Abstract P6-08-07: Gain and amplification of RAC1 GTP-ase in BC: Explaining alterations in patients by experiments using TNBC model

Abstract INTRODUCTION: RAC1-GTPase which transduces signals from cell surface integrins, have been implicated in metastasis. We reported that Wnt-beta-catenin pathway (WP) that signals metastasis (BMC Cancer, 2013), is one of the salient genetic features of Triple-Negative Breast Cancer (TNBC) (PlosOne, 2013).AIM: We demonstrated that TNBC cells acquire integrin-directed metastasis-associated (ID-MA) phenotypes following an upregulation of the WP (Oncotarget, In Press). Here we examined how WP signals are transduced in the context of ID-MA phenotypes in TNBC.METHOD: We documented gain and amplification of RAC1 gene in Breast Invasive Carcinoma subtypes from cBioPortal. The outcome for RFS was studied in the Hungarian ER-ve BC cohort.Mechanistically, we studied fibronectin-directed (1) migration, (2) matrigel- invasion, (3) RAC1 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin 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, RESULTS: The collective percentage of gain and amplification of RAC1 were (1) 31% of total 1105 breast invasive carcinoma samples, (2) 29% of total 594 ER+ve samples, (3) 39% of total 174 ER-ve samples, (4) 38% of total 120 HER2+ve samples and (4)35% of total 82 TNBC samples (brca/tcga/pub2015; Cell 2015).In invasive ductal BC subtypes, gain and amplification of RAC1 were (1) 32% of total 201 Luminal A samples, (2) 37% of total 122 PAM50 Luminal B samples, (3) 47% of total 51 PAM50 Her2-enriched samples and (4) 33% of total 107 PAM50 Basal-like samples. In invasive lobular cancers, gain and amplification of RAC1 were 24% of total 127 samples.Involvement of WP in different TNBC cells was tested following stimulation by LWnt3ACM and Wnt3Arecombinant protein and different inhibitors of WP by both qRT-PCR and WB for beta-catenin, active beta-catenin, cMYC, cyclin D1and WP specific several stem cell markers. The WP attenuation, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration & invasion, (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 fibronectin-induced ID-MA phenotypes following WP stimulation by LWnt3ACM and Wnt3Arecombinant protein. High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort.CONCLUSION:In TNBC model, the activation of RAC1 signals downstream of WP mediated ID-MA phenotypes. The identification of the functional relationship between RAC1 signaling and the WP activation in the control of ID-MA mechanistically explains how the activation of WP in TNBC is associated with the high metastatic incidences and a dismal outcome. Citation Format: Dey N, Carlson JH, Jepperson T, Willis S, De P, Leyland-Jones B. Gain and amplification of RAC1 GTP-ase in BC: Explaining alterations in patients by experiments using TNBC model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-07.