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.

The c-Jun N-terminal kinases, JNK1/2 are members of the mitogen activated protein kinase (MAPK) family that can mediate inflammatory responses, cell differentiation, cell proliferation, and/or cell death. In breast cancer, JNK signaling has been associated with increased proliferation, angiogenesis, and tumor metastasis. Also, inhibition of JNK has been shown to promote tumor cytostasis, but this inhibition also attenuates the cytotoxic effects of certain chemotherapeutics, calling into question the therapeutic potential of JNK inhibitors. In this study, we hypothesized that JNK1/2 inhibition induced autophagy, as a mechanism of protection against chemotherapy- and endocrine-induced apoptosis. Autophagy is a normal physiologic process required for the elimination of damaged mitochondria and misfolded proteins that can be induced in cancer cells under certain stresses such as hypoxia and drug insult to provide protection from death. To characterize the role of JNK1/2 in autophagy, we utilized two ERα expressing human breast cancer cell lines, T47-D and MCF-7. Autophagy flux experiments were performed in which the steady state levels of the autophagy protein LC3 II were analyzed in control cells versus cells undergoing JNK1/2 inhibition with the non-reversible JNK-IN8 inhibitor. JNK-IN8 was used as a single agent or in combination with estradiol and/or antiestrogen treatment. These studies reproducibly identified JNK1/2 inhibition as a mechanism of autophagy activation. The induction of autophagy correlated with sustained inhibition of proliferation as determined by cell counts, MTT, and clonogenic assays, and induction of senescence (in T47-D cells) as determined by the expression of β-galactosidase. In contrast, induction of apoptosis was not a major outcome of JNK1/2 inhibition. These pre-clinical studies provide strong evidence that JNK1/2 is an attractive molecular target to improve the treatment of endocrine responsive breast cancer, but they also emphasize that cells are surviving by autophagy and/or senescence. Because cell survival by autophagy and/or senescence could ultimately lead to breast cancer relapse, ongoing studies are addressing approaches to effectively kill these surviving cell populations. Citation Format: Kyler Herrington, Carol Joseph, Samar Abdulmoneim, Allison Lewis, Jingwen Cai, Hannah Youngblood, Yutao Liu, Patricia Schoenlein. Targeting JNK1/2 induces a potent cytostatic response in estrogen receptor expressing breast cancer cells, with induction of autophagy and senescence as measurable outcomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1231.

Antiestrogen resistance is still a major clinical challenge preventing the eradication of estrogen receptor positive (ER+) breast cancer. A major goal of our laboratory is to identify targeted therapies that can be combined with antiestrogen treatment to block the emergence of antiestrogen resistant breast cancer. Toward this goal, we previously determined that the catabolic process of autophagy facilitates the emergence of antiestrogen resistance (Sammadar et al., Molecular Cancer Ther. 9, 2008) and identified cathepsin L as a key lysosomal protease required for antiestrogen induced pro-survival autophagy ER+ MCF-7cells (Periyasamy-Thandavan et al., Autophagy. 6:19-35, 2010). Thus, we hypothesize that blocking cathepsin L expression / activity is one approach to targeting pro-survival autophagy during antiestrogen treatment of breast cancer. In support of this hypothesis, we now show that antiestrogen resistant TR5 cells show increased levels of active cathepsin L compared to the levels in the parent antiestrogen sensitive MCF-7 cells. The upregulation of cathepsin L expression is consistent with the fact that TR5 cells utilized autophagy to survive long-term 4-hydroxytamoxifen (4-OHT) selection. Withdrawal of 4-OHT selection during TR5 passage does not lead to a reduction in cathepsin L levels, suggesting that the increased expression of cathepsin L is a stable genetic or epigenetic alteration. Elevated expression of active (phosphorylated) MAPK1/2 is also present in TR5 cells and targeting MEK1/MAPK1/2 with the selective inhibitor U0126 consistently reduced the levels of active cathepsin L. We further established a key role for MEK1/MAPK1/2 in the regulation of cathepsin L in the parent MCF-7 cells by performing cathepsin L activity assays. Cells treated with 4-OHT showed increased cathepsin L activity compared to E2-treated control cells; whereas, cathepsin L activity in cells treated with 4-OHT + UO126 was significantly reduced compared to the levels in 4-OHT-treated cells. This reduction in cathepsin L by U0126-mediated blockade of MEK1/MAPK1/2 correlated directly to “impaired” autophagic flux and increased BimEL-dependent apoptosis in the antiestrogen-treated cell populations and was mediated, at least in part, via transcriptional regulation of cathepsin L as determined by quantitative PCR. Further, MEK1/MAPK1/2 up-regulation of cathepsin L is selective, as cathepsin B activity is not reduced by MEK1 targeting. These studies provide evidence that the targeting MEK1/MAPK1/2 in combination with antiestrogen treatment has the potential to reduce cathepsin-L mediated pro-survival autophagy in ER+ breast cancer. Citation Format: Annie Liu, Carol Joseph, Jesse Wayson, Timothy Summers, Haifeng Cai, Patricia V. Schoenlein. MEK1:MAPK1/2 targeting attenuates pro-survival autophagy and enhances antiestrogen-induced apoptosis in breast cancer cells via a cathepsin L-dependent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1337.

No abstract