Abstract 104: Identification of the molecular components required for the protumorigenic effect of MSLN in pancreatic cancer
Authors: Avula Leela Rani , Rudloff Michael , Arons Danielle , El-Behaedi Salma , Zhang Xianyu , Guerin Theresa , Kozlov Serguei , Alewine Christine 
Published: August 2018

Abstract
Mesothelin (MSLN) is a GPI-linked protein expressed in &gt;90% of pancreatic adenocarcinomas (PDAC) but not in healthy pancreas nor in the parenchyma of other vital organs. Due to this differential expression, multiple antibody-based therapies are targeting MSLN. The physiologic role of MSLN is yet unknown. The MSLN gene encodes for a precursor protein which is cleaved to produce mature MSLN protein and soluble Megakaryocyte Potentiating Factor (MPF), both of which can be secreted or shed. Previous reports suggested that overexpression of MSLN precursor protein increases tumorigenicity and metastatic potential of PDAC. Here, we have used CRISPR-Cas9 gene editing to delete MSLN from the KLM-1 pancreatic cancer cell line (ΔMSLN) and have examined the growth of these cells compared to mock deleted cells (Mock). The ΔMSLN and Mock cells grew at the same rate both in cell culture and as subcutaneous tumors in nude mice, but ΔMSLN had significantly impaired growth when inoculated into the mouse intraperitoneal (IP) cavity as well as when injected orthotopically into the pancreas. Histological analysis (H&E, Ki67 and Trichrome) showed no significant gross differences between ΔMSLN and Mock IP tumors. RNA deep sequencing analysis comparing ΔMSLN and Mock cell lines showed strong upregulation of MUC-4 in KO cells. Restoration of WT MSLN expression in ΔMSLN cells restored IP growth, however, transduction of a Y318A point mutant could not. Point mutation of Y318 diminished ability of shed MSLN to interact with KLM-1 cells, presumably by impairing association with MUC-16, the only known binding partner of MSLN. To determine the role of mature MSLN and MPF in tumorigenicity, we expressed these proteins separately in ΔMSLN cells and found that neither could restore IP growth, although mature MSLN was appropriately expressed on the cell surface and MPF secreted. This suggests either that both proteins are required for tumorigenicity or that when synthesized individually they are incorrectly processed by the cell. To examine the role of MSLN in tumorigenesis, MSLN KO mouse were bred into the KPC genetically engineered mouse model of PDAC, which spontaneously develops autochthonous tumors. Unlike PDAC patients, KPC typically die from complications of their primary tumors. Loss of MSLN had no effect on the survival of these mice. Effect of MSLN loss on metastasis in this model is currently being evaluated. In summary, we have demonstrated that MSLN enhances the IP growth of PDAC and defined key regions of MSLN important for this activity.
Citation Format: Leela Rani Avula, Michael Rudloff, Danielle Arons, Salma El-Behaedi, Xianyu Zhang, Theresa Guerin, Serguei Kozlov, Christine Alewine. Identification of the molecular components required for the protumorigenic effect of MSLN in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 104.