C2ORF7, PHI32, LOC642393, GPHN, L0C440567, ABHD5, MRPL34, COX6A1, COX4NB, IFNGR1, JAGN1, COQ% C60RF72, RPP14, C3, GK, PRPS1, HBLD1, SPA1, PIN13, G6PD, GPI, TBL2, DBI, AK2, XBP1, GNPAT, CBR3, TIMM8A, SLC5A6, TXNDC14, TIMM17A, PSMA1, PPARG, MRPL12, LRRC59, COX17, PLA2G12A, UQCR, TOMM40, ESRRA, NDUFAB1, ETFB, ACO2, PRDX3, ACADM, IVD, SORBS1, CYCl, MRPS34, PDIA6, SDHD, PEX11A, KIAA1161, ADAM12, MRPL18, MRPS2, MTX2, MRPL15, GRPEL1, NDUFA8, ALDOA, TIMM23, PPA1, 5RP44, ORMDL3, MGST3, TMEM97, MRP526, BCAP31, ARL6IP2, TIMM9, ECHS1, ATP5G1, PSMA5, POR, PC, PDCD8, DNAIB9, LPIN1, ZNRF2, CRAT, FAM73B, NRIP1, PTRF, LMAN2, FDX1

-51-STANDARD

NAME
IDX_TSA_UP_CL 15033539 Burton OR, Nagarajan R, Peterson CA, McGehee USTERS REJr

- 52 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
ADIP_MFLUF' 'rnsigclb V2.5 '24-Mar-08 .c2:1138 Mouse

- 53 -STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
ADIP JAFF_UF' GENE _SYMBOL c2

- 54 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
ADIP_DIFF3JF1 121 John Newman Washington University Upregulated in mature adipocytes following differentiation from 3T3-L1 fibroblasts

- 55 -STANDARD
FULL DESCRIPTION
NAME
ADIP_DlFF_UF' AB - Troglitazone (TGZ) a member of the thiazolidineclione class of anti-diabetic compounds and a peroxisome proliferator activator receptor-gamma (PPAR-gamma) agonist restores systemic insulin sensitivity and improves the full insulin resistance syndrome in vivo. The mechanisms underlying its in vivo function are not understood. Here we investigated the potential functional interaction between F'PAR-gamma and NF-kappaB in adipocytes. We show that TGZ selectively blocked tumor necrosis factor-alpha-induced and NF-kappaB-dependent repression of multiple adipocyte-specific genes and induction of growth phase and other genes. This occurs without interfering with NE-kappaB expression activation nuclear translocation or DNA binding and without suppressing NF-kappaB-dependent survival signals. Notably the expressions of some tumor necrosis factor-alpha-induced genes in adipocytes were unaffected by PPAR-gamma activation, in reporter gene assays in HeLa cells ectopic expression of PPAR-gamma abolished induction of a NF-kappaB-responsive reporter gene by the p65 subunit (RelA) of NF-kappaB and the inhibition was further enhanced in the presence of TGZ. Conversely overexpression of p65 inhibited induction of a PPAR-gamma-responsive reporter gene by activated PPAR-gamma in a dose-dependent manner. The inhibitory effect was independent of the presence of NF-kappaB-binding sites in the promoter region. Other NF-kappaB family members p50 and c-Rdl as well as the 5276A
mutant of p65 blocked PPAR-gamma-mediated gene transcription less effectively.
Thus p65 antagonizes the transcriptional regulatory activity of PPAR-gamma in adipocytes and PPAR-gamma activation can at least partially override the inhibitory effects of p65 on the expression of key adipocyte genes. Our data suggest that inhibition of NF-kappa6 activity is a mechanism by which PPAR-gamma agonists improve insulin sensitivity in vivo and that adipocyte NF-kappaB is a potential therapeutic target for obesity-linked type 2 diabetes.

- 56 -STANDARD
MEMBERS
ADIP_DFFUPNAME
1RX3, DGAT1, DCN, SLC2A4, TALD01, RASD1, ABCD2, PPARG, ACOX1, SCARB1, PFKFB1, ACSL1, ADIPOC), ACADS, ITGA6, PLA2G6, FAM62A, BCKDHA, NNMT, 1ST, ACADM, TPP2, SORBS1, SELENBP1, MUT, HIPK3, AP3S1, TAP2, PEX11A, CBX4, COL15A1, ECH1, CEBPG, ALAD, AATK, DHRS3, AQP7, ABCA1, ORM2, GOS2, CIDEC, HP, CP, REEP5, OB2, SERPINA3, IFNGR1, NFS1, CDKN2C, ME1, FUR, AUNS1, RNF11, REIN, FASN, GBP2, STAT1, DH1, PC, LTC4S, ENTPDS, LAMA4, DBI, RGS2, NRIP1, AK2, UCK1, NR1H3, IDH3G

- 57 -STANDARD
MEMBERS SYMBOLIZED
NAME
ADIP_DIFF_UF' RX3, DGAT1, DCN, SLC2A4, TALDOI, RASD1, ABCD2, PPARG, ACOX1, SCARB1, PFKFB1, ACSL1, ADIPOQ, ACADS, ITGA6, PLA2G6, FAM62A, BCKDHA, NNMT, 1ST, ACADM, TPP2, SORBS1, SELENBP1, MUT, HIPK3, AP3S1, TAP2, PEX11A, CBX4, COL15A1, ECH1, CEBPG, ALAD, AATK, DHRS3, AQP7, ABCA1, ORM2, GOS2, CIDEC, HP, CP, REEPS, 0B2, SERPINA3, IFNGR1, NFS1, CDKN2C, MEI, FUR, ALAS1, RNF11, REIN, FASN, GBP2, STAT1, DH1, PC, LTC4S, ENTPDS, LAMA4, DBI, RGS2, NRIP1, AK2, UCK1, NR1H3, IDH3G

- 58 -

59 STANDARD

NAME
ADIP_MFLUF' 12732648 Ruan H, Pownall Hi, Lodish HF

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
TISSUE _DEVELOP msigclb V2.5 '24-Mar-08 c5:449 Homo sapiens MENT
EPIDERMIS_DEV msigdb V23 24-Mar-08 6:1047 Homo sapiens ELOPMENT

- 60 -STANDARD
EXTERNAL DETAILS URL CHIP CATEGORY CODE
NAME
115SUEDEVELOP http://amigo.geneontology.org GENE _SYMBOL c5 MENT Lcgi:
biniamige/go.cgOview=details &search constraint=terms&cle pth=0&querv=60:0009888 EPIDERMIS_DEV http://amigo.geneontology.org GENE SYMBOL c5 ELOPMENT

biniatnigo/go,cgRview=cletais &search constraint=terms&de pth=0&query=G0:0008544

-61-STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
T15SUE_DEVELOP Gene Ontology Gene Ontology Genes annotated by the GO term MENT GO:0009888. The process whose specific outcome is the progression of a tissue over time, from its formation to the mature structure.
EPIDERMIS_DEV Gene Ontology Gene Ontology Genes annotated by the GO term ELOPMENT GO:0008544. The process whose specific outcome is the progression of the epidermis overtime, from its formation to the mature structure. The epidermis is the outer epithelial layer of a plant or animal, it may be a single layer that produces an extracellular material (e.g. the cuticle of arthropods) or a complex stratified squamous epithelium, as in the case of many vertebrate species.

- 62 -STANDARD
FULL DESCRIPTION
NAME
TISSUE DEVELOP
MENT
EPIDERMIS_DEV
ELOPMENT

- 63 -STANDARD
MEMBERS
NAME
115SUEDEVELOP DHCR24, PTF1A, HOXC11, LAMC1, RUNX2, Gill, NF1, 5ilT2, AP0A5, KLF4, MENT TR1M15, BMP4, KL, ERCC2, ERCC3, SPlNK5, ANKH, EDA, STX2, KRT6A, KRT6B, PROX1, TFAP2A, VAX2, ZBTB7B, SMURF1, COL5A2, KRT9, SASP, TGFB2, EYA2, GHSR, PPARD, GHRL, FST, COL13A1, RTN4Ri1, RTN4R12, KLK8, NME2, BMX, BTK, HCK, JAK2, MATK, MEST, SECTM1, T, TBX6, TCF15, TCF21, TIE1, GDF11, IKZFl, 1KZF3, ALDH3A2, ALOX12B, STS, ATP2A2, BNC1, BID, CDSN, COL1A1, C0i7A1, COL17A1, CRABP2, CTGF, DCT, DSP, EMP1, EVPL, FABP5, FGF7, FLOT2, GJB5, KRTAP5-9, KRT1, KRT2, KRT5, KRT10, KRT13, KRT14, KRT15, KRT16, KRT17, KRT31, KRT32, KRT34, KRT83, KRT85, LAMA3, LAMB3, LAMC2, PLOD1, KLK7, PTHLH, RBP2, S100A7, SPRR1A, SPRR1B, UGCG, WAS, FOXN1, PTCH2, SCEL, TGM5, HOXB13, CASP14, KLK5, POU2F3, ATP2C1, CALML5, SNAI2, CDK6, TVVIST2, IL20, BMP1, COL11A1, MGP, ALX1, AHSG, SRGN, TBX3, ACVR1, ZNF675, ACHE, GL12, 11_17F, ATP6V181, CASR, DMP1, DSPP, SPARC, STATH, 05TH., ATP6V0A4, TGM3, EPIDERMIS_DEV ALDH3A2, ALOX12B, STS, ATP2A2, BNC1, BID, CDSN, COL1A1, COL7A1, COL17A1, ELOPMENT CRABP2, CTGF, OCT, DSP, EMP1, EVPL, FABP5, FGF7, FLOT2, GJB5, KRTAP5-9, KRT1, KRT2, KRT5, KRT10, KR113, KRT14, KRT15, KRT16, KRT17, KRT31, KRT32, KRT34, KR183, KRT85, LAMA3, LAMB3, LAMC2, PLOD1, PPARD, KLK7, PTHLH, RBP2, S100A7, SPRR1A, SPRR1B, UGCG, WAS, FOXN1, PTCH2, SCEL, TGM5, HOXB13, CASP14, KLK5, POU2F3, ATP2C1, CALMLS, ERCC2, ERCC3, SPINK5, C015A2, DHCR24, KRT9, SASP, TGFB2, FST, NME2, Gill, 1120, TGM3

-64-STANDARD
MEMBERS SYMBOLIZED
NAME
TISSUE _DEVELOP DHCR24, PTF1A, HOXC11, LAMC1, RUNX2, Gill, NF1, 5ilT2, AP0A5, KLF4, MENT TR1M15, BMP4, KL, ERCC2, ERCC3, SPlNK5, ANKH, EDA, STX2, KRT6A, KRT6B, PROX1, TFAP2A, VAX2, ZBTB7B, SMURF1, COL5A2, KRT9, SASP, TGFB2, EYA2, GHSR, PPARD, GHRL, FST, COL13A1, RTN4Ri1, RTN4R12, KLK8, NME2, BMX, BTK, HCK, JAK2, MATK, MEST, SECTM1, T, TBX6, TCF15, TCF21, TIE1, GDF11, IKZFl, 1KZF3, ALDH3A2, ALOX12B, STS, ATP2A2, BNC1, BID, CDSN, COL1A1, C0i7A1, COL17A1, CRABP2, CTGF, DCT, DSP, EMP1, EVPL, FABP5, FGF7, FLOT2, GJB5, KRTAP5-9, KRT1, KRT2, KRT5, KRT10, KRT13, KRT14, KRT15, KRT16, KRT17, KRT31, KRT32, KRT34, KRT83, KRT85, LAMA3, LAMB3, LAMC2, PLOD1, KLK7, PTHLH, RBP2, S100A7, SPRR1A, SPRR1B, UGCG, WAS, FOXN1, PTCH2, SCEL, TGM5, HOXB13, CASP14, KLK5, POU2F3, ATP2C1, CALML5, SNAI2, CDK6, TVVIST2, IL20, BMP1, COL11A1, MGP, ALX1, AHSG, SRGN, TBX3, ACVR1, ZNF675, ACHE, GLI2, 11_17F, ATP6V181, CASR, DMP1, DSPP, SPARC, STATH, 05TH., ATP6V0A4, TGM3, EPIDERMIS_DEV ALDH3A2, ALOX12B, STS, ATP2A2, BNC1, BID, CDSN, COL1A1, COL7A1, COL17A1, ELOPMENT CRABP2, CTGF, OCT, DSP, EMP1, EVPL, FABP5, FGF7, FLOT2, GJB5, KRTAP5-9, KRT1, KRT2, KRT5, KRT10, KR113, KRT14, KRT15, KRT16, KRT17, KRT31, KRT32, KRT34, KR183, KRT85, LAMA3, LAMB3, LAMC2, PLOD1, PPARD, KLK7, PTHLH, RBP2, S100A7, SPRR1A, SPRR1B, UGCG, WAS, FOXN1, PTCH2, SCEL, TGM5, HOXB13, CASP14, KLK5, POU2F3, ATP2C1, CALMLS, ERCC2, ERCC3, SPINK5, C015A2, DHCR24, KRT9, SASP, TGFB2, FST, NME2, Gill, 1120, TGM3

- 65 -STANDARD
PMlD AUTHORS
NAME
TlSSUE_DEVELOP Ashburner M, Ball CA, Blake JA, Botstein D, MENT Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis 5, Matese JC, Richardson JE, Ringwald M, Ru bin GM, Sherlock G.
EPIDERMIS_DEV Ashburner M, Ball CA, Blake IA, Botstein D, ELOPMENT Butler H, Cherry JM, Davis AP, Dolinski K, Dwight 55, Eppig IT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis 5, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G.

- 66 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
MORPHOGENESI msigdb V2.5 '24-Mar-08 6:843 Homo sapiens S_OF_AN_EPITH
ELIUM
KERATINOCYTE_ msigdb V2.5 24-Mar-08 6:917 Homo sapiens DIFFERENTIATIO
ECTODERM_DEV msigdb V2.5 24-Mar-08 6:1745 Homo sapiens ELOPMENT

- 67 -STANDARD
EXTERNAL DETAILS URL CHIP CATEGORY CODE
NAME
MORPHOGENESI http://amigo.geneontology.org GENE SYMBOL c5 S_OF_AN_EPITH /cg--ELI UM biniamige/go.cgOview=details &search constraint=terms&cle pth=0&querv=60:0002009 KERATINOCYTE_ http://arnigo.geneontology.org, GENE_SYMBOL c5 DIFFERENTIATIO /cgi-N bin/arnigo/go,cgiNiew=detaHs &search constraintzterms&de pth=0&query=G0:0030216 ECTODERM_DEV http://arnigo.geneontology.org GENE SYMBOL c5 ELOPMENT

bin/arnigo/go.cgi?view----:details &search constraintzterms&de pth----0&query=G0:0007398

- 68 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
MORPHOGENESI Gene Ontology Gene Ontology Genes annotated by the GO term S_OF_AN_EPITH GO:0002009. The process by ELIUM which the anatomical structures of epithelia are generated and organized. Morphogenesis pertains to the creation of form.
An epithelium is a sheet of closely packed cells arranged in one or more layers, that covers the outer surfaces of the body or lines any internal cavity or tube.
KERATINOCYTE_ Gene Ontology Gene Ontology Genes annotated by the GO term DIFFERENTIATIO GO:0030216. The process whereby a relatively unspecialized cell acquires specialized features of a keratinocyte.
ECTODERM_DEV Gene Ontology Gene Ontology Genes annotated by the GO term ELOPMENT GO:0007398. The process whose specific outcome is the progression of the ectoderm over time, from its formation to the mature structure. In animal embryos, the ectoderm is the outer germ layer of the embryo, formed during gastrulation.

- 69 -STANDARD
FULL DESCRIPTION
NAME
MORPHOGENESI
S_OF_AN_EPITH
ELIUM
KERATINOCYTE_ DIFFERENTIATIO
ECTODERM_DEV
ELOPMENT

- 70 -STANDARD
MEMBERS
NAME
MORPHOGENESI TIMELESS, DMBT1, ELF3, KRT3, KRT4, UPK1B, UPK2, UPK3A, SP1NK5, UPK1A, EHF, S_OF_AN_EPITH CELSR1, VANGL2, GLI2, LM04, BCL10 ELIUM
KERATINOCYTE_ ANXA1, CSTA, DSP, EREG, EVPL, IVL, LOR, SPRR1A, SPRR1B, TGM1, TGM3, SCEL, DIFFERENTIATIO TXN1P, NME2, 1120 ECTODERM_DEV EDA, STX2, KRT6A, KRT6B, PROX1, TFAP2A, VAX2, ZBTB7B, SMURF1, ERCC2, ELOPMENT ERCC3, SPINK5, COL5A2, DHCR24, KRT9, SASP, TGF132, FST, NME2, ALDH3A2, ALOX128, STS, ATP2A2, BNC1, BTD, CDSN, COL1A1, COL7A1, COL17A1, CRABP2, CTGF, DCT, DSP, EMP1, EVPL, FABP5, FGF7, FLOT2, GJB5, KRTAP5-9, KRT1, KRT2, KRTS, KRT10, KR113, KRT14, KRT15, KRT16, KRT17, KRT31, KR132, KRT34, KRT83, KRT85, LAMA3, LAMB3, LAMC2, PLOD1, PPARD, KLK7, PTHLH, RBP2, 5100A7, SPRR1A, SPRR1B, UGCG, WAS, FOXN1, PTCH2, SCEL, TGM5, HOXB13, CASP14, KLK5, POU2F3, ATP2C1, CALML5, 11.20, TGM3

- 71 -STANDARD
MEMBERS SYMBOLIZED
NAME
MORPHOGENESI TIMELESS, DMBT1, ELF3, KRT3, KRT4, UPK1B, UPK2, UPK3A, SP1NK5, UPK1A, EHF, S_OF_AN_EPITH CELSR1, VANGL2, GLI2, LM04, Ba10 ELIUM
KERATINOCYTE_ ANXA1, CSTA, DSP, EREG, EVPL, IVL, LOR, SPRR1A, SPRR1B, TGM1, TGM3, SCEL, DIFFERENTIATIO TXN1P, NME2, 1120 ECTODERM_DEV EDA, STX2, KRT6A, KRT6B, PROX1, TFAP2A, VAX2, ZBTB7B, SMURF1, ERCC2, ELOPMENT ERCC3, SPINK5, COL5A2, DHCR24, KRT9, SASP, TGFI32, FST, NME2, ALDH3A2, ALOX128, STS, ATP2A2, BNC1, BTD, CDSN, COL1A1, COL7A1, COL17A1, CRABP2, CTGF, DCT, DSP, EMP1, EVPL, FABP5, FGF7, FLOT2, GJB5, KRTAP5-9, KRT1, KRT2, KRTS, KRT10, KR113, KRT14, KRT15, KRT16, KRT17, KRT31, KR132, KRT34, KRT83, KRT85, LAMA3, LAMB3, LAMC2, PLOD1, PPARD, KLK7, PTHLH, RBP2, 5100A7, SPRR1A, SPRR1B, UGCG, WAS, FOXN1, PTCH2, SCEL, TGM5, HOXB13, CASP14, KLK5, POU2F3, ATP2C1, CALML5, 11.20, TGM3

-72 -STANDARD
PMlD AUTHORS
NAME
MORPHOGENESI Ashburner M, Ball CA, Blake JA, Botstein D, S_OF_AN_EPITH Butler H, Cherry JM, Davis AP, Dolinski K, ELIUM Dwight S5, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis 5, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G.
KERATINOCYTE_ Ashburner M, Ball CA, Blake JA, Botstein D, DIFFERENTIATIO Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig _IT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G.
ECTODERM_DEV Ashburner M, Ball CA, Blake JA, Botstein D, ELOPMENT Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig IT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G.

- 73 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
EPITHELIAL _CELL msigclb V2.5 '24-Mar-08 .c5:448 Homo sapiens _DIFFERENTIATI
ON
VANTVEER_BREA msigdb V2.5 24-Mar-08 c2:825 Human ST_OUTCOME_G
00D_VS_POOR_ DN

- 74..

STANDARD
EXTERNAL DETAILS URL CHIP CATEGORY CODE
NAME
EKIHELIAL_CELL http://amigo.geneontology.org GENE _SYMBOL c5 _DIFFERENTIAll /cg ON biniamige/go.cgOview=details &search constraint=terms&cle pth=0&querv=60:0030855 VANTVEER_BREA GENE SYMBOL c2 ST_OUTCOME_G
00D_VS_POOR_ DN

- 75 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
EPITHELIAL SELL Gene Ontology Gene Ontology Genes annotated by the GO term _DIFFERENTIATI GO:0030855. The process ON whereby a relatively unspecialized cell acquires specialized features of an epithelial cell, any of the cells making up an epithelium.
VANTVEER_BREA Jean Junior Broad Institute Poor prognosis marker genes in ST_OUTCOME_G Breast Cancer (part of NKI-70) 00D_VS_POOR_ from Van't Veer et al DN

-76 -STANDARD
FULL DESCRIPTION
NAME
EPiTHELIALSELL
_DIFFERENTIATi ON
VANTVEER_BREA AB - Breast cancer patients with the same stage of disease can have markedly different ST_OUTCOME_G treatment responses and overall outcome. The strongest predictors for metastases (for 00D_VS_POOR_ example lymph node status and histological grade) fail to classify accurately breast tumours DN according to their clinical behaviour. Chemotherapy or hormonal therapy reduces the risk of distant metastases by approximately one-third however 70-80% of patients receiving this treatment would have survived without it. None of the signatures of breast cancer gene expression reported to date allow for patient-tailored therapy strategies.
Here we used DNA
microarray analysis on primary breast tumours of 117 young patients and applied supervised classification to identify a gene expression signature strongly predictive of a short interval to distant metastases ('poor prognosis signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative). in addition we established a signature that identifies tumours of BRCA1 carriers. The poor prognosis signature consists of genes regulating cell cycle invasion metastasis and angiogenesis. This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.

