Jessica L. Schwartz-Roberts

GX15-070 (Obatoclax) Induces Apoptosis and Inhibits Cathepsin D- and L–Mediated Autophagosomal Lysis in Antiestrogen-Resistant Breast Cancer Cells

In estrogen receptor–positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX15-070 has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here, we show that GX15-070 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 GX15-070 correlates with an accumulation of autophagic vacuoles. Formation of autophagosomes in GX15-070-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 GX15-070 treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulate. Moreover, GX15-070 exposure leads to a reduction in cathepsin D (CTSD) and L (CTSL1) protein expression that would otherwise digest autolysosome cargo. Thus, GX15-070 has dual roles in promoting cell death: (i) directly inhibiting antiapoptotic BCL2 family members, thereby inducing apoptosis; and (ii) inhibiting downstream CTSD and CTSL1 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 GX15-070-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer. Mol Cancer Ther; 12(4); 448–59. ©2013 AACR.

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

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.—Cook, K. L., Clarke, P. A. G., Parmar, J., Hu, R., Schwartz‐Roberts, J. L., Abu‐Asab, M., Wärri, A., Baumann, W. T., Clarke, R. Knockdown of estrogen receptor‐α induces autophagy and inhibits antiestrogen‐mediated unfolded protein response activation, promoting ROS‐induced breast cancer cell death. FASEB J. 28, 3891‐3905 (2014). www.fasebj.org

MYC regulates the unfolded protein response and glucose and glutamine uptake in endocrine resistant breast cancer

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.

Interferon Regulatory Factor-1 Signaling Regulates the Switch between Autophagy and Apoptosis to Determine Breast Cancer Cell Fate

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 affects 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 to exit the nucleus at the time when IRF1 nuclear localization occurred. Conversely, silencing IRF1 promoted autophagy by increasing BECN1 and blunting IGF1 receptor and mTOR survival signaling. Loss of IRF1 promoted resistance to antiestrogens, 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.

Linking autophagy with inflammation through IRF1 signaling in ER+ breast cancer

Resistance to antiestrogen therapy remains a critical determinant of mortality in patients affected by ER+ breast cancer. Our previous work identified autophagy and interferon regulatory factor 1 (IRF1) signaling as key regulators of this process. We have recently demonstrated a novel reciprocal interaction between IRF1 and ATG7, linking inflammation and autophagy.

Chapter 7 – Blockage of Lysosomal Degradation Is Detrimental to Cancer Cell Survival: Role of Autophagy Activation

Autophagy is a major catabolic process used by cells to remove superfluous or damaged proteins and organelles. In the final stages of autophagy, acidic organelles (lysosomes) act to degrade autophagic cargo and to facilitate their recycling. Little is known about how cancer cells undergoing autophagy, often as a consequence of stress, respond when lysosomal function is blocked. To elucidate this mechanism, several recent studies report that lysosomes and their hydrolytic proteases (cathepsins) play a critical role in autophagy and subsequent cancer progression. Our studies in breast cancer suggest that inhibition of cathepsins D and L using the BH3-mimetic, obatoclax, is effective in reducing the cell density of anti-estrogen sensitive and resistant breast cancer cells. Furthermore, blockage of cathepsin protein expression with obatoclax leads to the accumulation of autophagic vacuoles and impairs the ability of cells to use degraded material to restore homeostasis. While cancer cells are dependent on effective lysosomal function, neoplastic transformation induces changes in lysosomal volume, number, and protease activity. Recent reports suggest that pro-oncogenic changes render cancer cells more susceptible to lysosomal-associated death pathways. A number of distinct stimuli have been shown to permeabilize the lysosomal membrane, leading to the release of hydrolases into the cytosol and ultimately cell death. Thus, changes in cathepsin and lysosomal membrane permeabilization (LMP) regulation during cancer cell progression suggest that strategies targeting this cellular compartment may be exploited to improve outcomes for cancer patients.

