Abstract P4-02-11: A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer
Authors: Wyatt Garhett L , Crump Lyndsey S , Lyons Traci R , Porter Weston W 
Published: March 2022

Abstract
BACKGROUND: Estrogen Receptor (ER) + breast cancers (BC) comprise over 70% of BC cases and can be targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases commonly become diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastasis. We have identified a tumor suppressor, Singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits EMT and metastasis, and is downregulated in the progression of breast disease. Our previous studies found that loss of SIM2s expression results in downregulation of ESR1 and increased basal markers in the MCF7 (ER+BC) cell line. Likewise, Semaphorin 7a (SEMA7A) expression is associated with decreased overall survival in ER+BC patients. Patients with SEMA7Ahi tumors treated with Tamoxifen exhibit shortened relapse free survival. SEMA7A is a downstream target of both NFKB and PTGS2/COX-2; and our previously published results establish a cross-talk between NFKB and SIM2s to regulate PTGS2 via interactions with the AKT pathway. Moreover, COX-2 inhibition is associated with better prognosis for breast cancer patients. Therefore, we are investigating how SIM2s may repress AKT signaling in SEMA7A driven ER resistance and metastasis. Methods: We performed in silico analysis of the SEMA7A promoter using the ConTra web server. For in vitro assays, we examined estrogen dependent differences in SIM2s, ER, and SP1 binding utilizing luciferase reporter assays using intact promoter and SIM2s site deletion mutants (Invitrogen) in MCF7 cells. Using immunoblotting and qPCR analysis we also assessed expression of SEMA7A, ESR1, PI3K/AKT signaling genes and EMT associated genes in Crispr/Cas9 SIM2s knockout (SIM2KO) MCF7 cells. Treatment of MCF7 cells with exogenous SEMA7A was used to observe possible changes in SIM2s expression and SIM2s promoter activity. For in vivo assays, we used immunohistochemistry (IHC) to assess SIM2s in SEMA7A low and high expressing MCF7 xenografts. To examine the relationship between SIM2s and ER, SEMA7A-/- (KO) mice were crossed with C57/BL6 MMTV-PYMT mice. Results: In silico analysis of the SEMA7A promoter via ConTra revealed four potential SIM2 response elements containing the SIM2s central midline elements (CME) binding motif and highly conserved estrogen response element half-sites adjacent to two specificity protein 1 consensus bindings sites. Luciferase reporter assays in MCF7 cells confirmed SIM2s represses basal and estrogen induced-SEMA7A promoter activity. Moreover, SIM2s was unable to repress the SEMA7A promoter activity with CME mutation. MCF7 SIM2KO cells exhibit increased SEMA7A promoter activity, SEMA7A, AKT signaling, EMT signatures and decreased ESR1, CDH1, and PTEN protein and mRNA expression. Both SIM2s expression and SIM2s promoter activity are significantly decreased with the addition of exogenous SEMA7A. Moreover, MCF7 xenografts reveal reciprocal expression of SIM2s and SEMA7A. Additionally in MMTV-PyMT mice, SIM2s expression is lost as tumors transition from pre-malignant to invasive phenotypes, yet we observe maintenance of SIM2s and ER in SEMA7A-/-;PyMT mice. Conclusion: These findings establish a regulatory relationship between SIM2s and SEMA7A in ER+BC. SIM2s functions to downregulate SEMA7A mediated pro-tumor signaling and maintain ER expression, but can be turned off to allow for resistance in ER+BC. Given the observed reciprocal expression, our study suggests a SIM2s/SEMA7A switch which may confer resistance in ER+BC via AKT signaling through downregulation of PTEN. Thus, a SIM2s/SEMA7A switch may act as a prognostic indicator to provide therapeutic advantages in resistant ER+BC metastasis.
Citation Format: Garhett L Wyatt, Lyndsey S Crump, Traci R Lyons, Weston W Porter. A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-11.