1. Cancer-inducing genes - CRGs (cooperation response genes) Paper Presentation 01.12.2008 Nadine Sündermann

2. Cancer-inducing genes Oncogenes and/or the loss of tumour suppressor genes generally cooperate with each other in transforming cells into cancer cells A new study identifies a list of genes synergistically regulated by the Ras oncogene and loss of the p53 tumour suppressor gene Many of these genes are essential for tumour formation in vivo, whereas few of the genes regulated by either Ras or p53 alone are important

3. Ras/p53 signalling

4. Oncogenic mutations in p53 and Ras Oncogenic mutations in transcription factor p53 and in the small GTPase protein Ras can transform normal cells into cancer cells Have individually limited effects on promoting cancer:  p53 mutation affects the expression of group A genes  Ras mutation modifies the expression of group B genes  both p53 and Ras are mutated in the same cell, they synergistically regulate a subset of genes (AB) known as cooperation response genes (CRGs), which turn out to be crucial mediators of tumour formation

5. The mutation spectrum of p53 The mutation spectrum of p53 in human cancer can help identify particular carcinogens and define the biochemical mechanisms responsible for the genetic lesions in DNA that cause human cancer

6. Establishment of a new approach to find genes important in tumorigenesis may also represent novel targets for tumour therapy

7. The strategy Identification of genes regulated synergistically by cooperating oncogenic mutations at genomic scale, mRNA expression profiles were used Comparison of young adult murine colon (YAMC) cells with YAMC cells expressing - mutant p53175H (mp53) - activated H-RasV12 (Ras) - both mutant proteins together (mp53/Ras)17 Method: Affymetrix microarrays and TaqMan low density array qPCR

8. Identification and characterization of CRGs Raw expression values (log2) of 538 differentially expressed genes (represented by 657 probe sets) for:  mp53 cells  Ras cells compared to YAMC ctrls  mp53/Ras cells rank-ordered according to synergy score Red and green indicate relative gene expression in the cells vs. YAMC cells purple or blue indicate the synergy score for each gene plotted synergy score of 0.9 or less defines CRGs. The cutoff is indicated by arrowheads or the threshold line

9. CRG expression in mp53/Ras cells as compared with YAMC cells, mp53 cells and Ras cells - data for 76 CRGs - Plots indicating fold change in CRG expression (log2)

10. CRG analysis CRGs encode proteins involved in regulation of cell signalling, transcription, apoptosis, metabolism, transport or adhesion a large proportion are misexpressed in human cancer for 47 out of 75 CRGs tested, co-regulation is found in primary human colon cancer and our murine colon cancer cell model altered expression of 29 CRGs has been reported in a variety of human cancer types

11. Differential expression and synergy scores of CRGs in mp53/Ras cells

12. Co-regulation of CRGs  Modulation of CRG expression has common features in malignant cell transformation of both murine and human cells -in mp53/Ras cells and human cancer on the basis of a literature survey for a variety of human cancers -two independent expression analyses of primary human colon cancers

13. Is tumour formation reduced after CRG perturbations? Genetic perturbation experiments were carried out using retrovirus-mediated reexpression of corresponding complementary DNAs (cDNAs) for downregulated genes and shRNA-dependent stable knockdown using multiple independent targets for overexpressed genes Injection of murine and human cancer cells with indicated perturbations into nude mice Measurement of Tumour volume weekly for 4 weeks after injection Number of injections (n) and significance levels as compared to matched controls are indicated

14. Reversal of the changes in CRG expression significantly reduced tumour formation by mp53/Ras cells in 14 out of 24 cases  indicates a critical role in malignant transformation for a surprisingly large fraction of these genes

15. Synergistic response of downstream genes to oncogenic mutations Distribution of gene perturbations over the set of genes differentially expressed in mp53/Ras cells, rank-ordered by synergy score Colourcoded bars indicate perturbed genes; CRG cutoff synergy score (0.9) is indicated by a horizontal line  malignant transformation strongly relies on the class of genes synergistically regulated by cooperating oncogenic mutations

16. CRG perturbations reduce tumour formation of both mp53/Ras and human cancer cells Plac8 knockdown is functionally rescued by expression of shRNA-resistant Plac8

17. CRG perturbations reduce tumour formation of both mp53/Ras and human cancer cells

19. CRG perturbation - conclusion CRG perturbations reduce tumour formation of both mp53/Ras and human cancer cells Perturbations of CRGs in human cancer cells (Fig. 4b, d, f,) had similarly strong tumour inhibitory effects to those in the genetically tractable murine mp53/Ras cells genetic perturbations disrupt tumour formation downstream of the initiating oncogenic mutations

20. Summary efficacy of CRG perturbations performed in human colon cancer cells comparable to murine colon cell transformation model  suggesting dependence of the malignant state on a similar set of genes in both backgrounds human cancer cells carry oncogenic mutations in genes in addition to Ras or Raf and p53 suggesting that CRGs may have a large involvement in the generation and maintenance of the cancer cell phenotype in a variety of contexts  CRGs may provide a valuable source for identification by rational means of the much sought ‘Achilles’ heel’ in human cancer

21. Thank you for your attention!!