Glutamine metabolism and the unfolded protein response in MYC-driven breast cancer

Antiestrogens are used to treat estrogen receptor positive (ER+) breast tumors that constitute 70% of all breast cancer cases. Unfortunately, acquired resistance to antiestrogen therapy remains a critical clinical obstacle. Here we show that human breast cancer cells and rat mammary tumors that have acquired resistance to antiestrogens express increased levels of MYC, a major regulator of both glutamine and glucose. Glutamine metabolism and glucose uptake were elevated in ER+ antiestrogen resistant cells (LCC9) compared with sensitive cells (LCC1). Inhibition of MYC, with siRNA or small molecule inhibitor, reduced cell viability and uptake of both glutamine and glucose in resistant cells. In resistant cells, MYC expression controlled protein levels of glutamine, glutamate and glucose transporters as well as GLUL and GLS, two enzymes that promote glutamate-glutamine inter-conversion. Increased MYC function in resistant cells correlated with increased cellular sensitivity to deprivation of, and also inhibitors of, both glutamine and glucose. While apoptosis eliminated all resistant cells in glucose-only conditions beyond 72 h, in glutamine-only conditions, the unfolded protein response (UPR) via GRP78-IRE1a and activating JNK and increased CHOP, induced apoptosis in majority of the cells but promoted survival in some. The antiestrogen faslodex (FAS; ICI 182,780) significantly reduced glucose uptake in antiestrogen resistant cells compared with sensitive cells. Thus, our findings reveal unique roles for MYC in promoting metabolic flexibility in and promoting survival in antiestrogen resistant breast cancer cells via the UPR. Targeting glutamine and glucose metabolism pathways, therefore, may provide novel strategies in treating endocrine resistant breast cancers.

Abstract 679: Glutamine metabolism in MYC-driven antiestrogen resistant breast cancer cells confers metabolic flexibility through the unfolded protein response

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antiestrogens are used to treat estrogen receptor positive (ER+) breast tumors that constitute 70% of all breast cancer cases. Unfortunately, acquired resistance to antiestrogen therapy remains a critical clinical obstacle. Here we show that human breast cancer cells and rat mammary tumors that have acquired resistance to antiestrogens express increased levels of MYC, a major regulator of both glutamine and glucose. Glutamine metabolism and glucose uptake were elevated in ER+ antiestrogen resistant cells (LCC9) compared with sensitive cells (LCC1). Inhibition of MYC, with siRNA or small molecule inhibitor, reduced cell viability and uptake of both glutamine and glucose in resistant cells. In resistant cells, MYC expression controlled protein levels of glutamine, glutamate and glucose transporters as well as GLUL and GLS, two enzymes that promote glutamate-glutamine inter-conversion. Increased MYC function in resistant cells correlated with increased cellular sensitivity to deprivation of, and also inhibitors of, both glutamine and glucose. While apoptosis eliminated all resistant cells in glucose-only conditions beyond 72 h, in glutamine-only conditions, the unfolded protein response (UPR) via GRP78-IRE1α and activating JNK and increased CHOP, induced apoptosis in majority of the cells but promoted survival in some. The antiestrogen faslodex (FAS; ICI 182,780) significantly reduced glucose uptake in antiestrogen resistant cells compared with sensitive cells. Thus, our findings reveal unique roles for MYC in promoting metabolic flexibility in and promoting survival in antiestrogen resistant breast cancer cells via the UPR. Targeting glutamine and glucose metabolism pathways, therefore, may provide novel strategies in treating endocrine resistant breast cancers. Citation Format: Ayesha N. Shajahan-Haq, Katherine L. Cook, Jessica L. Schwartz-Roberts, Ahreej E. Eltayeb, Diane M. Demas, Anni M. Warri, Leena A. Hilakivi-Clarke, Robert Clarke. Glutamine metabolism in MYC-driven antiestrogen resistant breast cancer cells confers metabolic flexibility through the unfolded protein response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 679. doi:10.1158/1538-7445.AM2014-679

Blockage of Lysosomal Degradation is Detrimental to Cancer Cell Survival: Role of Autophagy Activation

