Stromal Carcinoma Associated Fibroblasts Promote Drug Resistance of Human Pancreatic Cancer Cells by Modulation of ROS via CXCR4/CXCL12 Signaling

Abstract

Pancreatic cancer is one of the most aggressive malignancies, with a 5-year overall survival of less than 5%. Tumor drug resistance to conventional chemotherapy, such as Gemcitabine, is often a significant contributor to poor overall survival. One of the common mechanisms of Gemcitabine resistance is activation of cell signaling via increased phosphorylation of mitogen-Activated kinase (MAP) kinases, leading to increased tumor sur- vival and reduced sensitivity to chemotherapeutic agents. A growing body of evidence suggests that the CXCL12/CXCR4 signal transduction axis in the tumor microenvironment is an important mediator of tumor migration, growth, and drug resistance. We hypothesized that stromal cells such as carcinoma-associated fibroblasts (CAFs), an important cellular component of the tumor microenvironment (TME), play a contributory role in the growth, invasiveness, and drug response of pancreatic cancer cells (PCCs) by activating CXCL12/CXCR4-mediated signal transduction. To test this, we used an in vitro model system to study the growth, invasion, and drug response of human PCCs in the presence or absence of in vitro generated CAFs and their precursors, the mesenchymal stem cells (MSCs). Functional studies demonstrated that co-culture of PCCs with CAFs led to signifi- cant increase in tumor cell invasion, which was abrogated by blockade of CXCR4 by plerixafor (AMD3100), a CXCR4 antagonist, and by siRNA- mediated knockdown of CXCR4 in CAFs. Further, we examined the effect of AMD3100 on the chemoresistance of PCCs to Gemcitabine. Our results indicated that AMD3100 reversed Gemcitabine-mediated chemoresistance of PCCs cells in the presence of CAFs or CAFconditioned media. In co- culture with pancreatic cancer cells, CAFs induced activation of MAPK signaling pathways and enhanced transcription of Mn-superoxide dismutase (SOD), glucose 6-phosphate dehydrogenase (G6PD), and catalase, genes involved in reactive oxygen species (ROS) pathways. Using Phloretin (a natural flavonoid found in apple leaves), a ROS inhibitor, we observed attenuation of MAPK signaling, SOD activity, and reversal of drug resistance in CAF-exposed pancreatic cancer cells. Phloretin in combination with Gem- citabine produced remarkable antitumor effects in a tumor xenograft model in immune compromised animals. These findings implicate CXCL12/CXCR4- dependent MAPK signaling and ROS pathways in CAF-mediated modulation of the growth, migration, and drug resistance of PCCs and provide a rationale for developing combination therapies for control of pancreatic cancer.