Presentations

Abstract

Introduction: SIAH is an evolutionarily highly E3 ubiquitin ligase that plays an important signaling gatekeeper role downstream of the EGFR/HER2/RAS pathway. SIAH is found to be upregulated in a myriad of chemo-resistant and metastatic cancer subtypes; particularly pancreatic ductal adenocarcinoma (PDAC) and triple negative breast cancer (TNBC). Our lab has found that SIAH functions as a potent drug target, and it is a tumor-specific, therapy-responsive, and prognostic biomarker in human malignancy in vitro and in vivo. The major aim of this research is to characterize a novel molecular mechanism of how SIAH inhibition changes cell shape, alters mechano-sensing, reduces cell adhesion, and rearranges cytoskeleton organization in SIAH-deficient cancer cells. In particularly, we have focused on validating and quantifying molecular changes of phospho-YAP, phospho-cofilin, and collagen I proteins in MDA-MB-468, MDA-MB-231, and MiaPaCa (human TNBC/PDAC cancer cell lines). In this study, we aim to delineate the functional roles of these aforementioned protein phosphorylation in regulating mechano-sensing, cell adhesion, cell shape, and cytoskeletal re-organization in SIAH-proficient and SIAH-deficient cancer cell line models in a pairwise fashion.

Methods: The state-of-the-art reverse protein phase array (RPPA) technology in combination with immunofluorescence (IF), Flow, and western blots (WB) were used to document and quantify the molecular changes in phospho-YAP, phospho-cofilin, and collagen I proteins in several human cancer cell lines. The protein lysates were prepared by inducing the expression of our home-made SIAH inhibitor by doxycycline utilizing a Tet-ON/OFF system in human cancer cell lines for three days and seven days, and the control cell lysates were collected in parallel without doxycycline induction. The samples were run in triplicate. RPPA was utilized to quantify the altered expression of these phosphorylated proteins utilizing a nitrocellulose coated plate dotted with these corresponding pair-matched cell lysates. The RPPA assays were performed by our collaborators at GMU. We performed the additional Western blots/IF/FACS assays to verify and validate the molecular changes of these protein targets in these DOX (+/-)-treated cancer cell lines independently at ODU VHS. Their altered protein expression was then quantified and calculated by FACS and WB by Image J, and statistical analyses were performed by the paired and unpaired student t-tests using the Prism software.

Results: We found that phospho-cofilin was upregulated, and phospho-YAP were downregulated in a few selected SIAH-deficient cancer cell lines in response to DOX (+) induction as compared to the SIAH-proficient control cells (-DOX). These detected molecular changes were consistent with the altered cellular shape, increased cell detachment, and changed cell morphology observed in these SIAH-mutant cancer cells in response to DOX (+) induction. Further investigations will be conducted to identify synergistic changes in other key signaling molecules and regulatory proteins in the Hippo/Yap/Cofilin collagen type 1 signaling pathway in hopes of providing a molecular insight and signaling crosstalk of pathway interaction and feedback regulation of EGFR/HER2/RAS/SIAH with mechano-sensing, cell adhesion, and cytoskeletal signaling pathways in SIAH-proficient and SIAH-deficient cancer cells in response to DOX (-/+) induction in a comprehensive pairwise study

Conclusion: The results indicate that expression of SIAH inhibitor changes the cell shape and cytoskeletal dynamics across multiple malignant cancer cell lines. Increased levels of phospho-cofilin suggest that SIAH inhibition may disrupt actin microfilament dynamics, alter cell shape, reduce cell motility and metastasis. Furthermore, changes in the Hippo/YAP pathway indicate that the mechano-sensing pathway may be altered in response to SIAHloss of function. Our preliminary results showed decreased cell adhesion, increased cell detachment, and altered cell shape in respond to the blockade of the EGFR/RAS/SIAH pathway. 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 human cancer cells in cancer.