ROCK2 Confers Acquired Gemcitabine Resistance in Pancreatic Cancer Cells by Upregulating Transcription Factor ZEB1

Abstract

Background: Gemcitabine resistance is a major clinical challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC). The increasing attention to Rho-associated coil protein kinase 2 (ROCK2)-mediated chemotherapy resistance in a variety of cancers reminded us to explore the effect of ROCK2 signalnaling in the acquired gemcitabine-resistant pancreatic cancer cells (GR cells). Methods: The relative expression of related proteins and mRNA were detected by western blot and real-time PCR, respectively. Short hairpin RNA (shRNA) was used to silencing related genes. The double luciferase reporter gene experiment was used to explore the activation of related gene promoter. Chromatin immunoprecipitation technique (CHIP) was used to detect the ability of transcription factor binding to the promoter. Many other cell based assays were adopted to explore the mechanism of ROCK2 on resistance of GR cells to gemcitabine. Findings: Pharmacological inhibition or gene silencing of ROCK2 markedly sensitized GR cells to gemcitabine by suppressing the expression of zinc-finger-enhancer binding protein 1 (ZEB1). Mechanically, ROCK2-induced sp1 phosphorylation at Thr-453 enhanced the ability of sp1 binding to ZEB1 promoter regions in a p38-dependent manner. Moreover, transcriptional activation of ZEB1 facilitated GR cells to repair gemcitabine-mediated DNA damage via ATM/p-CHK1 signaling pathway. Interpretation: ROCK2 contributed to EMT-induced gemcitabine resistance in pancreatic cancer cells and might provided strong evidence for the clinical application of fasudil, a ROCK2 inhibitor, in gemcitabine-refractory PDAC. Funding Statement: This work was supported by the National Natural Science Foundation of China (No.81372268, No.81672816 and No.81872337), the Program for Jiangsu Province Innovative Research (KYLX16_1120), the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province (No. BK20130026), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. ZJ11173). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all animal experimental procedures were approved by Experimentation Ethics Review Committee of China Pharmaceutical.