University of New South Wales, Sydney, Australia Silencing Heat Shock Proteins 27 and 47 Inhibit Platelet-Derived Growth Factor (PDGF)-Induced Pancreatic Stellate Cell Proliferation: Implication in Pancreatic Cancer Progression Youkhana J, Liu J, Yang L, Xu Z, Biankin A, Goldstein D, Wilson J, Apte MV and Phillips PA. Pancreatic Cancer Translational Research Team, Pancreatic Research Group University of New South Wales, Sydney, Australia

Pancreatic Cancer Fourth leading cause of cancer related death in Western Societies 5-year survival rate < 5% Current treatments have limited efficacy Alternative approaches are needed to improve the outcome of this condition

Pancreatic Cancer Tumour Stroma It is now well established that activated PSCs are critical players in the development of fibrosis in two major pancreatic diseases, chronic pancreatitis and pancreatic cancer Stroma

Interaction between Cancer Cells and Pancreatic Stellate Cells Fibrosis Pancreatic Cancer Cells  Proliferation  Apoptosis  Migration  Invasion/Metastasis  Chemoresistance Pancreatic Stellate Cells  Activation  Proliferation  ECM deposition  Migration  Pancreatic Tumor Progression and Metastasis

Platelet Derived Growth Factor (PDGF) Induces PSC Activation Pancreatic Cancer Cells PDGF Quiescent PSC (Vitamin-A storing phenotype) Activated PSC (Myofibroblast-like phenotype)  ECM production  Migration  Proliferation Heat shock proteins play a role in these functions in other cell types

Heat Shock Proteins (HSPs) Classified into six major families: Large molecular weight HSPs HSP90 HSP70 HSP60 HSP40 Small HSPs

HSPs and Pancreatic Diseases Protective against acute pancreatitis Anti-apoptotic effects on pancreatic cancer cells No previous studies have examined the role of HSPs in the stroma of pancreatic cancer

Heat Shock Proteins and PSCs PSCs express HSPs (27, 47, 70 and 90) HSP expression is increased in hPSCs exposed to PDGF 3) Cancer-associated hPSCs exhibit increased HSPs compared to normal hPSCs 4) Silencing HSP47 in hPSCs inhibits collagen secretion

Aim To determine the effect of silencing HSPs (27, 47, 70 or 90) on cancer-associated human PSC migration and proliferation

Pancreatic Adenocarcinoma Cancer-Associated hPSCs (CA-hPSCs) Methods Pancreatic Adenocarcinoma Cancer-Associated hPSCs (CA-hPSCs) Mock (L2K alone) or non-silencing siRNA (100nM) or HSP siRNA (100nM) 48h Post-transfection PDGF (10ng/ml) 48h 48h Proliferation (Cell Counting Kit-8, Dojindo) Migration (Chemotactic PDGF; modified Boyden chamber)

Silencing of HSP27 in CA-hPSCs using siRNA * *p<0.05 compared to ns-siRNA; n=3

Effect of HSP27 siRNA on Migration of CA-hPSCs * * * * *p<0.05 compared to media alone; n=4

Silencing of HSP47 in CA-hPSCs using siRNA * *p<0.05 vs ns-siRNA; n=3

Effect of HSP47 siRNA on Migration of CA-hPSCs * * * *p<0.05 compared to media alone; n=3

Effect of HSP27 siRNA on Proliferation of CA-hPSCs # # ** #p<0.001 vs media alone ; **p<0.001 vs ns-siRNA+PDGF; n=4

Effect of HSP47 siRNA on Proliferation of CA-hPSCs # # ** #p<0.001 vs media alone ; **p<0.001 vs ns-siRNA+PDGF; n=4

Effect of HSP70 or HSP90 siRNA on Proliferation of CA-hPSCs # # # # #p<0.001 vs media alone; n=4

HSP47 siRNA Reduces Cell Cycle Gene Expression in PSCs Treated with PDGF Preliminary Results: Cell Cycle PCR Array (SAB Biosciences) PSCs + HSP47 siRNA + PDGF vs PSCs + ns-siRNA + PDGF

HSP 27 and 47 Expression in Human Pancreatic Ductal Adenocarcinoma 200X Magnification Isotype Control HSP47 HSP27

Conclusions 1. Silencing HSP27 or HSP47 had no effect on PDGF-induced migration. 2. Suppression of HSP27 or HSP47 (but not HSP70 or HSP90) inhibits PDGF-induced PSC proliferation.

Implication Modulation of HSPs in PSCs may represent a novel approach to influence PSC function and PSC interactions with cancer cells

Acknowledgments Pancreatic Research Group Prof Minoti Apte Prof Jeremy Wilson A/Prof Ron Pirola Prof David Goldstein Prof Andrew Biankin Janet Youkhana Jie Liu Narada Kiriella Susan Yang Zhihong Xu Eva Fiala-Beer Dr Alain Vonlaufen Funding Cancer Council NSW Innovator Grant Pancreatic Cancer Network Ramaciotti Foundation Cure Cancer Australia Cancer Institute NSW Fellowship