Knockdown of estrogen receptor‐α induces autophagy and inhibits antiestrogen‐mediated unfolded protein response activation, promoting ROS‐induced breast cancer cell death

Abstract

Approximately 70% of all newly diagnosed breast cancers express estrogen receptor (ER)-α. Although inhibiting ER action using targeted therapies such as fulvestrant (ICI) is often effective, later emergence of antiestrogen resistance limits clinical use. We used antiestrogen-sensitive and -resistant cells to determine the effect of antiestrogens/ERα on regulating autophagy and unfolded protein response (UPR) signaling. Knockdown of ERα significantly increased the sensitivity of LCC1 cells (sensitive) and also resensitized LCC9 cells (resistant) to antiestrogen drugs. Interestingly, ERα knockdown, but not ICI, reduced nuclear factor (erythroid-derived 2)-like (NRF)-2 (UPR-induced antioxidant protein) and increased cytosolic kelch-like ECH-associated protein (KEAP)-1 (NRF2 inhibitor), consistent with the observed increase in ROS production. Furthermore, autophagy induction by antiestrogens was prosurvival but did not prevent ERα knockdown-mediated death. We built a novel mathematical model to elucidate the interactions among UPR, autophagy, ER signaling, and ROS regulation of breast cancer cell survival. The experimentally validated mathematical model explains the counterintuitive result that knocking down the main target of ICI (ERα) increased the effectiveness of ICI. Specifically, the model indicated that ERα is no longer present in excess and that the effect on proliferation from further reductions in its level by ICI cannot be compensated for by increased autophagy. The stimulation of signaling that can confer resistance suggests that combining autophagy or UPR inhibitors with antiestrogens would reduce the development of resistance in some breast cancers.

Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER+ Breast Cancer Cells

“…However, in the presence of ICI, further reduction in ERα expression occurred and this was associated with a significant decrease in cell viability in the LCC9 suggesting that ERα protein in the shH19-ETR cells had become susceptible to ICI-induced protein degradation. A previous report by Cook et al [12] also indicated that decreased ERα renders LCC9 cells susceptible to ICI where they observed a 50% decrease in relative cell density after 3 days of ICI treatment. Based on our finding that H19 regulation of ERα is reciprocated by ERα, we posit that the sensitization of ERα-knocked down LCC9 cells to ICI reported by Cook et.…”

“…Based on our finding that H19 regulation of ERα is reciprocated by ERα, we posit that the sensitization of ERα-knocked down LCC9 cells to ICI reported by Cook et. al., [12] was due to decreased H19 levels. However, here we report >90% cell loss in shH19-LCC9 cells exposed to ICI for 3 days.…”

“…Several mutations in the ERα gene have been identified that confer ligandindependent activity of ERα [10,11]. Interestingly, decreasing ERα expression in the LCC9 cells results in cell death [12] however, mechanisms that can contribute to maintaining ERα expression in these ETR cells remains unknown.…”

KEAP1 Genetic Polymorphisms Associate with Breast Cancer Risk and Survival Outcomes

“…Estrogen also reportedly acts on the ERa ligandbinding domain and inhibits NQO1 promoter activity; therefore, inhibition of estrogen signaling leads to activation of NRF2-dependent NQO1 transcription (46). Breast cancer cells transfected with ERa shRNA exhibit reduced nuclear NRF2 localization as well as cytoplasmic KEAP1 accumulation, which would bind any remaining NRF2, preventing antioxidant response and allowing ROS generation (47). Among patients with endometrioid endometrial adenocarcinoma who received adjuvant (CT and/or RT) treatment, heterozygous rs1048290 minor allele carriers (analyzed from tumor tissue) have previously been found to show shorter progression-free survival compared with CC homozygotes (25).…”

FOXO factors and breast cancer: outfoxing endocrine resistance

“…Within breast cancer cells GRP78 controls the autophagic response via signaling through the 5 0 -AMP-activated protein kinase and mammalian target of rapamycin (mTOR) signaling pathways (Cook et al 2012). Cook et al (2014) observed that treatments such as tamoxifen and fulvestrant can stimulate the pro-survival UPR and autophagy signaling in breast cancer cells.…”