-77-STANDARD
MEMBERS
NAME
EKIHELIALSELL DMBTI, ELF3, KRT3, KRT4, UPKIB, UPK2, UPK3A, SPINK5, UPKIA, EHF
_DIFFERENTIA-11 ON
VANTVEER_BREA PRC1, MGAT4A, 113, LCHN, BM037, RAD21, FU23468, EXT1, CCNB2, FUI0549, ST_OUTCOME_G RAB6B, STK15, NMU, IVICM6, TRIP13, L2DTL, PFKP, PRO2000, GGH, L0056901, 00D_VS_POOR_ HRB, FU10461, DCK, FBX05, FU20354, CCNE2, ECT2, CENPA, AD024, 0CI3, DN PCTK1, EZH2, ADM, BN1P3, FU10134, BM039, PSMD2, L0057110, CP, PRAME, H5U54999, FLTI, SMC4L1, CENPF, LAPIS, DKFZP761H171, BUB1, DEGS, FU12443, TK1, ASNS, RFC4, FU21924, DKFZP434B168, GMPS, PSMD7, CK52, FU11190, ESM1, MAD2L1, PGKI, MMP9, PIR, NSI-BP, COL4A2, MAA0175, CDC258, ORC6L, KIAA0042, SM-20, KIAA0008, VEGF, AKAP2, MPI, RRM2, TFRC, PTDSSI, 1GFBP5, HMG4, KIAA1104, FLI22341, SERF1A, NMB, FU10511, DKFZP564D0462, MCCC1, CTSL2, GNAZ, CA9, DKFZp762E1312, SACS, PK428, FU10156, STX1A, 5T7, CTRS, MRCS, SLC2A3, NDRGI, STK3, FU22477, MAPRE2, L0051203, NSAPI, OXCT, MTMR2, UCH37, HEC, TMEFFI, GBE1, FU10901, STK6

- 78 -STANDARD
MEMBERS SYMBOLIZED
NAME
EPITHELIAL SELL DMBTI, ELF3, KRT3, KRT4, UPKIB, UPK2, UPK3A, SPINK5, UPKIA, EHF
_DIFFERENTIATI
ON
VANTVEER_BREA PRC1, MGAT4A, 113, LCHN, BM037, RAD21, FU23468, EXT1, CCNB2, FUI0549, ST_OUTCOME_G RAB6B, STK15, NMU, IV1CM6, TRIP13, L2DTL, PFKP, PRO2000, GGH, L0056901, 00D_VS_POOR_ HRB, FU10461, DCK, FBX05, FU20354, CCNE2, ECT2, CENPA, AD024, 0CI3, DN PCTK1, EZH2, ADM, BNIP3, FU10134, BM039, PSMD2, L0057110, CP, PRAME, H5U54999, FLTI, SMC4L1, CENPF, LAPIS, DKFZP761H171, BUB1, DEGS, FU12443, TK1, ASNS, RFC4, FU21924, DKFZP434B168, GMPS, P5MD7, CK52, FU11190, ESM1, MAD2L1, PGKI, MMP9, PIR, NSI-BP, COL4A2, KIAA0175, CDC258, ORC6L, KIAA0042, SM-20, KIAA0008, VEGF, AKAP2, MPI, RRM2, TFRC, PTDSSI, IGFBP5, HMG4, KIAA1104, FLI22341, SERF1A, NMB, FU10511, DKFZP564D0462, MCCC1, CTSL2, GNAZ, CA9, DKFZp762E1312, SACS, PK428, FU10156, STX1A, 5T7, CTRS, BIRC5, SLC2A3, NDRGI, STK3, FU22477, MAPRE2, L0051203, NSAPI, OXCT, MTMR2, UCH37, HEC, TMEFFI, GBE1, FU10901, STK6

- 79 -STANDARD PMID AUTHORS
NAME
EPlTHELIALSELL Ashburner M, Ball CA, Blake JA, Botstein D, _DIFFERENTIATl Butler H, Cherry JM, Davis AP, Dolinski K, ON Dwight S5, Eppig JT, Harris MA, Hill DP, Issei-Tarver L, Kasarskis A, Lewis 5, Matese JC, Richardson JE, Ringwald M, Rlibin GM, Sherlock G.
VANTVEER_BREA 11823860 van 't Veer LJ, Dai H, van de Vijver Ml, He YD, Hart ST_OUTCOME_G AA, Mao M, Peterse HL, van der Kooy K, Marton 00D_VS_POOR_ Ml, Witteveen AT, Schreiber GJ, Kerkhoven RM, DN Roberts C, Linsley PS, Bernards R, Friend SH

- 80 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
BRCA_PROGNOS msigclb V2.5 '24-Mar-08 .c2:1215 Human I5_NEG

-81-STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
BRCA_PROGNOS GENE _SYMBOL c2 LS_NEG

- 82 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
BRCA_PROGNOS 121 John Newman Washington University Genes whose expression is 15_NEG consistently negatively correlated with breast cancer outcomes - higher expression is associated with metastasis and poor prognosis

- 83 -STANDARD
FULL DESCRIPTION
NAME
BRCA_PROGNOS AB - Breast cancer patients with the same stage of disease can have markedly different treatment responses and overall outcome. The strongest predictors for metastases (for example lymph node status and histological grade) fail to classify accurately breast tumours according to their clinical behaviour. Chemotherapy or hormonal therapy reduces the risk of distant metastases by approximately one-third however 70-80% of patients receiving this treatment would have survived without it. None of the signatures of breast cancer gene expression reported to date allow for patient-tailored therapy strategies.
Here we used DNA
microarray analysis on primary breast tumours of 117 young patients and applied supervised classification to identify a gene expression signature strongly predictive of a short interval to distant metastases ('poor prognosis' signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative). In addition we established a signature that identifies tumours of BRCA1 carriers. The poor prognosis signature consists of genes regulating cell cycle invasion metastasis and angiogenesis. This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.

-84 -STANDARD
MEMBERS
NAME
BRCA_PROGNOS SMC4, C1ORF106, ASPM, GMPS, F'SMD7, PRC1, Kff21A, C160RF61, CKS2, ESM1, IS_NEG MGAT4A, PGK1, MADal, LRP12, PAQR3, RAD21, EXT1, MMP9, PM, CCNB2, DTL, C014A2, AURKA, KNTC2, DLG7, NMU, MCM6, IVNS1ABP, TRIP13, PTPLB, MTDH, PFKP, DEGS1, CDC25B, ORC6L, SYNCR1P, SPBC25, GGH, HRB, VEGF, CENPN, AVTL2, TFRC, DCK, PTDSS1, INTS7, FBX05, IGFBP5, HMGB3, ECT2, KIF14, NMB, FAM64A, UCHL5, PIMM1, ATAD2, CENPA, MCCC1, PCTK1, CTS12, GNAZ, CA9, EZH2, ADM, BNIP3, SACS, PLEKHAl, PSMD2, OXCT1, PRAME, FLT, GPR126, TSPYL5, CTPS, GPSM2, TMEM45A, MRPL13, D1APH3, ARMC1, NDRG1, CDC42BPA, STK3, DEPDC1, C200RF46, BUB1, MAPRE2, DKFZP762E1312, MTMR2, MELK, STMN1, SLC7A1, GBE1, RRAGD, ASNS, RFC4

- 85 -STANDARD
MEMBERS SYMBOLIZED
NAME
BRCA_PROGNOS SMC4, C1ORF106, ASPM, GMPS, F'SMD7, PRC1, KIF21A, C160RF61, CKS2, ESM1, IS_NEG MGAT4A, PGK1, MAD2L1, LRP12, PAQR3, RAD21, EXT1, MMP9, PR, CCNB2, DTL, C014A2, AURKA, KNTC2, DLG7, NMU, MCM6, IVNS1ABP, TRIP13, PTPLB, MTDH, PFKP, DEGS1, CDC25B, ORC6L, SYNCRIP, SPBC25, GGH, HRB, VEGF, CENPN, AYTL2, TFRC, DCK, PTDSS1, INTS7, FBX05, IGFBPS, HMGB3, ECT2, KIF14, NMB, FAM64A, UCHL5, PITRM1, ATAD2, CENPA, MCCC1, PCTK1, CTS12, GNAZ, CA9, EZH2, ADM, BNIP3, SACS, PLEKHAl, PSMD2, OXCT1, PRAME, FLT, GPR126, TSPYL5, CTPS, GPSM2, TMEM45A, MRPL13, DIAPH3, ARMC1, NDRG1, CDC42BPA, STK3, DEPDC1, C200RF46, BUB1, MAPRE2, DKFZP762E1312, MTMR2, MELK, STMN1, SLC7A1, GBE1, RRAGD, ASNS, RFC4

- 86 -STANDARD

NAME
BRCA_PROGNOS 11823860 van 't Veer Li, Da i H, van de Vijver MJ, He YD, Hart 1S_NEG AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bemards R, Friend SH

- 87 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
HCC_SURVIVAL_ msiulla V2.5 '24-Mar-08 c2:785 Human GOODJS_POOR
DN

- 88 -STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
HCC_SURVIVAL_ SECLACCESSION c2 GOOD_VS_POOR
DN

- 89 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
HCC_SURVIVAL_ Yujin Hoshida Broad institute Genes highly expressed in GOOD_VS_POOR hepatocellular carcinoma with DN poor survival.

- 90 -STANDARD
FULL DESCRIPTION
NAME
HCC_SURVIVAL_ GOOD_VS_POOR
DN

- 91 -STANDARD
MEMBERS
NAME
HCC_SURVIVAL_ CSDA, ENIGMA, RPS3, RWDD1, MTHFD2, LAMM, FU23468, NSF, YWHAH, DSG2, GOOD_VS_POOR HIST2H4, HSPC133, AGRN, RPL35, LSM8, MCM6, MGC20486, CDW92, ATP5C1, DN 1CAP-1A, C6or183, BUB3, ATP281, PRO2000, RPL12, VCL, HRB, TCTEL1, FU10036, YWHAQ, NTS, SERPINHI, FU10697, EEF1E1, FU20354, ECT2, MCM2, NAPILl, KIAA1764, CGI-130, CTBP2, MGC5627, Cl9orf2, AD024, DUSP18, LAMR1, CGI-62, BZW2, HRMT1L1, LXN, DNCL1, RPS3A, L0C148213, CALM2, BCAT1, UBA2, TES, TETRAN, TD-60, ACTR3, HMGB2, MCM7, KRT10, CENPF, MMD, MARCKS, JJAZ1, KHDRBS1, RAN, TMSB4X, HNRPC, CCNA2, HRMT1L2, RP136A, 5100A6, ARHGAP18, DYRK2, TSSC3, 5PF45, SET, MYH2, CCNB1, C20orf35, ODC1, FUI0468, SNRPG, LYAR, RBX1, CKS2, SLBP, KRT19, L0051685, NOL5A, NALP2, USP1, CG1421, RPL27, HDAC2, M6C18216, PBX1, FBL, CCT6A, PLP2, NP, CBX3, DLG7, BAF53A, CCI5, NCBP2, PELI1, RPS18, HSPC150, ZNF138, FU14761, UBE2D1, TERA, FU10407, PTMA, FABP5, RPL9, SLC34A1, TSPAN-3, RPL38, H1F1A, RP56, AWN, H1ST1H4C, KIAA0101, RPS9, K-ALPHA-1, RPS5, MGC24665, L0C256112, PDC195, SMC211, ANXA3, ROD1, DEK, HIST1H4A, MRPL42, RALA, HNRPA1, IER3, RPS7, CLIC1, RPE, KLF5, RP511P1, MAPRE1, ARHE, RAB32, CD164, NFE213, UK, SNRPB2, RPL17, CRFG, TOPBP1, MSH6, HMGN1, HN1, CDC10, PCNA, MGC45594, YWHAB, OLIG2, RPL31, KIF5B, OPLAFI, DDX48, B3GNT5, CDK4, KNTC1, RGS2, ANXA4, Hes4, SMT3H2, D10S170, ITPR3, FU10901, PFDN4, CD2AP, NPM1

- 92 -STANDARD
MEMBERS SYMBOLIZED
NAME
HCC_SURVIVAL_ CSDA, ENIGMA, RPS3, RWDD1, MTHFD2, LAMM, FU23468, NSF, YWHAH, DSG2, GOOD_VS_POOR HIST2H4, HSPC133, AGRN, RPL35, LSM8, MCM6, MGC20486, CDW92, ATP5C1, DN 1CAP-1A, C6or183, BUB3, ATP281, PRO2000, RPL12, VCL, HRB, TCTEL1, FU10036, YWHAQ, NTS, SERPINHI, FU10697, EEF1E1, FU20354, ECT2, MCM2, NAPILl, KIAA1764, CGI-130, CTBP2, MGC5627, Cl9orf2, AD024, DUSP18, LAMR1, CGI-62, BZW2, HRMT1L1, LXN, DNCL1, RPS3A, L0C148213, CALM2, BCAT1, UBA2, TES, TETRAN, TD-60, ACTR3, HMGB2, MCM7, KRT10, CENPF, MMD, MARCKS, JJAZ1, KHDRBS1, RAN, TMSB4X, HNRPC, CCNA2, HRMT1L2, RP136A, 5100A6, ARHGAP18, DYRK2, TSSC3, 5PF45, SET, MYH2, CCNB1, C20orf35, ODC1, FUI0468, SNRPG, LYAR, RBX1, CKS2, SLBP, KRT19, L0051685, NOL5A, NALP2, USP1, CG1421, RPL27, HDAC2, M6C18216, PBX1, FBL, CCT6A, PLP2, NP, CBX3, DLG7, BAF53A, CCI5, NCBP2, PELI1, RPS18, HSPC150, ZNF138, FU14761, UBE2D1, TERA, FU10407, PTMA, FABP5, RPL9, SLC34A1, TSPAN-3, RPL38, H1F1A, RP56, AWN, H1ST1H4C, KIAA0101, RPS9, K-ALPHA-1, RPS5, MGC24665, L0C256112, PDCD5, SMC211, ANXA3, ROD1, DEK, HIST1H4A, MRPL42, RALA, HNRPA1, IER3, RPS7, CLIC1, RPE, KLF5, RP511P1, MAPRE1, ARHE, RAB32, CD164, NFE213, UK, SNRPB2, RPL17, CRFG, TOPBP1, MSH6, HMGN1, HN1, CDC10, PCNA, MGC45594, YWHAB, OLIG2, RPL31, KIF5B, OPLAFI, DDX48, B3GNT5, CDK4, KNTC1, RGS2, ANXA4, Hes4, SMT3H2, D10S170, ITPR3, FU10901, PFDN4, CD2AP, NPM1

- 93 -STANDARD
PMM AUTHORS
NAME
HCC_SURVIVAL_ GOOD_VS_POOR
DN

- 94 -STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
LIJETALys_WT msigdb V2.5 '24-Mar-08 c2:888 Human KIDNEY DN
HSA05222_SMAL msigdb V2.5 24-Mar-OS c2:1990 Homo sapiens L_CELLLUNG_C
ANCER

- 95 -STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
LIJETALys_WT AFFYMETRIX c2 KIDNEY DN
HSA05222_SMAL http://www.kegg.jadhget- GENE SYMBOL c2 L_CELLLUNG_C bin/show pathway?H5A05222 ANCER

- 96 -STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
LIJETALys_WT Kevin Vogelsang Broad institute These are genes identified by KIDNEY DN
simple statistical criteria as differing in their mRNA
expresssion between VVTs and fetal kidneys HIGH
HSA05222_SMAL KEGG KEGG Genes involved in small cell lung 1.._CELLLUNG_C cancer ANCER

- 97 -STANDARD
FULL DESCRIPTION
NAME
LIJETALys_WT AB - Wilms' tumor (WT) has been considered a prototype for arrested cellular differentiation KIDNEY DN in cancer but previous studies have relied on selected markers.
We have now performed an unbiased survey of gene expression in WTs using oligonucleotide microarrays.
Statistical criteria identified 357 genes as differentially expressed between WTs and fetal kidneys. This set contained 124 matches to genes on a microarray used by Stuart and colleagues (Stuart RO Bush KT Nigam SK Changes in global gene expression patterns during development and maturation of the rat kidney. Proc Natl Acad Sci USA 2001 98 5649-5654) to establish genes with stage-specific expression in the developing rat kidney. Mapping between the two data sets showed that WTs systematically overexpressed genes corresponding to the earliest stage of inetanephric development and underexpressed genes corresponding to later stages.
Automated clustering identified a smaller group of 27 genes that were highly expressed in WTs compared to fetal kidney and heterologous tumor and normal tissues. This signature set was enriched in genes encoding transcription factors. Four of these PAX2 EYA1 HBF2 and HOXA11 are essential for cell survival and proliferation in early metanephric development whereas others including SIX? MOX1 and SALL2 are predicted to act at this stage. SIX? and SALL2 proteins were expressed in the condensing mesenchyme in normal human fetal kidneys but were absent (SIX?) or reduced (SALL2) in cells at other developmental stages.
These data imply that the blasterna in WTs has progressed to the committed stage in the mesenchymal-epithelial transition where it is partially arrested in differentiation. The WT-signature set also contained the Wnt receptor FZD7 the tumor antigen PRAME the imprinted gene NNAT and the metastasis-associated transcription factor ElAF.
HSA05222_SMAL
L_CELLLUNG_C
ANCER

- 98 -STANDARD
MEMBERS
NAME
LIJETALys_WT 40004 at, 35260 at, 904_s_at, 1383_4 36491 at, 36597_4 1782_s_at, 35141_4 _KIDNEY_DN 34878 at 38875 rat 36206,A, 40074_4 40726_at, 36581 at 2003_s_at, 572_at, 32081 at, 39425_4 31872_at, 38753_at, 40512 at, 37585_4 39073_4 36671 at, 32598 at, 33255_4, 40117 at, 34563 at, 39230 at, 37663 at, 34402 at, 32842 at, 2053_4 39642 at, 39519 at, 419_4 39715_4 603_4 40634_4 35200_4 1833 at, 41050 at, 39426 at, 36837 at, 34843_at, 34314_at, 38724 at, 36813_4 39045 at, 37809 at, 37564 at, 604 at, 349_g_at, 35699 at, 32120 at, 37193 at, 33324_s_at, 40412_at, 37686_s_at, 37171_4 34851_4 38424 at, 37739_at, 32263 at, 38251_4 975_4 361178 at, 40641 at, 1985 sat, 32617_at, 32190Lat, 1599 at 38158 at 33852 at 36932 at 41626_at, 41352_4 41569_4 38728_at, 34829_at, 860Lat, 1031_at, 40465_at, 32644_at, 35615_4 34950_at, 39109_at, 40690 at, 38414 at, 36898_r_at, 41403 at, 35657 at, 36650 at, 32102_4, 1592 at, 37231_at, 38863_4 37508Wat, 38951_4 36603_at, 40774_at, 37283_ot, 37347_at, 40145_4 38834_at, 1287_at, 37605_at, 40827_at, 36189 at, 36192 at, 35829_4 38032 at, 32261_4 40575_at, 32589 at, 39175_4 37293_4 37985_4 33266_at, 39372_4 38847_4 38116_at, 35969_at, 33222 at, 36611_4 33929 at, 212 at, 798 at, 37971 at, 35694 at, 1521 at, 1860 at, 41099 at, 34783_s_at, 41583_4 649_s_at, 1235_at, 1057_4 32696_4 38313_at, 39342_at, 37073 at, 38105_at, 1584_at, 1721_g_at, 1884_s_at, 36010_at, 36059_at, 37567_at, 37774_at, 35312_at, 41395_4 32790_4 38358_at, 37302_at, 31838_at, 37305_at, 1058_at, 39337_4 38977_4 34726_4 35745_f_at, 40407_at, 38573_at, 1544_at, 38702_at, 36815 at, 2093_s_at HSA05222_SMAL AKT3, CDK2, CDK4, CDK6, CDKN1B, CDKN2B, LAMC3, PIAS3, CHUK, CKS1B, L_CELL JUNG_C COL4A1, COL4A2, C014A4, COL4A6, E2F1, E2F2, E2F3, AKT1, AKT2, FHIT, LAMB4, ANCER FN1, PIK3135, LAMA1, APAF1, BIRC2, BIRC3, B1RC4, IKBKB, 1TGA6, ITGA2, ITGA2B, 1TGA3, 1TGAV, ITGB1, LAMA2, LAMA3, LAMA4, LAMAS, IAMBI, LAMB2, LAMB3, LAMC1, LAMC2, MAX, MYC, NFKB1, NFKB2, NFKBIA, NOS1, NOS2A, NOS3, PIAS4, PIK3CA, P1K3CB, PHOCD, PIK3CG, PIK3R1, PIK3R2, CYCS, PTEN, PTGS2, PTK2, RARB, RB1, CCND1, BC12, RELA, BCL2L1, RXRA, RXRB, RXRG, SKP2, TP53, TRAF1, TRAF2, TRAF3, TRAF5, TRAF6, CASP9, P1K3R3, IKBKG, PIAS1, CCNE1, P1AS2, CCNE2, TRAF4

- 99 -STANDARD
MEMBERS SYMBOLIZED
NAME
LIJETALys_WT MNI, AURKB, BIM, DDX23, SPAG5, MU, ABCA2, CKSIB, FENI, MTH FD2, _KIDNEY_DN PRI M2A, CKAPS, NME1, TIAI, SLCI6A1, CCNB2, CHSTI, ILF2, AURKA, TXNRDI, GPCI, TOP2A, MCM6, RNASEH2A, PLK4, MARS, TPS3BP2, BRCA1, TRIP13, UNG, TMSLB, BUB3, XPOT, COL2A1, PFKP, CDK2, KPNA2 /// MGC40489 /, RRM1, KIAA0692, LMNBI., KIAA1794, NOLC1, KIF23, NELL2, MSH2, WASF3, STRAP, SRPKI, PRPF40A /// 1OC64245, CACNB3, MCM2, NAPIL1, CRABP2, BCL7A, BOPI /ll 1OC653119, CHAFIA, CCND2, EZH2, EYAI, PRIM CDH2, DDX52, FZD7, CENPF, ST6GAL1, RFC5, H2AFZ, CXCR4, LRP4, CDC2, SIX1, MELK, NUP188, SV2A, 1I63, NUP205, PTTG1, ASNS, IGSF4, FLJ11021, SMC4, YWHAZ, TARBP2, ZNF516, MEOXI, YAF2, CKS2, ADIPOR2, MAD2L1, NR2C1, DLGS, ESPL1, ELOVL2, CH NI, GTF3C2, MIZF, FADS2, DLG7, HMGA2, KIFC1, NCBP2, FADSI, CDKN3, DKCI, FAM62A, MPHOSPH9, C120RF24, EN01 /// SNRPF, BUBIB, BAZIA, MKI67, SALL2, POLE3, KIAA0101, SNRPA1, BAZIB, KIF14, GREBI, KIAA0515, NASP, SCRNI, GARS, TCERGI, SHMT2, CCT3, CIT, TPX2, TIMELESS, SS18, CDC25C, COL13A1, PCBP2, SACS, MLXIP, MAP4K4, SSRPI, KIF2C, IARS, UK, TOPBPI, CDC20, BTAF1, PBX3, MSH6, DDXI, KIF11, XRCC5, MYL6B, SEP16, PPP2R2A, YARS, PCNA, APOBEC3B, DNAJC9, ACPI, HOXA9, STMNI, PARP1, ZN F423, GCNILI, TAF5, ROR2 HSA05222_SMAL AKT3, CDK2, CDK4, CDK6, CDKNIB, CDKN2B, LAMC3, PIAS3, CH UK, CKSIB, L_CELLLUNG_C C014A1, COL4A2, C014A4, COL4A6, E2F1, E2F2, E2F3, AKT1, AKT2, FH
IT, LAMB4, ANCER FNI, PIK3R5, LAMM, APAF1, BIRC2, BIRC3, BIRC4, IKBKB, ITGA6, ITGA2, ITGA2B, ITGA3, ITGAV, ITGB1, LAMA2, LAMA3, LAMA4, LAMAS, LAMM, LAMB2, LAMB3, LAMCI, LAMC2, MAX, MYC, NFKBI, NFKB2, NFKBIA, NOS1, NOS2A, NOS3, PIAS4, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, CYCS, PTEN, PTGS2, PTK2, RARB, RB1, CCN D1, BC12, RELA, BCL2LI, RXRA, RXRB, RXRG, SKP2, TP53, TRAFI, TRAF2, TRAF3, TRAF5, TRAF6, CASP9, PIK3R3, IKBKG, PIAS1, CCN El, PIAS2, CCNE2, TRAF4 - -STANDARD
PMlD AUTHORS
NAME
LIJETALys_WT 12057921 Li CM, GUO M, Borczuk A, Powell CA, Wei M, KIDNEY DN
Thaker HM, Friedman R, Klein U, Tycko B
HSA05222_SMAL Kanehisa, M., Araki, M., Goto, S., Hattori, L_CELLLUNG_C M., Hirakawa, M., ltoh, M., Katayama, T., ANCER Kawashirna, S., Okuda, S., Tokimatsu, T., Yarnanishi, Y.

