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Gene Expression and Cancer Presentation: Inna Weiner.

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1 Gene Expression and Cancer Presentation: Inna Weiner

2 Cancer Cellular level: over–proliferation of the cell Tissue level: cells deviate from their natural place in the tissue and spread 3 main principles: –Tumors are mono-clonal –DNA mutations (6-7 usually) –Selection (from bad to worse)

3 Cellular mechanisms in cancer Signaling pathways damage Tumor cells uncontrolled proliferation Growth factors constitutive activity Constitutive up/down regulation DNA repair problem Apoptosis mechanism not active Cells acquire metastatic potential …

4 Primary Tumor

5 Cancer – metastatic pathway

6 Articles A molecular signature of metastasis in primary solid tumors. S. Ramaswamy et al. Nature Genetics, 2002 Robustness, scalability, and integration of a wound- response gene expression signature in predicting breast cancer survival. H. Y. Chang et al. PNAS, 2005 An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis. A. Sweet-Cordero et al. Nature Genetics, 2004

7 A molecular signature of metastasis in primary solid tumors Sridhar Ramaswamy, Ken N. Ross, Eric S. Lander & Todd R. Golub Nature Genetics, December 2002

8 Motivation for Predicting Metastasis Metastasis (Greek: change of the state): spread of cancer from its primary site to other places in the body (e.g., brain, liver) Metastasis is the principal event leading to death in individuals with cancer

9 Model of Metastasis Most primary tumor cells have low metastatic potential Rare cells (estimated at less than 1 in 10,000,000) within large primary tumors acquire metastatic capacity through somatic mutation

10 Metastatic Phenotype Has the ability to –migrate from the primary tumor –survive in blood or lymphatic circulation –invade distant tissues –establish distant metastatic nodules Supported by animal models

11 Setup 12 metastatic adenocarcinoma nodules of diverse origin (lung, breast, prostate, colorectal, uterus, ovary) 64 primary adenocarcinomas representing the same spectrum of tumor types

12 Hypothesis: a gene-expression program of metastasis may already be present in the bulk of some primary tumors at the time of diagnosis

13 Hypothesis testing 62 stage I/II primary lung adenocarcinomas Hierarchical clustering in the space 128 metastases-derived genes

14 Clinical Outcome Prediction 128 pre-defined genes 17 unique genes nearest the centroids of the two lung cancer clusters all genes

15 Generality of metastatic signature

16 17-gene metastatic signature Upregulation: Protein translation apparatus

17 17-gene metastatic signature Upregulation: Non-epithelial components of the tumor

18 17-gene metastatic signature Downregulation: Antigene presenting cell

19 17-gene metastatic signature Downregulation: Tumor suppressor

20 Novel Model of Metastasis Prevailing Model: incidence of metastasis is related to the number of cells susceptible to metastasis-promoting mutations, and hence to tumor size New Model: the propensity to metastasize reflects the predominant genetic state of a primary tumor selection process favoring the metastatic phenotype rare metastatic phenotype consequence of particular mechanisms of transformation metastasis-potential tumor

21 Critical View The authors did not prove that there is a single cell with all metastatic functions Maybe a small fraction of primary tumors (the biggest?) did acquire metastatic-potential cells

22 Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival H. Y. Chang, D. S. A. Nuyten, J. B. Sneddon, T. Hastie, R. Tibshirani, T. Sørlie, H. Dai, Y. D. He, L. J. van’t Veer, H. Bartelink, M. van de Rijn, P. O. Brown, and M. J. van de Vijver PNAS, March 8, 2005

23 Chang et al (2004), PLoS Hypothesis: Molecular program of normal wound healing might play an important role in cancer metastasis Procedure: Measured gene expression of serum response of cultured fibroblasts from 10 anatomic sites in vitro Result: Identified a set of “core serum response” genes and their canonical expression profile in fibroblasts activated with serum

24 512 core serum response genes were identified and were considered representative of a ‘‘wound’’ signature

