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.

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 Beta 1 (ERB1) increases response to chemotherapy but is opposed by ERB4, which it preferentially dimerizes with. The role of ERB1 and ERB4 in influencing chemotherapy sensitivity has never been studied before. CAS9/CRSPR was used to truncate ERB1 Ligand Binding Domain (LBD) and knock down the exon unique to ERB4. We show that the truncated ERB1 LBD in a variety of mutant p53 TNBC cell lines, where ERB1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERB4 knockdown cell line was sensitized to Paclitaxel. We further show that ERB1 LBD truncation as well as treatment with ERB1 antagonist 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 ERB4 regulate these stem cell markers like SOX2, OCT4, and Nanog in an opposing manner; and we further show that this regulation is mediated by HIF. We show the upregulation of cancer cell stemness due to ERB1 LBD truncation is attenuated when HIF1/2a is knocked down by siRNA. Finally we show, an increase in the frequency of breast cancer stem cell population due to ERB1 antagonist using both ALDEFLUOR and SORE6 reporter 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 ERß1 with agonists and inactivation of ERB4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemo resistant TNBC patients. Citation Format: Ayesha Bano, Jessica Stevens, Paulomi S. Modi, Anders Strom, Jan-Ake Gustafsson. Estrogen receptor β4 regulates chemotherapy resistance and induces cancer stem cells in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3072.