1. CONTEXT SPECIFIC ROLE OF DEUBIQUITYLASE ENZYME, USP9X, IN HEAD AND NECK CANCER Devathri Nanayakkara Eskitis Institute for Drug Discovery Griffith University

2. Head and neck cancer

3. Sixth most common cancer

4. Head and neck cancer Sixth most common cancer Five year survival rate after diagnosis, 50%

5. Head and neck cancer Sixth most common cancer Five year survival rate after diagnosis, 50% Drug resistance Early tumors - asymptomatic

6. Head and neck cancer Sixth most common cancer Five year survival rate after diagnosis, 50% Drug resistance Early tumors - asymptomatic “Need to study the underlying molecular pathways unveiling potential detection markers and drug targets”

7. In a recent study, Characterized the somatic mutation landscape of OSCC-GB

9. - Found Five new genes associated with OSCC-GB

10. -Among them was USP9X -22% harboured copy number loss and truncating mutations -Role as tumor suppressor

11. USP9X Deubiquitylating enzyme – Family of cysteine and metalloproteases

12. USP9X Deubiquitylating enzyme – Family of cysteine and metalloproteases http://legacy.butler.edu/biology/faculty-staff/research-interests/jkowalski/research-in-the-kowalski-lab/

13. Murtaza et al, 2015

14. USP9X in cancer

16. Breast cancer Colorectal cancer Bladder cancer Oral cancer Brain cancer Pancreatic cancer Prostate cancer Lung cancer Lymphoma

17. In this study, Aims : To evaluate the role of USP9X in an in vitro system To elucidate the molecular mechanisms USP9X is involved in

18. How? In vitro cell lines – SCC15, CAL27, FaDu and Detroit 562

19. Immunoblotting to probe for USP9X expression All 4 cell lines express USP9X SCC15FaDu Detroit 562 CAL27 USP9X 290 kDa β tubulin 51 kDa

20. Knockdown approach – siRNA

21. Immunoblotting to probe for USP9X protein levels 72 h after siRNA treatment USP9X is efficiently knocked down in all four cell lines NT USP9X NT USP9X NT USP9X NT USP9X SCC15 CAL27 FaDu Detroit 562 siRNA USP9X 290 kDa Β tubulin 51 kDa

22. Effect on cell aspects – Cell proliferation CyQUANT Assay

23. time In absence of USP9X, a decrease in cell numbers was observed CyQUANT Analysis of cell proliferation following siRNA treatment to knockdown USP9X SCC15 Detroit 562 CAL27 FaDu * * * * * * * * * P value < 0.05

24. Decrease in cell numbers, – Apoptosis?

25. No elevation in apoptosis detected upon depletion of USP9X β tubulin 51 kDa Cleaved PARP-1 89 kDa siRNA NT USP9X NT USP9X NT USP9X NT USP9X NT USP9X NT USP9X time 48h 96h 144h 48h 96h 144h SCC15 CAL27 FaDu Detroit 562 Immunoblotting for cleaved PARP-1

26. Decrease in cell numbers in absence of USP9X prompts a role of a

27. Decrease in cell numbers in absence of USP9X prompts a role of a “tumor promoter”

28. Contradicts predicted oncosupressive role Context specific

29. Decrease in cell numbers in absence of USP9X prompts a role of a “tumor promoter” Contradicts predicted oncosupressive role Context specific – Pancreatic cancer

30. Decrease in cell numbers in absence of USP9X prompts a role of a “tumor promoter” Contradicts predicted oncosupressive role Context specific – Pancreatic cancer

31. Decrease in cell numbers in absence of USP9X prompts a role of a “tumor promoter” Contradicts predicted oncosupressive role Context specific – Pancreatic cancer Tumor suppressor

32. Decrease in cell numbers in absence of USP9X prompts a role of a “tumor promoter” Contradicts predicted oncosupressive role Context specific – Pancreatic cancer Tumor suppressor

33. Decrease in cell numbers in absence of USP9X prompts a role of a “tumor promoter” Contradicts predicted oncosupressive role Context specific – Pancreatic cancer Tumor suppressor Tumor promotor

34. To further confirm, Overexpression of USP9X

35. To further confirm, Overexpression of USP9X Usp9x cDNA

36. To further confirm, Overexpression of USP9X Usp9x cDNA Linearized the plasmid Lipofectamine 2000 After 3 days: antibiotic selection (12 days) To establish stable cell lines,

37. USP9X is ectopically expressed in all four cell lines V5 SCC15CAL27FaDuDetroit 562scc15CAL27FaDuDetroit 562 pDEST51 USP9X pDEST51 β tubulin Immunoblotting to detect ectopic expression of USP9X

38. Ectopic USP9X protein expression increased cell proliferation CyQUANT Analysis of cell proliferation following ectopic expression of USP9X SCC15 Detroit 562 CAL27 FaDu * * * * ** * * * * * * * * P value < 0.05

39. Cell numbers are directly proportional to level of USP9X protein

40. Cell numbers are directly proportional to level of USP9X protein has an oncogenic role

41. Molecular mechanism regulated by USP9X

42. Murtaza et al, 2015

43. Molecular mechanism regulated by USP9X – mTOR – Wnt – Notch Regulates cell proliferation Known USP9X substrates

44. Molecular mechanism regulated by USP9X? – mTOR – Wnt – Notch CyclinD1, c-MYC, HES1

45. Molecular mechanism regulated by USP9X? – mTOR – Wnt – Notch Quantitate the RNA levels by qPCR CyclinD1, c-MYC, HES1

46. RNA extraction cDNA qPCR

47. Fold change of target genes 144 h after knockdown of USP9X SCC15 CAL27 FaDuDetroit 562

48. Fold change of target genes 144 h after knockdown of USP9X SCC15 CAL27 FaDuDetroit 562

49. Fold change of target genes 144 h after knockdown of USP9X SCC15 CAL27 FaDuDetroit 562

50. SCC15 CAL27 FaDu Detroit 562 Fold change of target genes 144 h after ectopic expression of USP9X

51. Consistently HES1 expression correlated with cell proliferation measured by CyQUANT assay USP9X seems to positively regulate notch pathway

52. Consistently HES1 expression correlated with cell proliferation measured by CyQUANT assay USP9X seems to positively regulate notch pathway Hypothesis: USP9X regulates proliferation of head and neck cancer cells through notch pathway

53. Conclusions USP9X depletion caused a decrease in cell proliferation Ectopic expression of USP9X led to increase in cell proliferation USP9X positively regulates Notch pathway

54. ACKNOWLEDGEMENT Stephen George Nicholas Saunders Wood lab members Funding: Griffith University

55. THANK YOU!