25 Chang et al (2004): Identified Annotations of Genes Matrix remodeling Cytoskeletal rearrangement Cell–cell signaling Angiogenesis Cell motility Likely to contribute to cancer invasion and metastasis

26 Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival H. Y. Chang, D. S. A. Nuyten, J. B. Sneddon, T. Hastie, R. Tibshirani, T. Sørlie, H. Dai, Y. D. He, L. J. van’t Veer, H. Bartelink, M. van de Rijn, P. O. Brown, and M. J. van de Vijver PNAS, March 8, 2005

27 Performance of “wound-response” signature 295 breast cancer samples using 442 available core serum response genes

28 Chang et al (2004): Clinical Outcome Prediction

29 Scalable Prognostic Score Problem: Hierarchical clustering provides biologically arbitrary threshold Solution: Create the centroid of the differential expression in response to serum in cultured fibroblasts from 10 anatomic sites (Chang, 2004) Score = corr (centroid, new example)

30 Improving Clinical Decision Making 30% of women with early breast cancer develop metastasis For young women chemotherapy increases 10 year survival at ~10% Chemotherapy does not benefit for 89-93% of all breast cancer patients

31 Summary Mechanism-driven approach to prognostic biomarker discovery on a genome scale Uncovered the catalog of genes involved in a potentially new cellular process that defines the clinical biology of breast cancer –pathogenic mechanisms –potential targets for treatment New findings applicable for clinical decision making

32 Cancer course, I. Ben-Neria

33 The MAP-K cascade : Protein-Protein interactions bridging the plasma membrane and the nucleus Cancer course, I. Ben-Neria

34 RAS Activation RAS is oncogenic due to constitutive activation in the GTP-bound form

35 An oncogenic KRAS2 expression signature identified by cross- species gene-expression analysis. A. Sweet-Cordero, S. Mukherjee, A Subramanian, H. You, J.J. Roix, C. Ladd-Acosta, T. R. Golub and T.Jacks Nature Genetics, December 2004

36 Why use animal models? Initiated by single well- characterized event Discover novel pathways obscure in human data Endogenous activation of oncogenes in vivo is distinct from overexpression in vitro

37 Experimental Setup Goal: build animal model for human lung adenocarcinoma Create KrasLA mouse model: Mice with sporadically activated Kras2 through spontaneous homologous recombination Mice develop lung adenoma Through time acquire characteristics similar to human tumor: nuclear atypia and high mitotic index

38 Gene Set Enrichment Analysis (GSEA) Is Rank-Ordered Gene List (from human analysis) enriched in independent a priori defined Gene set (from mouse model)?

39 Gene Set Enrichment Analysis (GSEA)

40 Comparison of Gene Expression in mouse and human lung cancer Using GSEA was found –Differentially expressed genes in KrasLA mouse model were significantly enriched in Human Lung Adenocarcinoma but not in other lung subtypes –NNK mouse model (induced by chemical mutogen) adenoma and carcinoma did not provide enriched Differentially Expressed Gene Set Mouse tumor from KrasLA and NNK model were not distinguishable histologically

41 Oncogenic KRAS2 signature 89 differentialy expressed genes (upregulated) in KrasLA mouse model that contributed maximally to the GSEA score in human data set

42 KRAS2 signature verification (1) KRAS2 signature is enriched in pancreatic adenocarcinoma KRAS2 mutation occurs in >90% of pancreatic adenocarcinomas  Link between KRAS2 signature and mutation of KRAS2

43 KRAS2 signature verification (2) Real-time PCR analysis of expression of selected KRAS2 signature genes (in human cell lines)

44 KRAS2 signature verification (3) Knock-down of KRAS2 in human lung cancer cell line

45 Summary Integrative analysis of mouse model and human cancer can –Validate the animal model –Extract an evidence of oncogene-specific program –Compare several models against human cancer types In this research were identified many potential effectors of KRAS2 –New directions for anti-Ras pathway therapeutic strategies

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