Abstract 4918: A Rho-directed ROCK:mDia2 signaling axis drives morphological plasticity and invasive transitions in EOC spheroids.

Epithelial ovarian cancer (EOC) is often detected late after it has metastasized within the peritoneal cavity. EOC cells are typically detected in the peritoneal ascites as single cells, multi-cell clusters or highly ordered, compacted spheroids. Compacted EOC spheroids represent a more invasive and tumorigenic population of ovarian tumor cells both in vitro and in vivo and are highly chemoresistant. Yet little is known about molecular mechanisms triggering an invasive transition within single cells emanating from these clinically dangerous structures that are fundamental to EOC dissemination and disease progression. In this regard, the field of molecular players driving EOC spheroid adhesion/migration/invasion has remained relatively static over the last decade. However, spheroid formation and ensuing invasion requires tight regulation of the actin cytoskeleton; therefore, a thorough understanding of the contribution of proteins regulating the spheroid F-actin cytoskeleton in both cell-cell adhesions and adhesion to/transmigration through peritoneal mesothelium is essential for progress in therapy of metastatic EOC. The mDia formin protein family has emerged as key regulators of cytoskeletal dynamics during cancer cell division and migration. mDia2 assembles non-branched F-actin networks underlying cell-cell junctions and is critical for driving single cell mesenchymal motility. Loss of mDia2 expression and/or function drives an amoeboid morphological transition that enhanced cell invasion and metastasis in breast and prostate cancer models. Our data strongly implicate a tightly controlled network of cytoskeletal proteins including the GTPase RhoA and its effectors mDia2 formin and ROCK in driving highly adaptable single cell motility from EOC spheroids within three-dimensional (3D) matrices. Here we show that mDia2 is involved not only in EOC spheroid growth and formation, yet is enriched in the invasive cellular front of spheroids embedded in 3D collagen gels. However, mDia2 disruption significantly enhances invasive dissemination of amoeboid single cells from EOC spheroids embedded in collagen gels. ROCK, which promotes myosin-based cellular contractility and amoeboid motility programs, similarly played a role in spheroid formation, yet its disruption via Y27632 treatment significantly inhibited spheroid invasion while driving an elongated cellular morphology. Treatment of invasive mDia2-depleted EOC spheroids with Y27632 blocked invasion more effectively than Y27632 treatment alone, indicating that under conditions of mDia2 depletion, invasion is dependent upon Rho-directed ROCK activity. Collectively, these data indicate interplay between a Rho-directed ROCK and mDia2 signaling axis in driving invasive transitions and morphological plasticity in EOC spheroids. Citation Format: Krista Pettee, Andrea Nestor-Kalinoski, Kathryn M. Eisenmann. A Rho-directed ROCK:mDia2 signaling axis drives morphological plasticity and invasive transitions in EOC spheroids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4918. doi:10.1158/1538-7445.AM2013-4918