Project Summary Triple negative breast cancer (TNBC) remains a complex unmet medical need because of its heterogeneity, poor prognosis, and its potential to grow rapidly and/or metastasize especially following therapeutic intervention. The response of TNBCs to various therapeutic interventions including tyrosine kinase inhibitors (TKIs) is generally poor. Our published and ongoing studies have implicated the Ca2+ dependent membrane binding Annexin A6 (AnxA6) in a wide range of cellular functions including cell growth and motility that define tumor progression, metastasis and chemo-resistance. We have now shown that AnxA6 is a tumor suppressor in TNBC and that the pro-tumorigenic properties of low AnxA6 and the pro-invasive functions of high AnxA6 TNBC cells are mediated at least in part, by AnxA6 modulated Ca2+ influx and activation of GRF2. Chronic treatment of AnxA6-low but not AnxA6 high TNBC cells with TKIs leads to AnxA6 upregulation and accumulation of cholesterol in late endosomes as a novel mechanism for acquired resistance of AnxA6 low TNBCs to these drugs. Furthermore, reduced expression of AnxA6 is more relevant in TNBC compared to non-TNBC and may be used as a reliable biomarker for response to chemotherapy and as an independent predictor of TNBC relapse after chemotherapy. Interestingly, the reciprocal expression of AnxA6 and GRF2 is clinically relevant and semi-quantitative assessment of the ratio of GRF2:AnxA6 can be used to delineate rapidly growing from highly invasive TNBCs. Together, this suggests that AnxA6 plays a critical role in TNBC progression, metastasis and resistance to therapeutic interventions, but the mechanisms underlying the chronic TKI induced reactivation and the pro- invasive properties of AnxA6 in TNBC remain poorly understood. We hypothesize that the pro-invasive properties of AnxA6 are mediated by extracellular and/or intracellular pools of AnxA6 via AnxA6-modulated interaction of GRF2 with Rho GTPases; and that reactivation of AnxA6 expression is triggered by inhibition of Ca2+ mobilizing RTKs via potent inhibition of Ca2+ entry channels and/or modification of specific histone marks. To test this we will determine the mechanisms underlying TKI-induced reactivation of AnxA6 and the effects of AnxA6 reactivation in TNBC progression and metastasis in Aim 1; and in Aim 2, we will determine the mechanisms underlying the pro-invasive properties of AnxA6 in basal-like TNBC. Data from this study will lead to a better understanding of how TNBC cells circumvent the effects of chronic treatment with TKIs to become even more aggressive and/or invasive, key attributes associated with TNBC patient mortality.

Project Narrative The potential for triple negative breast cancer (TNBC) to relapse and/or spread to other sites after therapeutic interventions depends on the ability of the tumor cells to reactivate growth and motility signals. In this study, we will determine how Annexin A6 promotes TNBC invasiveness and whether the increased expression of Annexin A6 following chronic tyrosine kinase inhibitor (TKI) treatment underlies the poor response of TNBCs to these drugs. Data from this study will improve our understanding of how TNBC cells circumvent the effects of chronic treatment with TKIs to become even more aggressive and/or invasive, key attributes which are associated with TNBC patient mortality.