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This study identifies macroautophagy as a key mechanism of cell survival in estrogen receptor–positive (ER+) breast cancer cells undergoing treatment with 4-hydroxytamoxifen (4-OHT). This selective ER modifier is an active metabolite of tamoxifen commonly used for the treatment of breast cancer. Our study provides the following key findings: (a) only 20% to 25% of breast cancer cells treated with 4-OHT in vitro die via caspase-dependent cell death; more typically, the antiestrogen-treated ER+ breast cancer cells express increased levels of macroautophagy and are viable; (b) 4-OHT–induced cell death, but not 4-OHT–induced macroautophagy, can be blocked by the pan-caspase inhibitor z-VAD-fmk, providing strong evidence that these two outcomes of antiestrogen treatment are not linked in an obligatory manner; (c) 4-OHT–resistant cells selected from ER+ breast cancer cells show an increased ability to undergo antiestrogen-induced macroautophagy without induction of caspase-dependent cell death; and (d) 4-OHT, when used in combination with inhibitors of autophagosome function, induces robust, caspase-dependent apoptosis of ER+, 4-OHT–resistant breast cancer cells. To our knowledge, these studies provide the first evidence that macroautophagy plays a critical role in the development of antiestrogen resistance. We propose that targeting autophagosome function will improve the efficacy of hormonal treatment of ER+ breast cancer. [Mol Cancer Ther 2008;7(9):2977–87]

Supplementary Fig. S1 from A role for macroautophagy in protection against 4-hydroxytamoxifen–induced cell death and the development of antiestrogen resistance

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL‑dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro‑apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro‑apoptotic action in an antiestrogen‑resistant breast cancer cell model. In addition, the present study identified a pro‑survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen‑resistant breast cancer cells survive EGFR targeting by pro‑survival autophagy. These pre‑clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro‑survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL‑dependent apoptosis.

EGFR is upregulated commonly in multiple cancer types, including breast cancer. However, the multiple role(s) of EGFR in breast cancer are not fully elucidated. In this study, we characterized a unique antiestrogen resistant breast cancer subline and provide compelling data that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitated escape from senescence, autophagy, and BimEL-dependent apoptosis. Our studies were conducted with the antiestrogen resistant TR5 cell line established by our laboratory, using a step-wise drug selection with the antiestrogen 4-hydroxytamoxifen (4-OHT) of the estrogen receptor α (ER+) MCF-7 breast cancer cells. A preliminary microarray comparing mRNA expression between 4-OHT-selected TR5 cells and MCF-7 parent cells identified upregulation of the epidermal growth factor receptor (EGFR). Western blot showed a greater than 5-fold upregulation of EGFR expression and downregulation of estrogen receptor alpha (ERα) compared to the levels in MCF-7 and T47-D (antiestrogen sensitive). Selective inhibition of EGFR, phosphorylated at Tyr 1068, was achieved with erlotinib. Inhibition blocked MEK1/MAPK1/2 signaling, which was also upregulated in 4-OHT selected TR5 cells, with detectable increases in the pro-apoptotic BH3 protein BimEL, and a fifty percent reduction in cell viability. However, a high number of cells survived erlotinib therapy due to autophagy induction determined to be cytoprotective. Autophagy induction was identified by analyzing the expression/turnover of autophagy proteins LC3-II and p62, a standard approach to ascertain autophagy levels in cells. Further, erlotinib treatment induced a 2-fold increase in the number of senescent cells in the surviving population. Senescent cells were identified by β-galactosidase staining of erlotinib-treated cells utilizing confocal microscopy. Interestingly, blockade of autophagy with the lysosomotrophic compound chloroquine enhanced the senescence cell population, suggesting that either autophagy blocked senescence induction or lysosome impairment increased senescence. Mechanistically, we have identified phosphorylated AMP-activated protein kinase (pAMPK) as being elevated under conditions of erlotinib treatment, and reduced in 4-OHT-selected TR5 cells relative to MCF-7 parent cells. This correlation supports the hypothesis that pAMPK was downregulated during 4-OHT selection of TR5 cells by elevated EGFR/MEK/MAPK1/2 signaling to allow surviving cells escape senescence induction and/or anti-proliferative autophagy. Overall we propose that EGFR mediated downregulation of pAMPK is a major regulatory pathway in breast cancer cells surviving endocrine treatment. Our current studies are dissecting the role of EGFR-mediated AMPK regulation with somatic cell-based studies and pharmacological intervention. Citation Format: Mackenzie L. Hagan, Michael McGrath, Carol Joseph, Allison Lewis, John R. Barrett, Muthusamy Thangaraju, David Gewirtz, Patricia V. Schoenlein. EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB133.

Our long-term goal is to elucidate the role of JNK/Jun signaling in breast cancer cell autophagy induced by anti-estrogen treatment and/or chemotherapy in ERα expressing and TNBC cells, respectively. In this study, we analyzed the role of JNK/Jun signaling in regulating the high basal autophagy levels in TNBC. To do so, we utilized the highly metastatic murine 4T1 and the human MDA-MB-231 TNBC models. In addition, we performed autophagic flux assays after treating breast cancer cells with JNK-IN8, a non-reversible small molecule JNK1/2 inhibitor. Treatments were performed for various times in the presence and absence of chloroquine (CQ) to allow analysis of steady state levels of atg8 (LC3-II) and sequestosome-1 (p62). These LC3-II and p62 proteins are involved in autophagosome formation and function, and are catabolized during autolysosomal turnover. To impair this process, CQ is a lysosomotropic agent that raises the pH of the lysosome and blocks such turnover. We also determined the effect of JNK-IN-8 on 4T1 and MDA-MB-231 cell number (proliferation). These studies determined that JNK-IN8 was an effective inhibitor of autophagic flux in the TNBC cells when used at concentrations that effectively blocked cJun phosphorylation, the downstream readout of JNK-IN8 efficacy used for our experiments. Blockade of autophagic flux by JNK-1/2 inhibition in TNBC was in stark contrast to results obtained with JNK-IN-8 treatment of ERα expressing MCF-7 and T47-D breast cancer cells, in which JNK-IN-8 induced a robust autophagy response with elevations in the steady state levels and flux of LC3-II and p62. Induction of autophagy by JNK-IN-8 in ERα expressing breast cancer cells occurred concomitantly with a substantial reduction in cell proliferation but minimal induction of apoptosis. Overall, these pre-clinical in vitro studies support the following conclusions: (1) JNK1/2 targeting in TNBC effectively blocks autophagic flux and may be particularly effective when used in combination with chemotherapies that induce pro-survival autophagy which is a current focus of our studies; and (2) JNK1/2 targeting in ERα expressing breast cancers has the potential to mediate a pro-survival autophagic activity in response to endocrine therapy or chemotherapy and should be cautiously approached. Ongoing studies are aimed at understanding the underlying mechanisms of the differential effects of JNK/Jun signaling on autophagy in TNBC versus ERα expressing breast cancer. Such an understanding should provide important information to aid in the ongoing interest in utilizing JNK1/2 as a molecular target for improved treatment of TNBC. Citation Format: Michael K. McGrath, McKenzie L. Hagan, Timmothy Summers, Jesse Wayson, Carol Joseph, Amanda C. Barrett, John T. Barrett, Patricia V. Schoenlein. Effects of JNK/Jun blockade on breast cancer cell autophagy are dependent on breast cancer subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3006.