DNMTi in combination with PARPi inhibits aberrant Wnt/beta-catenin signaling and tenasin-C pathways, cancer stemness and metastasis in triple negative breast cancer

Triple-negative breast cancer (TNBC) has a higher rate of metastasis; a poor prognosis and survival compared with other breast cancer types. Poly-ADP ribose polymerase inhibitors (PARPis) are used to treat TNBC patients that harbor germline BRCA1/2 mutations, inducing synthetic lethality, but responses are not durable. We previously reported that PARPis in combination with DNA methyltransferase inhibitors (DNMTis) exert synergistic cytotoxicity in TNBC, independent of BRCA mutations, but the effects of these drugs on metastasis and stemness, which are associated with poor survival outcomes, are not known. Aberrant Wnt/beta-catenin signaling in TNBC is known to drive cancer stemness, metastasis, and resistance to apoptosis and chemotherapy. Genome-wide transcriptomic analysis in TNBC cell line MDA MB 231 demonstrated that combining DNMTis azacytidine (AZA) and PARPis talazoparib (TAL) down-regulated cancer stemness and metastases pathways, and key leading-edge genes including those involved in Wnt/beta-catenin signaling and tenasin-C (TNC), a multimodular glycoprotein that promotes the migration of cancer cells, were decreased. The effects of this drug combination on cell migration were functionally validated using scratch and transwell migration and invasion assays in multiple TNBC cell lines, including MDA MB231 and SUM159, and patient-derived organoids (N=3). Xenograft studies of MDA MB 231 by tail vein and SC injection showed decreased metastasis to the lung with this drug combination. Notably, stem cell assays, including spheroid assays and stem cell markers, SOX2, ALDH1a1 and CD44, were also decreased with AZA and TAL treatment. Notably, we show for the first time in TNBC that Beta-catenin/TCF12 transcriptionally regulates TNC by binding to its promoter region and that inhibition or KD of WNT/Beta catenin or TNC expression decreases the cell migration, metastasis and stemness, mimicking the effects of the drug combination in TNBC cells. Taken together, our results show for the first time that PARPi and DNMTi combination therapy targets WNT/Beta-catenin signaling and TNC regulation in driving aggressive disease, metastasis, stemness and poor survival in TNBC.