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
FLOTHOSASP8A msigdb V2.5 '24-Mar-08 c2:1101 Human PLMRD_DIFF
CANCER_NEOPL msigdb V2.5 24-Mar-08 c2:1234 Human ASTIC_META_UP

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
F LOTH O_CASP 8A AFFYMETRIX c2 PLMRD_DIFF
CANCER_NEOPL GENE SYMBOL c2 ASIK_META_UP

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
FLOTHOSASF18A Kevin Vogelsang Broad Institute Genes significantly associated PLMRD_DIFF with MRD on day 46 CANCER_NEOPL 121 John Newman Washington University Sixty-seven genes commonly ASTiC_META_UP upregulated in cancer relative to normal tissue, from a meta analysis of the OncoMine gene expression database STANDARD
FULL DESCRIPTION
NAME
FLOTHO_CASP8A AB - In childhood acute lymphoblastic leukemia (ALL) early response to treatment is a PLMRD_DIFF powerful prognostic indicator. To identify genes associated with this response we analyzed gene expression of diagnostic lymphoblasts from 189 children with ALL and compared the findings with minimal residual disease (MRD) levels on days 19 and 46 of remission induction treatment. After excluding genes associated with genetic subgroups we identified 17 genes that were significantly associated with MRD. The caspase 8-associated protein 2 (CASP8AP2) gene was studied further because of its reported role in apoptosis and glucocorticoid signaling. In a separate cohort of 99 patients not included in the comparison of gene expression profiles and MRD low levels of CASP8AP2 expression predicted a lower event-free survival (P = .02) and a higher rate of leukemia relapse (P = .01) and were an independent predictor of outcome. High levels of CASP8AP2 expression were associated with a greater propensity of leukemic lymphoblasts to undergo apoptosis. We conclude that measurement of CASP8AP2 expression at diagnosis offers a means to identify patients whose leukemic cells are highly susceptible to chemotherapy. Therefore this gene is a strong candidate for inclusion in gene expression arrays specifically designed for leukemia diagnosis.
CANCER_NEOPL AB - Many studies have used DNA microarrays to identify the gene expression signatures of ASTiC_META_UP human cancer yet the critical features of these often unmanageably large signatures remain elusive. To address this we developed a statistical method comparative rnetaprofiling which identifies and assesses the intersection of multiple gene expression signatures from a diverse collection of microarray data sets. We collected and analyzed 40 published cancer microarray data sets comprising 38 million gene expression measurements from >3 700 cancer samples. From this we characterized a common transcriptional profile that is universally activated in most cancer types relative to the normal tissues from which they arose likely reflecting essential transcriptional features of neoplastic transformation. In addition we characterized a transcriptional profile that is commonly activated in various types of undifferentiated cancer suggesting common molecular mechanisms by which cancer cells progress and avoid differentiation. Finally we validated these transcriptional profiles on independent data sets.

STANDARD
MEMBERS
NAME
F LOTH 0_CASF18A 200081_5_4 220657_4 218268_4 200937_5_4 217747_5_4 218986 sat, P2M RD_D I FF 207894 sat, 215177_5_4 218586_4 201429_5_4 200019_s_at, 201904_5_4 208904_5_4 201337_s_at, 209499_x_at, 203963 at 204599_5_4 209543_5_4 209288_5_4 218736_s_at, 209732_4 209510_4 203422_at, 202326_4 211937_4 208724_5_4 211717_4 21804 ix at, 222201_s_at, 212202_5_4 200034_5_4 212042_x_at, 213075_at, 209502 sat 217098_5_4 200927_5_4 218562 sat, 209760_4 212509_5_4 208330_4 218033 sat, 203544 sat, 221646_5_4 212419_4 205888_5_4 201259_5_4 219165_at, 207979_5_4 202393_5_4 215717_s_at, 211927_x_at, 212773_5_at, 217281_x_at, 216520_5_4 213890_x_at, 200005 at, 200025_5_4 216444_4 207940_x_at, 200024 at, 202649_x_at, 217915_5_4 200038_5_4 218084_x_at, 221726 at, 204102_5_4 217719_4 219364_4 204804 at, 214351_x_at, 206142_4 200716_x_at, 201325_5_4 218115_at, 208856_x_at, 221718_5_4 206890_4 209795_at, 38158_4 200949_x_at, 203702_5_4 203276_at, 206502_5_at, 204426_4 200632_s_at CANCER_NEOPL TSTA3, FAP, CKS2, MTHFD2, NME1, SOX4, SNRPF, HNRPA2B1, TRAF4, SSR1, AS11C_META_UP MMP9, ILF2, CBX3, I FNGR2, CCT5, HSPD1, NCBP2, TOP2A, SSBP1, TARS, CDKN3, LDHA, PRDX4, CANX, PPP2R5C, PTMA, RBM4, MRPS12, PSMC4, KPNA2, SMARCA4, KIAA0101, KDELFQ, UBE2S, PLK1, CRI P2, MCM3, G3BP, NONO, E2F5, TPX2, TGIF, CCT4, PAFAH1B3, H DAC1, AHCY, 1ARS, PSME2, HSPE1, OGT, DVL3, COPB2, MRPL3, COL1A2, TUBB, A RMET, PAICS, ACLY, CDC2, SNRPE, DDX48, NUP205, SDHC, RFC4 STANDARD
MEMBERS SYMBOLIZED
NAME
FLOTHOSASP8A OLFML2A, VAMP3, KIAA0922, ESPL1, RPL27, ZNF96, STAM, C150RF15, BA1AP2, P2_MRD_DIFF MRPL28, ZNF135, ZDHHC11, ITGA6, POLD1, CD8B, SYPL1, EIF3S7, EEF1G /fi L00654007, CA12, TMED2, RPLPO, LMNB1, RPS16, LGP2, CDC42EP3, RPS6, CNR1, CTDSPL, RPS9, TBC1D15, RPS5, SMURF2, FBN2, AKAPI3, EEF2, TTLL4, SLC38A2, SNN, RF'15, TOMM20, ElF4B, RPL6, F'DLIM2, FAU, KLF10, RPL13A, RAB1A, TMEM87A, ElF3S6IP, TPT1, TRIM21, RPL7 /// LOC653702 1, INSM1, KLHL11, RPL17, TCL6, NDRG1, ANKRD40, MXRA7, FLJ20035, IL12RB1, RPL22, PLK1 /il RPL37A, CD69, TNFSFI3 /1/ TNFSFI2-, A5F1B, RPS28 111 L00645899, PALMD, C100RF56, RPSI9, RP113 /// 10C388344, CD34, RPS20, RNF139, TMEM57, C200RF20, EMP1, EHMT2, CASP8AP2, CLEC2B, ALX4, FXYD5, JAKMIP2, RAB14 CANCER_NEOPL TSTA3, FAP, CKS2, MTHFD2, NME1, SOX4, SNRPF, HNRPA2B1, TRAF4, SSR1, ASTIC_META_UP MMP9, 111'2, CBX3, IFNGR2, CCT5, HSPD1, NCBP2, TOP2A, SSBP1, TARS, CDKN3, LDHA, PRDX4, CANX, PPP2R5C, PTMA, RBM4, MRPS12, PSMC4, KPNA2, SMARCA4, KIAA0101, KDELR2, UBE2S, PLK1, CRIP2, MCM3, G3BP, NONO, E2F5, TPX2, TGIF, CCT4, PAFAH1B3, HDAC1, AHCY, IARS, PSME2, HSPE1, OGT, DVL3, COPB2, MRPL3, COL1A2, TUBB, ARMET, PAICS, ACLY, CDC2, SNRPE, DDX48, NUP205, SDHC, RFC4 STANDARD
PMiD AUTHORS
NAME
FLOTHOSASP8A 16627760 Flotho C, Coustan-Smith E, Pei D, iwamoto S.
PLMRD_DIFF Song G, Cheng C, Pui CH, Downing JR, Campana CANCER_NEOPL 15184677 Rhodes DR, Yu J, Shanker K, Deshpande N, AS11C_META_UP Varambally R, Ghosh Di, Barrette T, Pandey A, Chinnaiyan AM

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
CANCER_UNDIFF msigdb V2.5 '24-Mar-08 .c2:1235 Human ERENTIATED JVIE
TA_UP
STEMCELLSOM msigdb V2.5 24-Mar-08 c2:1645 Mouse MON_UP

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
CANCER_UNDIFF GENE SYMBOL c2 ERENTIATED_ME
TA_UP
STEMCELLSOM GENE SYMBOL c2 MON_UP

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
CANCER_UNDIFF 121 John Newman Washington University Sixty-nine genes commoniy ERENT1ATED_ME upregulated in undifferentiated TA_UP cancer relative to well -differentiated cancer, from a meta-analysis of the OncoMine gene expression database STEMCELLSOIVI LH John Newman Washington University Enriched in mouse embryonic, MON_UP neural and hematopoietic stem cells, compared to differentiated brain and bone marrow cells STANDARD
FULL DESCRIPTION
NAME
CANCER_UNDIFF AB - Many studies have used DNA microarrays to identify the gene expression signatures of ERENTIATED_ME human cancer yet the critical features of these often unmanageably large signatures remain TA_UP elusive. To address this we developed a statistical method comparative metaprofiling which identifies and assesses the intersection of multiple gene expression signatures from a diverse collection of microarray data sets. We collected and analyzed 40 published cancer microarray data sets comprising 38 million gene expression measurements from >3 700 cancer samples. From this we characterized a common transcriptional profile that is universally activated in most cancer types relative to the normal tissues from which they arose likely reflecting essential transcriptional features of neoplastic transformation. In addition we characterized a transcriptional profile that is commonly activated in various types of undifferentiated cancer suggesting common molecular mechanisms by which cancer cells progress and avoid differentiation. Finally we validated these transcriptional profiles on independent data sets.
STEMCELLSOIVI AB - The transcriptional profiles of mouse embryonic neural and hematopoietic stem cells MON_UP were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells and several of these genes are clustered in the genome. When compared to differentiated cell types stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown.
Embryonic and neural stem cells have many similarities at the transcriptional level.
These results provide a foundation for a more detailed understanding of stem cell biology.

STANDARD
MEMBERS
NAME
CANCER_UNDIFF PSMD14, CKS1B, 062, FOXMl, MTHFD2, MAD2L1, F130, NME1, NCAPD2, ERENT1ATED_ME SLC16A1, RAD21, MYBL2, ILF2, CCT6A, YBX1, DLG7, TOP2A, MCM6, ADRM1, TA_UP SSBP1, H2AFX, CDKN3, GCLM, PRDX4, TRIP13, CDC6, NCAPH, ElF2S2, GGH, 10:23, KPNA2, OPM1, KIAA0101, TMSB10, POLR2K, UBE2S, KIF14, MCM2, MCM3, CENPA, GARS, CEBPG, EZH2, PSMD2, CISL, BIRC5, KIF2C, SLC7A5, GPSM2, CDC20, HMGB2, H2AFZ, CCNA2, TUBB4, COL1A2, PCNA, TUBB, PSMB7, RPA3, GAS6, CDC2, UBE2C, MELK, SEC61B, CCNB1, TAP1, CXCL9, NUDT1, RFC4 STEMCELLSOM UBE2T, LAS1L, PRPSAP1, MDFIC, ERCC5, CPXMl, WDR43, PANX3, FHL1, ZNF639, MON_UP DPHS, RYK, PSMD11, MRPSIO, STAM, MK167IP, TXNDC9, TXNRD1, E1F4EBP1, BLZFl, YWHAH, SNX12, WDR55, CAD, 1TGA6, RPS6KB1, TOM1L1, PLA266, MTMRIO, YAP1, SNRPC, XPOT, C40RF28, C120RF45, CHD1, SFRS6, ELOVL6, TGIF2, KLHL7, PSMD12, ADAM9, RFFL, MSH2, PEX7, SOCS2, PPP1R2, TBC1D15, MRPL17, DTYMK, ARCN1, WTAP, RARSL, USP10, CTTN, PCF11, SLC38A2, C190RF2, MPHOSPHIO, CTBP2, TBRG4, SLC4A7, ZZZ3, GAB1, C170RF79, ANKRD17, PPIC, RABGGTB, NUP35, COPS7A, LAPTM4A, SMARCAD1, KCNAB3, RCN?, FKBP11, C1RH1A, MRPL34, TR1P6, EPRS, TARSL1, STRN3, JAGN1, MARCH7, IARS2, XP01, RPP14, ZFX, MRPL3, CRTAP, XTP3TPA, RRN3, ZNF644, PAFAH2, GCAT, TiPl, SFRS3, CRSP3, FU11021, CBR3, ALDH7A1, ZNF213, USP9X, MPDU1, YES?, P1GX, FU31951, GSTA4, RNF138, LSG1, DICER?, ITGB1, RAB18, SEC23A, MRPL45, WBP5, SMAD2, LIMA1, COPS4, NDUFAB1, C200RF6, GCLM, SMAD1, GNL2, IMPAD1, KIF2A, ACADM, STXBP3, TPP2, MAP4K3, ZCCHC10, SUCLG2, BACH1, TEAD2, UBE2D2, FKBP9, GNB1, ZMYM4, E1F4G2, GRWD1, MRPS2, ElF3S1, MTERFD1, GARS, ARIH1, ZNF281, GAS2, PTPN2, UMPS, PHTF2, PPA1, FBX038, PRPF6, SEC23IP, LAPTM4B, FBX08, UPP1, MRS, RNF4, STATIP1, PLS3, RASA1, DDX1, RPUSD4, XRCC5, RPL22, PKD2, NOP5/NOP58, TXNL1, MDFI, YWHAB, ROCK2, DNAJB6, RAD23B, UTP20, MRPS31, ZMAT3, PDCD2, RSL1D1, CDKN1A, SH3D19, NDUFAF1 STANDARD
MEMBERS SYMBOLIZED
NAME
CANCER_UNDIFF PSMD14, CKS1B, 062, FOXMl, MTHFD2, MAD2L1, FI3O, NME1, NCAPD2, ERENTIATED_ME SLC16A1, RAD21, MYBL2, ILF2, CCT6A, YBX1, DLG7, TOP2A, MCM6, ADRM1, TA_UP SSBP1, H2AFX, CDKN3, GCLM, PRDX4, TRIP13, CDC6, NCAPH, El F2S2, GGH, 10:23, KPNA2, OPM1, KIAA0101, TMSB10, POLR2K, UBE2S, KIF14, MCM2, MCM3, CENPA, GARS, CEBPG, EZH2, PSMD2, CISL, BIRC5, KIF2C, SLC7A5, GPSM2, CDC20, HMGB2, H2AFZ, CCNA2, TUBB4, COL1A2, PCNA, TUBB, PSMB7, RPA3, GAS6, CDC2, UBE2C, MELK, SEC61B, CCNB1, TAP1, CXCL9, NUDT1, RFC4 STEMCELLSOM UBE2T, LAS1L, PRPSAP1, MDFIC, ERCC5, CPXMl, WDR43, PANX3, FHL1, ZNF639, MON_UP DPHS, RYK, PSMD11, MRPSIO, STAM, MKI671P, TXNDC9, TXNRD1, E1F4EBP1, BLZFl, YWHAH, SNX12, WDR55, CAD, ITGA6, RPS6KB1, TOM1L1, PLA266, MTMRIO, YAP1, SNRPC, XPOT, C40RF28, C120RF45, CHD1, SFRS6, ELOVL6, TGIF2, KLHL7, PSMD12, ADAM9, RFFL, MSH2, PEX7, SOCS2, PPP1R2, TBC1D15, MRPL17, DTYMK, ARCN1, WTAP, RARSL, USP10, CTTN, PCF11, SLC38A2, C190RF2, MPHOSPHIO, CTBP2, TBRG4, SLC4A7, ZZZ3, GAB1, C170RF79, ANKRD17, PPIC, RABGGTB, NUP35, COPS7A, LAPTM4A, SMARCAD1, KCNAB3, RCN?, FKBP11, C1RH1A, MRPL34, TRIP6, EPRS, TARSL1, STRN3, JAGN1, MARCH7, IARS2, XP01, RPP14, ZFX, MRPL3, CRTAP, XTP3TPA, RRN3, ZNF644, PAFAH2, GCAT, SFRS3, CRSP3, FU11021, CBR3, ALDH7A1, ZNF213, USP9X, MPDU1, YES?, PIGX, FU31951, GSTA4, RNF138, LSG1, DICER?, ITGB1, RAB18, SEC23A, MRPL45, WBP5, SMAD2, LIMA1, COPS4, NDUFAB1, C200RF6, GCLM, SMAD1, GNL2, IMPAD1, KIF2A, ACADM, STXBP3, TPP2, MAP4K3, KCHC10, SUCLG2, BACH1, TEAD2, UBE2D2, FKBP9, GNB1, ZMYM4, E1F4G2, GRWD1, MRPS2, El F3S1, MTERFD1, GARS, AR11-11, ZNF281, GAS2, PTPN2, UMPS, PHTF2, PPA1, FBX038, PRPF6, SEC23IP, LAPTM4B, FBX08, UPP1, MRS, RNF4, STATIP1, PLS3, RASA1, DDX1, RPUSD4, XRCC5, RPL22, PKD2, NOP5/NOP58, TXNL1, MDFI, YWHAB, ROCK2, DNAJB6, RAD23B, UTP20, MRPS31, ZMAT3, PDCD2, RSL1D1, CDKN1A, SH3D19, NDUFAF1 STANDARD
PMiD AUTHORS
NAME
CANCER_UNDIFF 15184677 Rhodes DR, Yli J, Shanker K, Deshpancle N, ERENT1ATED_ME Varambally R, Ghosh D, Barrette T, Pandey A, TA_UP Chinnaiyan AM
STEMCELLCOM 12228720 Ramalho-Santos M, 'loon S. Matsuzaki V. Mulligan MON_UP RC, Melton DA

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NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
BHATTACHARYA msigclb V2.5 '24-Mar-08 .c2:726 Human ESC UP

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
BHATTACHARYA GENE _SYMBOL c2 ESC UP

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
BRATTACHARYA Kate Stafford Broad Institute Genes upregulatecl in _ESC_UP undifferentiated human embryonic stem cells.

STANDARD
FULL DESCRIPTION
NAME
BRATTACHARYA AB - Human embryonic stem (huES) cells have the ability to differentiate into a variety of cell _ESC_UP lineages and potentially provide a source of differentiated cells for many therapeutic uses.
However little is known about the mechanism of differentiation of huES cells and factors regulating cell development. We have used high-quality microarrays containing seventy-base pair oligonucleoticies to examine gene expression in 6 of the 11 available huES
cell lines. Expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All 6 cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient > 0,85).A common subset of 92 genes was identified that included Nanog GTCM-1 con nexin 43 (GJA1) act-4 and TDGF1 (cripto). Gene expression was confirmed by a variety of techniques including comparison with databases reverse transcriptase-polymerase chain reaction focused cDNA microarrays and immunocytochemistry. Comparison with published "sternness" genes revealed a limited overlap suggesting little similarity with other stem cell populations. Several novel ES cell-specific expressed sequence tags were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES
cells and provide a unique set of markers to profile and better understand the biology of huES cells.

