Mitogen activated protein kinases (MAPKs) are key players in relaying extracellular signals to the cell nucleus and are often overexpressed in aggressive and endocrine-resistant breast cancers. Our previous studies (Madak-Erdogan et al. Mol Cell Biol 31:226,2011) revealed that one such kinase, ERK2, was recruited to distal enhancers of estradiol(E2)-regulated genes together with estrogen receptor alpha (ERα) and collaborated with ERα to regulate gene expression and cell proliferation programs in response to hormone. In the current study, we identified ERK5, a close relative of ERK2, as a factor that is recruited to the transcription start site (TSS) of E2-stimulated genes. Use of dominant negative and constitutively active forms, as well as small molecule inhibitors of ERK5 and MEK5, revealed that activation of ERK5 by upstream kinase MEK5 and activity of ERK5 itself was required for recruitment of the kinase to the chromatin. Immunofluorescence studies demonstrated that ERK5 localized to transcription factories in the nucleus upon cell treatment with E2. Inhibitors of ERK5 completely abrogated E2-dependent cell proliferation and colony formation for all of the ERα-positive breast cancer cell lines studied. In contrast to what we observed in ERα-positive cells, in ERα-negative breast cancer cell lines we did not detect any nuclear ERK5; instead, we found ERK5 localized to the actin cytoskeleton, especially to sites where substantial remodeling takes place. Upon expression of ERα in these ERα-negative cells, we observed re-localization of ERK5 to the nucleus and a decrease in actin reorganization associated with reduced cell motility and invasion. Abrogation of ERα expression using siRNA in ERα-positive cell lines caused loss of nuclear ERK5 and relocalized this kinase to the actin cytoskeleton. Our studies with tamoxifen resistant cell lines revealed that ERK5 localization to the nucleus was reduced and localization to the actin cytoskeleton increased as resistance progressed, suggesting a correlation between ERα activity, ERK5 localization and motility of breast cancer cells. Our data reveal that ERα elicits nuclear localization of ERK5, diminishing ERK5 localization to regions of high actin remodeling, thereby possibly accounting for the generally lower metastatic potential that is characteristic of many ERα-positive breast cancer cells. (Supported by grants T32 ES007326 and P50 AT006268 from NIH, and a grant from The Breast Cancer Research Foundation.) Citation Format: Zeynep Madak Erdogan, Anna Bergamaschi, Rosa Ventrella, Hailing Lu, Benita S. Katzenellenbogen. Estrogen receptor-α dictates the subcellular localization of ERK5 to control differential proliferation and invasion programs of breast cancer cells. [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 1316. doi:10.1158/1538-7445.AM2013-1316
Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to prevent or delay recurrence of ERα (+) tumors. The objective of this study was to elucidate the role of mitochondrial pathways that are activated in the presence of single therapies and undermine therapy effectiveness in ER (+) breast cancers. In our previous studies, we identified Exportin 1 (XPO1), a nuclear export protein, as an important player in endocrine resistance progression. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using a combination of transcriptomics, kinase arrays, metabolomics and metabolic flux experiments, we identified glutamine metabolism pathways to be rewired during endocrine resistance. In limited media conditions mimicking nutrient deprived tumor microenvironment, endocrine resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine. The effect of glutamine was dependent on conversion of the glutamine to glutamate and mitochondrial complex 1 activity. In order to examine metabolites that might result in the observed phenotype we performed GC/MS whole metabolite profiling and identified aminoacid metabolism pathways to be upregulated when cells were treated with SXR. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine-resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα (+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences. Citation Format: Eylem Kulkoyluoglu Cotul, Brandi Patrice Smith, Hua Chang, Yosef Landesman, Kevin Duong, Zeynep Madak Erdogan. Role of mitochondria and glutamine metabolism in therapy resistance: Combined targeting of estrogen receptor alpha and exportin 1 in endocrine resistant breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1013.
Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing a potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis, with a 5-year relative overall survival rate of just 24% for patients with metastatic disease. Previously, we described an increased reliance on NAD+ metabolism in metastatic breast cancer (MBC) cells, which are driven by clonal selection from monotherapy resistance and the metastatic organ microenvironment. In the current study, we investigated if inhibition of NAMPT to block NAD+ production would synergize with standard of care therapies for ER+ MBC. Combination therapy of the NAMPT Inhibitor, KPT9274 and the ERα agonist Fulvestrant (Fulv) synergistically reduced tumor burden in liver metastasis in vitro (combination index < 1 in cell viability assays) and in vivo in mouse xenograft models. A synergistic effect was not observed when KPT-9274 was combined with Palbociclib or Tamoxifen, nor Fulv with other metabolic inhibitors. RNA-Seq analysis showed that NAMPT inhibition restored antiestrogenic activity of Fulv. Metabolomics analysis showed that NAMPT inhibition reduced abundance of metabolites associated with glucose and lipid metabolism. Mechanistically, chromatin immunoprecipitation showed that NAMPT is recruited to ERα binding sites in MBC xenografts and PDX tumors. Targeting metabolic adaptations in endocrine-resistant metastatic breast tumors is a novel approach that could lead to new combined therapies that reduce mortality. Citation Format: Ayca N. Mogol, Christine Chien, Qianying Zuo, Akshara S. Raghavendra, Debu Tripathy, Yosef Landesman, Zeynep Madak Erdogan. ERα and NAMPT crosstalk at enhancers drives Fulvestrant resistance in metastatic breast tumors [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 2698.
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