Abstract

BackgroundCancer cell phenotypes evolve over the course of a tumors treatment. The phenotypes that emerge and disappear over time will be specific to each drug regimen and type of cancer. Chemotherapy remains one of the most common and effective treatments for metastatic breast cancer patients; however, resistance to chemotherapy inevitably emerges. Cancer chemotherapy treatment regimens are not designed to target emerging chemo-resistance, despite its clear importance in progressive cancer. This study focuses on finding sequential treatment strategies that target acquired chemo-resistant states and optimize response to chemotherapy. MethodsIn this study, we used heterogeneous tumor samples from patients to identify subclones resistant to chemotherapy. Using flow cytometry for stem cell markers and DNA sequencing to define subclonal population changes, we measured the enrichment of cancer stem cell-like (CSL) phenotypes in subclones that survive chemotherapy. We then analyzed breast cancer patient tumor organoids and cell line acquisition of CSL traits following chemotherapy, as well as the ability of different drugs to reverse acquired resistance, using flow cytometry, mammosphere assays, and single cell RNA-sequencing analysis. ResultsWe show that in progressive estrogen receptor positive (ER+) metastatic breast cancer patients, resistant tumor subclones that emerge following chemotherapy have increased CSL abundance. Further, in vitro organoid growth of ER+ patient cancer cells also shows that chemotherapy treatment leads to increased abundance of ALDH+/CD44+ CSL cells. Chemotherapy induced CSL abundance is blocked by treatment with a pan-HDAC inhibitor, belinostat. Further, belinostat treatment diminished both mammosphere formation and size following chemotherapy, also indicating a decrease in progenitor CSL traits. HDAC inhibitors specific to class IIa (HDAC4, HDAC5) and IIb (HDAC6) were shown to primarily reverse the chemo-resistant CSL state. Single-cell RNA sequencing analysis with patient samples showed that HDAC targets and MYC signaling were promoted by chemotherapy and inhibited upon HDAC inhibitor treatment. ConclusionThese findings indicate that HDAC inhibition can block chemotherapy-induced drug resistant phenotypes with one-two punch strategy in refractory breast cancer cells.