Abstract 1449: The molecular dissection of pro-tumorigenic activity of mesothelin in pancreatic cancer

Abstract

Mesothelin (MSLN) is a cancer-specific antigen that is differentially expressed in many human cancers, including malignant mesothelioma and pancreatic, ovarian, and lung adenocarcinomas. The MSLN gene encodes a 71 kD precursor protein, that is reportedly cleaved by the intracellular protease furin to produce a 31 kD N-terminal fragment called megakaryocyte potentiating factor (MPF) and mature 40 kD MSLN C-terminal fragment. Mature MSLN is localized to the cell surface by a GPI-linkage. We synthesized a series of mutant MSLN constructs containing sequence modifications predicted to disrupt the canonical signaling sequences for furin cleavage and GPI-linkage. These constructs were then expressed in a MSLN knock-out pancreatic cancer cell line. We used flow cytometry to detect cell surface MSLN expression and immunoblotting for MSLN proteins to assess the effect of mutation. We expected that a 23aa C-terminal truncation that removed a predicted hydrophobic signaling sequence for GPI-linkage would eliminate plasma membrane insertion of MSLN. However, this truncation did not suppress cell surface expression. A deeper 31aa truncation which eliminated two potential anchoring sites at Ser599 and Ser606 was required to successfully eliminate surface expression. We are currently evaluating the growth of this pancreatic cancer cell line expressing GPI-truncated soluble MSLN in vitro and in vivo. Our results suggest that MSLN may have an alternate means for membrane insertion besides GPI-linkage. For the panel of furin site mutants, non-conservative mutations were made to disrupt the reported consensus recognition motif for furin 290-RPRFRR-295 (R290H, R292Q, RR294-5GG) or the entire motif region was deleted (289_315del). Mutants were also made to disrupt the putative furin cleavage site (R286A) and neighboring basic residues that were potential protease cleavage sites (R282A, K306Q, K307Q). These furin site mutants were expected to eliminate cleavage of the 71 kD precursor protein to 40 kD mature MSLN. We found that all mutants did increase levels of precursor but did not ablate formation of mature MSLN. Similarly, expression of WT full-length MSLN into the LoVo cell line, which lacks furin activity, still resulted in formation of 40 kD mature MSLN predominately. In conclusion, we found that precursor processing to mature MSLN still took place despite extensive mutation of the putative furin cleavage site and in the absence of active furin. These results suggest that additional proteases besides furin can perform MSLN precursor cleavage. Citation Format: Sarah Mary Joseph, Leela Rani Avula, Xianyu Zhang, Christine Campo Alewine. The molecular dissection of pro-tumorigenic activity of mesothelin in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1449.

Eicosapentaenoic Acid Rescues L-Type Ca²⁺ Channel Remodeling Caused by Saturated Fatty Acids via Both Free Fatty Acid Receptor 4-Dependent and -Independent Pathways in Cardiomyocytes

“…The major findings of this study were that (1) the long-term application of OAPA decreased Cav1.2 mRNA/protein, ICa.L and the spontaneous beating of cardiomyocyte, (2) EPA application reversed the remodeling of Cav1.2 channel caused by OAPA, (3) an FFAR4 agonist TUG-891 reversed expression of Cav1.2 and CREB mRNA caused by OAPA, (4) an FFAR4 antagonist AH-7614 abolished the effects of EPA on Cav1.2 and CREB mRNA caused by OAPA, (5) OAPA increased ROS production, while the action was eliminated by EPA, (6) an ROS generator H2O2 decreased the expression of Cav1.2 and CREB, which was prevented EPA, and (7) suppressions of Cav1.2 and CREB mRNA by OAPA was prevented by an ROS scavenger NAC.…”

“…A large number of epidemiologic studies demonstrate that higher saturated fat intake is associated with an increased risk of sudden cardiac death, suggesting that the effects of dietary saturated fat may be usually sufficient to cause heart diseases [1][2][3][4]. High levels of circulating saturated fatty acids are also associated with diabetes, obesity, and hyperlipidemia [5,6]. In the heart, the accumulation of saturated fatty acids has been proposed to play a role in the development of heart failure and arrhythmias [6][7][8].…”

“…High levels of circulating saturated fatty acids are also associated with diabetes, obesity, and hyperlipidemia [5,6]. In the heart, the accumulation of saturated fatty acids has been proposed to play a role in the development of heart failure and arrhythmias [6][7][8]. The two major circulating fatty acids are saturated palmitic acid (C16:0) and monounsaturated oleic acid (C18:1) [9,10].…”