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.

BackgroundAbout 70% of all breast cancers are estrogen receptor alpha positive (ER+) and are treated with antiestrogens. However, 50% of ER + tumors develop resistance to these drugs (endocrine resistance). In endocrine resistant cells, an adaptive pathway called the unfolded protein response (UPR) is elevated that allows cells to tolerate stress more efficiently than in sensitive cells. While the precise mechanism remains unclear, the UPR can trigger both pro-survival and pro-death outcomes that depend on the nature and magnitude of the stress. In this study, we identified MYC, an oncoprotein that is upregulated in endocrine resistant breast cancer, as a regulator of the UPR in glucose-deprived conditions.MethodsER+ human breast cancer cell lines (LCC1, LCC1, LY2 and LCC9) and rat mammary tumors were used to confirm upregulation of MYC in endocrine resistance. To evaluate functional relevance of proteins, siRNA-mediated inhibition or small molecule inhibitors were used. Cell density/number was evaluated with crystal violet assay; cell cycle and apoptosis were measured by flow cytometry. Relative quantification of glutamine metabolites were determined by mass spectrometry. Signaling molecules of the UPR, apoptosis or autophagy pathways were investigated by western blotting.ResultsIncreased MYC function in resistant cells correlated with increased dependency on glutamine and glucose for survival. Inhibition of MYC reduced cell growth and uptake of both glucose and glutamine in resistant cells. Interestingly, in glucose-deprived conditions, glutamine induced apoptosis and necrosis, arrested autophagy, and triggered the unfolded protein response (UPR) though GRP78-IRE1α with two possible outcomes: (i) inhibition of cell growth by JNK activation in most cells and, (ii) promotion of cell growth by spliced XBP1 in the minority of cells. These disparate effects are regulated, at different signaling junctions, by MYC more robustly in resistant cells.ConclusionsEndocrine resistant cells overexpress MYC and are better adapted to withstand periods of glucose deprivation and can use glutamine in the short term to maintain adequate metabolism to support cell survival. Our findings reveal a unique role for MYC in regulating cell fate through the UPR, and suggest that targeting glutamine metabolism may be a novel strategy in endocrine resistant breast cancer.

In estrogen receptor positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (GX; obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here we show that GX is more effective in reducing the cell density of antiestrogen resistant breast cancer cells versus sensitive cells, and that this increased sensitivity of resistant cells to GX correlates with an accumulation of autophagic vacuoles. Formation of autophagosomes in GX-treated cells was verified by changes in expression of the lipidation of microtubule-associated protein-1 light chain-3 and both confocal and transmission electron microscopy. While GX treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulate. Moreover, GX exposure leads to a reduction in cathepsin D and L protein expression that would otherwise digest autolysosome cargo. Thus, GX has dual roles in promoting cell death: (1) directly inhibiting antiapoptotic BCL2 family members, thereby inducing apoptosis; and (2) inhibiting downstream cathepsins D and L protein expression to limit the ability of cells to use degraded material to fuel cellular metabolism and restore homeostasis. Our data highlight a new mechanism of GX-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer.

Interferon regulatory factor-1 (IRF1) is a tumor suppressor that regulates cell fate in several cell types. Here we report an inverse correlation in expression of nuclear IRF1 and the autophagy regulator ATG7 in human breast cancer cells that directly impacts their cell fate. In mice harboring mutant Atg7, nuclear IRF1 was increased in mammary tumors, spleen, and kidney. Mechanistic investigations identified ATG7 and the cell death modulator Beclin-1 (BECN1) as negative regulators of IRF1. Silencing ATG7 or BECN1 caused estrogen receptor-α (ERα) to exit the nucleus at the time when IRF1 nuclear localization occurred. Conversely, silencing IRF1 promoted autophagy by increasing BECN1 and blunting IGF-1 receptor and mTOR survival signaling. Loss of IRF1 promoted resistance to anti-estrogens, whereas combined silencing of ATG7 and IRF1 restored sensitivity to these agents. Using a mathematical model to prompt signaling hypotheses, we developed evidence that ATG7 silencing could resensitize IRF1-attenuated cells to apoptosis through mechanisms that involve other estrogen-regulated genes. Overall, our work shows how inhibiting the autophagy proteins ATG7 and BECN1 can regulate IRF1 dependent and independent signaling pathways in ways that engender a new therapeutic strategy to attack breast cancer.