1. 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

2. 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

3. 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

4. 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

5. 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

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

7. 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

8. 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

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

10. 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)

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

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

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

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

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

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

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

18. 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

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

20. 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.

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

23. 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