Presentation on theme: "Gene Expression and Cancer"— Presentation transcript:
1Gene Expression and Cancer Presentation: Inna Weiner
2Cancer Cellular level: over–proliferation of the cell Tissue level: cells deviate from their natural place in the tissue and spread3 main principles:Tumors are mono-clonalDNA mutations (6-7 usually)Selection (from bad to worse)
3Cellular mechanisms in cancer Signaling pathways damageTumor cells uncontrolled proliferationGrowth factors constitutive activityConstitutive up/down regulationDNA repair problemApoptosis mechanism not activeCells acquire metastatic potential…
5Cancer – metastatic pathway דה-אדהיז'ן - הפסקת התאחיזה בין התאים ברקמה (ירידה בביטוי גנים של תאחיזה בין תאית)פלישה - רכישת כושר פולשנות . יצירת אנזימים שיכולים לעכל את הקרום הבסיסי.תנועה - רכישת כושר תנועתיות – חריגה מהנישה הטבעית שלהם.חדירה לכלי הדם או הלימפה (Intravasation). הפיזור של התאים בכל הגוף הוא מיידי. מאפשר פיזור נרחב של גרורות [אין ספציפיות לגידול, אולם יש העדפות מסוימות. הכיצד?]החלצות מכלי הדם (Extravasation) ברקמת המטרה. יצירת מושבה של גרורות.
6Articles A molecular signature of metastasis in primary solid tumors. S. Ramaswamy et al. Nature Genetics, 2002Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival.H. Y. Chang et al. PNAS, 2005An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis.A. Sweet-Cordero et al. Nature Genetics, 2004
7A molecular signature of metastasis in primary solid tumors Sridhar Ramaswamy, Ken N. Ross, Eric S. Lander & Todd R. GolubNature Genetics, December 2002
8Motivation 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 cancerCancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body.
9Model of MetastasisMost primary tumor cells have low metastatic potentialRare cells (estimated at less than 1 in 10,000,000) within large primary tumors acquire metastatic capacity through somatic mutation
10Metastatic Phenotype Has the ability to migrate from the primary tumor survive in blood or lymphatic circulationinvade distant tissuesestablish distant metastatic nodulesSupported by animal modelsThe successful metastatic cancer cell must leave the primary tumor, enter the lymphatic and blood circulation, survive within the circulation, overcome host defenses, extravasate and grow as a vascularized of tumor cells, but less than 0.01% of these cells develop into metastasis.
11Setup12 metastatic adenocarcinoma nodules of diverse origin (lung, breast, prostate, colorectal, uterus, ovary)64 primary adenocarcinomas representing the same spectrum of tumor types
12Hypothesis:a gene-expression program of metastasis may already be present in the bulk of some primary tumors at the time of diagnosisThe arrow - these tumors being misclassified as metastases (see Web Note Aand Web Table A). This observation suggested the hypothesisthat a gene-expression program of metastasis may already be presentin the bulk of some primary tumors at the time of diagnosis.
13Hypothesis testing 62 stage I/II primary lung adenocarcinomas Hierarchical clustering in the space 128 metastases-derived genesa, Hierarchical clustering of 62 primary lung adenocarcinomas using 128 metastases-derived genes defined two predominant primary-tumor groups in the resulting dendrogram. Colorgram depicts high (red) and low (blue) relative levels of gene expression. Vertical bar (left) indicates genes that were originally expressed in primary tumors (black) or metastases (red). Horizontal bar (top) indicates samples from individuals whose cancer was observed to be non-recurrent (black) or recurrent (red).
14Clinical Outcome Prediction 128 pre-defined genesall genes17 unique genes nearest the centroids of the two lung cancer clustersKaplan–Meier analyses
15Generality of metastatic signature Kaplan–Meier analyses of cluster-defined primary-tumor subsets60 medulloblastomaslarge B-cell lymphoma10 (P = 0.497; Fig. 3d), consistent with the idea that hematopoietic tumors have specialized mechanisms for navigating the hematologic and lymphoid compartments.
1617-gene metastatic signature Upregulation: Protein translation apparatusNotably, the large stromal component of the signature would have been missed had only malignantepithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.
1717-gene metastatic signature Upregulation: Non-epithelial components of the tumorNotably, the large stromal component of the signature would have been missed had only malignantepithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.
1817-gene metastatic signature Notably, the large stromal component of the signature would have been missed had only malignantepithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.Downregulation: Antigene presenting cell
1917-gene metastatic signature Notably, the large stromal component of the signature would have been missed had only malignantepithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.Downregulation: Tumor suppressor
20Novel Model of Metastasis Prevailing Model: incidence of metastasis is related to the number of cells susceptible to metastasis-promoting mutations, and hence to tumor sizeNew Model: the propensity to metastasize reflects the predominant genetic state of a primary tumorA metastatic tumor may be more efficient at producing metastasis because it has a higher proportion of the rare metastatic cell or because the average tumor cell in the colony has a stronger metastatic fitness.selection process favoring the metastatic phenotyperare metastatic phenotypeconsequence of particular mechanisms of transformationmetastasis-potential tumor
21Critical ViewThe authors did not prove that there is a single cell with all metastatic functionsMaybe a small fraction of primary tumors (the biggest?) did acquire metastatic-potential cells-- some individuals harboring metastasis can survive many years despite a large metastatic burden, whereas others will succumb rapidly to aggressive metastatic disease-- host/stromal cells
22Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survivalH. 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 VijverPNAS, March 8, 2005
23Chang et al (2004), PLoSHypothesis:Molecular program of normal wound healing might play an important role in cancer metastasisProcedure:Measured gene expression of serum response of cultured fibroblasts from 10 anatomic sites in vitroResult:Identified a set of “core serum response” genes and their canonical expression profile in fibroblasts activated with serumBlood plasma is the liquid component of blood, in which the blood cells are suspended. Serum is the same as blood plasma except that clotting factors (such as fibrin) have been removed (Wikipedia)
24of a ‘‘wound’’ signature 512 core serum response genes were identified and were considered representativeof a ‘‘wound’’ signature(A) The fibroblast common serum response.Genes with expression changes that demonstrate coordinate induction or repression by serum in fibroblasts from ten anatomic sites are shown. Representative genes with probable function in cell cycle progression (orange), matrix remodeling (blue), cytoskeletal rearrangement (red), and cell–cell signaling (black) are highlighted by colored text on the right. Three fetal lung fibroblast samples, cultured in low serum, which showed the most divergent expression patterns among these samples (in part due to altered regulation of lipid biosynthetic genes, are indicated by blue branches.(B) Identification of cell cycle-regulated genes in the common serum response signature. The expression pattern of each of the genes in (A)during HeLa cell cycle over 46 h after synchronization by double thymidine block is shown.
