Abstract A35: SOX9 induces chemo-resistance in pancreatic cancer cells and its high expression predicts poor prognosis
Authors: Shingo Kagawa, Taku Higasihara, Hideyuki Yoshitomi, Shigetsugu Takano, Hiroaki Shimizu, Masayuki Ohtsuka, Atsushi Kato, Katsunori Furukawa, Masaru Miyazaki
Journal: Cancer Research (2016)

Abstract Introduction: Although surgical resection still remains the most important treatment for pancreatic cancer, there is a growing body of evidence showing the importance of chemotherapy. On the other hand, despite the advances in chemotherapy, the outcome of patients with pancreatic cancer remains far from satisfactory. These facts suggest that the understanding of the molecular mechanisms underlying the chemo-resistance of pancreatic cancer is urgently needed for developing more effective chemotherapies. To identify the factors that regulate chemo-resistance in pancreatic cancer, we focused on a transcription factor, Sex determining region Y box 9 (Sox9), that maintains progenitor cells during pancreatic organogenesis and carcinogenesis. The purpose of this study is to evaluate the roles of Sox9 in pancreatic cancer. Methods: Pancreatic cancer tissue were obtained from 106 consecutive patients who underwent curative surgical resection in the Department of General Surgery, Chiba University Hospital, Japan, from June 2006 to October 2011. We analyzed these primary invasive pancreatic ductal adenocarcinoma (PDAC) tissues by immunohistochemistry of Sox9. Human pancreatic cancer cell lines, PANC-1, Sox9 high expressing cell, and BxPC-3, Sox9 low expressing cell were used for in vitro analysis. Sox9 was inhibited by siRNAs. Cell cytotoxicity assay was done with Gemcitabine and 5-FU by PANC-1 and BxPC-3 cells. To elucidate the mechanism of chemo-resistance, we focused on the stemness of Sox9 expressing cells and performed sphere formation assays and flow cytometry analysis. Gene expression was determined by quantitative RT-PCR and Western blotting. The expression levels of CD44 and CD24, which are thought to be stem cell markers of pancreatic cancer, were examined by flow cytometry analysis. Tet-On Inducible Sox9 shRNA transfected PANC-1 cells were established and injected subcutaneously into the bilateral flanks of mouse. Xenograft tumors were assessed four weeks after injection. Results: Expression of Sox9 predicts poor survival in PDAC. Sox9 high expressing patients showed poorer prognosis than Sox9 low expressing patients. Median overall survival time and 3-year survival rate was 25.3 months and 21.1% for the Sox9 high group, and not reached and 62.9% for the low group, respectively (p=0.011). The multivariate overall survival time analysis showed Sox9 expression level as an independent prognostic factor (HR 0.344; 95%CI 0.183-0.647; p=0.0009), along with histological grade, distant metastasis, arterial invasion, residual tumor, and adjuvant chemotherapy. Sox9 high expressing PANC-1 cells showed chemo-resistance against GEM and 5-FU treatments. PANC-1 cells with suppressed Sox9 expression by siRNA showed decreased cell numbers when they were treated with GEM. These data implicated that Sox9 induced chemo-resistance in pancreatic cancer cells. PANC-1 cells, which have strong Sox9 expression, successfully formed spheres during culture on ultra-low attachment plates. The knockdown of Sox9 expression in PANC-1 cells significantly decreased sphere formation rates, compared with cells treated with negative siRNA. In PANC-1 cells, knockdown of Sox9 by siRNA significantly decreased the proportion of the CD44highCD24high cells compared with cells treated with negative control siRNA (p=0.0005). Sox9 knockdown can reduce tumor initiation capacity in vivo analysis. Conclusions: These data indicate that Sox9 plays an important role in chemo-resistance by the induction of stemness in pancreatic cancer cells. Citation Format: Shingo Kagawa, Taku Higasihara, Hideyuki Yoshitomi, Shigetsugu Takano, Hiroaki Shimizu, Masayuki Ohtsuka, Atsushi Kato, Katsunori Furukawa, Masaru Miyazaki.{Authors}. SOX9 induces chemo-resistance in pancreatic cancer cells and its high expression predicts poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A35.