Abstract 1359: How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC

Abstract

Abstract Introduction: We reported that Wnt Pathway (WP) upregulation, one of the salient genetic features of metastatic TNBC, controls the acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes of TNBC cells (Dey et al., 2013). RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. Aim: Here we tested the role of the mechanism of involvement of RAC1 in the regulation of WP-mediated ID-MA phenotypes in TNBC cells and examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Method: Oncoprints showed that the major alterations of RAC1 gene in TNBC were constituted by the amplification/gain of the gene. The collective percentage of alteration(s) in RAC1 in ER+ve BC was 35% as compared to 57% in ER-ve BC (brca/tcga/pub2015). Using pharmacological agents (sulindac sulfide), genetic tools (siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed migration and various motility parameters (actin dynamics, filopodia and lamellipodia by confocal microscopy) in the context of fibronectin-directed RAC1 and Cdc42 activation. Result: Breaking down by molecular subtypes of BC, RAC1 gene alterations are observed in 20% in PAM50 Luminal A, 35% in PAM50 Luminal B, 37% in PAM50 HER2 enriched and 40% in PAM50 Basal-like. For the samples set of TCGA, Cell 2015, RAC1 gene was altered in 42% total while it was altered 37% in PAM50 Luminal A, 57% in PAM50 Luminal B, 55% in PAM50 HER2 enriched and 60% in PAM50 basal-like. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) altered actin dynamics and (d) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report (De et al., 2016) that WP upregulation stimulates ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in an RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Citation Format: Nandini Dey, Jennifer H. Carlson, Casey Williams, Tyler Jepperson, Pradip De, Brian Leyland-Jones. How does RAC1 GTP-ase signal Wnt-beta-catenin pathway-mediated integrin-directed metastasis-associated tumor cell phenotypes in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1359. doi:10.1158/1538-7445.AM2017-1359