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
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.

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
Cancer 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.

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
The 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.

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
The arrow - these tumors being misclassified as metastases (see Web Note A
and Web Table A). This observation suggested the hypothesis
that 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
a, 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).

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

15. Generality of metastatic signature
Kaplan–Meier analyses of cluster-
defined primary-tumor subsets
60 medulloblastomas
large 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.

16. 17-gene metastatic signature
Upregulation: Protein translation apparatus
Notably, the large stromal component of the signature would have been missed had only malignant
epithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.

17. 17-gene metastatic signature
Upregulation: Non-epithelial components of the tumor
Notably, the large stromal component of the signature would have been missed had only malignant
epithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.

18. 17-gene metastatic signature
Notably, the large stromal component of the signature would have been missed had only malignant
epithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.
Downregulation: Antigene presenting cell

19. 17-gene metastatic signature
Notably, the large stromal component of the signature would have been missed had only malignant
epithelial cells been isolated (for example, by laser capture microdissection) before expression profiling.
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
A 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 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
-- 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

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
Blood 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)

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

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
NIH - five molecular subtypes, SG - discovered by directly fitting to survival data, and one based on an in vitro model of a wound response
185 patients did not receive chemotherapy
10% undertreated
30% overtreated

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
Small 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

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
Official 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.

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
Validation by Gene Expression Profile

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
Conclusion: the found genes were NOT merely cancer/proliferation signature.
Conclusion: mouse models that seem similar may differ on gene expression level.

41. Oncogenic KRAS2 signature
89 differentialy expressed genes (upregulated) in KrasLA mouse model that contributed maximally to the GSEA score in human data set
Boston: 185 tumor, 17 normal. Ann Arbor: 85 adenocarcinoma, 10 normal
Each gene is only moderately changed in human.
As a collection the entire set achieves statistical significance at p < 0.05.

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
 Link between 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