Estrogen Receptor β4 Regulates Chemotherapy Resistance and Induces Cancer Stem Cells in Triple Negative Breast Cancer

Abstract

Triple Negative Breast Cancer (TNBC) has the worst prognosis among all breast cancers, and survival in patients with recurrence is rarely beyond 12 months due to acquired resistance to chemotherapy, which is the standard of care for these patients. Our hypothesis is that Estrogen Receptor β1 (ERβ1) increases response to chemotherapy but is opposed by ERβ4, which it preferentially dimerizes with. The role of ERβ1 and ERβ4 in influencing chemotherapy sensitivity has never been studied before. CRISPR/CAS9 was used to truncate ERβ1 Ligand Binding Domain (LBD) and knock down the exon unique to ERβ4. We show that the truncated ERβ1 LBD in a variety of mutant p53 TNBC cell lines, where ERβ1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERβ4 knockdown cell line was sensitized to Paclitaxel. We further show that ERβ1 LBD truncation, as well as treatment with ERβ1 antagonist 2-phenyl-3-(4-hydroxyphenyl)-5,7-bis(trifluoromethyl)-pyrazolo[1,5-a] pyrimidine (PHTPP), leads to increase in the drug efflux transporters. Hypoxia Inducible Factors (HIFs) activate factors involved in pluripotency and regulate the stem cell phenotype, both in normal and cancer cells. Here we show that the ERβ1 and ERβ4 regulate these stem cell markers like SOX2, OCT4, and Nanog in an opposing manner; and we further show that this regulation is mediated by HIFs. We show the increase of cancer cell stemness due to ERβ1 LBD truncation is attenuated when HIF1/2α is knocked down by siRNA. Finally, we show an increase in the breast cancer stem cell population due to ERβ1 antagonist using both ALDEFLUORTM and SOX2/OCT4 response element (SORE6) reporters in SUM159 and MDA-MB-231 cell lines. Since most TNBC cancers are ERβ4 positive, while only a small proportion of TNBC patients are ERβ1 positive, we believe that simultaneous activation of ERβ1 with agonists and inactivation of ERβ4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemotherapy resistant TNBC patients.