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.

<p>Supplementary Materials, Methods, and References</p>

<p>Supplementary Figures and Tables</p>

Rhabdomyosarcoma (RMS) is tumor of the muscle and is the most common soft tissue cancer in children and teens, with approximately 400 to 500 new cases every year in the United States. There are two main subtypes of rhabdomyosarcoma: 1) Alveolar rhabdomyosarcoma (fusion-positive RMS FP-RMS), is classified primarily by the presence of the fusion between PAX3/PAX7 and FOXO1 proteins. 2) Embryonal rhabdomyosarcoma (fusion-negative FN-RMS) is the more common subtype. The survival rate for patients with RMS is 70%, but at relapse it is less than 30%. The standard of care for this disease includes radiation, chemotherapy and surgery. To better understand how RMS tumors survive therapy, we sought to define the role of SNAI2 in the response to radiation treatment. We identified SNAI2 as a critical oncogene that protects RMS tumors from radiation induced apoptosis by repressing pro-apoptotic BIM expression (Cancer Research 2021). SNAI2 additionally prevents FN-RMS tumors and cells from undergoing myogenic differentiation (Nature Comm. 2021). We also showed that MEK maintained SNAI2 post translationally in RAS mutant FN-RMS tumors and combining MEK inhibitor with radiation led to complete tumor regression and delayed event free survival in xenograft and PDX models. However, rare tumor cells grow back after a 2-4 weeks delay (Molecular Cancer Therapeutics, 2022). Hence, we hypothesized that there are additional genes or pathways in cells lacking SNAI2 that are co-opted to drive relapse disease. We have identified that CDKN1A or p21 expression is robustly increased in SNAI2 null cells. CDKN1A is thought to be a tumor suppressor in RMS and other cancers. However, our preliminary data indicate that CDKN1A is an oncogene and in RMS patients, high expression in tumors is associated with poor outcome. We found that p21 expression in combination with SNAI2 can predict resistance to radiation across RMS cell lines. We next developed control RD, Rh30 and Rh18 cells either CDKN1A or both CDKN1A and SNAI2 ablated using CRISPR/Cas9 reagents with control cells expressing control gRNAs. Ablation of CDKN1A while not effecting proliferation led to a significant increase in apoptosis post-radiation and this effect was increased in SNAI2/CDKN1A double knockout cells. Further, in colony forming assays we find that double ablated cells form significantly fewer colonies than control and single knockout cells. Rather unexpectedly, loss of CDKN1A, SNAI2, or CDKN1A/SNAI2 led to a G2/M cell cycle block, indicating that the cell cycle effects of p21 might not be responsible for the phenotypes observed. Ongoing xenograft experiments will determine if the effect we observe in vitro will also translate in vivo. Additionally, we are performing experiments to test how SNAI2 modulates CDKN1A expression. In summary, our study identifies CDKN1A as an oncogene that protects SNAI2 deficient cells from ionizing radiation, thus enabling the survival or rare relapse driving tumor cells. Citation Format: Paulomi S. Modi, Long Wang, Prethish Sreenivas, Myron Ignatius. A SNAI2/CDKN1A pathway protects rhabdomyosarcoma tumors from radiation [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 1465.