The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box bind-ing homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast can-cers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemother-apeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies. In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT mark-ers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.

Abstract Extracellular signal-regulated kinase (ERK) 5, a member of mitogen activated protein kinase (MAPK) family, is an emerging target in cancer therapeutics. Activation of ERK5 via overexpression induces EMT and hormone-independent growth of breast cancer. EMT leads to the loss of cell polarity, downregulation of E-cadherin, and upregulation of mesenchymal markers snail, zinc-finger E-box binding homeobox (ZEB1), and vimentin. EMT is also associated with drug resistance. Although ERK1/2 and ERK5 activation is known to mediate EMT, the effect of ERK1/2 and ERK5 inhibition on mesenchymal to epithelial transition (MET), the reverse of EMT, is poorly understood in cancer.Triple negative breast cancer (TNBC) cells have a mesenchymal phenotype and show poor sensitivity to chemotherapy agents. The loss of estrogen, progesterone hormone receptors and human epidermal growth factor receptors (HER2) contributes to the aggressive state of the disease and lack of targeted therapy. Activation of the intracellular signaling pathways such as the MAPK pathway mediates tumorigenesis in TNBCs. MEK1/2 inhibitors have been successful clinical drug candidates; however, there is emerging evidence that the activation of the MEK5-ERK5 pathway mediates resistance to the MEK1/2 inhibitors in several BRAF and KRAS-mutant cancers. The effect of MEK1/2 inhibition on MEK5-mediated EMT, cell survival, and migration in TNBC is less well understood. Therefore, we hypothesize that dual inhibition of the ERK1/2 and ERK5 pathways is a relevant strategy to target TNBCs.In the present study, the effect of dual ERK1/2 and ERK5 inhibition on MET, cell viability, migration, and anchorage-independent growth was evaluated in TNBC. ERK1/2 and ERK5 activities were modulated via pharmacological inhibitors and molecular tools. Cell morphology and protein expression of EMT markers E-cadherin, ZEB1, snail, and vimentin were evaluated. XMD8-92, an ERK5 inhibitor was found to synergize with doxorubicin in lung and cervical cancer cells. Therefore, the effect of dual ERK1/2 and ERK5 on doxorubicin sensitivity was evaluated. Citation Format: Akshita Bhatt, Thomas Wright, Van Barnes, Suravi Chakrabarty, Patrick Flaherty, Matthew Burow, Jane Cavanaugh. Targeting the ERK5 and ERK1/2 pathways simultaneously induces mesenchymal to epithelial transition in TNBC [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 5042.

Triple negative breast cancer is characterized by the loss of hormone receptors and lack of targeted therapy. Most invasive cancers, including triple negative (TNBC) breast cancer have a mesenchymal phenotype, which is associated with increased chemoresistance. Activation of the MEK1/2 and MEK5 pathways plays a crucial role in the activation of the epithelial to mesenchymal transition program and increases the survival, proliferation, and migration of the cancer cells. Disruption of actin skeleton via ras and src mediated activation of extracellular regulated kinase 1/2 (ERK1/2) and ERK5 is reported, indicating their role in oncogenic transformation. Moreover, inhibition of either pathway results in a compensatory increase in the PI3K/AKT pathway. These crosstalk mechanisms are involved in mediating therapeutic drug resistance. MDA-MB-231, a BRAF and KRAS mutant TNBC cell line has more than 90% of high CD44+/CD24-/low stem cell population, and high ERK5 and ERK1/2 expression; hence it was used as the model for our experiments. Moreover, MDA-MB-231-ERK5-KO cells were utilized to confirm the mechanism of action. To target TNBC, known inhibitors of the MEK1/2 pathway: trametinib, an FDA approved drug for BRAF mutant melanoma, VX-11-e, an ERK2 inhibitor, and XMD-8-92, an ERK5 inhibitor were used in combination with the chemotherapeutic drugs paclitaxel and doxorubicin to examine cell viability. We have shown that dual inhibition of the ERK5 and AKT signaling pathways synergistically reduces TNBC cell viability and enhances sensitivity of the cells to paclitaxel. A series of novel quinazoline derivatives was generated to dually target the ERK5 and the AKT pathway. The effect of novel quinazolines on cell viability in combination with the chemotherapeutic agent paclitaxel was examined in the TNBC cells and some encouraging results were obtained. The overall significance of this research is to enhance the anti-cancer activity of chemotherapeutic agents and reduce off-target toxicity by dose-reduction strategy. Citation Format: Akshita B. Bhatt, Thomas D. Wright, Saloni Patel, Suravi Chakrabarty, Van Barnes, Matthew Burow, Patrick T. Flaherty, Jane Cavanaugh. Inhibition of the MAPK pathways enhances the sensitivity of triple negative breast cancer cells to chemotherapeutic drugs [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B078. doi:10.1158/1535-7163.TARG-19-B078