STANDARD
MEMBERS
NAME
BRATTACHARYA ARLB, ZNF257, BRIX, KRT18, MTHFD2, MAA1573, STK12, ZNF43, SNRPF, SLC16A1, _ESC_UP RP524, HDAC2, DSG2, MP-2, HMGY, MGC27165, TNNT1, MAD212, HSSG1, RPL4, MGST1, FLJ12581, RPLPO, FABP5, P1TX2, HNRPA1(C20orf168), ELOVL6, GJA1, HSPA4, GDF3, SERPINH1, LRRN1, ZNF117, HNRPAB, SMS, KPNA2, PPAT, ACTC, KRT8, C20orf129, Ribosomai4OALarninreceptor, SPS, TDGF1, NBR2, RPL7, PSIP1, NASP, CRABP2, TUBB-5, CYP26A1, UN-28, RPL6, CCTB, GAL, CRABP1, TUBB-4, NME2, EPRS, LDHB, GSH1, LAPTM4B, CAW, NS, MTHFD1, CCNC, TD-60, RMGB2, SSB, Cl5orf15, Jade-1, Numatrin, CDC2, PODXL, E1F4A1, PSMA2, SFRP2, RAMP, IDH1, LEFTB, SET, CCNB1, PSMA3, TK1, DDX21, PTTG1, RPL24, C20orfl/TPX2, K1F4A, POU5F1/Oct4, IMPDH2, SEMA6A, NPM1 STANDARD
MEMBERS SYMBOLIZED
NAME
BHATTACHARYA ARLB, ZNF257, BRIX, KRT18, MTHFD2, KIAA1573, STK12, ZNF43, SNRPF, SLC16A1, _ESC_UP RP524, HDAC2, DSG2, IMP-2, HMGIY, MGC27165, TNNT1, MAD212, HSSG1, RPL4, MGST1, FLJ12581, RPLPO, FABP5, P1TX2, HNRPA1(C20orf168), ELOVL6, GJA1, HSPA4, GDF3, SERPINH1, LRRN1, ZNF117, HNRPAB, SMS, KPNA2, PPAT, ACTC, KRT8, C20orf129, RibosomaI40ALarninreceptor, SPS, TDGF1, NBR2, RPL7, PSIP1, NASP, CRABP2, TUBB-5, CYP26A1, UN-28, RPL6, CCTB, GAL, CRABP1, TUBB-4, NME2, EPRS, LDHB, GSH1, LAPTM4B, CAW, NS, MTHFD1, CCNC, TD-60, HMGB2, SSB, Cl5orf15, Jade-1, Numatrin, CDC2, PODXL, EIF4A1, PSMA2, SFRP2, RAMP, IDH1, LEFTB, SET, CCNB1, PSMA3, TK1, DDX21, PTTG1, RPL24, C20orfl/TPX2, KIF4A, POU5F1/Oct4, IMPDH2, SEMA6A, NPM1 STANDARD
PMiD AUTHORS
NAME
BRATTACHARYA 15070671 Bhattacharya B, Miura T, Brandenberger R, Mejido _ESC_UP J, LLIO Y, Yang AX, Joshi BH, Ginis L Thies RS, Arra M, Lyons I, Condie BG, Itskovitz-Eldor J, Rao MS, Puri RK

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
BROWN_MYELOI msigclb V2.5 '24-Mar-08 .c2:931 Human D_PROLILAND_ SELF RENEWAL

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
BROWN_MYELU AFFYMETRIX c2 D_PROLILAND_ SELF RENEWAL

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
BROWN_MYELOI Kevin Vogelsang Broad Institute Genes associated with Myeloid D_PROLIF_AND_ cell proliferation and self-SELF_RENEWAL renewal STANDARD
FULL DESCRIPTION
NAME
BROWN_MYELOl AB - Mechanisms controlling the balance between proliferation and self-renewal versus D_PROULAND_ growth suppression and differentiation during normal and leukemic myelopoiesis are not SELF RENEWAL understood. We have used the bi-potent FDB1 myeloid cell line model which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor having differential signaling and leukemogenic activity.
This model is suited to large-scale gene-profiling and we have used a factorial time-course design to generate a substantial and powerful data set. Linear modeling was used to identify gene-expression changes associated with continued proliferation differentiation or leukemic receptor signaling. We focused on the changing transcription factor profile defined a set of novel genes with potential to regulate myeloid growth and differentiation and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene-expression changes associated specifically with the leukemic GM-CSF receptor mutant V449E. Signaling from this receptor mutant down-regulates CCAATienhancer-binding protein alpha (C/EBPalpha) target genes and generates changes characteristic of a specific acute myeloid leukemia signature defined previously by gene-expression profiling and associated with CJEBPalpha mutations.

STANDARD
MEMBERS
NAME
BROWN_MYELCA Zfpnla2, Mg11, Ankrd10, U66667, BC011467, Papss2, Nt5c3, Zfpnla4, F2r, H2-Ebl, D_PROULAND_ Kcnk5, 712375, Ailey, U26789, Aqp9, Nme1, 2810026P18Rik, Z12540, Trim28, SIcla5, SELF RENEWAL 2810028N01Rik, Bn1, Rcor2, Ppef2, 2410004N09R1k, Pcx, Cpa3, M55696, Ide, Meplb, 4932438H23Rik, Tnfsf11, Fcerla, Oact2, Ivnslabp, Sfrs7, H2-Ea, BC003986, Txk, 5eptl, Msh2, Npml, Maga, Mfng, Gata2, Tyms-ps, 181001412M, Fbxw4, Cct6a, U54534, 712266, Sic7a8, Npi, P2ry10, Nsun2, Clqbp, Gnal, Wdr43, 49305551311Rik, Eif4e, Odd, Tcrb-V8.2, Nripl, Pdk, 5830474E16R1k, Bzw2, Cdh17, F2d3, Cdk4, Gtpbp4, Lama5, 712248, Trf, 2310066E14R1k, Rpol-2, Hsp105, 712249, Ppan, BCO29169, Sodl, 4933415F23R1k, Nrgn, St6gall, 4930485D02Rik, Rcll, 8430438D04Rik, Timm8a, 5t7, Slc16al, Fut7, D13Wsul77e, Tcrb-V13, 722043, Akr1c12, 712552, 1300007C21R1k, Rgs5, Akr1c13, 712259, Stc2, 503, Nol5a, Rp123, 5830405N2ORik, Ptpn9, Pgrmcl, Xpot, RuvbI2, Psatl, 712246, Mgat5, S1c7a5, Trim37, Spint2, Msi2h, Nutf2, Mcpt8, Cbfa2t3h, Gait, Emx2, Erh, Apexl, Nars, 5f3b3, H2-Aa, F9, Ncl, Galnact2, H2-Ab1, S1c22a3, Fkbp4, Kka15, Tex292, Sox4, Gas5, 1500012F01Rik, 4930535E02Rik, D13Ertd275e, Rab27b, Smarccl, Myb, Fut8, Ctia2a, Ssr3, Hspdl, No6, Shmt2, Rif1, Aasdhppt, MGI:1929091, NM 026110, Hmgnl, Cak, Osbp13, Cyplial, Gpr56, Nolcl, Ifitm3, C330027104R1k, Cd48, Serpina3g STANDARD
MEMBERS SYMBOLIZED
NAME
BROWN_MYELOI Zfpnla2, Mg11, Ankrc110, U66667, BC011467, Papss2, Nt5c3, Zfpnla4, F2r, H2-Ebl, D_PROULAND_ Kcnk5, 712375, Ailey, U26789, Aqp9, Nme1, 2810026P18Rik, Z12540, Trim28, 51cla5, SELF RENEWAL 2810028N01Rik, Binl, Rcor2, Ppef2, 2410004N09R1k, Pcx, Cpa3, M55696, Ide, Meplb, 49324381-123Rik, Tnfsf11, Fcerla, Oact2, Ivnslabp, Sfrs7, H2-Ea, BC003986, Txk, Sept', Msh2, Npml, Maga, Mfng, Gata2, Tyms-ps, 1810014L12Rik, Fbxw4, Cct6a, U54534, 712266, Sic7a8, Npl, P2ry10, Nsun2, Clqbp, Gnal, Wdr43, 49305551311Rik, Eif4e, Odd, Tcrb-V8.2, Nripl, Pdir, 5830474E16R1k, Bzw2, Ccih17, F2d3, Cdk4, Gtpbp4, Lama5, Z12248, Trf, 2310066E14R1k, Rpol-2, Hsp105, 712249, Ppan, BCO29169, Sodl, 4933415F23R1k, Nrgn, St6gall, 4930485D02Rik, Rcll, 8430438D04Rik, Timm8a, 5t7, Slc16al, Fut7, D13Wsul77e, Tcrb-V13, 722043, Akr1c12, 712552, 1300007C21R1k, Rgs5, Akr1c13, 712259, Stc2, SytI3, Nol5a, RpI23, 5830405N2ORik, Ptpn9, Pgrmcl, Xpot, RuvbI2, Psatl, 712246, Mgat5, S1c7a5, Trim37, Spint2, Msi2h, Nutf2, Mcpt8, Cbfa2t3h, Gait, Emx2, Erh, Apexl, Nars, 5f3b3, H2-Aa, F9, Ncl, Galnact2, H2-Ab1, S1c22a3, Fkbp4, KIra15, Tex292, Sox4, Gas5, 1500012F01Rik, 4930535E02Rik, D13Ertd275e, Rab27b, Smarccl, Myb, Fut8, Ctla2a, Ssr3, Hspdl, NoI5, Shmt2, Rif1, Aasdhppt, MGI:1929091, NM 026110, Htngnl, CaIr, Osbp13, Cyplial, Gpr56, Nolcl, Ifitm3, C330027104R1k, Cd48, Serpina3g STANDARD
PMiD AUTHORS
NAME
BROWN_MYELOl 16769770 Brown AL, Wilkinson CR, Waterman SR, Kok CH, D_PROULAND_ Salerno DG, Diakiw SM, Reynolds B, Scott HS, Tsykin SELF RENEWAL A, Glonek GF, Goodall al, Solomon Pi, Gonda Ti, D'Andrea PJ

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
IDX_TSA_DN_CL msigclb V2.5 24-Mar-08 c2:1498 Mouse ADIPOGENESIS_ msigdb V2.5 24-Mar-OS c2:1132 Human H MSC_CLA5S8_ DN

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
1DX_TSA_DN_CL GENE SYMBOL c2 ADIPOGENESIS_ GENE SYMBOL c2 H MSC_CLASS8_ DN

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
IDX_TSA_DN_CL 121 John Newman Washington University Progressively down-regulated USTER2 from 8-96 hours during differentiation of 3T3-11 fibroblasts into adipocytes with IDX (insulin, dexamethasone and isobutylxanthine), vs. fibroblasts treated with IDX TSA to prevent differentiation (cluster 2) ADIPOGENESIS_ 121 John Newman Washington University Down-regulated 144 days FIMSC_CLASS8_ following the differentiation of DN human bone marrow mesenchymal stem cells (hMSC) into adipocytes, versus untreated hMSC cells (Class VIII) STANDARD
FULL DESCRIPTION
NAME
IDX_TSA_ON_CL AB - During cellular differentiation and development it is recognized that many complex USTER2 molecular mechanisms as well as precise patterns of differentially expressed genes occur in directing precursor cells toward a given lineage. Using microarray-based technology we examined gene expression across the course of 3T3-L1 aclipocyte differentiation. Total cellular RNA was isolated at times 0 2 8 16 24 48 and 96 h following treatment with either standard hormonal inducers of differentiation insulin dexamethasone isobutylmethylxanthine (IDX) or IDX plus trichostatin A (TsA) a histone deacetylase inhibitor and potent adipogenic inhibitor. cRNA was synthesized from cellular RNA and hybridized to high density Affymetrix MG_U74Av2 microarray gene chips containing 12 488 cDNA/Expressed Sequence Tags (ESTs) probe sets. From the IDX-only treated cells all probe sets that were either unchanged or differentially expressed less than 2-fold throughout differentiation with respect to time 0 preadipocytes were excluded from further analyses.
This selection resulted in a net of 1686 transcripts 859 were increased in expression and 827 were decreased in expression at least 2-fold across differentiation. To focus in on genes that were more specific to differentiation the same analysis was performed on IDX
plus TsA-treated non-differentiating cells and all probe sets from the iDX-only group that exhibited similar expression profiles in the non-differentiating TsA-treated group were excluded leaving a total of 1016 transcripts that were regulated only under differentiating conditions.
Six hundred and thirty-six of these transcripts were elevated at least 2-fold and 380 exhibited a decrease in expression relative to time 0 preadipocytes. This group of genes was further analyzed using hierarchical clustering and self-organizing maps and resulted in the identification of numerous genes not previously known to be regulated during adipocyte differentiation. Many of these genes may well represent novel adipogenic mediators and markers of adipogenesis.
ADIPOGENESIS_ AB - Human bone marrow mesenchymal stem cells (hMSCs) give rise to adipocytes in HMSC_CLASS8_ response to adipogenic hormones. An in-house cDNA microarray representing 3400 genes DN was employed to characterize the modulation of genes involved in this process. A total of 197 genes showed temporal gene expression changes during adipogenesis including genes encoding transcriptional regulators and signaling molecules. Semi-quantitative RT-PCR
analyses confirmed differential expression at the transcriptional level of several genes identified by cDNA microarray screening. Cluster analysis of the genes regulated during the late phase (from day 7 to day 14) of hMSC adipogenesis indicated that these changes are well correlated with data previously reported for murine preadipocytes.
However during the early phase (day 1-day 5) the modulations of genes differed from those reported for the preadipocytes. These data provide novel information on the molecular mechanisms required for lineage commitment and maturation accompanying adipogenesis of hMSC.

STANDARD
MEMBERS
NAME
IDX_TSA_DN_CL USP22, PUP, PTG1S, PPGB, OGN, RPS6KA4, ANXA5, NCAM1, ACTA2, WBP5, BDNF, USTER2 CD99, FGF7, PRDX4, MAP1LC3B, TW1ST2, LEPROT, LOX, 11NP1, PDLIM7, FAM91A1, SLC38A4, VCL, PGCP, ASAH1, PLYNB2, TMSB10, GNB1, PHLDB2, LAMP2, ITGAV, 100653503, ANXA3, DSTN, MYH9, EMP3, GBA, CMTM3, F2R, PRSS23, PLA2G7, RAB31, SCPEPI, VCAM1, ZFP36L2, F'DPN, BAX, EPPB9, 51CIA4, CD276, 51C7A5, SERPINF1, TES, C014A5, PRAF2, TSPAN6, CAP1, PLD3, SMAD6, NO02, MSN, SKI, SERTAD1, L0C440525, FXYD5, CAPG
ADIPOGENESIS_ CYR61, COL15A1, SCYE1, RGS4, BASP1, 1GFBP3, IER2, TNFRSF11B, HCK, SLC7A5, HMSC_CLASS8_ SERPINEL TMEM47, HAPLN1, THBS2, C5ORF13, ABI3BP, LOX, LRP3, C019A3, MYH11, DN 1TGA3, SRPK2, STK381, GDF15, SERP1NE2, NT5E, SMAD3, ZNF133, ACTG2, TPM1, KRR1, POSTN

STANDARD
MEMBERS SYMBOLIZED
NAME
IDX_TSA_DN_CL USP22, PUP, PTGIS, PPGB, OGN, RPS6KA4, ANXA5, NCAM1, ACTA2, WBPS, BDNF, USTER2 CD99, FGF7, PRDX4, MAP1LC3B, TWIST2, LEPROT, LOX, TINP1, PDLIM7, FAIV191A1, SLC38A4, VCL, PGCP, ASAH1, PLYNB2, TMSB10, GNB1, PHLDB2, LAMP2, ITGAV, 100653503, ANXA3, DSTN, MYH9, EMP3, GBA, CMTM3, F2R, PRSS23, PLA2G7, RAB31, SCPEPI, VCAM1, ZFP36L2, F'DPN, BAX, EPPB9, 51CIA4, CD276, 51C7A5, SERPINF1, TES, C014A5, PRAF2, TSPAN6, CAP1, PLD3, SMAD6, NO02, MSN, SKI, SERTAD1, L0C440525, FXYD5, CAPG
ADIPOGENESIS_ CYR61, COL15A1, SCYE1, RGS4, BASP1, IGFBP3, IER2, TNFRSF11B, HCK, SLC7A5, HMSC_CLASS8_ SERPINE1, TMEM47, HAPLN1, THBS2, C5ORF13, ABI3BP, LOX, LRP3, C019A3, MYH11, DN ITGA3, SRPK2, STK381, GDF15, SERPINE2, NT5E, SMAD3, ZNF133, ACTG2, TPM1, KRR1, POSTN

STANDARD PMD AUTHORS
NAME
IDX_TSA_DN_CL 15033539 Burton OR, Nagarajan R, Peterson CA, McGehee RE
USTER2 Jr ADIPOGENESIS_ 12646203 Nakamura T, Shiojima S, Hirai Y, lwama T, Tsuruzoe HMSC_CLASS8_ N, Hirasawa A, Katsuma S. Tsujimoto G
DN

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
CTNNK_oncoge msigclb V2.5 '24-Mar-08 .c2:1791 Human nic_signature WNTJARGETS msigdb V2.5 24-Mar-08 c2:802 Human STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
CTNNBl_oncoge HQUI33_Plus_2 c2 nic_signature WNTJARGETS http://www.stanforcl.ecluhnu SEQ_ACCESSION c2 sse/wntwindow.htmI

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
CTNNBl_oncoge Arthur Liberzon Broad Institute Genes selected in supervised nic_signature analyses to discriminate cells expressing activated beta-catenin (CTNNB1) oncogerie from control cells expressing GFP.
WNTJARGETS Yujin Hoshicla Broad Institute WNT target genes from literatures STANDARD
FULL DESCRIPTION
NAME
CTNNBl_oncoge The authors used primary mammary epithelial cell cultures (HMECs) to develop a series of nic_signature pathway signatures. The authors used recombinant adenoviruses to force expression of human activated beta-catenin in an otherwise quiescent cell, thereby specifically isolating the subsequent events as defined by the activation of/deregulation of this single pathway.
RNA from multiple independent infections was collected for DNA microarray analysis using Affymetrix HG_U133_Plus2 array. Gene expression signature that reflects the activity of the CTNNB1-induced pathway was identified using supervised classification methods described in [PMID: 11562467]. The analysis selects a set of genes for which the expression levels are most highly correlated with the classification of HMEC samples into beta-catenin-activated/deregulated versus control (forced expression of green fluorescence protein).
WNTJARGETS

STANDARD
MEMBERS
NAME
CTNNBl_oncoge 235009_4 211671_5_4 213350_4 1560318_4 212355 at, 223139_5_4 nic_signature 1569594_a_at, 207700_5_4 222760_4 203509_4 217277_4 1557081 at, 209257_5_4 216563_4 1554260_a_at, 226799_4 212560_4 203147_5_4 211343_5_4 244287 at, 212794_5_4 235388_at, 222122_5_4 225098 at, 213637_at, 225097_4 236241_4 226094_4 241464_5_4 207002_5_4 1555673_4 201865_x_at, 213328_4 208859_5_4 228315_4 223380_5_4 1558173_a_at, 225116_4 222728_5_4 232094_4 244677_4 1555920_4 218796 at, 233204_.4 229422_4 213850_5_4 209318_x_at, 229958_4 222696_4 212044_5_4 210057_at, 208901_5_4 224250_5_4 212996_5_4 229846_5_4 203255_4 210118_5_4 241617_x_at, 212492_5_4 221900_4 212994_4 228180_4 208953_4 210178_x_at, 225021_4 60474_4 222667_5_4 206108_5_4 200842_5_4 203304_4 204048_5_4 209457_4 235209_4 215646_5_4 1568408_x_at, 232231_4 1555945_5_4 214814_4 212263 at, 1562416_4 222747_5_at, 212177_4 206504_at, 202648_4 213478_4 213352_4 219024_4 210355_4 202643_5_4 212692_5_at, 222227_4 222834_5_4 229115_4 212420_4 218150_4 208900_s_at 227475_at, 244075_at WNT _TARGETS PLAUR, FOSL1, FGF18, ATOH1, CD44, JUN, NRCAM, FST, EFNB1, MET, CCND1, MMP7, B1RC5, FOXN1, FZD7, GSF4C, LEF1, VEGF, PPARD, CLDN1, MMP26, 1D2 1D2, BMP4, GAST

STANDARD
MEMBERS SYMBOLIZED
NAME
CTNNBl_oncoge FAM44A, NR3C1, RPS11, ARHGAP29, KIAA0323, DHX36, SDCCAG1, NCOA3, ZNF703, nic_signature SORL1, RBM25, SMC3, ANKRD12, FRYL, C110RF32, TRIM14, C0L13A1, SFRS12, KIAA1033, CHD9, THOC2, AB12, HIPK2, MED31, PIK3C2A, FU27365, PLAGL1, KRTAP2-1, NEK1, ATRX/1110C642995, LAT52, LUZF'1, J0SD3, C150RF29, PERI, CBX3, C200RF42, FU11903, NRD1, SFR521P, C80RF61, AX1N2, RPL27A, SMG1, TOP1, SECISBP2, C210RF108, MAPKAP1, FBX011, lIlA, JMJD2B, COL8A2, 5MU1, LARP5, FUSIP1///
L00642558, ZNF532, ASH1L, SFRS6, EPRS, BAMBI, PHACTR2, DUSP5, RPESP, C5PG2, RUNX2, FAM120A, YTHDC1, QKI, FLNB, SCML1, C6ORF111, CYP24A1, RPS19, KIAA1026, TMCC1, PLEKHAl, PTHLH, TNFAIP3, LRBA, ZNF236, GNG12, DYNC1H1, ELF1, ARLSA, FOXQ1, LOC158160 WNTJARGETS PLAUR, FOSL1, FGF18, ATOH1, CD44, JUN, NRCAM, FST, EFNB1, MET, CCND1, MMP7, BIRCS, FOXN1, FZD7, IGSF4C, LEF1, VEGF, PPARD, CLDN1, MMP26, 1D2/1/1D2B, ID2, BMP4, GAST

STANDARD
PMlD AUTHORS
NAME
CTNNBl_oncoge 16273092 Bild Aft Yao 6, Chang JT, Wang Q, Potti A, nic_signature Chasse D, Joshi MB, Harpole D, Lancaster IM, Berchuck A, Olson JA Jr, Marks JR, Dressman HK, West M, Nevins JR.
WNTJARGETS

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
TGFBETA_C4_UP msigclb V2.5 '24-Mar-08 .c2:1662 Human STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
TGFBETA_Cil_UP GENE _SYMBOL c2 STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
TGFBETA_C4_UP 121 John Newman 'Washington University Upreguiated by TGF-beta treatment of skin fibroblasts, cluster 4 STANDARD
FULL DESCRIPTION
NAME
TOFBETA_C4 _UP AB - Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (I-OF-beta) signaling through the Smad pathway little progress has been made in the identification of direct target genes. In this report using cDNA
microarrays we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target we postulated that it had to meet the following criteria (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach we have identified COL1A2 COL3A1 COL6A1COL6A3 and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2 but not COL6A2 as novel Smad targets. Together these results represent a significant step toward the identification of novel early-induced Smad-dependent TGF-beta target genes in fibroblasts.

STANDARD
MEMBERS
NAME
TGFBETA_Cil_UP THBS1, EPHB2, COL16A1, MMP17, MMF'1, TNC, MTA1, PAK2, LAMM, RAC1, THBS2 STANDARD
MEMBERS SYMBOLIZED
NAME
TGFBETA_Cil_UP THBS1, EPHB2, COL16A1, MMP17, MMF'1, TNC, MTA1, PAK2, LAMB1, RAC1, THBS2 STANDARD
PMiD AUTHORS
NAME
TGFBETA_Cil_UP 11279127 Verrecchia F, Chu ML, Mauviel A

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
TGFBETAJARLY msigclb V2.5 '24-Mar-08 .c2:1664 Human Up STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
TGFBETAJARLY GENE SYMBOL c2 up STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
TGFBETAJARLY 121 John Newman 'Washington University Upreguiated by TGF-beta UP treatment of skin fibroblasts at 30 min (dusters 1-3) STANDARD
FULL DESCRIPTION
NAME
TOFBETAJARLY AB - Despite major advances in the understanding of the intimate mechanisms of UP transforming growth factor-beta (I-OF-beta) signaling through the Smad pathway little progress has been made in the identification of direct target genes. In this report using cDNA
microarrays we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target we postulated that it had to meet the following criteria (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach we have identified COL1A2 COL3A1 COL6A1COL6A3 and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2 but not COL6A2 as novel Smad targets. Together these results represent a significant step toward the identification of novel early-induced Smad-dependent TGF-beta target genes in fibroblasts.

