Human Cancer and mTOR

1. Human Cancer and mTOR Ronald Crandall

2. Overview Background Hypothesis Experimental Design & Expected Results Conclusion

3. Cancer “Cancer is the uncontrolled growth of abnormal cells in the body. Cancerous cells are also called malignant cells.” - NCBI Mutation inactivates DNA repair gene Mutation of proto-oncogene creates an oncogene Mutation inactivates several more tumor suppressor genes National Cancer Institute

4. Stages of Carcinogenesis Grade 1 - Initiation Grade 2 -Promotion Grade 3 - Progression Shah, K.V. and Howley, P.M.,“Papillomaviruses” in Virology (Fields, B.N., ed.), 1996, p. 2090.

5. mTOR in Cancer Constitutive activation of mTOR can cause cancer Occurs through mutations in mTORor upstream signals.

6. Study Question What stage in cancer does mTOR most effect? Shah, K.V. and Howley, P.M.,“Papillomaviruses” in Virology (Fields, B.N., ed.), 1996, p. 2090.

7. Overview Background Hypothesis Experimental Design & Expected Results Conclusion

8. Hypothesis Constitutively active mTORacts as a promoter of skin epithelium cancer carcinogenesis.

9. Overview Background Hypothesis Experimental Design & Expected Results Conclusion

10. Experiment Overview

11. Model Organism - Mouse Human and Mouse mTOR share 98.9% amino acid similarity.  ClustalW2 Tree

12. Experiment 1 Steps 1) Generate transgenic mice with constitutively active expression of mTOR in basal epithelial cells • Using human keratin 14 transcriptional promoter 2) Use chemical cancer initiator and promoter through skin painting to determine role of mTOR in cancer progression. k14 promoter mTOR

13. Generate transgenic mice steps Create vector with human keratin 14 promoter and mTOR Microinject DNA into mouse ES cells and put in blastocyst. Biopsy mice and genotype for vector product, if established mate founder mice to create transgenic line

14. Experiment 1 Steps 1) Generate transgenic mice with constitutively active expression of mTOR in basal epithelial cells • Using human keratin 14 transcriptional promoter 2) Use chemical cancer initiator and promoter through skin painting to determine role of mTOR in cancer progression. k14 promoter mTOR

15. Determining the role mTOR plays in cancer progression DMBA – mutagen can only initiate TPA – growth promoter Shah, K.V. and Howley, P.M.,“Papillomaviruses” in Virology (Fields, B.N., ed.), 1996, p. 2090.

16. Treatment Groups DMBA TPA DMBA + TPA Skin painting on both the transgenic and wild type mice for each treatment group.

17. Expected Results

18. Experiment Overview

19. Experiment 2 Steps • Surgically remove tumors from treatment groups • If available include cancers of each stage from treatments • Analyze Transcriptome and proteome • MudPIT • Ribosomal Profiling

20. Expression levels of downstream signals from mTORwould increase in tumors with constitutively active mTOR Proteins S6K1 4E-BP1 eIF4E SREBP1 http://dx.doi.org/10.1016/j.cell.2012.03.017

21. Full proteome and transcriptome profiling categories http://dx.doi.org/10.1016/j.cell.2012.03.017

22. Ribosomal profiling of stage 3 tumors

23. Ribosomal profiling of control epidermal tissue

24. Conclusions mTOR is a promoter in the progression of cancer. Constitutively active mTOR shows increased expression of immediate downstream signaling of S6K1, 4E-BP1, eIF4E, and SREBP1. Ribosomal profiling of constitutively active mTORshows increased cell growth, lipid synthesis, metabolism and proliferative signaling.

25. mTOR future research Test potential mTORinhibitor drug candidates on the cell lines at each stage in cancer progression. GWAS on mTORpolymorphisms and cancer

26. Questions?