Characterizing drivers of invasiveness in basal-like triple negative breast cancer

PURPOSE: Triple-negative breast cancer (TNBC) is characterized by aggressive tumor growth, resistance to treatment, higher likelihood of relapse, and overall worse prognosis when compared to other breast cancer subtypes. The TNBC tumor microenvironment is molecularly heterogeneous, containing various numbers of rapidly growing basal-like (BSL), and invasive mesenchymal-like (MSL) stromal and TNBC cells. Annexin A6 (AnxA6) is a multifunctional calcium-dependent scaffolding protein that is highly expressed in MSL TNBC cells but expressed at low levels in BSL TNBC, and is implicated in cell proliferation, motility, and drug resistance. It is currently unclear how altered AnxA6 levels influence the invasiveness of MSL and BSL TNBC cells. We hypothesize that altered expression of AnxA6 differentially contributes to the invasiveness of BSL and MSL TNBC subgroups. METHODS: Short hairpin RNAs were used to downregulate AnxA6 in parental (PAR) subpopulations of model MSL (BT-549) and BSL (MDA-468) TNBC cell lines. The invasive subpopulations (INV) of these cell lines with or without AnxA6 downregulation were isolated from control and AnxA6 downregulated cells by sequential invasion assays in Boyden chambers. The parental and invasive subpopulations were then assessed for markers of invasiveness, cancer stem cell (CSC) expression, drug sensitivity, oncoprotein expression, and differential gene expression. RESULTS: Invasiveness increased AnxA6-dependently in BSL TNBC INV subpopulations compared to the PAR population. Sensitivity to doxorubicin was decreased in the INV subpopulations of both MSL and BSL TNBC. Flow cytometry revealed an increase in CD49f+/EpCAM+ CSC expression among BSL TNBC INV subpopulations. Carbonic anhydrase IX (CA9) was found upregulated only in the INV subpopulations of BSL TNBC, and that downregulation of CA9 results in decreased invasiveness and viability within BSL TNBC INV subpopulations. RNA sequencing and RT-qPCR validation has revealed an upregulation of EDN2 and PDXN among INV BSL TNBC subpopulations, while BIRC3 and PARP9 were found to be upregulated in MSL TNBC INV subpopulations. CONCLUSION: Our data suggest that the invasive subpopulations of both TNBC subgroups differentially modify key oncogenes with respect to their parental controls. Altered AnxA6 expression is associated with differential invasive potential and sensitivity to chemotherapy for MSL versus BSL TNBC cells. Sequential invasion assays have successfully isolated invasive subpopulations that upregulate TNBC subgroup-dependent biomarkers. Targeting the upregulated genes and proteins identified in this study may yield promising results in the future for treating TNBC metastatic disease.