STANDARD
MEMBERS
NAME
TGFBETAJARLY MMP3, MARCKSL1, IGF2R, CD44, EPHB3, PXN, SPARC, PLAT, FN1, IGFBP3, RHOA, UP MMP19, PAK1, NID1, TIMP1, RHOQ, SERPINE1, CSPG2, CD59, RHOB, JUP, COL6A3, NOTCH 2, BSG, COL1A2, ZYX, ITGA3, TCF7L2, RND3, COL3A1, CDH6, WNT2B, ADAM9, HSPG2, ITGB5, RHOG, ICAM1, K3FBP5, LAMA4, DVL1J ARHGENA, T6B2, LRP1, C016A1, 1GFBP2, IRRC17, MMP14 STANDARD
MEMBERS SYMBOLIZED
NAME
TGFBETAJARLY MMP3, MARCKSL1, IGF2R, CD44, EPHB3, PXN, SPARC, PLAT, FN1, IGFBP3, RHOA, UP MMP19, PAK1, NID1, TIMP1, RHOQ, SERPINE1, CSPG2, CD59, RHOB, JUP, COL6A3, NOTCH 2, BSG, COL1A2, ZYX, ITGA3, TCF7L2, RND3, COL3A1, CDH6, WNT2B, ADAM9, HSPG2, ITGB5, RHOG, ICAM1, I3FBP5, LAMA4, DVL1J ARHGDIA, IT6B2, LRP1, C016A1, IGFBP2, IRRC17, MMP14 STANDARD
PMiD AUTHORS
NAME
TGFBETAJARLY 11279127 Verrecchia F, Chu ML, Mauviel A
UP

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
TGFBETAALLU msigclb V2.5 '24-Mar-08 .c2:1658 Human RAS_ONCOGENI msigdb V2.5 Mar 24, 2008 c2:1789 Human C_S1GNATURE

STANDARD
EXTERNAL DETAILS URI CHP CATEGORY CODE
NAME
TGFBETA_ALLU GENE _SYMBOL c2 RAS_ONCOGEN I FIQU133Plus_2 c2 C_SIGNATURE

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
TGFBETAALLU 121 John Newman Washington University Upreguiated by TGF-beta treatment of skin fibroblasts, at any time point RAS_ONCOGENI Arthur Liberzon Broad Institute Genes selected in supervised C_SIGNATURE analyses to discriminate cells expressing activated H-Ras oncogene from control cells expressing GFP.

STANDARD
FULL DESCRIPTION
NAME
TOFBETAALLU AB - Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (I-OF-beta) signaling through the Smad pathway little progress has been made in the identification of direct target genes. In this report using cDNA
microarrays we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target we postulated that it had to meet the following criteria (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach we have identified COL1A2 COL3A1 COL6A1 COL6A3 and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2 but not COL6A2 as novel Smad targets. Together these results represent a significant step toward the identification of novel early-induced Smad-dependent TGF-beta target genes in fibroblasts.
RAS_ONCOGENI The authors used primary mammalian epithelial cell cultures (HMECs) to develop a series of C_S1GNATURE pathway signatures. The authors used recombinant adenoviruses to force expression of human activated H-Ras in an otherwise quisecent cell, thereby specifically isolating the subsequent events as defined by the activation/deregulation of this single pathway. RNA
from multiple independent infections was collected for DNA microarray analysis using Affymetrix FIG J.I133_Plus_2 array. Gene expression signature that reflects the activity of the RAS-induced pathway was identified using supervised classification methods described in [PM: 11562467]. The analysis selects a set of genes for which the expression levels are most highly correlated with the classification of HMEC samples into H-Ras-activated/deregulated versus control (forced expression of green fluorescence protein).

STANDARD
MEMBERS
NAME
TGEBETA_ALL_U MMP3, MARCKSL1, IGF2R, LAMB1, SF'ARC, FN1, ITGA4, SMO, MMF'19, ITGB8, ITGA5, NID1, TIMP1, SEMA3F, RHOQ, CTNNB1, MMP2, SERPINE1, EPHB2, C0116A1, EPHA2, INC, JUP, ITGA3, TCF712, COL3A1, CDH6, WNT2B, ADAM9, DSP, HPG2, ARHGAP1, ITGB5, IGFBP5, ARHGDIA, LRP1, GFBP2, CTNNA1, LRRC17, MMP14, NE01, EFNA5, ITGB3, EPHB3, CD44, IGEBP4, TNFRSF1A, RAC1, PXN, PLAT, COL8A1, WNT8B, 1GFBP3, RHOA, EPHB4, MMP1, PAK1, MTA1, THBS2, C5PG2, MMP17, CD59, DVL3, RHOB, COL6A3, NOTCH2, BSG, MMP11, COL1A2, ZYX, RN D3, THB51, RHOG, ICAM1, LAMA4, DV Li, PAK2, ITGB2, COL6A1, FGD1 RAS_ONCOGENI 201286_4 235077 at, 1554997_a_at, 1558517 sat, 228498 at, 225950_4 226177_4 C_SIGNATURE 1568513_x_at, 219403._s_at, 213030_s_at, 229817_4 201490 sat, 209720_s_at, 212242_4 217608 at, 226120_4 1552648_a_at, 202332_4 46665_4 212943_4 204720_5_4 215707_s_at, 203625_x_at, 209453_at, 202696_at, 225612_s_at, 209281_5_4 224480_5_4 215243_5_4 229004_4 221840_4 238058 at 228527_s_at, 218451_4 1555950_a_at, 204614_4 1553995_a_at, 38149_4 37028_4 225544_4 229676_4 215210..5_4 203821_4 1558378_a_at, 226034_4 240991_4 205015_5_4 229872_s_at, 227510_x_at, 238063_4 223309iLat, 209598_4 207945_5_4 222881_4 210638_5_4 235263_4 206156_4 228846_at, 221489._s_at, 221009_s_at, 220658..5_4 205032_4 201044_x_at, 215977_x_at, 204014_4 205895_5_4 231735_5_4 205014_4 220949_5_4 226275_4 215101_5_at, 208613_5_4 201631..5_4 211467_5_4 218796_4 228046_at, 200756_x_at, 218736_5_4 209193_4 230779_4 1553722_s_at, 215071_5_4 205290_5_4 1552575_a_at, STANDARD
MEMBERS SYMBOLIZED
NAME
TGFBETAALLU MMP3, MARCKSL1, IGF2R, LAMB1, SF'ARC, FN1, ITGA4, SMO, MMF'I9, ITGB8, ITGA5, NID1, TIMP1, SEMA3F, RHOQ, CTNNB1, MMP2, SERPINE1, EPHB2, C0L16A1, EPHA2, INC, JUP, ITGA3, TCF712, COL3A1, CDH6, WNT2B, ADAM9, DSP, HPG2, ARHGAP1, ITGB5, IGFBP5, ARHGDIA, LRP1, IGFBP2, CTNNA1, LRRC17, MMP14, NE01, EFNA5, ITGB3, EPHB3, CD44, IGFBP4, TNFRSF1A, RAC1, PXN, PLAT, COL8A1, WNT813, IGFBP3, RHOA, EPHB4, MMP1, PAK1, MTA1, THBS2, C5PG2, MMP17, CD59, DVL3, RHOB, COL6A3, NOTCH2, BSG, MMP11, COL1A2, ZYX, RN D3, THB51, RHOG, ICAM1, LAMA4, DV Li, PAK2, ITGB2, COL6A1, FGD1 RAS_ONCOGENI SDC1, MEG3, PTG52, LRRC8C, SAMD8, GLTP, PRSS1, HPSE, PLXNA2, ZNF608, PPIF, SERPINB3, C_SIGNATURE TUBA', P18SRP, TTC8, TNFRSF10A, CSNK1E, SEMA4C, KIAA0528, DNAJC6, PRNP, SKP2, SLC9A1, OX5R1, B3GNT5, ATP281, LPAAT-THETA, GIB3, PTPRE, 5LC25A37, CDCP1, CD55, SERP1NB2, NT5E, ARHGAP25, PPP1R15A, TBX3, PAPD1, DLST
DISTP, HBEGF, C140RF78, NDR61, TGFA, L0C440667 /// L0C440, PR01073, IMEM154, PNPLA8, PNMA2, CSNK1D, FBX09, DKEZP434A0131, G3135, MXD1, SPRY4 /// LOC653170, ANGPTL4, ARNTL2, ITGA2, DUSP1, GK, DU5P4, NOLC1, FGFBP1, C70RF49, CXCL5, FLNB, ER3, NFIB, C200RF42, L0C152485, CALU, PALMD, PIM1, TNRC6B, RNF152, HIST1H2AC, BMP2, C60RF141, CXCL3, CCL20, ABCA1, IL8, RAPH1, DLL1, FAM83A, SOCS1, PVR, SDC4, UAP1, EPHA2, CYP27131, SLC2A3, EHD1, KLF6, VEGF, DUSP6, SERPINB1, C160RF74, BCL6, ZNF192, PIK3CD, TNRC6A, TRIB1, IL1A, MLL3, G052, GTPBP2, PLAUR, ZNF273, ANKRD38, LYN, RPRC1, TGFB2, EGR1, PHLDA1, TFPI2, DUSP5, EPA51, 5LC6A15, RNU3IP2, CASP1, CHST11, PNLIPRP3, MALL, DDX17, LATSI, INPP1, FAM46B, P13, CASP2, ITPR3, LRAT, SH2D5, LAMA3, FN1, RUNX1, LDLR, IER2, STANDARD

NAME
TGFBETAALLU 11279127 Verrecchia F, Chu ML, Mauviel A
RAS_ONCOGENI 16273092 Bild Aft Yao G, Chang JT, Wang Q, Potti A, Chasse 0, C_S1GNATURE Josh' MB, Harp le D, Lancaster JM, Berchuck A, Olson JA Jr, Marks JR, Dressman HK, West M, Nevins IR.

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
RAS_ONCOGENI
C_SIGNATURE
(continued) STANDARD
EXTERNAL DETAILS URI CHiP CATEGORY CODE
NAME
RAS_ONCOGEN I
C_SIGNATURE
(continued) STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
RAS_ONCOGENI
C_SIGNATURE
(continued) STANDARD
FULL DESCRIPTION
NAME
RAS_ONCOGENI
C_SIGNATURE
(continued) STANDARD
MEMBERS
NAME
RAS_ONCOGENI 207850,A, 205476 at, 203504_s_at, 211506_s_at, 225188_at, 204748 at, 224215_5,A, C_S1GNATURE 238741 at 210001_s_at, 214443A, 212930A, 202071 at, 209340A, 230778,A, (continued) 203499_at, 205676 at, 202499_s_at, 209039_x_at, 208614_s_at, 224606_at, 210513_s_at, 208891,A, 212268_at, 242509 at, 203140_at, 206579 at, 203879,A, 34734 sat, 202241_at, 210118_s_at, 1557158_s_at, 213524_s_at, 221050_s_at, 211924_s_at, 215239_x_at, 229125_at, 202626_s_at, 230820_at, 217943_s_at, 209908_s_at, 201694_s_at, 217997_at, 209278_s_at, 204882_at, 209457_at, 201925_s_at, 230711_at, 206376_at, 213572_s_at, 204133_at, 206011_at, 226372_at, 1558846_at, 209373_at, 204015_s_at, 208151_x_at, 1570425_s_at, 208719_s_at, 202794_at, 229518_at, 244025A, 205016_at, 203691_at, 226032_at, 201189_s_at, 220317_at, 230973_at, 228726,A, 234608_at, 214701_s_at, 211181_x_at, 202067_s_at, 229949_at, 202081_at, 219563_at, 212408,A, 204855 at, 223195_s_at, 204679 at, 205289 at, 1556773 at, 234725_s_at, 209124_at, 205266_at, 241495_at, 200797_s_at, 212096_s_at, 212171_x_at, 208002_s_at, 214866_at, 38037_at, 223333_s_at, 202828_s_at, 213358_at, 210512_s_at, 235390_at, 210732_s_at, 225316_at, 208785_s_at, 1566968_at, 230603_at, 1553581_s_at, 1554835_a_at, 217173_s_at, 39402_at, 208893_s_at, 226808_at, 219235_s_at, 201531_at, 227180,A, 201188_s_at, 204420 at, 220407_s_at, 203263_s_at, 201861_s_at, 202711 at, 202859_x_at, 230323_s_at, 208960_s_at, 216867_s_at, 41386_i_at, 211756_at, 209427_at, 1560017_at, 216236_s_at, 209774_x_at, 201666_at, 231067_s_at, 204470_at, 227755_at, 201287_s_at, 205490_x_at, 203234_at, 218368_s_at, 228834_at, 201473_at, 203939_at, 208553_at, 202436_s_at, 223834_at, 204823_at, 203946_s_at, 212983_at, 203417_at, 226863 at, 207243 sat, 227458 at, 208961_s_at, 21993 Sat, 211620_x_at, 241464 sat, 1555167_s_at, 204457_s_at, 221773_at, 212658_at, 205180_s_at, STANDARD
MEMBERS SYMBOLIZED
NAME
RAS_ONCOGENI C140RF139, TOR1A1P1, SERPINB5, SESN2, KCNK1, PTHLH, SEMA4B, MYD88, LIF, CCNIL
C_SIGNATURE MCL1, MTUS1, ACOT7, MMP14, KIAA0802, LGALS8, MFSD2, MAP1LC3B, SPRY4, C0L27A1, (continued) L1Bõ L00643641, PHACTR4, ZFP36, ELOVL7, FOSLL ARHGEF9, LRRFIP1, EFNBL TMEM45B, PDGFA, ilviJD3, SMTN, TMTC3, CXCL2, TIMPL AKAP12, CXCL1, UPPL TNFRSF12A, TOB1, JUNB, HIST1H1E, CYP1B1, CD274, NAV3, ARG2, HRAS, MFAP2, FAM110C, CALM2, ADAMTS5, FLI27365, PBEF1, GASL ELK3, LHFPL2õ ADAMS, SAT1, S100A6, EDG4, HOXC6, NFKBIZ, FGFR2, RBMS3, GRHL2, PHLDA2, ARHGAP27, MBOAT2, MRGPRX3, FLJ43663, CYP2R1, CCNAL EREG, TCF7L2, C19ORF10, DENND2C, 5LC16A3, PIA51, GLCCIL TOP1, FOS, TPM1, HK2, PPBP, HMGN3, KIAA1718, MBNL2, IRX2, EPHA4, COL12A1, FLRT3, NR6A1, 10C203274, SLC20A1, KLF5, L00641799 /// L00641, LRIG3õ GDF15, STX1A, IL13RA2, TIA1, TRIM22, TNFRSF10B, SFN, XIST, LRP8, MIDI, SRRM2, TNS4, IL11, MED25, ODC1, MDHL ZBED2, KIAA1754, ST5, ULBP2, ADRB2, PTX3, DKK3, CLCF1, KCNN4, EFNA5, VANGL2, HNRPH1, TSC22D1, FOXQ1 STANDARD

NAME
RAS_ONCOGENI
C_S1GNATURE
(continued) STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
RAS_ONCOGENI
C_SIGNATURE
(continued) STANDARD
EXTERNAL DETAILS URI CHiP CATEGORY CODE
NAME
RAS_ONCOGEN I
C_SIGNATURE
(continued) STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
RAS_ONCOGENI
C_SIGNATURE
(continued) STANDARD
FULL DESCRIPTION
NAME
RAS_ONCOGEN I
C_SIGNATURE
(continued) STANDARD
MEMBERS
NAME
RAS_ONCOGENI 230333 at, 201489 at, 208892_s_at, 228923 at, 206722 sat, 206858_s_at, 229545 at, C_S1GNATURE 223217 sat, 219267 at, 203639_s_at, 225756_at, 240245 at, 219388 at, 209803 sat, (continued) 227057_at, 226726 at, 1553293_at, 239331 at, 227109 at, 205899 at, 225189_s_at, 205767 at, 201926 sat, 216035_x_at, 216483_s_at, 200632 sat, 230769 at, 231775_at, 227288 at, 202856 sat, 160020 at, 211182_x_at, 217864_s_at, 225706.3t, 208901_s_at, 209189_at, 238688 at, 202934 at, 211527_x_at, 214146_s_at, 205067_at, 209377 sat, 217996 at, 227404_s_at, 221778_at, 232138 at, 228462 at, 227449 at, 234951_s_at, 219250_s_at, 60474_at, 232478_at, 207390_s_at, 217279iLat, 232034_at, 205179_s_at, 201920_at, 209212_s_at, 215667_x_at, 226908_at, 204678_s_at, 221577_x_at, 204729_s_at, 223196_s_at, 206172_at, 201447_at, 213293_s_at, 210405_x_at, 209260 at 243712_at, 225611_at, 202014_at, 232158 _x_at, 208433_s_at, 203636_at, 1554671_a_at, 222265 at, 206924 at, 1553993_s_at, 219039 at, 200790 at, 235374 at, 219836 at, 210845_s_at, 225582_at, 202440_s_at, 202068_s_at, 221291_at, 242899_at, 206170_at, 214845_s_at, 206157_at, 228314_at, 236656_s_at, 232947_at, 218000_s_at, 202435_s_at, 219500_at, 227364_at, 204401_at, 210355_at, 201041_s_at, 1559360_at, 226029_at, 213472_at, 208786_s_at, 215111_s_at, 227475_at STANDARD
MEMBERS SYMBOLIZED
NAME
RAS_ONCOGEN I
C_SIGNATURE
(continued) STANDARD

NAME
RAS_ONCOGENI
C_S1GNATURE
(continued) STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
SRC_ONCOGENI msigclb V2.5 '24-Mar-08 .c2:1790 Human C_SIGNATURE
SCHUMACHER_ msigdb V2.5 24-Mar-08 c2:762 Human MYC_UP

STANDARD
EXTERNAL DETAILS URL CHIP CATEGORY CODE
NAME
SRC_ONCOGEN I HG_U133_Plus_2 c2 C_SIGNATURE
SCHUMACHER_ Hu6800 c2 MYC_UP

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
SRC_ONCOGENI Arthur Liberzon Broad Institute Genes selected in supervised C_SIGNATURE analyses to discriminate cells expressing c-Src oncogene from control cells expressing GFP.
SCHUMACHER_ Yujin Hoshida Broad Institute Genes up-regulated by MYC in MYC_UP P493-6 (B-cell) STANDARD
FULL DESCRIPTION
NAME
SRC_ONCOGENI The authors used primary mammary epithelial cell cultures (HMECs) to develop a series of C_SIGNATURE pathway signatures. The authors used recombinant adenoviruses to force expression of human c-Src in an otherwise quiescent cell, thereby specifically isolating the subsequent events as defined by the activation of/deregulation of this single pathway.
RNA from multiple independent infections was collected for DNA microarray analysis using Affymetrix HG_U133_Plus_2 array. Gene expression signature that reflects the activity of the SRC-induced pathway was identified using supervised classification methods described in [PMID:
11562467]. The analysis selects a set of genes for which the expression levels are most highly correlated with the classification of HMEC samples into c-Src-activated/cleregulated versus control (forced expression of green fluorescence protein).
SCHUMACHER_ AB - The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes MYC_UP growth proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis lipid metabolism protein turnover/folding nucleotide/DNA synthesis transport nucleolus function/ANA binding transcription and splicing oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

STANDARD
MEMBERS
NAME
SRC_ONCOGENI 206414 sat, 242082_4 229101_4 218796_4 213350_4 202506_4 213069_4 C_S1GNATURE 230304_4 213485_s_at, 229582_4 1552797_s_at, 227921_4 212044 sat, 213865_at, 202245 at 224321_4 203301_s_at, 224250_s_at, 202569_s_at, 212560 at 241617_x_at, 211756 at, 1556006_s_at, 1556499_s_at, 212492_s_at, 209537 at, 221900_at, 1568680_s_at, 222667_s_at, 204614_at, 37028 at, 225640 at, 225461_at, 218397_at, 206011 at 201447 at, 229666 sat, 201128_s_at, 213279 at 228955 at 204847 at, 206591 at, 1568408_x_at, 212435_at, 212928 at, 1554021_a_at, 219181 at, 201879 at, 200908_s_at, 209773_s_at, 1558211_s_at, 220687_at, 31874_at, 204404_at, 231866_at, 235423_at, 1556773_at, 213262_at, 219571_s_at, 238933_at, 202648 at, 202643_s_at, 213352_at, 226065_at, 221284_s_at, 213243_at, 206976_s_at, 213056_at, 215867_x_at, 235392_at, 213164_at, 236251_at, 201737_s_at SCHUMACHER_ UCK2, MEST, NME1, SLC16A1, JTV1, LRP8, MAC30, L0056902, SORD, POLD2, ACSL1, MYC_UP PRDX4, LDHA, ZNF239, BOP1, RRS1, KIAA0020, DHODH, FABP5, RANBP1, SLC39A14, CYP51A1, NOLC1, FXN, TFRC, AKAP1, NEFH, PRPS2, POLR2H, AEBP1, UCHL3, SLC20A1, CTPS, AHCY, TARBP1, FKBP4, ARS, MTHED1, DDX10, ATP1B3, CTSC, HSPE1, AUH, SLC39A6, VRK1, MRPL3, EBNA1BP2, PAICS, RABEPK, PBEF1, PYCR1, ABCE1, GRSF1, STANDARD
MEMBERS SYMBOLIZED
NAME
SRC_ONCOGENI DDEF2, MMAB, 10C150166, C200RF42, RPS11, SSFA2, HEG1, ABCC10, C180RF37, C_SIGNATURE PROM2, RPL27A, DCBLD2, LSS, TMEFF2, DMTF1, SECISBP2, MARK3, C110RF32, PTHLH, CSNK1A1, COL1A1, iNLID2B, EXTL2, COL8A2, YTHDC2, ASH1L, SERPINB2, PPP1R15A, L0C401504, EHMT1, FANCL, CASP1, TIA1, CSTF3, ACLY, OHRS1, ZBTB11, RAG1, TRIM33, TSPYL4, ZNF12, LIPG, ARIH1, RPLP2, RRM2, SRC, GAS2L1, SLC12A2, IN PEP, SACS, RPS19, TNFAIP3, TMCC1, PRICKLE1, VPS1313, HSPH1, FRMD4B, CA12, IRS1, SLC5A3, ITGAV, MARCH6 SCHUMACHER_ UCK2, MEST, NME1, SLC16A1, JTV1, LRP8, MAC30, L0056902, SORD, POLD2, ACSL1, MYC_UP PRDX4, LDHA, ZNF239, OP1, RRS1, KIAA0020, DHODH, FABP5, RANBP1, SLC39A14, CYP51A1, NOLC1, FXN, TFRC, AKAP1, NEFH, PRPS2, POLR2H, AEBP1, UCHL3, SLC20A1, CTPS, AHCY, TARBP1, FKBP4, IARS, MTHFD1, DDX10, ATP1B3, CTSC, HSPE1, AUH, SLC39A6, VRK1, MRPL3, EBNA1BP2, PAICS, RABEPK, PBEF1, PYCR1, ABCE1, GRSF1, STANDARD
PMlD AUTHORS
NAME
SRC_ONCOGENI 16273092 Bild Aft Yao G, Chang JT, Wang Q, Potti A, C_SIGNATURE Chasse D, Joshi MB, Harpole D, Lancaster IM, Berchuck A, Olson JA Jr, Marks JR, Dressman HK, West M, Nevins JR.
SCHUMACHER_ 11139609 Schuhmacher M, Kohlhuber F, HoIzel M, Kaiser C, MYC_UP Burtscher H, Jarsch M, Bornkamm GW, Laux G, Polack A, Weidie UH, Eick D

