Abstract 1004: Investigating the role of Cdc42 in the developing mouse mammary gland

Abstract

Abstract Rho GTPases coordinate and transmit extracellular signals to control downstream effectors. The Rho GTPase cell division cycle 42 (Cdc42) is overexpressed and hyperactivated in human breast cancers. Cdc42 regulates cell polarity, mitosis, adhesion, migration, and stem cell function. These same cellular processes also function in normal mammary gland development. Exactly which functions of Cdc42 are crucial for normal mammary gland (MG) development and its precise role in mammary tumor formation and progression remain unknown. We hypothesize that Cdc42 plays critical and pleiotropic roles in normal MG development and tumorigenesis through regulation of the signal transduction pathways that are involved in cell cycle progression, cell polarity determination, cell motility, and cell adhesion. To investigate the molecular mechanisms by which Cdc42 regulates MG development, we studied the effects of Cdc42 deletion on morphogenesis of primary mammary epithelial cells (MECs). Adeno-cre viral transduction was used to introduce cre recombinase into Cdc42fl/fl primary MECs, and effects on morphogenesis were assessed using a three-dimensional mammary morphogenesis assay. Transduction efficiency ranged from 50-80% and ∼70% percent knockdown of Cdc42 expression was achieved. Control Adeno-GFP transduced MECs developed into polarized mammary acini whereas morphogenesis was markedly perturbed in the Cdc42 deficient MECs. Cdc42 knockout MECs formed small (30 µm in diameter compared to 45 µm in the control structures, p < .001, n = 28), disorganized structures lacking polarity and lumens (99% vs. 8% in the controls, n > 30). Markers of normal development including polarity markers, pERM and α6; adhesion, ZO-1; and downstream signaling, pkc-ζ, displayed aberrant expression patterns in the knockout structures. Interestingly, Cdc42 deficient structures contained invasive cells, which were rarely seen in control acini (41% vs 7%, n=80), suggesting that loss of Cdc42 induces aberrant migration. Cdk4 levels were also reduced in Cdc42 deficient cells and cell cycle analysis by flow cytometry demonstrated an increase in the percentage of cells in G1, suggesting that loss of Cdc42 inhibits MEC proliferation by blocking the transition from G1 to S phase. In addition, analysis of an S phase marker, phospho-histone H3, was performed at an early time point (3 days) in morphogenesis. Only 35% of the transduced knockout structures expressed phospho-histone H3, compared to 100% in the controls (n>20). These studies demonstrate a vital role for Cdc42 in the processes of growth, adherence, and polarity establishment during mammary gland development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1004.