Functional Genomics : PTPRF-Mediated Contact Inhibition in HCC development

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1 Functional Genomics : PTPRF-Mediated Contact Inhibition in HCC development Sen-Yung Hsieh, MD, PhD Liver Research Unit Chang Gung Memorial Hospital Taoyuan, Taiwan

2 Aims To identify genes controlling tumor initiation of human hepatoma from human kinome and phosphatome

5 PTPRF: a suppressor of tumor initiation in HCC?

6 Rabi et al., Hepatology sins siptprf sins siptprf Hep3B HepG sins siptprf sins siptprf Huh SK-Hep

7 Relative cell counts/stationary phase Rabi et al., Hepatology 2014 Silencing of PTPRF enhances cell proliferation and increases total cell numbers Control shptprf-1 shptprf-2 P<0.001 P<0.02 P<0.002 HepG2 Huh7 SKHep1

anchorage-independent cell growth 70 60 50 shluci 40

8 Colony number Rabi et al., Hepatology 2014 Silencing of PTPRF enhances anchorage-independent cell growth shluci clone-1 clone-2 10 shptprf 0 shluci shptprf Soft-agar assays for tumor sphere formation

9 shptprf control Volume of tumor (mm 3 ) Rabi et al., Hepatology 2014 Silencing of PTPRF enhances xenograft tumor formation shptprf shluci Control Time (wk)

Relative cell counts/stationary phase Colony number Rabi et al.

phosphatase mutant, suppresses proliferation and tumorigenecity 3.

50 P<0.01 P<0.0001 70 60 50 40 SKHep1 1.00 30 0.50 0.

10 Relative cell counts/stationary phase Colony number Rabi et al., Hepatology 2014 Ectopic expression of wild-type PTPRF, but not the phosphatase mutant, suppresses proliferation and tumorigenecity 3.00 Vector PTPRF-wd PTPRF-mt P< Vector PTPRF-wd PTPRF-mt P<0.01 P< SKHep HepG2 Huh7 SKHep Vector wd mt SK-control SK-LAR/WT SK-LAR-C/S The biological functions depend on its phosphatase activities!

11 Summary PTPRF suppresses cell proliferation and tumor initiation c/w a TSG Suppression of tumor formation depends on its phosphatase activities PTPRF suppresses total cell mass involved in contact inhibition??? Rabi et al., Hepatology 2014

12 Hanahan & Weinberg 2011

13 Hypothesis PTPRF PTPRF functions as a negative regulator to prevent overproliferation A negative feedback loop prevents hyperproliferation and tumor formation? Growth stimuli Cell proliferation? PTPRF

14 Does cell proliferation induce PTPRF expression? Rabi et al., Hepatology 2014

15 Is PTPRF induced due to increased cell density? Rabi et al., Hepatology 2014

16 Is PTPRF induced by cell-cell contact? PTPRF GAPDH Cell density (10 5 /well) EGTA mm mm 5 mm x 10 5 /well EGTA: 0 mm 4.5 x 10 5 /well EGTA: 5 mm Rabi et al., Hepatology 2014

17 Tentative conclusion PTPRF suppresses tumor formation PTPRF suppresses cell proliferation and total cell mass during proliferation PTPRF is induced by cell-cell contact during proliferation Loss of PTPRF function hyperproliferation and tumor initiation Growth stimuli Growth stimuli Cell proliferation Cell proliferation Overgrowth & tumor initiation PTPRF PTPRF Rabi et al., Hepatology 2014

18 How PTPRF suppresses cell proliferation? Cell proliferation Cell-cell contact? PTPRF?? growth signaling

19 PTPRF inhibits ERK phosphorylation/activation in response to growth stimuli Rabi et al., Hepatology 2014

20 Rabi et al., Hepatology 2014 PTPRF inhibits ERK-dependent signaling RTK RAS RAF MEK ERK elf4e RSK MYC MNK

21 PTPRF knockdown greatly enhances ERK-MYC growth signaling Rabi et al., Hepatology 2014

22 Rabi et al., Hepatology 2014 ERK1/2 activation required for MYC activation Cell proliferation Cell-cell contact MYC PTPRF? ERK RTK growth signaling

23 How PTPRF suppresses ERK-MYC signaling? Cell proliferation Cell-cell contact MYC PTPRF? ERK RTK growth signaling

24 Immunoprecipitation Rabi et al., Hepatology 2014

25 Reciprocal immunoprecipitation anti-ptprf IP: IgG Anti-SRC HepG2 Huh7 HepG2 Huh7 Proximity ligation assays IP: PTPRF IgG PP2A IB: PTPRF IB: PTPRF SRC PP2A PP2AC HepG2 Huh7 HepG2 Huh7 Abs: PTPFR+SRC PTPRF+ACTB PTPRF+PP2A Rabi et al., Hepatology 2014

26 Rabi et al., Hepatology 2014

27 A novel PTPRF-mediated contact inhibition pathway Cell-cell contact Restricted proliferation Cell proliferation Tumor initiation Hyperproliferation Cell-cell contact PTPRF MYC MYC PTPRF SRC PP2A ERK ERK RTK RTK Aberrant growth signaling

28 Relative IHC score Case 1 2 PTPRF downregulated in ~40% of HCC upregulated in ~22% of HCC N N T P = P = P = T 0.00 Tumor stage I II II III III Non-tumor N T N T N T N T N T N T N T N T N T N T N T N T N T N T nl PTPRF b- Actin PTPRF b-actin HCC

29 Rabi et al., Hepatology 2014 The PTPRF gene frequently deleted in HCC (20.5%) cases PTPRF: 1p34.2 ( ) Chromosome 1 Gene copy variation was determined by using array CGH

30 Rabi et al., Hepatology 2014 PTPRF downregulated lower survival upregulated lower recurrence & higher survival % 100 Disease-free survival % 100 Overall survival P = P = Upregulation: yes Upregulation: no M Downregulation: yes Downregulation: no M

31 Summary The PTPRF gene Frequently deleted in human HCC ~20.5% Frequently downregulated in human HCC ~40% Frequently downregulated in human CRC ~100% Frequently downregulated in human GC ~66% PTPRF downregulation Prediction of worse clinical outcomes PTPRF is a TSG!

32 Conclusions A novel PTPRF-SRC/PP2A-ERK-MYC negativefeedback loop prevents overproliferation of cells The PTPRF-ERK-MYC pathway may involve in tissue homeostasis Disruption of this contact inhibition mechanism leads overproliferation and tumor initiation

Functional characterisation of hepatitis B viral X protein/microrna-21 interaction in HBVassociated hepatocellular carcinoma

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