Abstract A94: CXCR2 macromolecular complex in pancreatic cancer: A potential therapeutic target in tumor growth.

Background: The signaling mediated by the CXC-chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of pancreatic cancer, one of the most lethal malignancies. CXCR2 possesses a consensus PDZ motif at carboxyl termini. A variety of PDZ scaffold proteins have been documented to orchestrate the formation/regulation of multi-protein signaling complexes at plasma membrane. Therefore, the PDZ motif of CXCR2 could, theoretically, mediate potential interactions with certain PDZ scaffold proteins which might cluster CXCR2 with other relevant signaling molecules into macromolecular signaling complex to transduce signals into cell interior and thereafter regulate cell behavior with higher specificity and efficiency. Aim: The purpose of this study is to define the role of the potential PDZ-mediated CXCR2 macromolecular complex in pancreatic cancer progression and to explore novel therapeutic targets for pancreatic cancer. Methods: Human pancreatic cancer cells, HPAC and Colo357, were used in our study. GST pull-down assays were performed to detect the interaction between GST-PDZ scaffold fusion proteins and endogenous CXCR2 or PLC-β3 (one of the downstream molecules in CXCR2 signaling) in pancreatic cancer cells. Co-immunoprecipitation assays were performed to investigate the existence of endogenous CXCR2 macromolecular complex in pancreatic cancer cells. To evaluate the effect of NHERF1 knockdown on the formation of the CXCR2 macromolecular complex and cellular functions in pancreatic cancer cells, Lentivirus-based shRNAs targeting NHERF1 were generated and used to transduce the pancreatic cancer cells. MTT assay and invasion assay was used to assess the proliferation and invasiveness of pancreatic cancer cells, respectively. Recombinant Adeno-Associated Virus (rAAV) was used to transduce the HPAC cells with the exogenous CXCR2 C-tail gene (containing the PDZ motif), and the transduced HPAC cells were used in a subcutaneous xenograft model to assess the tumor growth in vivo. This in vivo human cancer mouse model was used to evaluate the therapeutic potential of disrupting CXCR2 PDZ motif-mediated interaction as a potential approach for pancreatic cancer treatment. Results: GST pull down assays demonstrated that both CXCR2 and PLC-β3 preferentially interacts with PDZ scaffold protein NHERF1 in a direct and PDZ motif-dependent manner. Co-immunoprecipitation assays revealed that an endogenous CXCR2 macromolecular complex (CXCR2-NHERF1-PLC-β3) exists in both Colo357 and HPAC cells. Furthermore, disrupting this complex, by gene delivery or peptide delivery of exogenous CXCR2 C-tail, significantly inhibits the proliferation and invasive potency of both Colo357 and HPAC cells. Knockdown of NHERF1 impedes formation of the CXCR2 macromolecular complex in pancreatic cancer cells, and significantly reduces cell proliferation and invasion. In addition, gene delivery of CXCR2 C-tail sequence by rAAV2 potently suppresses tumor growth in vivo. Conclusion: Our data demonstrated a physical and functional coupling of CXCR2 and its downstream effector PLC-β3 mediated by NHERF1, forming a macromolecular signaling complex for efficient and specific CXCR2 signaling in pancreatic cancer. Targeting the CXCR2 macromolecular complex may represent a novel and effective therapeutic strategy against pancreatic cancer. Citation Format: Shuo Wang, Yanning Wu, Yuning Hou, Marcello P. Castelvetere, Xiaoqing Guan, Jacob J. Oblak, Sanjeev Banerjee, Theresa M. Filtz, Fazul H. Sarkar, Chunying Li. CXCR2 macromolecular complex in pancreatic cancer: A potential therapeutic target in tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A94.