Abstract P3-11-15: A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells

Abstract

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance is due to upregulation of growth factor pathways (i.e., HER2 and EGFR) and increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells (LTLT-Ca). However, many postmenopausal women with AI-resistance tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor (ER+) positive and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer using a combination approach of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation and motility. To test this hypothesis, T47Darom cells (T47D cells which stably overexpress aromatase) and T47DaromLR cells (T47Darom cells that are letrozole resistant) were treated with lapatinib, glyceollin, or the combination. To evaluate viability, resazurin assays were performed and results demonstrated that glyceollin and/or lapatinib had no effect on proliferation in the T47Darom cells. However, glyceollin alone and glyceollin + lapatinib inhibited proliferation of T47DaromLR cells by 46% and 59%, respectively. Cell survival was assessed by colony formation assays and glyceollin caused a 33% reduction in cell survival and glyceollin + lapatinib caused a 60% reduction in the colony formation of T47Darom cells. Interestingly, in T47DaromLR cells glyceollin and the combination significantly inhibited cell survival by 94% alone and 96%. To further evaluate changes that occur as AI sensitive cells transition to AI resistance, a quantitative proteomic analysis of the whole cell lysates of T47DaromLR (AI-resistant) versus T47Darom cells (AI-sensitive) was performed to identify significant protein expression changes. Results demonstrated a 3.195-fold-increase (p<0.01) in protein disulfide isomerase (P4HB) and a 0.484-fold decrease (p<0.01) in peptidyl-prolyl cis-trans isomerase (FKBP4) which was validated by immunoblotting. While the molecular mechanism by which P4HB contributes to tumorigenesis and metastasis is unknown, it has been demonstrated to be involved in the proliferation, survival, and metastasis of several types of cancer cells. FKBP4 is also of interest as it has been shown to be involved in mitotic arrest and apoptotic signaling. To interrogate the effect of combination therapy on HER effector signaling cascades, western blot analyses were performed to establish the endogenous expression of various proteins. Results demonstrated ERα expression was increased while HER2 was decreased in T47Darom cells compared to T47DaromLR cells. The protein expression findings from the T47D variant cell lines followed a similar trend as those previously reported in LTLT-Ca cells and their AI-sensitive counterparts. Taken together, these results suggest that dual inhibition using glyceollins and lapatinib may have potential as a novel combination therapy approach for postmenopausal patients with hormone-dependent, letrozole-resistant breast tumors. This work was also supported by NIH grant 1SC1GM126617 awarded to SL Tilghman. Citation Format: Rashidra R Walker, Jankiben Patel, A. Michael Davidson, Karen Gallegos, Syreeta L Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-15.