Abstract 2637: Investigation of cell shape, focal adhesion, and cytoskeletal changes induced by SIAH inhibition in malignant human cancer cell lines

Abstract INTRODUCTION: SIAH is an evolutionarily conserved E3 ubiquitin ligase that plays an important gatekeeper role downstream of the tumor-driving EGFR/HER2/RAS pathway. SIAH expression is prognostic in human cancer, including pancreatic ductal adenocarcinoma (PDAC) and triple negative breast cancer (TNBC). Our lab has demonstrated that SIAH is a potent cancer target. Here, we found a novel molecular mechanism of how SIAH inhibition changes cell shape, focal adhesion, mechano-sensing, and cytoskeleton organization in SIAH-deficient cancer cells. In this study, we focus on validating and quantifying the changes of phospho-YAP, phospho-cofilin, and collagen I proteins in MDA-MB-468, MDA-MB-231, and MiaPaCa cancer cell lines. METHODS: The reverse protein phase array (RPPA) technology in combination with immunofluorescence (IF), Flow, and western blots (WB) were used to determine the SIAH inhibitor-induced changes in phospho-YAP, phospho-cofilin, and collagen I proteins in human PDAC and TNBC cell lines. Triplicated RPPA assays were performed to quantify the altered expression of these phosphorylated proteins utilizing a nitrocellulose coated plate dotted with these pair-matched cell lysates with SIAH proficiency and deficiency in triplicates. We then performed WB/IF/FACS assays to verify the changes of these protein expression in the DOX (+/-) cancer cell lines independently. RESULTS: We found that phospho-cofilin was upregulated, and phospho-YAP were downregulated in a few selected SIAH-deficient cancer cell lines as compared to their SIAH-proficient cancer cells. These molecular changes were consistent with the altered cellular shape, increased cell detachment, and altered cytoskeleton observed in the SIAH-mutant cells. Further investigations will be conducted to identify the crosstalk of the Hippo/Yap/Cofilin/Collagen signaling pathways with the EGFR/RAS/SIAH signaling pathway in human cancer cells. CONCLUSION: Our results showed that SIAH deficiency altered the cell shape, adhesion, and cytoskeletal dynamics in malignant cancer cells. Increased phospho-cofilin expression suggested that SIAH inhibition may disrupt actin cytoskeleton organization, alter cell shape, decrease focal adhesion, increase cell detachment, and impede cell motility. The changes in the Hippo/YAP pathway indicate that the mechano-sensing pathway may be altered in SIAH mutant cells. These documented changes in the Hippo/YAP/Cofilin/Collagen Type 1 pathways may provide a novel mechanism to explain tumor suppression phenotype of SIAH-deficient cancer cells in xenograft mouse models. Citation Format: Daniel J. McWilliams, Jonathan M. Baker, Amy H. Tang, Emanuel Petricoin, Julia Wulfkuhle, Andrew Howell. Investigation of cell shape, focal adhesion, and cytoskeletal changes induced by SIAH inhibition in malignant human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2637.