25Chang et al (2004): Identified Annotations of Genes Matrix remodelingCytoskeletal rearrangementCell–cell signalingAngiogenesisCell motilityLikely to contribute to cancer invasion and metastasis
26Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survivalH. 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 VijverPNAS, March 8, 2005
27Performance of “wound-response” signature 295 breast cancer samples using 442 available core serum response genes
29Scalable Prognostic Score Problem: Hierarchical clustering provides biologically arbitrary thresholdSolution: 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)
30Improving Clinical Decision Making 30% of women with early breast cancer develop metastasisFor young women chemotherapy increases 10 year survival at ~10%Chemotherapy does not benefit for 89-93% of all breast cancer patientsNIH - five molecular subtypes, SG - discovered by directly fitting to survival data, and one based on an in vitro model of a wound response185 patients did not receive chemotherapy10% undertreated30% overtreated
31SummaryMechanism-driven approach to prognostic biomarker discovery on a genome scaleUncovered the catalog of genes involved in a potentially new cellular process that defines the clinical biology of breast cancerpathogenic mechanismspotential targets for treatmentNew findings applicable for clinical decision making
33The MAP-K cascade : Protein-Protein interactions bridging the plasma membrane and the nucleus Cancer course, I. Ben-Neria
34RAS ActivationSmall G proteins are monomeric G proteins with molecular weight of kDa. Like heterotrimeric G proteins, their activity depends on the binding of GTP.Cycling of the Ras protein between active and inactive states. Like other G proteins, Ras can switch between GTP-bound and GDP-bound states. Transition from the GDP-bound to the GTP-bound state is catalyzed by guanine nucleotide exchange factor (GEF) which induces exchange between the bound GDP and the cellular GTP. The reverse transition is catalyzed by a GTPase-activating protein (GAP) which induces hydrolysis of the bound GTP.RAS is oncogenic due to constitutive activation in the GTP-bound form
35An 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.JacksNature Genetics, December 2004Official Symbol: KRAS and Name: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog Summary: This gene, a Kirsten ras oncogene homolog from the mammalian ras gene family, encodes a protein that is a member of the small GTPase superfamily. A single amino acid substitution is responsible for an activating mutation. The transforming protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma. Alternative splicing leads to variants encoding two isoforms that differ in the C-terminal region.
36Why use animal models? Initiated by single well-characterized event Discover novel pathways obscure in human dataEndogenous activation of oncogenes in vivo is distinct from overexpression in vitroValidation by Gene Expression Profile
37Experimental SetupGoal: build animal model for human lung adenocarcinomaCreate KrasLA mouse model: Mice with sporadically activated Kras2 through spontaneous homologous recombinationMice develop lung adenomaThrough time acquire characteristics similar to human tumor: nuclear atypia and high mitotic index
38Gene Set Enrichment Analysis (GSEA) Is Rank-Ordered Gene List (from human analysis) enriched in independent a priori defined Gene set (from mouse model)?
40Comparison of Gene Expression in mouse and human lung cancer Using GSEA was foundDifferentially expressed genes in KrasLA mouse model were significantly enriched in Human Lung Adenocarcinoma but not in other lung subtypesNNK mouse model (induced by chemical mutogen) adenoma and carcinoma did not provide enriched Differentially Expressed Gene SetMouse tumor from KrasLA and NNK model were not distinguishable histologicallyConclusion: the found genes were NOT merely cancer/proliferation signature.Conclusion: mouse models that seem similar may differ on gene expression level.
41Oncogenic KRAS2 signature 89 differentialy expressed genes (upregulated) in KrasLA mouse model that contributed maximally to the GSEA score in human data setBoston: 185 tumor, 17 normal. Ann Arbor: 85 adenocarcinoma, 10 normalEach gene is only moderately changed in human.As a collection the entire set achieves statistical significance at p < 0.05.
42KRAS2 signature verification (1) KRAS2 signature is enriched in pancreatic adenocarcinomaKRAS2 mutation occurs in >90% of pancreatic adenocarcinomas Link between KRAS2 signature and mutation of KRAS2 Link between signature and mutation of KRAS2
43KRAS2 signature verification (2) Real-time PCR analysis of expression of selected KRAS2 signature genes (in human cell lines)
44KRAS2 signature verification (3) Knock-down of KRAS2 in human lung cancer cell line
45Summary Integrative analysis of mouse model and human cancer can Validate the animal modelExtract an evidence of oncogene-specific programCompare several models against human cancer typesIn this research were identified many potential effectors of KRAS2New directions for anti-Ras pathway therapeutic strategies