Devathri Nanayakkara Eskitis Institute for Drug Discovery Griffith University Head and neck cancer Head and neck cancer Sixth most common cancer Head and neck cancer Sixth most common cancer ID: 302988
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CONTEXT SPECIFIC ROLE OF DEUBIQUITYLASE ENZYME, USP9X, IN HEAD AND NECK CANCER
Devathri NanayakkaraEskitis Institute for Drug DiscoveryGriffith University Slide2
Head and neck cancerSlide3
Head and neck cancer
Sixth most common cancerSlide4
Head and neck cancer
Sixth most common cancerFive year survival rate after diagnosis, 50%Slide5
Head and neck cancer
Sixth most common cancerFive year survival rate after diagnosis, 50%Drug resistanceEarly tumors - asymptomaticSlide6
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”Slide7
In a recent study,
Characterized the somatic mutation landscape of OSCC-GBSlide8Slide9
- Found Five new genes associated with OSCC-GBSlide10
Among them was USP9X
22% harboured copy number loss and truncating mutations Role as tumor suppressor Slide11
USP9XDeubiquitylating
enzymeFamily of cysteine and metalloproteasesSlide12
USP9XDeubiquitylating
enzymeFamily of cysteine and metalloproteaseshttp://legacy.butler.edu/biology/faculty-staff/research-interests/jkowalski/research-in-the-kowalski-lab/Slide13
Murtaza
et al, 2015Slide14
USP9X in cancerSlide15
USP9X in cancerSlide16
USP9X in cancer
Breast cancer
Colorectal cancer
Bladder cancer
Oral cancer
Brain cancer
Pancreatic cancer
Prostate cancer
Lung cancer
L
ymphomaSlide17
In this study,
Aims :To evaluate the role of USP9X in an in vitro systemTo elucidate the molecular mechanisms USP9X is involved inSlide18
How?
In vitro cell lines SCC15, CAL27, FaDu and Detroit 562Slide19
Immunoblotting to probe for USP9X expression
All 4 cell lines express USP9XSCC15FaDuDetroit 562CAL27USP9X
290 kDaβ tubulin51
kDaSlide20
Knockdown approach siRNASlide21
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
kDaSlide22
Effect on cell aspectsCell proliferationCyQUANT
AssaySlide23
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 < 0.05Slide24
Decrease in cell numbers,Apoptosis?Slide25
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-1Slide26
Decrease in cell numbers in absence of USP9X prompts a role of aSlide27
Decrease in cell numbers in absence of USP9X prompts a role of a “
tumor promoter”Slide28
Decrease in cell numbers in absence of USP9X prompts a role of a
“tumor promoter”Contradicts predicted oncosupressive roleContext specificSlide29
Decrease in cell numbers in absence of USP9X prompts a role of a
“tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerSlide30
Decrease in cell numbers in absence of USP9X prompts a role of a
“tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerSlide31
Decrease in cell numbers in absence of USP9X prompts a role of a
“tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerTumor suppressorSlide32
Decrease in cell numbers in absence of USP9X prompts a role of a
“tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerTumor suppressorSlide33
Decrease in cell numbers in absence of USP9X prompts a role of a
“tumor promoter”Contradicts predicted oncosupressive roleContext specificPancreatic cancerTumor suppressorTumor promotor Slide34
To further confirm,Overexpression of USP9XSlide35
To further confirm,Overexpression of USP9X
Usp9x cDNASlide36
To further confirm,Overexpression of USP9X
Usp9x cDNALinearized the plasmid
Lipofectamine
2000
After 3 days: antibiotic selection
(12 days)
To establish
stable cell lines
,Slide37
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 USP9XSlide38
Ectopic USP9X protein expression increased cell proliferation
CyQUANT Analysis of cell proliferation following ectopic expression of USP9XSCC15Detroit 562CAL27FaDu
*
*
*
*
*
*
*
*
*
*
*
*
*
* P value < 0.05Slide39
Cell numbers are directly proportional to level of USP9X protein
Slide40
Cell numbers are directly proportional to level of USP9X protein
has an oncogenic roleSlide41
Molecular mechanism regulated by USP9XSlide42
Molecular mechanism regulated by USP9X
Murtaza et al, 2015Slide43
Molecular mechanism regulated by USP9XmTOR
Wnt NotchRegulates cell proliferationKnown USP9X substratesSlide44
Molecular mechanism regulated by USP9X?mTOR
Wnt NotchCyclinD1, c-MYC, HES1Slide45
Molecular mechanism regulated by USP9X?mTOR
Wnt NotchQuantitate the RNA levels by qPCRCyclinD1, c-MYC, HES1Slide46
RNA extraction
cDNAqPCRSlide47
Fold change of target genes 144 h after
knockdown of USP9XSCC15CAL27FaDu
Detroit 562Slide48
Fold change of target genes 144 h after
knockdown of USP9X
SCC15
CAL27
FaDu
Detroit 562Slide49
Fold change of target genes 144 h after
knockdown of USP9X
SCC15
CAL27
FaDu
Detroit 562Slide50
SCC15
CAL27
FaDu
Detroit 562
Fold change of target genes 144 h after ectopic expression of USP9XSlide51
Consistently HES1 expression correlated with cell proliferation measured by
CyQUANT assay USP9X seems to positively regulate notch pathwaySlide52
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 pathwaySlide53
ConclusionsUSP9X depletion caused a decrease in cell proliferation
Ectopic expression of USP9X led to increase in cell proliferationUSP9X positively regulates Notch pathwaySlide54
ACKNOWLEDGEMENT
StephenGeorgeNicholas SaundersWood lab membersFunding: Griffith UniversitySlide55
THANK YOU!