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
IGH_NIH3T3_UP 'rnsigclb V2.5 '24-Mar-08 .c2:1522 Mouse SERUM JIBROBL msigdb V2.5 24-Mar-08 c2:1640 Human AST_CELLCYCLE

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE

_AH3T3_UP GENE SYMBOL c2 SERUM JIBROBL GENE SYMBOL c2 AST_CELLCYCLE

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
IGF1NH3T3UP 121 John Newman Washington University Up-regulated by treatment with IGF1 of NIFI3T3 cells overexpressing IGF1R (Tables 14-3) SERUM JIBROBL La John Newman Washington University Cell-cycle dependent genes AST_CELLCYCLE regulated following exposure to serum in a variety of human fibrobiast cell lines STANDARD
FULL DESCRIPTION
NAME
IGFl_N1ll3T3_UP AB - The IGF-1 receptor and the related insulin receptor are similar in structure and activate many of the same postreceptor signaling pathways yet they mediate distinct biological functions. It is still not understood how the specificity of insulin vs. IGF-1 signaling is controlled. In this study we have used cDNA microarrays to monitor the gene expression patterns that are regulated by insulin and 1GF-1. Mouse fibroblast NIH-3T3 cells expressing either the wild-type human IGF receptor or the insulin receptor were stimulated with either IGF-1 or insulin respectively. Thirty genes 27 of which were not previously known to be IGF-1 responsive were up-regulated by IGF-1 but not by insulin. Nine genes none of which was previously known to be insulin responsive were up-regulated by insulin but not by iGF-1. The IGF- and insulin-induced regulation of 10 of these genes was confirmed by Northern blot analysis. Interestingly more than half of the genes up-regulated by IGF-1 are associated with mitogenesis and differentiation whereas none of the genes specifically up-regulated by insulin are associated with these processes. Our results indicate that under the conditions used in this study IGF-1 is a more potent activator of the mitogenic pathway than insulin in mouse fibroblast NIH-3T3 cells.
SERUM_FIBROBL AB - Cancer invasion and metastasis have been likened to wound healing gone awry. Despite AST_CELLCYCLE parallels in cellular behavior between cancer progression and wound healing the molecular relationships between these two processes and their prognostic implications are unclear. In this study based on gene expression profiles of fibroblasts from ten anatomic sites we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors allowing us to identify tumors with gene expression signatures suggestive of active wounds.
Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves by tumor-associated fibroblasts or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage persist during treatment and predict increased risk of metastasis and death in breast lung and gastric carcinomas. Thus the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing as well as a powerful predictor of the clinical course in several common carcinomas.

STANDARD
MEMBERS
NAME
1GF1 _NH-13T3_UP CYR61, GPD2, CSDA, SMAD5, PVALB, H3F3B, TWIST1, TNFRSF1A, ER3, SLC25A5, SOX2, ETF1, MYH3, WEE1, IL3RA, CSF1, SLC20A1, RBM13, ITGA5, PHLDA1, NFE2L2, 114R, KIF1A, NAB2, GDNF, TUBB2B, FOXC2, ZFP90, MGP, VHL, SFRS2, NT5E, DAXX, TAGLN, SFRS3, L00653441 SERUM JIBROBL UBE2T, CDKL5, SPAG5, CKS1B, FEN1, RECQL4, PR1M2A, CDCA8, AMD1, MPHOSPH1, ASTSELLCYCLE CCNB2, ILF2, MCM5, CASP3, KIAA1333, AURKA, YWHAH, TIMP1, TOP2A, MCM8, MCM6, TACC3, TYMS, H2AFX, CENPQ, TRIP13, CDC6, TUBB2C, CKAP2, NCAPH, FBXL20, FAM83D, MCM4, RRM1, LMNB1, KIAA1794, GMNN, WDR51A, KIF23, ABCC5, H2-ALPHA, DHFR, PLK1, CKAP2L, 1TGB3, CENPA, EX01, EZH2, PRIM1, OITA, PBK, TUBA1, CENPF, MLF1IP, BUB1, UHRF1, RAD51AP1, CCNA2, GINS3, ASF1B, FAM111B, HIST1H2AC, CDCA1, PSRC1, CDC2, KIAA1370, MELK, PTTG1, RFC4, NCAPD3, SMC4, HMMR, WSB1, GTSE1, LYAR, SCML1, ANKRD10, CKS2, FOXMl, MAD211, NALP2, USP1, MET, PPM, DONSON, CDCA5, DLG7, KIFC1, ESCO2, CDCA7, MLLT6, CDKN3, DEPDC1B, MAPK13, HELLS, G1NS2, SDC1, CDC25A, C130RF3, ANP32E, RRM2, C7ORF41, ANLN, MBOAT1, KIAA0101, CTNNA1, FAM64A, ATAD2, SGCD, FANCA, TWIN, PWP1, RFC2, TPX2, CCDC99, CDC25C, L0C441052, PHTF2, EFHC1, FANCG, TNCRNA, PAQR4, HN1, RANGAP1, CCNF, PCNA, TUBB, RP114105.2, DKFZP762E1312, MND1, FL#25416, UBE2C, BARD1, CENPM, KNTC1, ADAMTS1, FAM72A, GAS213 STANDARD
MEMBERS SYMBOLIZED
NAME
IGF1 _NIH3T3_UP CYR61, GPD2, CSDA, SMAD5, PVALB, H3F3B, TWIST1, TNFRSF1A, ER3, SLC25A5, SOX2, ETF1, MYH3, WEE1, IL3RA, CSF1, SLC20A1, RBM13, ITGA5, PHLDA1, NFE2L2, 114R, KIF1A, NAB2, GDNF, TUBB2B, FOXC2, ZFP90, MGP, VHL, SFRS2, NT5E, DAXX, TAGLN, SFRS3, L00653441 SERUM JIBROBL UBE2T, CDKL5, SPAG5, CKS1B, FEN1, RECQL4, PRIM2A, CDCA8, AMD1, MPHOSPH1, ASTSELLCYCLE CCNB2, ILF2, MCM5, CASP3, KIAA1333, AURKA, YWHAH, TIMP1, TOP2A, MCM8, MCM6, TACC3, TYMS, 1-12AFX, CENPQ, TRIP13, CDC6, TUBB2C, CKAP2, NCAPH, FBXL20, FAM83D, MCM4, RRM1, LMNB1, KIAA1794, GMNN, WDR51A, KIF23, ABCC5, H2-ALPHA, DHFR, PLK1, CKAP2L, ITGB3, CENPA, EX01, EZH2, PRIM1, CIITA, PBK, TUBA1, CENPF, MLF1IP, BUB1, UHRF1, RAD51AP1, CCNA2, GINS3, ASF1B, FAM111B, HIST1H2AC, CDCA1, PSRC1, CDC2, KIAA1370, MELK, PTTG1, RFC4, NCAPD3, SMC4, HMMR, GTSE1, LYAR, SCML1, ANKRD10, CKS2, FOXMl, MAD211, NALP2, USP1, MET, PPIH, DONSON, CDCA5, DLG7, KIFC1, ESCO2, CDCA7, MLLT6, CDKN3, DEPDC1B, MAPK13, HELLS, GINS2, SDC1, CDC25A, C130RF3, ANP32E, RRM2, C7ORF41, ANLN, MBOAT1, KIAA0101, CTNNA1, FAM64A, ATAD2, SGCD, FANCA, TIPIN, PWP1, RFC2, TPX2, CCDC99, CDC25C, L0C441052, PHTF2, EFHC1, FANCG, TNCRNA, PAQR4, HN1, RANGAP1, CCNF, PCNA, TUBB, RP114105.2, 0KFZP762E1312, MND1, FLI25416, UBE2C, BARD1, CENPM, KNTC1, ADAMTS1, FAM72A, GAS213 STANDARD
PMiD AUTHORS
NAME
IGF1_NiH3T3_UP 11606465 Dupont J, Khan 1, Qu BH, Metzier P. Heiman 1, LeRoith D
SERUM JIBROBL 14737219 Chang HY, Sneddon JB, Alizadeh AA, Sood R, West AST_CELLCYCLE RB, Montgomery K, Chi, van de Rijn M, Botstein D, Brown PO

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
SERUM_FIBROBL msigclb V2.5 '24-Mar-08 .c2:1642 Human AST_CORE_UP

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
SERUM_FIBROBL GENE _SYMBOL c2 MI-SO RE_U P

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
SERUM_FIBROBL 121 John Newman Washington University Core group of genes consistently AST_CORE_UP up-regulated following exposure to serum in a variety of human fibroblast cell lines (higher expression in activated cells, not cell-cycle dependent) STANDARD
FULL DESCRIPTION
NAME
SERUNI_FIBROBL AB - Cancer invasion and metastasis have been likened to wound healing gone awry. Despite AST_CORE_UP parallels in cellular behavior between cancer progression and wound healing the molecular relationships between these two processes and their prognostic implications are unclear. In this study based on gene expression profiles of fibroblasts from ten anatomic sites we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors allowing us to identify tumors with gene expression signatures suggestive of active wounds.
Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves by tumor-associated fibroblasts or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage persist during treatment and predict increased risk of metastasis and death in breast lung and gastric carcinomas. Thus the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing as well as a powerful predictor of the clinical course in several common carcinomas.

STANDARD
MEMBERS
NAME
SERUM_FIBROBL SPFH1, UCK2, FARSLB, CENPJ, VIL2, C8ORF13, ACTC1, MAP3K8, ACTL6A, CBX1, AST_CORE_UP ElF4EBP1, CHEK1, ITGA6, SNRPC, CDK2, RANBP1, GGH, SNRPB, C6ORF173, DBNDD1, C6ORF55, LSM3, PSMD12, DHFR, BRIP1, CORO1C, DTYMK, FARSLA, FLNC, UBE2J1, SPAG17, LPAL2, DUT, PSMC3, NUP35, STX3, DCBLD2, L7R, WDR77, H2AFZ, 51C25A40, PLOD2, ElF4G1, TIMM50, RPN1, NUDT15, SFRS2, MSN, C30RF26, SNRPE, NCLN, STK17A, KIAA0090, NUDT1, WDHD1, PITPNC1, SNRPD1, TDP1, UAP1, FCRLA, MRPS16, MRP528, MET, EN01, 10056902, NLN, MRPL12, POLE2, HSPC111, RAB3B, SDC1, PIS, CDCA4, TPRKB, HNRPA3, POLE3, PHF19, WDR54, SNRPA1, TPM2, DDX11, EPHB1, NOLA2, NUPL1, ANKRD32, PCAA4, FU10292, IRRC40, PLAUR, 5LC25A5, NUP85, COQ2, SSR3, PNN, HMGN2, RFC3, BRCA2, SAR1B, GNG11, TXNL2, RPP40, NDRG1, C110RF24, MKKS, STRA13, RBMX, RNF41, HNRPR, EBNA1BP2, DCLRE1B, DNAJC9, GPLD1, PGM2, PSMA7, HYLS1, HAS2, TMEM48, PSMD14, LYPLA2, SMC2, JTV1, F3, DYNLT1, TMEM130, RNASEH2A, NPTN, MT3, C1ORF41, DLEU1, DLEU2, ID3, PFKP, CENPN, CEP78, HNRPAB, SIV1S, DCK, ID2, ST3GAL6, 1PO4, RUVBL2, CLCF1, NUP93, INFRSF12A, MGC42105, PSMD2, PCSK7, BCCIP, SNRPA, TUBA1, MTHFD1, ALKBH7, MNAT1, MCM7, CCDC5, MLF1IP, C130RF1, COPB2, MCTS1, IFRD2, UCRC, SH3BP5L, SFR510, TFP12, LYAR, C160RF61, RNF138, GSTCD, MRPL37, FAM33A, EMP2, CRSP8, MYBL2, PPIH, RGS13, CCT5, C190RF48, WSB2, TOMM40, PFN1, PAX9, PDL1M7, PTPLB, C120RF24, FABPS, HMGB1, MT1F, EXOSC8, CSMD1, SMURF2, POLR3K, KRR1, LMNB2, C1ORF33, C180RF24, MCM3, RUVI31.1, UMPS, MAPRE1, LCTL, C160RF34, NR1P3, NUP107, CCND3, AADACL1 STANDARD
MEMBERS SYMBOLIZED
NAME
SERUM_FIBROBL SPFH1, UCK2, FARSLB, CENPJ, VIL2, C8ORF13, ACTC1, MAP3K8, ACTL6A, CBX1, AST_CORE_UP ElF4EBP1, CHEK1, ITGA6, SNRPC, CDK2, RANBP1, GGH, SNRPB, C6ORF173, DBNDD1, C6ORF55, LSM3, PSMD12, DHFR, BRIP1, CORO1C, DTYMK, FARSLA, FLNC, UBE2J1, SPAG17, LPAL2, DUT, PSMC3, NUP35, STX3, DCBLD2, L7R, WDR77, H2AFZ, 51C25A40, PLOD2, ElF4G1, TIMM50, RPN1, NUDT15, SFRS2, MSN, C30RF26, SNRPE, NCLN, STK17A, KIAA0090, NUDT1, WDHD1, PITPNC1, SNRPD1, TDP1, UAP1, FCRLA, MRPS16, MRP528, MET, EN01, 10056902, NLN, MRPL12, POLE2, HSPC111, RAB3B, SDC1, PIS, CDCA4, TPRKB, HNRPA3, POLE3, PHF19, WDR54, SNRPA1, TPM2, DDX11, EPHB1, NOLA2, NUPL1, ANKRD32, PDIA4, FU10292, IRRC40, PLAUR, 5LC25A5, NUP85, COQ2, SSR3, PNN, HMGN2, RFC3, BRCA2, SAR1B, GNG11, TXNL2, RPP40, NDRG1, C110RF24, MKKS, STRA13, RBMX, RNF41, HNRPR, EBNA1BP2, DCLRE1B, DNAJC9, GPLD1, PGM2, PSMA7, HYLS1, HAS2, TMEM48, PSMD14, LYPLA2, SMC2, JTV1, F3, DYNLT1, TMEM130, RNASEH2A, NPTN, MT3, C1ORF41, DLEU1, DLEU2, ID3, PFKP, CENPN, CEP78, HNRPAB, SIV1S, DCK, ID2, ST3GAL6, IP04, RUVBL2, CLCF1, NUP93, INFRSF12A, MGC42105, PSMD2, PCSK7, BCCIP, SNRPA, TUBA1, MTHFD1, ALKBH7, MNAT1, MCM7, CCDC5, MLF1IP, C130RF1, COPB2, MCTS1, IFRD2, UCRC, SH3BP5L, SFR510, TFPI2, LYAR, C160RF61, RNF138, GSTCD, MRPL37, FAM33A, EMP2, CRSP8, MYBL2, PPIH, RGS13, CCT5, C190RF48, WSB2, TOMM40, PFN1, PAX9, PDLIM7, PTPLB, C120RF24, FABPS, HMGB1, MT1F, EXOSC8, CSMD1, SMURF2, POLR3K, KRR1, LMNB2, C1ORF33, C180RF24, MCM3, RUVI31.1, UMPS, MAPRE1, LCTL, C160RF34, NRIP3, NUP107, CCND3, AADACL1 STANDARD
PMiD AUTHORS
NAME
SERUM_FIBROBL 14737219 Chang HY, Sneddon JB, Alizadeh AA, Sood R, West AST_CORE_UP RB, Montgomery K, Chi, van de Rijn M, Botstein D, Brown PO

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
CHANG_SERUM_ msigdb V2.5 24-Mar-08 c2:823 Human RESPONSE UP
OLDAGE_DN msigdb V2.5 24-Mar-OS c2:1583 Human STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
CHANG_SERUM_ GENE SYMBOL c2 RESPONSE UP
OLDAGE_DN GENE SYMBOL c2 STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
CRANG_SERUM_ 'Jean Junior Broad institute CSR (Serum Response) signature RESPONSE UP for activated genes (Stanford) OLDAGE_DN 121 John Newman Washington University Downreguiated in fibroblasts from old individuals, compared to young STANDARD
FULL DESCRIPTION
NAME
CRANG_SERUM_ AB - Cancer invasion and metastasis have been likened to wound healing gone awry. Despite RESPONSE UP parallels in cellular behavior between cancer progression and wound healing the molecular relationships between these two processes and their prognostic implications are unclear. In this study based on gene expression profiles of fibroblasts from ten anatomic sites we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors allowing us to identify tumors with gene expression signatures suggestive of active wounds.
Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves by tumor-associated fibroblasts or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage persist during treatment and predict increased risk of metastasis and death in breast lung and gastric carcinomas. Thus the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing as well as a powerful predictor of the clinical course in several common carcinomas.
OLDAGLDN AB - Messenger RNA levels were measured in actively dividing fibroblasts isolated from young middle-age and old-age humans and humans with progeria a rare genetic disorder characterized by accelerated aging. Genes whose expression is associated with age-related phenotypes and diseases were identified. The data also suggest that an underlying mechanism of the aging process involves increasing errors in the mitotic machinery of dividing cells in the postreproductive stage of life. We propose that this dysfunction leads to chromosomal pathologies that result in rnisregulation of genes involved in the aging process.

STANDARD
MEMBERS
NAME
CHANG_SERUM_ UMPK, ENIGMA, SDFR1, CENPJ, VIL2, MAP3K8, MGC3101, CBX1, ElF4E13P1, CHEK1, RESPONSE UP KIAA0095, ITGA6, HSU79274, SNRPC, MGC10200, CDK2, STK18, SNRPB, GGH, LSM3, PSMD12, PDAP1, FLJ10036, MGC11266, FARSL, MCT4, DHFR, KIAA1363, BRIP1, CORO1C, MGC13170, FLNC, VDAC1, UBE2J1, LSM4, OUT, PSMC3, ARHC, HRI, BM039, IL7R, H2AFZ, MY16, L0051128, C1or133, PLOD2, ElF4G1, RPN1, SFRS2, MSN, C6orf55, SNRPE, STK17A, NUTF2, KIAA0090, NUDT1, SSSCA1, PITPNC1, SNRPD1, LOC115106, LOXL2, UAP1, MGC10974, MRPS16, CGI-121, MRPS28, MET, EN01, 10056902, NLN, MRPL12, L0056926, POLE2, HSPC111, FLJ10407, SDC1, COP56, PTS, MGC14480, CDCA4, FU32915, SNRPA1, HN1L, TPM2, EPHB1, KE04, NOLA2, SRM, NUPL1, FL110292, C13orf1, PLAUR, SLC25A5, SSR3, PNN, HMGN2, BRCA2, MYCBP, RFC3, GNG11, TXNL2, ESDN, FU30532, MKKS, RBMX, HNRPR, EBNA1BP2, RNF41, DCLRE1B, PAICS, TIM5OL, 01P2, GPLD1, PSMA7, HAS2, DKF4761.11417, P5MD14, NME1, Cllorf14, SLC16A1, JTV1, FU23468, FU20331, PCNT1, F3, L0C129401, PA2G4, RNASEH2A, MT3, DLEU1, FU10983, ID3, DEW, PFKP, TCTEL1, CL640, EEF1E1, DCK, SMS, KIAA1720, TAG LW, ID2, PO4, FRSB, DC13, COTL1, TNFRSF12A, PSMD2, L0C201562, PCSK7, BCCIP, C1lorf24, SNRPA, TUBA?, MTHFD1, MCM7, MNAT1, MEP50, IFRD2, FU12643, SFRS10, C8orf13, TFPI2, HRB2, LYAR, RNF138, MRPL37, RNASEP1, HNRPA2B1, EMP2, MYBL2, PPIH, C0X17, ERP70, TUBG1, CCT5, BAF53A, WSB2, MYBL1, TOMM40, PFN1, RBM14, MP4, HMGB1, MT1F, CKLF, MTH2, DKFZP727G051, TPM1, MGC4825, SMURF2, POLR3K, SMC2L1, LMNB2, MCM3, FU12953, RUVB11, L0051668, AND-1, UMPS, MAPRE1, MGC4308, PLG, TPI1, TCEB1, NUP107, ADAMTS1, L0C93081 OLDAGLDN HAS2, CENPA, NASP, CKS1B, PPP1CC, SAFB, FOXMl, PSMC6, APPBP1, CKAP5, CDH11, 0DX39, MYBL2, PSMD11, CSE1L, KIF2C, H2AFX, CDC20, PSMC2, CTSC, HMGB2, HADH2, PTGS2, CENPF, UGCG, H2AFZ, KIF11, NUP88, CDC25B, 118, CCNA2, RANBP1, CCNF, ATR, PSMD12, LIBE2C, PSMA2, BARD?, KIF23, PARP1, PKMYT1, PSMA3, KIF14, MCM2, FBN2, POSTN, PLK1 STANDARD
MEMBERS SYMBOLIZED
NAME
CHANG_SERUM_ HAS2, PSMD14, CENRI, VIL2, C80RF13, NME1, 51C16A1, FM, F3, MAP3K8, CBX1, RESPONSE UP ElF4EBP1, PA2G4, RNASEH2A, CHEK1, MT3, ITGA6, DIEU1, SNRPC, ID3, DIEU2, PFKP, CDK2, SNRPB, GGH, C60RF55, LSM3, PDAP1, PSMD12, EEF1E1, SMS, DCK, DHFR, BRIP1, TAGLN, CORO1C, 1D2, IP04, FLNC, VDAC1, UBE2J1, 15M4, OUT, PSMC3, COT11, TNFRSF12A, PSMD2, PCSK7, L7R, BCCIP, SNRPA, TUBA1, MTHFD1, MNAT1, MCM7, H2AFZ, C130RF1, MYL6, IFRD2, PLOD2, E1F4G1, RPN1, SFRS10, SFRS2, MSN, SNRPE, 51K17A, NUTF2, KIAA0090, NUDT1, TFPI2, SSSCA1, PITPNC1, SNRPD1, [VAR, 10X12, UAP1, RNF138, MRP516, MRPI37, HNRPA2B1, EMP2, MRPS28, MET, EN01, PPIH, MYBL2, 10056902, NIN, MRP112, COX17, CCT5, TUBG1, MYBL1, WSB2, TOMM40, PFN1, HSPC111, POLE2, RBM14, SDC1, COPS6, MP4, HMGB1, PTS, CDCA4, MT1F, CKLF, SNRPA1, TPM2, TPM1, SMURF2, EPHB1, POLR3K, NOLA2, SRM, NUPL1, LMNB2, C1ORF33, FU10292, MCM3, PLAUR, RUVB11, 5LC25A5, PNN, 55R3, UMPS, HMGN2, BRCA2, MYCBP, RFC3, MAPRE1, GNG11, TXNL2, PIG, C110RF24, MKKS, RBMX, TPI1, HNRPR, EBNA1BP2, RNF41, DCLRE1B, PAICS, TCEB1, NUP107, ADAMTS1, GPLD1, PSMA7 OLDAGLDN HAS2, CENPA, NASP, CKS1B, PPP1CC, SAFB, FOXMl, PSMC6, APPBP1, CKAPS, CDH11, 0DX39, MYBL2, PSMD11, CSE1L, KIF2C, H2AFX, CDC20, PSMC2, CTSC, HMGB2, HADH2, PTGS2, CENPF, UGCG, H2AFZ, K1F11, NUP88, CDC25B, 118, CCNA2, RANBP1, CCNF, AIR, PSMD12, LIBE2C, PSMA2, BARD1, KIF23, PARP1, PKMYT1, PSMA3, KIF14, MCM2, FBN2, POSTN, PLK1 STANDARD
PMiD AUTHORS
NAME
CHANG_SERUM_ 14737219 Chang HY, Sneddon JB, Alizadeh AA, Sood R, West RESPONSE UP RB, Montgomery K, Chi, van de Rijn M, Botstein Brown PO
OLDAGE_DN 10741968 Ly DH, Lockhart DJ, Lerner RA, Schultz PG