Autophagy is a major catabolic process used by cells to remove superfluous or damaged proteins and organelles. In the final stages of autophagy, acidic organelles (lysosomes) act to degrade autophagic cargo and to facilitate their recycling. Little is known about how cancer cells undergoing autophagy, often as a consequence of stress, respond when lysosomal function is blocked. To elucidate this mechanism, several recent studies report that lysosomes and their hydrolytic proteases (cathepsins) play a critical role in autophagy and subsequent cancer progression. Our studies in breast cancer suggest that inhibition of cathepsins D and L using the BH3-mimetic, obatoclax, is effective in reducing the cell density of anti-estrogen sensitive and resistant breast cancer cells. Furthermore, blockage of cathepsin protein expression with obatoclax leads to the accumulation of autophagic vacuoles and impairs the ability of cells to use degraded material to restore homeostasis. While cancer cells are dependent on effective lysosomal function, neoplastic transformation induces changes in lysosomal volume, number, and protease activity. Recent reports suggest that pro-oncogenic changes render cancer cells more susceptible to lysosomal-associated death pathways. A number of distinct stimuli have been shown to permeabilize the lysosomal membrane, leading to the release of hydrolases into the cytosol and, ultimately, cell death. Thus, changes in cathepsin and lysosomal membrane permeabilization (LMP) regulation during cancer cell progression suggest that strategies targeting this cellular compartment may be exploited to improve outcomes for cancer patients.

Abstract 1677: Inhibition of autophagy proteins ATG7 and BECN1 correlates with increased interferon regulatory factor-1 (IRF1) protein expression through STAT1 phosphorylation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Approximately two-thirds of newly diagnosed invasive breast tumors express the estrogen receptor-α protein (ER+) and most will be treated with an endocrine therapy such as an antiestrogen or aromatase inhibitor. Despite the widespread clinical efficacy of antiestrogens in the treatment of ER+ breast cancers, approximately half of these women will exhibit de novo or acquired resistance to endocrine therapies. One mechanism by which breast cancer cells evade antiestrogen-induced cell death is through stimulation of autophagy. Autophagy is a catabolic process in which cells digest subcellular organelles in an attempt to maintain homeostasis. Evidence for increased autophagy has been shown to protect cancer cells against various therapies, including radiotherapy and the antiestrogen, tamoxifen. Previous studies from our lab have also established interferon regulatory factor-1 (IRF1) as a tumor suppressor gene and mediator of antiestrogen sensitivity. High mRNA levels of IRF1 in breast cancer core biopsies correlated significantly with a pathologic complete response after neoadjuvant systemic treatment. However, the link between autophagy and this immune function gene is largely unknown. Here, we show that reduced expression of autophagy-related protein, ATG7, correlates with increased nuclear IRF1 protein in 107 human breast cancer core biopsies. Knockout of a single ATG7 allele in mice induced IRF1 activation in both mammary and spleen tissue compared to wild-type mice. In antiestrogen sensitive and resistant breast cancer cells, knockdown of ATG7 and BECN1, but not ATG5, using siRNA resulted in increased IRF1 protein expression. We go on to show that increased IRF1 expression is an effect of increased phosphorylation of STAT1, suggesting that autophagy inhibits the interferon signaling pathway. Using the contrary approach, we found that inhibition of IRF1 using siRNA inhibits the lipidation of LC3 and autophagosome formation. Furthermore, antiestrogen sensitive LCC1 cells become resistant to the growth inhibitory effects of faslodex (Fulvestrant; ICI182780;ICI), 3-methyladenine, and doxorubicin following knockdown of IRF1. Overexpression of IRF1 in antiestrogen resistant LCC9 cells inhibits ATG7 protein expression and induces mitochondrial outer membrane permeability. Thus, the disruption of autophagy machinery in breast cancer cells activates the interferon signaling pathway to induce apoptosis and breast cancer cell death. Citation Format: Jessica L. Schwartz-Roberts, Katherine L. Cook, Ayesha N. Shajahan, Margaret Axelrod, Anni Warri, Robert Clarke. Inhibition of autophagy proteins ATG7 and BECN1 correlates with increased interferon regulatory factor-1 (IRF1) protein expression through STAT1 phosphorylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2013-1677