LAUSR

High expression of the single-pass transmembrane sialomucin, podocalyxin, has been shown by many groups to correlate with poor disease outcome in a number of solid tumor types, including colorectal, ovarian, pancreatic and breast cancers. We had previously identified that high podocalyxin expression in invasive ductal breast carcinoma was an independent indicator of poor survival (Somasiri et al., 2004, Cancer Res. 64:15). Further analysis of these tumor samples revealed that this decrease in survival occurred without the loss of membranous, junctional E-cadherin, suggesting that these tumors may have invaded collectively without necessitating an overt epithelial to mesenchymal transition (EMT). Experimentally, forced over-expression of podocalyxin in polarity-disrupted human MCF7 breast cancer cells drives the formation of invasive orthotopic xenograft tumors and elongated, cohesive, and E-Cadherin-expressing spheroids in three-dimensional (3D) culture as compared to control (Graves et al., 2016, Breast Canc. Res. 18:11). Further, treatment of these podocalyxin-overexpressing MCF7 cells with the myosin inhibitor, blebbistatin, and the small molecule inhibitor of ezrin-actin binding, NSC668394, resulted in decreased collective invasion and migration, respectively. Together these data suggest that podocalyxin, through interactions with the actin cytoskeleton via its cytoplasmic tail binding partners, can facilitate increased collective epithelial tumor cell motility, at least in some contexts. To test this hypothesis, we generated podocalyxin null MCF7 clones and cell populations using CRISPR-Cas9 genome editing and reconstituted these cells with mutant forms of podocalyxin that are unable to interact with the scaffolding proteins NHERF and/or ezrin and hence with the actin cytoskeleton. Preliminary results from 3D culture and live imaging of these mutant podocalyxin-expressing cells suggests that loss of podocalyxin9s cytoplasmic tail results in decreased spheroid invasion that may be a result of deficiencies in actomyosin contractility. Hence, increased expression and mislocalization of podocalyxin may facilitate aberrant interactions with the actin cytoskeleton and contractile machinery, driving enhanced cell motility and, in certain tumor microenvironments, promote collective tumor invasion. Citation Format: Erin M. Bell, Marcia L. Graves, Pamela Dean, Kelly M. McNagny, Calvin D. Roskelley. Characterizing the role of podocalyxin9s cytoplasmic tail domain in collective tumor invasion [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 4911.