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
P21_1)53_ANY_D msigclb V2.5 '24-Mar-08 .c2:1601 Human STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
P21253_ANY_D GENE _SYMBOL c2 STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
P21P53_ANY_D 121 John Newman 'Washington University Down-ref.,,uIated at any timepoint (4-24 hrs) following ectopic expression of p21 (CDKN1A) in OvCa cells, p53-dependent STANDARD
FULL DESCRIPTION
NAME
P21P53_ANY_D AB - In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line 2774 and the derivative cell lines 2774qw1 and 2774qw2. We observed that rAcl-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells.
Surprisingly overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines.
Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX BCL2 and caspase 3 genes specific in rAd-p53-induced apoptotic cells was not altered in rAd-p21-induced apoptotic cells suggesting p21(WAF1/CIP÷-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells Infected with rAd-p21 on 60 000 cDNA microarrays identified 159 genes in response to p21(WAF1/C1P1) expression in at least one time point with 2.5-fold change as a cutoff.
Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAFIJOP1)-induced apoptosis. Furthermore promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.

STANDARD
MEMBERS
NAME
P21253_ANY_D UBE2T, AURKB, NCAPG, ASPM, PRC1, TUBA3, SMC2, PARP2, HNRPA281, AURKA, B1RC2, DLG7, MCM6, TYMS, TUBB3, CDC258, MCM4, KIAA1794, VEGF, IVIKI67, ANLN, C140RF106, MCM2, CYR61, ATAD2, MCM3, CEP55, TRIM44, EX01, TPX2, CCDC99, SERPINI2, ZBTB5, PBK, ZNF84, TTK, HMGB2, MCM7, TCN2, RACGAPI, BUB1, TUBB4, FAT2, CDC2, UBE2C, KNTC1, WDHD1, NCAPD3, NPM1 STANDARD
MEMBERS SYMBOLIZED
NAME
P21253_ANY_D UBE2T, AURKB, NCAPG, ASPM, PRC1, TUBA3, SMC2, PARP2, HNRPA281, AURKA, BIRC2, DLG7, MCM6, TYMS, TUBB3, CDC258, MCM4, KIAA1794, VEGF, IVIKI67, ANLN, C140RF106, MCM2, CYR61, ATAD2, MCM3, CEP55, TRIM44, EX01, TPX2, CCDC99, SERPINI2, ZBTB5, PBK, ZNF84, TTK, HMGB2, MCM7, TCN2, RACGAPI, BUB1, TUBB4, FAT2, CDC2, UBE2C, KNTC1, WDHD1, NCAPD3, NPM1 STANDARD

NAME
P21 _P53_ANY_D 12138103 Wu Q, Kirschmeier P, Hockenberry T, Yang TV, Brassard DL, Wang L, McClanahan T, Black S, Rini G, Musco ML, Mirza A, Liu STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
P21_1)53 _EARLY msigclb V2.5 '24-Mar-08 .c2:1602 Human _DN

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
P21253 _EARLY GENE _SYMBOL c2 _LA

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
P21J53_EARLY 121 John Newman Washington University Down-regulated at early _DN timepoints (4-8 hrs) following ectopic expression of p21 (CDKN1A) in OvCa cells, p53-dependent STANDARD
FULL DESCRIPTION
NAME
P21P53_EARLY AB - In this study we used adenovirus vector-mediated transduction of either the p53 gene _DN (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line 2774 and the derivative cell lines 2774qw1 and 2774qw2. We observed that rAcl-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells.
Surprisingly overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines.
Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX BCL2 and caspase 3 genes specific in rAd-p53-induced apoptotic cells was not altered in rAd-p21-induced apoptotic cells suggesting p21(WAF1/CIP÷-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells Infected with rAd-p21 on 60 000 cDNA microarrays identified 159 genes in response to p21(WAF1/C1P1) expression in at least one time point with 2.5-fold change as a cutoff.
Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAFIJOP1)-induced apoptosis. Furthermore promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.

STANDARD
MEMBERS
NAME
P21 J53_EARLY CYR61, NCAPG, TCN2, RACGAP1, PRCI, TPX2, CDC2, UBE2C, 114M67, AURKA, MRC2, _DN DLG7, 11-K

STANDARD
MEMBERS SYMBOLIZED
NAME
P21 J53_EARLY CYR61, NCAPG, TCN2, RACGAP1, PRCI, TPX2, CDC2, UBE2C, 114M67, AURKA, MRC2, _DN DLG7, TTK

STANDARD

NAME
P21 J53_EARLY 12138103 Wu Q, Kirschmeier P, Hockenberry T, Yang TV, _DN Brassard DL, Wang L, McClanahan T, Black S, Rini G, Musco ML, Mirza A, Liu STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
P21 J)53_MIDDL msigdb V2.5 24-Mar-08 c2:1604 Human E_DN
HSA04512_ECM msigdb V2.5 24-Mar-08 c2:1939 Homo sapiens _RECEPTOR_INT
ERACTION

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
P21253_MIDDL GENE SYMBOL c2 E_DN
HSA04512ECM http://www.kegg.jadhget- GENE SYMBOL c2 _RECEPTOR _INT bin/show pathway?H5A04512 STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
P21P53_MIDDL 121 John Newman Washington University Down-regulated at intermediate E_DN timepoints (12-16 hrs) following ectopic expression of p21 (CDKN1A) in OvCa cells, p53-dependent FISA04512_ECM KEGG KEGG Genes involved in ECM-receptor _RECEPTOR_INT interaction ERACTION

STANDARD
FULL DESCRIPTION
NAME
P21P53_MIDDL AB - In this study we used adenovirus vector-mediated transduction of either the p53 gene E_DN (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line 2774 and the derivative cell lines 2774qw1 and 2774qw2. We observed that rAcl-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells.
Surprisingly overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines.
Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX BCL2 and caspase 3 genes specific in rAd-p53-induced apoptotic cells was not altered in rAd-p21-induced apoptotic cells suggesting p21(WAF1/CIP÷-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells Infected with rAd-p21 on 60 000 cDNA microarrays identified 159 genes in response to p21(WAF1/C1P1) expression in at least one time point with 2.5-fold change as a cutoff.
Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAFIJOP1)-induced apoptosis. Furthermore promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.
FISA04512_ECM
_RECEPTOR_INT
ERACTION

STANDARD
MEMBERS
NAME
P21P53_MIDDL UBE2T, AURKB, ASPM, MCM3, ATAD2, CEP55, TUBA3, 5MC2, EX01, CCDC99, PBK, E_DN ZNF84, TYMS, TUBB3, HMGB2, MCM7, BUB1, CDC25B, TUBB4, MCM4, VEGF, ANLN, KNTC1, WDHD1, NCAPD3 HSA04512_ECM LAMC3, CHAD, COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A4, COL4A6, _RECEPTOR _INT COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL11A1, COL11A2, COL6A6, DAG1, ERACT1ON LAMB4, ITGAll, FNDC3A, SV2C, FN1, FNDC5, GP1BA, GP1BB, GP5, GP9, LAMA1, HMMR, HSPG2, TNC, BSP, 1TGA6, ITGA1, TGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA7, iTGA9, ITGAV, ITGB1, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, ITGB8, AGRN, LAMA2, LAMA3, LAMA4, LAMAS, LAMB1, LAMB2, LAMB3, LAMC1, LAMC2, COL5A3, GP6, RELN, SDC1, SDC2, 50C4, TNN, FNDC.4, 5PP1, THBS1, THBS2, THB53, THBS4, TNR, TNXB, VTN, VWF, FNDC1, ITGA10, ITGA8, CD36, CD44, CD47, SDC3, SV2B, SV2A

STANDARD
MEMBERS SYMBOLIZED
NAME
P21P53_MIDDL UBE2T, AURKB, ASPM, MCM3, ATAD2, CEP55, TUBA3, 5MC2, EX01, CCDC99, PBK, E_DN ZNF84, TYMS, TUBB3, HMGB2, MCM7, BUB1, CDC25B, TUBB4, MCM4, VEGF, ANLN, KNTC1, WDHD1, NCAPD3 HSA04512_ECM LAMC3, CHAD, COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A4, COL4A6, _RECEPTOR _INT COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL11A1, COL11A2, COL6A6, DAG1, ERACT1ON LAMB4, ITGAll, FNDC3A, SV2C, FN1, FNDC5, GP1BA, GP1BB, GP5, GP9, LAMA1, HMMR, HSPG2, TNC, BSP, ITGA6, ITGA1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA7, ITGA9, ITGAV, ITGB1, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, ITGB8, AGRN, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMB3, LAMC1, LAMC2, COL5A3, GP6, RELN, SDC1, SDC2, 50C4, TNN, FNDC.4, 5PP1, THBS1, THBS2, THB53, THBS4, TNR, TNXB, VTN, VWF, FNDC1, ITGA10, ITGA8, CD36, CD44, CD47, SDC3, SV2B, SV2A

STANDARD

NAME
P21 _P53_MIDDL 12138103 Wu Q, Kirschmeier P, Hockenberry T, Yang TV, E_DN Brassard DL, Wang L, McClanahan T, Black S, Rini G, Musco ML, Mirza A, Liu HSA04512_ECM Kanehisa, M., Araki, M., Goto, S., Hattori, M., _RECEPTOR_INT Hirakawa, M., ltoh, M., Katayama, T., ERACT1ON Kawashirna, S., Okuda, S., Tokimatsu, T., Yarnanishi, Y.

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
MENSE_HYPDXI msigclb V2.5 '24-Mar-08 .c2:915 Human A_UP

STANDARD
EXTERNAL DETAILS URI CHIP CATEGORY CODE
NAME
MENSE_HYPDX1 AFFYMETRIX c2 A_UP

STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
MENSE_HYPDX1 Kevin Vogelsang Broad institute List of Hypoxia-induced genes A_UP found in both Astrocytes and HeLa Cell STANDARD
FULL DESCRIPTION
NAME
MENSE_HYPDXI AB - Oxygen is vital for the development and survival of mammals.
In response to hypoxia A_UP the brain initiates numerous adaptive responses at the organ level as well as at the molecular and cellular levels including the alteration of gene expression.
Astrocytes play critical roles in the proper functioning of the brain thus the manner in which astrocytes respond to hypoxia is likely important in determining the outcome of brain hypoxia. Here we used microarray gene expression profiling and data-analysis algorithms to identify and analyze hypoxia-responsive genes in primary human astrocytes. We also compared gene expression patterns in astrocytes with those in human HeLa cells and pulmonary artery endothelial cells (ECs). Remarkably in astrocytes five times as many genes were induced as suppressed whereas in HeLa and pulmonary ECs as many as or more genes were suppressed than induced. More genes encoding hypoxia-inducible functions such as glycolytic enzymes and angiogenic growth factors were strongly induced in astrocytes compared with HeLa cells.
Furthermore gene ontology and computational algorithms revealed that many target genes of the EGF and insulin signaling pathways and the transcriptional regulators Myc Jun and p53 were selectively altered by hypoxia in astrocytes. indeed Western blot analysis confirmed that two major signal transducers mediating insulin and EGF action Akt and MEK1./2 were activated by hypoxia in astrocytes. These results provide a global view of the signaling and regulatory network mediating oxygen regulation in human astrocytes.

STANDARD
MEMBERS
NAME
MENSE_HYPDXI 228500_4 229879_4 227299_4 211162_x_at, 239474_4 226390_4 226099_4 A_UP 215078_4 203282_4 212665 at, 220482_5_4 226452_4 201848_5_4 202934_4 224708_4 207543,5_4 219888_4 244604,A, 235592_4 227368,A, 201169_s_at, 223746,A, 218651_5_4 242669_4 1554008_at, 217739_5_4 202140_5_4 226348_4 227868,A, 1556715,A, 221845 sat, 209984_4 214978_5_4 59625,A, 213397_x_at, 205158,A, 203439_5_4 218498_5_at, 202672.5_4 227501,A, 221478_4 213861_5_4 1565906_4 211974_x_at, 218507_4 201627_5_4 203725_4 203574_4 220942_x_at, 230710,A, 222646_5_4 202014_4 202733_4 235850_4 231242_4 202912_4 242310_4 214073_at, 204298_5_4 201313_4 215446_5_4 243659_4 36711_at, 210426_x_at, 217047_5_4 238482_4 221567_4 209122_4 236513_4 216236_5_4 239159_4 207079_5_4 212722_5_at, 221985_at, 1553976_a_at, 212501_4 218325_5_at, 202498_5_4 224314_5_4 225898 at, 227539_4 232293_4 202129_5_4 214482_4 232628_4 241342_A, 202464_5_4 235226_4 45714_.4 227337_4 242449_at, 207785_5_at, 202364_4 202022_at, 242523_4 242758,_x_at, 221479_5_4 1556357_5_4 223046_at, 205141_4 217738_4 224602_4 212496_5_4 224345_x_at, 208308_5_,4 212689_5_4 204284,A, 200737 at, 1558164_5_4 234970_4 203973_5_4 221009_s_at, 1556697_at, 1555167_5_4 221841_5_4 210513_5_4 206307_5_4 203192_4 228499_4 226863_at, 209566_4 202620_5_4 235737_4 202973_x_at, 211527_x_4 200632_5_4 223193_x_at, 236545A, 221497_x_at STANDARD
MEMBERS SYMBOLIZED
NAME
MENSE_HYPDX1 ATF3, FAM119B, PRPSAP1, PEX13, CDC2L6, RBPSUH, TMEM65, RNASE4, CEBPD, ABCB6, A_UP HK2, PFKFB3, MTAC2D1, SPAG4, JIVLID1A, ELL2, PDK1, RORA, 50D2, GOSR2, UFM1, iMiD2C, L0C285513, LCORL, NFIL3, CTTN, IVIGC21644, OSMR, STARD4, HCFC1R1, PTDSR, ADM, BNIP3, ERO1L, GADD45A, ERICH1, STC2, FAM110C, FOXD1, AN G /1/
RNASE4, RAB40C, C30RF28, MAFF, PLOD2, PBEF1, L0C400027, GPI, ANGPTL4, BNIP3L, WSB1, SERGEF, PGK1, KIAA2013, THAP8, RP11-529110A, SLC6A6, EN02, PPP1R15A, SCD, KLF7, MXI1, L0C154761, LOX, WDR5B, CLPB, EGLN1, KLHL24, FAM13A1, VEGF, FNBP1L, WDR54, ALDOC, N013, P4HA1, iMiD2B, NADSYN1, BFILHB2, CCNI, CLK3, BHLHB3, TIPARP, HIG2, PBEF1/1/ L00646309, P4HA2, TSLP, DID01, PPP3CA, ADFP, MED6, CEBPB, INSIG1, NDRG1, SLC2A3, RIOK3, INSIG2, PPP1R3C, L0C401152, KLF4, PFKFB4, STK4, MPH, C6ORF166, ANKRD37, LARP6, PPFIA4, GBE1, ZBTB25 STANDARD

NAME
MENSE_HYPDX1 16507782 Mense SM, Sengupta A, Zhou M, Lan C, Bentsman A_UP G, Volsky Di, Zhang L

STANDARD
NAME VERSION BUILD DATE SYSTEMATIC NAME ORGANISM
NAME
POSITIVE_REGUL msigclb V2.5 '24-Mar-08 .c5:112 Homo sapiens ATION_OF_CELL
_PROLIFERATION
REGULATION OF rnsigdb V2.5 24-Mar-08 6:292 Homo sapiens _CELL_MIGRATI
ON

STANDARD
EXTERNAL DETAILS URL CHIP CATEGORY CODE
NAME
POSITIVE_REGUL http://amigo.geneontology.org GENE _SYMBOL c5 ATION_OF_CELL /cg--_PROLIFERATION biniamige/go.cgOview=details &search constraint=terms&cle pth=0&querv=60:0008284 REGULATION OF http://amigo.geneontology.ora GENE _SYMBOL c5 _CELL MIGRATI icgi-ON bin/amigo/go.cgi ?view.--cietaOs &search constraint=terms&de pth----0&query=G0:0030334 STANDARD CONTRIBUTOR
CONTRIBUTOR
BRIEF DESCRIPTION
NAME ORGANIZATION
POSITIVE_REGUL Gene Ontology Gene Ontology Genes annotated by the GO term ATION_OF_CELL GO:0008284. Any process that _PROLIFERATION activates or increases the rate or extent of cell proliferation.
REGULATION OF Gene Ontology Gene Ontology Genes annotated by the GO term _CELL_MIGRATI GO:0030334. Any process that ON modulates the frequency, rate or extent of cell migration.

STANDARD
FULL DESCRIPTION
NAME
POSIT1VE_REGUL
ATION_OF_CELL
_PROLIFERATION
REGULATION OF
_CELL_MIGRATI
ON

STANDARD
MEMBERS
NAME
POSMVE_REGUL ADRA1D, ADRA2A, ALOX12, RHOG, BCGF1, BNC1, BTC, CAPN1, CAPNS1, CCKBR, CD86, ATION_OF_CELL CD47, CD81, CDC25B, CDK2, CHRM1, CHRNA7, CSF1, CSF3, CTF1, DDX11, DHPS, EDG3, _PROLIFERATION EDN1, EGR4, PTK2B, FGF4, FGF7, FIGF, FLT1, FLT3, FLT3LG, FLT4, GLI1, HCLS1, HOXC10, HTR1A, IGF1, IGF1R, IL2, IL3, IL61 IL7, IL9, ILBRB, IL11, ILURB1, IL12RB2, IL15, CXCL10, LIF, LRP5, LYN, MATK, MST1R, MYC, NAP1L1, NOL1, PDGFA, PGF, POU3F2, PRTN3, PTN, REG1A, TSPAN31, CLEC11A, CCL14, CXCL5, SLAMF1, SSR1, TBX2, TBX3, TDGF1, TGFB2, TGFBR1, TGFBR2, TIMP1, TSHR, K, TNFSF4, VEGFA, VEGFB, VIP, VIPR1, FOSL1, CDC7, CLIL3, CDC2L5, TNFSF13, TNFR5F11A, FGF18, NRP1, CDC123, TBRG4, EDGS, GLP2R, CIA01, PBEF1, DNAJA2, TORG1, STAMBP, TNFSF13B, FGFR1OP, TBC1D8, MCTS1, SERTAD1, DERL2, TWIN, SIRPG, MARK4, PDF, IL31RA, SPDYA, FGF10, CD3E, CD28, IL4, NCK1, PTPRC, NCK2, IL21, CD276, ANG, CDH13, SCG2, TNFSF12, AGGF1, ELA2, EREG, EGFR, ERBB2, IAMBI, LAMC1, NME1, NME2, TGFA, EPGN, LAMA1, EBI3, CD24, IL12B, IL18, ICOSLG, BMI1, CDK4, CDK6, CDKN1A, NDUFS4, SPHK1 REGULATION OF ABI3, RTN4, PARD6B, NEXN, NF1, ACVRL1, ALOX15B, NF2, PTEN, SHH, TBXS, THY1, _CELL_MIGRATI VCL, GTPBP4, CLIC4, BMP10, CENTD3, MIA3, CDH13, EGFR, IAMBI, TDGF1, TRIP6, ON SPHK1, BCAR1, ANGPTL3, PLO, AMOT

STANDARD
MEMBERS SYMBOLIZED
NAME
POSMVE_REGUL ADRA1D, ADRA2A, ALOX12, RHOG, BCGF1, BNC1, BTC, CAPN1, CAPNS1, CCKBR, CD86, ATION_OF_CELL CD47, CD81, CDC25B, CDK2, CHRM1, CHRNA7, CSF1, CSF3, CTF1, DDX11, DHPS, EDG3, _PROLIFERATION EDN1, EGR4, PTK2B, FGF4, FGF7, FIGF, FLT1, FLT3, FLT3LG, FLT4, GLI1, HCLS1, HOXC10, HTR1A, IGF1, IGF1R, IL2, IL3, IL61 IL7, IL9, ILBRB, IL11, ILURB1, IL12RB2, IL15, CXCL10, LIF, LRP5, LYN, MATK, MST1R, MYC, NAP1L1, NOLL, PDGFA, PGF, POU3F2, PRTN3, PTN, REG1A, TSPAN31, CLEC11A, CCL14, CXCL5, SLAMF1, SSR1, TBX2, TBX3, TDGF1, TGFB2, TGFBR1, TGFBR2, TIMP1, TSHR, K, TNFSF4, VEGFA, VEGFB, VIP, VIPR1, FOSL1, CDC7, CLIL3, CDC2L5, TNFSF13, TNFR5F11A, FGF18, NRP1, CDC123, TBRG4, EDGS, GLP2R, CIA01, PBEF1, DNAJA2, TORG1, STAMBP, TNFSF13B, FGFR1OP, TBC1D8, MCTS1, SERTAD1, DERL2, TWIN, SIRPG, MARK4, PDF, IL31RA, SPDYA, FGF10, CD3E, CD28, IL4, NCK1, PTPRC, NCK2, IL21, CD276, ANG, CDH13, SCG2, TNFSF12, AGGF1, ELA2, EREG, EGFR, ERBB2, IAMBI, LAMQ, NME1, NME2, TGFA, EPGN, LAMA1, EBI3, CD24, IL12B, IL18, ICOSLG, BMI1, CDK4, CDK6, CDKN1A, NDUFS4, SPHK1 REGULATION OF ABI3, RTN4, PARD6B, NEXN, NF1, ACVRL1, ALOX15B, NF2, PTEN, SHH, TBXS, THY1, _CELL_MIGRATI VCL, GTPBP4, CLIC4, BMP10, CENTD3, MIA3, CDH13, EGFR, LAMM, TDGF1, TRIP6, ON SPHK1, BCAR1, ANGPTL3, PLO, AMOT

STANDARD
PMID AUTHORS
NAME
POSITIVE_REGUL Ashburner M, Bail CA, Blake JA, Botstein D, Butler ATION_OF_CELL H, Cherry JM, Davis AP, Dolinski K, Dwight SS, _PROLIFERATION Eppig .1T, Harris MA, Hill DP, Issel-Taryer L, Kasarskis A, Lewis 5, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G.
REGULATION OF Ashburner M, Ball CA, Blake JA, Botstein D, Butler _CELL_MIGRATI H, Cherry JM, Davis AP, Dolinski K, Dwight SS, ON Eppig JT, Harris MA, Hill DP, lssei-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G.

