CONTEXT SPECIFIC ROLE OF DEUBIQUITYLASE ENZYME USP9X IN HEAD AND NECK CANCER Devathri Nanayakkara Eskitis Institute for Drug Discovery Griffith University Head and neck cancer Head and neck cancer ID: 768819
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CONTEXT SPECIFIC ROLE OF DEUBIQUITYLASE ENZYME, USP9X, IN HEAD AND NECK CANCER Devathri NanayakkaraEskitis Institute for Drug DiscoveryGriffith University
Head and neck cancer
Head and neck cancer Sixth most common cancer
Head and neck cancer Sixth most common cancerFive year survival rate after diagnosis, 50%
Head and neck cancer Sixth most common cancerFive year survival rate after diagnosis, 50%Drug resistanceEarly tumors - asymptomatic
Head and neck cancer Sixth most common cancerFive year survival rate after diagnosis, 50%Drug resistanceEarly tumors - asymptomatic“Need to study the underlying molecular pathways unveiling potential detection markers and drug targets”
In a recent study, Characterized the somatic mutation landscape of OSCC-GB
- Found Five new genes associated with OSCC-GB
Among them was USP9X 22% harboured copy number loss and truncating mutations Role as tumor suppressor
USP9XDeubiquitylating enzymeFamily of cysteine and metalloproteases
USP9XDeubiquitylating enzymeFamily of cysteine and metalloproteaseshttp://legacy.butler.edu/biology/faculty-staff/research-interests/jkowalski/research-in-the-kowalski-lab/
Murtaza et al, 2015
USP9X in cancer
USP9X in cancer
USP9X in cancer Breast cancer Colorectal cancer Bladder cancer Oral cancer Brain cancer Pancreatic cancer Prostate cancer Lung cancer L ymphoma
In this study, Aims :To evaluate the role of USP9X in an in vitro systemTo elucidate the molecular mechanisms USP9X is involved in
How? In vitro cell lines SCC15, CAL27, FaDu and Detroit 562
Immunoblotting to probe for USP9X expression All 4 cell lines express USP9XSCC15FaDuDetroit 562CAL27USP9X 290 kDaβ tubulin51 kDa
Knockdown approach siRNA
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 siRNAUSP9X290 kDaΒ tubulin51 kDa
Effect on cell aspectsCell proliferationCyQUANT Assay
time timetimetimeIn absence of USP9X, a decrease in cell numbers was observed CyQUANT Analysis of cell proliferation following siRNA treatment to knockdown USP9XSCC15 Detroit 562 CAL27 FaDu * * * * * * * * * P value < 0.05
Decrease in cell numbers,Apoptosis?
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 NT USP9X NT USP9X NT USP9X NT USP9X NT USP9X NT USP9X time 48h 96h 144h 48h 96h 144h 48h 96h 144h 48h 96h 144h SCC15 CAL27 FaDu Detroit 562 Immunoblotting for cleaved PARP-1
Decrease in cell numbers in absence of USP9X prompts a role of a
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”Contradicts predicted oncosupressive roleContext specific
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancer
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancer
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerTumor suppressor
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerTumor suppressor
Decrease in cell numbers in absence of USP9X prompts a role of a “ tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerTumor suppressorTumor promotor
To further confirm,Overexpression of USP9X
To further confirm,Overexpression of USP9X Usp9x cDNA
To further confirm,Overexpression of USP9X Usp9x cDNALinearized the plasmid Lipofectamine 2000 After 3 days: antibiotic selection (12 days) To establish stable cell lines ,
USP9X is ectopically expressed in all four cell lines V5SCC15CAL27FaDuDetroit 562scc15CAL27 FaDu Detroit 562 pDEST51 USP9X pDEST51 β tubulin Immunoblotting to detect ectopic expression of USP9X
Ectopic USP9X protein expression increased cell proliferation CyQUANT Analysis of cell proliferation following ectopic expression of USP9XSCC15Detroit 562CAL27FaDu * * * * * * * * * * * * * * P value < 0.05
Cell numbers are directly proportional to level of USP9X protein
Cell numbers are directly proportional to level of USP9X protein has an oncogenic role
Molecular mechanism regulated by USP9X
Molecular mechanism regulated by USP9X Murtaza et al, 2015
Molecular mechanism regulated by USP9XmTOR Wnt NotchRegulates cell proliferationKnown USP9X substrates
Molecular mechanism regulated by USP9X?mTOR Wnt NotchCyclinD1, c-MYC, HES1
Molecular mechanism regulated by USP9X?mTOR Wnt NotchQuantitate the RNA levels by qPCRCyclinD1, c-MYC, HES1
RNA extraction cDNAqPCR
Fold change of target genes 144 h after knockdown of USP9XSCC15CAL27FaDu Detroit 562
Fold change of target genes 144 h after knockdown of USP9X SCC15 CAL27 FaDu Detroit 562
Fold change of target genes 144 h after knockdown of USP9X SCC15 CAL27 FaDu Detroit 562
SCC15 CAL27 FaDu Detroit 562 Fold change of target genes 144 h after ectopic expression of USP9X
Consistently HES1 expression correlated with cell proliferation measured by CyQUANT assay USP9X seems to positively regulate notch pathway
Consistently HES1 expression correlated with cell proliferation measured by CyQUANT assay USP9X seems to positively regulate notch pathwayHypothesis: USP9X regulates proliferation of head and neck cancer cells through notch pathway
ConclusionsUSP9X depletion caused a decrease in cell proliferation Ectopic expression of USP9X led to increase in cell proliferationUSP9X positively regulates Notch pathway
ACKNOWLEDGEMENT StephenGeorgeNicholas SaundersWood lab membersFunding: Griffith University
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