Abstract Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem cell-like (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling, particularly Notch1, is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch directly have been impaired by the systemic toxicity of the Gamma Secretase Inhibitors (GSI) used, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that target Notch signaling in breast cancer cells indirectly and selectively are a potentially attractive strategy. However, no such target has been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening method, we have identified a small molecule compound that targets MAP2K5 (MEK5) and decreases phosphorylation of MAPK7 (ERK5). Expression of ERK5 is associated with poor prognosis in TNBC. Consistent with ERK5KO cells, suppression of ERK5 phosphorylation decreased the amount of Notch1 and Jagged1 protein and mRNAs. More importantly, a selective MEK5 inhibitor, SC-181, reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells without suppressing T-cell proliferation. Treatment with nanomolar concentration of this compound decreased the number and size of mammospheres in a dose- dependent manner. Our preliminary results suggest that targeting the MEK5-ERK5 pathway is a promising strategy to selectively target Notch signaling in TNBC CSC without systemic Notch inhibition. Citation Format: Deniz A. Ucar-Bilyeu, Margarite D. MATOSSIAN, VAN Hoang Barnes, Fokhrul M. Hossain, Mohit Gupta, HOPE E. BURKS, THOMAS D. WRIGHT, Jane Cavanaugh, Patrick Flaherty, Matthew E. Burow, Lucio Miele. Targeting notch one notch above [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 967.

Many receptor‐mediated cellular processes progress through mitogen‐activated protein kinase (MAPK) signaling pathways, which are initiated by activation of a surface receptor and ultimately lead to a change in cell physiology. Previous research has shown that the influence of melatonin on osteoblast activity is mediated in part through MAPK/ERK kinases (MEK) 1/2 and MEK5. Here we explore the possible effects of MEK 1/2 and MEK5 disruption on the skeletal structure in rodents with diurnal melatonin rhythms. To investigate the influence of MAPK signaling on bone mineral density and trabecular bone phenotypes, one group of SCID mice (SC; n = 4) was injected daily for a period of 30 days with a dual MEK1/2 and MEK5 inhibitor, SC‐1‐151. A second control group (n = 4) was injected daily with DMSO vehicle for the same period. At the end of the experimental period mice were euthanized, and tibiae were dissected free of soft tissues and fixed in formalin. Tibia were scanned at 9.61 μm 3 voxel size for subsequent morphometric analyses. Regions of interest were identified on the scans and then analyzed for trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), bone volume fraction (BV/TV), and cortical bone mineral density (BMD) using ImageJ software. Resulting bone phenotypes were analyzed using Bayesian linear models. We found that the SC group showed credibly decreased BV/TV compared to the DMSO controls (Pr[SC < DMSO] = 0.83). However, comparisons of Tb.Th, Tb.Sp, and BMD indicated minimal credible differences between the SC and DMSO treatment groups. Overall, our results suggest that, while previous research has shown that disruption of the MAPK signaling pathway may have a negative effect on osteoblast activity, these effects may result in only subtle changes for trabecular bone phenotypes and no change in cortical bone mineralization. This project provides preliminary results for a larger‐scale project investigating the role of melatonin, MEK1/2, and MEK5 in bone formation. Support or Funding Information Research support: University of Missouri School of Medicine, National Institute of Arthritis and Musculoskeletal and Skin Diseases This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .