Abstract LB-016: Estrogen receptor stability is modulated by calpain activity in breast cancer cells

Abstract

Abstract Breast cancer is a disease that affects thousands of women worldwide, and despite a number of treatment options, death results from metastasis and disease resistance. Two-thirds of all breast cancer cases involve dysregulation of estrogen receptor (ER) signaling; thus, a number of treatment strategies employ using compounds that affect its activity. Included in these drugs is the selective ER modulator (SERM) tamoxifen, which binds ER, leading to its association with DNA and the recruitment of corepressor proteins that inhibit ER signaling. While therapies involving tamoxifen are useful in controlling the disease, resistance inevitably occurs, rendering these treatments ineffective. Thus, other compounds are used such as selective ER down regulators (SERDs), which leads to ubiquitin-mediated ER degradation and complete loss of its signaling. Because resistance to these compounds also occurs, other methods for regulating ER signaling must be explored. In this study, we report loss in ER protein expression in response to treatment with doxorubicin and MG132. We found that treatment of the human breast cancer cell lines MCF7 and T47D with doxorubicin plus MG132 leads to ER protein expression loss as demonstrated by western blotting. In contrast, no ER protein expression loss was observed by treatment with either drug alone. RNA-Seq analysis of cells treated with this drug combination demonstrated loss in downstream ER targets, confirming suppressed ER signaling. As MG132 inhibits both the proteasome and calpain activity, we next sought to determine which of these protease activities was involved. Cells were treated in the presence of the proteasome inhibitors bortezomib and lactacystin, and no effect on ER expression was found with doxorubicin treatment, suggesting that the proteasome does not play a role in the ER loss. In contrast, treatment with doxorubicin plus the calpain-specific inhibitors PD 150606 and EST led to ER protein expression loss, confirming that calpain activity is involved in ER protein regulation. Next, to determine which calpains were specifically involved in the loss of ER protein, we knocked down several calpain proteins found to be expressed in MCF7 cells including calpains 1, 7, 8, 9, and 13 and the regulatory subunit CAPNS1. While knockdown of calpains 1, 7, 8, 9, and 13 had no effect on ER protein expression in the presence of doxorubicin, knockdown of CAPNS1 led to a loss in ER that was similar to treatment with calpain inhibitors in the presence of doxorubicin. In conclusion, this study identifies a new pathway by which ER stability is affected i.e., DNA damage induction plus calpain inhibition, suggesting a novel chemotherapeutic treatment paradigm that may overcome resistance in patients with breast cancer. Thus, future studies will focus on identifying the proteins and mechanisms directly responsible for ER loss as well as analyzing preclinical in vivo mouse models. Citation Format: Shannon T. Bailey, Thomas Westerling, Myles Brown. Estrogen receptor stability is modulated by calpain activity in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-016. doi:10.1158/1538-7445.AM2015-LB-016