Taken together, the foregoing results define a differentiation hierarchy in a primary NSCLC culture and indicate that retinoic acid differentiation can partially reverse the tumorigenic profile.

Identification of Tumor Initiating Cells In Non Small Cell Lung Cancer Cell Xenografts Primary xenograft lines were prepared using female, athymic nu/nu (nude) and NOD-SCID mice (18-23 g) obtained from Charles River of Wilmington, Massachusetts, USA. To assess tumorigenic potential of sorted cells, the cells were implanted in 50% Matrigel (BD Biosciences) subcutaneously between the shoulder blades Typically, for H460T and HCC2429, 100 sorted cells were implanted per nude mouse. For the 37622 line. 2500 cells were implanted per nude mouse. For the 60257 line, 5000 cells were implanted per nod-scid mouse.
Tumors were measured at least once a week with tumor volume = 0.5 x (tumor width2) x (tumor length). Each implant line was propagated by explanting a fragment of the resulting xenograft into new animals and thus was maintained exclusively in vivo. In each line, the histology of the xenografts resembled that of the original tumor. Samples were cryopreserved so that experiments could be performed and repeated in low-passage xenografts.
Immunohistochemical analysis of primary implants was performed using standard techniques and revealed heterogeneous expression of 514. In multiple implant lines the highest 5T4 expression was observed at the tumor-stroma interface. In xenografts prepared using 37622 cells, a similar staining pattern was observed for vimentin, a marker of the epithelial-mesenchymal transition of differentiation. Vimentin was not detected in xenografts prepared using 60274 cells.
Heterogeneous 5T4 expression in xenografts was also observed by flow cytometry. Dissociated 37622 implants showed distinct 5T4high and 5T4Icw populations were evident among the viable human cells (Figure 8A). When a serum-free culture was established from 37622 xenografts, all cells expressed high 5T4 (Figure 8A), which was consistent with the culture conditions that promote stem cell growth. When these cells were re-implanted into animals, the resulting tumors were heterogeneous for 5T4 expression (Figure 8A).
To determine whether 5T4 expression was associated with higher tumorigenicity, as in H460T, primary implant xenografts were dissociated and FACS-isolated cells were implanted into animals. 5T4h'gh cells were more tumorigenic than 5Tew cells in 37622 and 60257 implant lines (Figure 8B).

Additional Biomarkers of Tumor Initiating Cells Cells harvested from cultured cell lines (as described in Examples 1 and 2) were resuspended in lysis buffer (QIAGEN of Valencia. California, USA) and total RNA was purified using QIAGEN RNEASYq columns following the manufacturer's instructions. For xenograft tumors (as described in Example 4), tumor samples were first disrupted by sonication in 3m1 ice-cold 4M
guanidinium/10% sodium acetate buffer (RNAGENTS(q), Promega of Madison, Wisconsin, USA), extracted 2X with phenol-chloroform-isoamyl alcohol (50:48.2) and RNA precipitated from aqueous phase using an equal volume of isopropanol. The precipitate was subsequently resuspended in lysis buffer (QIAGEN) and total RNA purified using QIAGEN RNEASY columns following the manufacturer's instructions.
cDNA was synthesized from 10pg of total RNA using the SUPERSCRIPT Kit (Gibco BRL of Gaithersburg, Maryland, USA) essentially as described by Byrne at al., in F. e. a. Ausubel, ed., Current Protocols in Molecular Biology, 2000, New York: John Wiley and Sons, Inc. First strand synthesis was carried out at 500C to prevent mispriming from ribosomal RNA and utilized a 17 RNA polymerase promoter containing poly-T primer (T7T24) for subsequent in vitro antisense RNA (cRNA) amplification and biotin labeling. cDNA was purified using GENECHIP sample cleanup module (Affymetrix of Santa Clara California, USA) following the manufacturer's instructions. In vitro 17 polymerase driven transcription reactions for synthesis and biotin labeling of antisense cRNA

utilized GENECHIP Expression 3'-Amplification Reagent kit (Affymetrix of Santa Clara California, USA) following the manufacturer's instructions, Synthesized cRNA was purified using QIAGEN RN EASY columns.
For each sample, 10pg of biotin-labeled cRNA was fragmented and hybridized to Human Genome U133+2 GENECHIP oligonucleotide arrays (Affymetrix of Santa Clara, California, USA) using buffers and conditions recommended by manufacturer. GENECHIP oligonucleoticle arrays were washed and stained with Streptavidin R-phycoerythrin (Molecular Probes of Eugene, Oregon, USA) using the GENECHIP Fluidics Station 450 and scanned with a Affymetrix GENECHIP Scanner 3000 (Affymetrix of Santa Clara, California, USA) following the manufacturer's instructions. Fluorescent data were collected and converted to gene specific signal intensities using MicroArray Suite 5.0 (MAS5) software where mean fluorescence difference between perfect match and single mismatch probe sets containing gene-specific oligonucleotides are used to determine mRNA signal intensity. For analysis, mean mRNA signal intensity of replicate samples was determined for each of the experimental groups. Genes were initially filtered to remove those probes where either all samples were called Absent by the MAS5 software. Mean signal intensity values were subsequently compared between experimental groups to identify genes with average fold change typically greater than 2-fold.
A number of genes were differentially expressed in tumor-initiating cells, including the following genes, which showed elevated expression in CD24-ItmCD44+ tumor-initiating cells: TGF13R111, Unc5D, PNPLA4, KCNJ2, GABRB3, DPYD, SPAG1, ICK, STC2, DEFfil, and predicted gene FI138736.
The gene expression profiles of the primary culture in growth and differentiation (see Example 3) were also compared to those of the H460T CO24-11mCD44+ and CD24highC044+ populations. A significant fraction of the genes that were expressed at higher levels during differentiation of the primary culture were also expressed at higher levels in the CD24hghCD44+ cells (FDR = 0.0015).
For statistical comparison of the H460T and 87426 data sets, the top 250 upregulated genes in the differentiated 87426 culture were compared in the H460T populations. Figure 7D shows the expression difference for genes that are above noise level in the H460T data set Statistical analysis yielded the False Discovery Rate of 0,0015. This analysis indicates that these very different experimental systems are physiological models of the differentiation hierarchy in NSCLC. The microarray data were confirmed by flow cytometry (Figures 7E-7F).
Accordingly, additional markers for enrichment or isolation of tumor initiating cells, either by positive selection, by low level expression, or by depletion of differentiated cells, include those set forth in Tables 1 and 2 (see Example 3).

Sox2 Regulates Differentiation of Lung Cancer Tumor Initiating Cells Gene expression profiling was performed on a panel of CD24=110wCD44+
clones to compare the clones that transitioned to CD24h`gh with the clones that were stable (>99% CD24-1'0w). CD24-43wCD444. cells were sorted from each clone and RNA was extracted for microarray analysis as described in Example 5.
Gene expression profiles for stable CD24-m wCD44+ clones and transitioning CD24wCD44+ clones were similar overall, but mRNA levels of some genes correlated with the transition efficiency. For example, Sox2 mRNA
levels were higher in the transitioning clones than in the stable clones (Figure 9A). Sox2 is a transcription factor that is required for pluripotency and self-renewal in stem cells (Avilion et al., Genes Dev., 2003, 171 126-140: Boyer et al., Cell. 2005, 122: 947-956) and can contribute to the induction of pluripotency in differentiated cells (Takahashi & Yamanaka, Cell, 2006, 126: 663-676). In parental H460T cells, Sox2 was expressed in the CD24"11CD44+ tumor-initiating cells but not in the CD24highCD44+ cells.
To test whether Sox2 could regulate the transition from CD24-'9'm to CD24"'' expression, exogenous Sox2 was introduced into stable CD24-"' "CD44+
clones. Expression vectors EX-T2547-M46 (Sox-2) and EX-M0425-M46 (Sox-11) from GeneCopoeia (Germantown; Maryland, USA) were introduced into H460T clones with the Amaxa nucleofector solution V, program T-020 (2 pg DNA
per 106 cells). Stable clones were transfected with Sox2-Flag, Sox11-Flag, empty vector, or no DNA. Forty-eight hours after transfection, G418 was added to 400 pg/ml, Cells were incubated in G418 for six days and subsequently without G418. Irrimunoblot analysis was performed as described in Example 2, which confirmed expression of the indicated transgenes (Figure 9B). After a 6-day selection in G418 and two additional weeks in culture, all three stable 1"CD44+ clones exhibited large fractions of CD24"hCD44+ cells after transfection with Sox2-Flag but not Soxl 1-Flag or empty vector (Figures 9C-9D).
These data indicated that Sox2 was sufficient to drive the transition from n" to CD24hgh, indicating a role in the differentiation of multipotent tumor-initiating cells.

Inhibition of Tumor Cell Growth Using Anti-5T4 Antibody/Drug Conjugates The CD2441'CD44+ population was more sensitive to an anti-5T4 antibody-drug conjugate than the CD24highCD44+ population in an cell viability assay and a colony growth assay. For each assay, antibody-calicheamicin AcBut-linked (AcBut AcBut-[4-(4-acetylphenoxy) butanoic acid]) conjugates were prepared as described (Hamann et al., Bloconjug. Chem., 2002, 13: 47-58).

The effect of anti-5T4 huH8 antibody-drug conjugate or anti-CD22 antibody-drug conjugate on sorted cells was assessed using a cellular viability indicator ((3-(4,5-dimethylthiazol-2-y1)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega of Madison, Wisconsin, USA) to determine the number of surviving cells following exposure to the drug treatment. Cells were sorted 18 hours prior to start of assay. Cells were seeded in 96-well microtiter plates at a density of 10000 cells per well and exposed to various concentrations of the drug. Following determination of the number of viable cells surviving hours of drug exposure, the 105.0 of each treatment was calculated based on the logistic regression parameters derived from the dose-response curves, IC50 values were calculated by logistic non-linear regression and are reported as the calicheamicin dimethyl hydrazide (CalichDMH) concentration from each treatment group that causes 50% loss of cell viability. CD24-ilmCD44r cells were more than ten-fold more sensitive to the anti-5T4-calicheamicin conjugate (Figure 10A). No difference between the two populations was observed when treated with anti-0O22-calicheamicin conjugate or calicheamicin alone (Figure 10A).
To perform a colony formation assay, cells were seeded in 24 well plate at a density of 5,000 cells per well. Twenty-four hours after seeding the cells were exposed to various concentrations of (0.000097, 0.000390, 0.00156, 0.00625, 0,025, 0.1, 0.4 ng calicheamicin equivalents/m1) of anti-5T4 H8-AcBut conjugate, anti-CD22 AcBut conjugate, or calicheamicin alone Seventy-two hours after the drug exposure, cells were trypsinized, counted and 200 cells were plated in 6 well plates. After 8 days, the colonies were fixed and stained with methylene blue. The number of colonies per well was counted using a Stereoscope. CD24-1 wCD44+ cells were more than ten-fold more sensitive to the conjugate (Figure 108). No difference between the two populations was observed when treated with anti-CD22-calicheamicin conjugate or calicheamicin alone (Figure 108).
To test whether 5T4 expression was directly associated with tumorigenic potential, H460T cells were sorted based upon 5-14hFgh and 5T41"' expression and implanted subcutaneously into mice. Tumors from 5T4hic'h cells were larger than tumors from the 5Tew cells (p<0.03; Figure 11). For this experiment, H460T
clonal line 24N-26 was used, which shows higher levels of 5T4 expression and increased resolution of 5Tegh and 5-1-ew expression as compared to the parental line.

Tumor Regression Using Anti-5T4 Antibody/Drug Conjugate Nude (for 37622) or nod-scid (for 60274) mice were injected subcutaneously between the shoulder blades with fragments of low-passage primary implants. When the tumors reached the mass of 0.2 to 0.5 g, the tumors were staged to ensure uniformity of the tumor mass between various treatment groups prior to the administration of therapy. Anti-5T4 huH8 antibody and anti-CD33 p67.6 antibody were conjugated to calicheamicin via an amide linker as described (Hamann et al., Bloconag. Chem , 2002, 131 40-46). The "amide"

linker restricts the release of calicheamicin to cells that internalize the antibody-drug conjugate (Hamann et al,, Bioconug. Chem., 2002, 13: 40-46). Antibody-drug conjugates or vehicle were each administered intraperitoneally in sterile saline (02 ml/mouse) on day 1 and the same treatment was repeated twice four days apart (Q4Dx3). The calicheamicin conjugates were administered at a dose of 160 pg/kg of CalichDMH. Tumors were measured at least once a week and their mass was as volume = 0.5 x (tumor width2)(tumor length). Mean tumor volume ( SEM) for each treatment group was calculated and compared to the vehicle-treated group for statistical significance using a one-sided t-test, with the error term for the t-test based on the pooled variance across all treatment groups. Tumor values for each treatment group were recorded up to 120 days after the initiation of treatment or until either tumor-bearing mice died or the tumors grew to 15% of the body weight at which time these mice were euthanized according to institutional regulations. The anti-CD33 conjugate served as control because these xenografts do not express CD33.
Treatment with anti-5T4-calicheamicin conjugate completely eradicated the 37622 xenografts, and no regrowth was observed through the end of the study, 120 days after the last dose (Figure 12A). Xenog rafts treated with vehicle or anti-CD33-calicheamicin conjugate grew into large tumors.
Similarly, treatment of 60274 xenografts with the anti-5T4-calicheamicin conjugate regressed the tumors significantly (Figure 126). In contrast, treatment of tumors with cisplatin at the maximum tolerable dose reduced tumor size transiently, and the tumors quickly regrew after completion of the dosing regimen (Figure 12C). 60274 cells express 5T4 at lower levels when compared to 37622 cells (Figure 13). These results demonstrated a specific effect of an anti-5T4 antibody-calicheamicin conjugate on growth inhibition of NSCLC primary implants with heterogeneous 5T4 expression.

Claims (34)

WHAT IS CLAIMED IS:

1. An isolated tumor-initiating cell population, which is derived from a tumor cell population, wherein the isolated tumor-initiating cell population comprises at least 90%
tumor-initiating cells, wherein the tumor-initiating cells (i) express 5T4 at a level that is at least 2-fold higher than non-tumorigenic cells of the same origin, (it) are tumorigenic, (iii) are capable of migration, (iv) are capable of self-renewal, and (v) generate tumors comprising non-tumorigenic cells.

2. The isolated tumor-initiating cell population of claim 1, which comprises at least 95% tumor-initiating cells.

3. The isolated tumor-initiating cell population of claim 1, wherein the tumor-initiating cells comprise less than about 50% of the tumor cell population from which it was derived.

4. An enriched tumor-initiating cell population derived from a tumor cell population comprising tumor-initiating cells and non-tumorigenic cells, wherein the tumor-initiating cells (i) express 5T4 at a level that is at least 2-fold higher than non-tumorigenic cells of the same origin, (ii) are tumorigenic, (iii) are capable of migration, (iv) are capable of self-renewal, (v) generate tumors comprising non-tumorigenic cells, and (vi) are enriched at least 2-fold compared to the tumor cell population.

5. The enriched tumor-initiating cell population of claim 4, wherein the tumor-initiating cells are enriched at least 5-fold compared to the tumor cell population.

6. The isolated tumor-initiating cell population of claim 1 or the enriched tumor-initiating cell population of claim 4, which expresses 5T4 at a level that is at least 5-fold higher than non-tumorigenic cells of the same origin.

7. The isolated tumor-initiating cell population of claim 1 or the enriched tumor-initiating cell population of claim 4, which further expresses CD24 at a level that is at least 5-fold lower than non-tumorigenic cells of the same origin, and/or which further expresses CD44.

8. The isolated tumor-initiating cell population of claim 1 or the enriched tumor-initiating cell population of claim 4, which is derived from a lung tumor.

9. The isolated tumor-initiating cell population of claim 1 or the enriched tumor-initiating cell population of claim 4, wherein a subpopulation of about 10 cells or less of the isolated tumor-initiating cell population has the capacity to form a palpable tumor.

10. A method of isolating or enriching a tumor-initiating cell population comprising:
(a) providing dissociated tumor cells, wherein a majority of the cells express at a low level and a minority of the cells express 5T4 at a high level;
(b) contacting the dissociated tumor cells with an agent that specifically binds to T4 ; and (c) selecting cells that specifically bind to the agent of (b) to an extent that shows a high level of 5T4 expression that is at least about 2-fold greater than the low level;
whereby a tumor-initiating cell population is isolated or enriched.

11. The method of claim 10, wherein the isolated or enriched tumor-initiating cell population comprises at least 95% tumor-initiating cells.

12. The method of claim 10, whereby the tumor-initiating cell population is enriched in tumor-initiating cells at least 2-fold when compared to the dissociated tumor cells.

13. The method of claim 11, wherein the dissociated tumor cells are lung cancer cells.

14. The method of claim 10, wherein the selecting cells is performed by flow cytometry, fluorescence activated cell sorting, panning, affinity column separation, or magnetic selection.

15. The method of claim 10, further comprising (d) contacting the dissociated tumor cells with an agent that specifically binds to CD44; and (e) selecting cells that specifically bind to the agent of (d) to an extent that shows expression of CD44.

16. The method of claim 10 or 15, further comprising:
(d) contacting the dissociated tumor cells with an agent that specifically binds to CD24; and (e) selecting cells that specifically bind to the agent of (d) to an extent that shows a low level of CD24 expression that is at least about 5-fold lower than non-tumorigenic cells of the same origin.

17. The method of claim 10 or 15, further comprising:
(d) contacting the dissociated tumor cells with an agent that specifically binds to CD24; and (e) depleting cells that specifically bind to the agent of (d) to an extent that shows a high level of CD24 expression that is at least about 5-fold greater than non-tumorigenic cells of the same origin.

18. The method of claim 10, further comprising:
(d) contacting the dissociated tumor cells with one or more agents that specifically binds to a differentiation marker expressed by the tumor cells; and (e) depleting the tumor-initiating cell population of cells that specifically bind to the one or more agents of (d).

19. An isolated or enriched tumor-initiating cell population isolated prepared according to the method of any one of claims 10-18.

20. A method of testing efficacy of a cancer drug or candidate cancer drug comprising:
(a) providing an isolated or enriched tumor-initiating cell population according to any one of claims 1-9 or 19;
(b) contacting the tumor-initiating cells with a cancer drug or a candidate cancer drug; and (c) observing a change in tumorigenic potential of the tumor- initiating cells following contacting the tumor-initiating cells with the cancer drug or candidate cancer drug.

21. The isolated tumor-initiating cell population of claim 3, wherein the tumor-initiating cells comprise less than about 33% of the tumor cell population from which it was derived.

22. The isolated tumor-initiating cell population of claim 21, wherein the tumor-initiating cells comprise less than about 25% of the tumor cell population from which it was derived.

23. The isolated tumor-initiating cell population of claim 22, wherein the tumor-initiating cells comprise less than about 15% of the tumor cell population from which it was derived.

24. The isolated tumor-initiating cell population of claim 23, wherein the tumor-initiating cells comprise less than about 10% of the tumor cell population from which it was derived.

25. The enriched tumor-initiating cell population of claim 5, wherein the tumor-initiating cells are enriched at least 10-fold compared to the tumor cell population.

26. The enriched tumor-initiating cell population of claim 25, wherein the tumor-initiating cells are enriched at least 50-fold compared to the tumor cell population.

27. The enriched tumor-initiating cell population of claim 26, wherein the tumor-initiating cells are enriched at least 100-fold compared to the tumor cell population.

28. The isolated tumor-initiating cell population or the enriched tumor-initiating cell population of claim 6, which expresses 5T4 at a level that is at least 10-fold higher than non-tumorigenic cells of the same origin.

29. The method of claim 10, wherein the agent that specifically binds to 5T4 is an anti-5T4 antibody.

30. The method of claim 12, whereby the tumor-initiating cell population is enriched in tumor-initiating cells at least 5-fold when compared to the dissociated tumor cells.

31. The method of claim 30, whereby the tumor-initiating cell population is enriched in tumor-initiating cells at least 10-fold when compared to the dissociated tumor cells.

32. The method of claim 15 wherein the agent that specifically binds to CD44 is an anti-CD44 antibody.

33. The method of claim 16 wherein the agent that specifically binds to CD24 is an anti-CD24 antibody.

34. The method of claim 17 wherein the agent that specifically binds to CD24 is an anti-CD24 antibody.