Abstract Background: Often, prolonged use of tamoxifen, an FDA approved SERM to treat ER+ breast cancer (BC), has untoward effects on the uterus and can lead to tamoxifen resistance. Novel tamoxifen-melatonin drug conjugates, C1-C5, with varying CH2- spacer lengths were developed (US Patent No. 8,785,501 B2) to offset tamoxifen-mediated tumor resistance and uterotropic actions. Two candidate compounds, C4 and C5, demonstrated anti-BC actions in MCF7, TNBC, and tamoxifen-resistant MCF7 cells (doi:10.1124/mol.119.116202). Methods: The goal of this study was to evaluate C4- and C5-mediated actions on estrogen resistant BC in vivo and ESR1 levels in the uterus in vivo using SCID/BEIGE mouse xenografts and western blot analyses. Results: Using CCK-8 assays, C4- and C5-mediated potency and efficacy to inhibit proliferation of ESR1 mutant (MCF7-luc Y537S, MCF7-luc D538G, MCF7-E380Q) and parental (MCF7-luc parental, MCF7-PE parental) lines revealed MCF7-lucD538G to be most sensitive to C4 (IC50= 3.8μM; 43% inhibition) and C5 (IC50=1.2μM; 26% inhibition). Preliminary testing using MCF7-lucD538G or parental cells bilaterally implanted into fat pads of SCID/BEIGE mice given additional estrogen or not demonstrated 60% to 90% greater tumor volumes compared to parental controls by 3 weeks, suggesting estrogen-independence. MCF7-luc D538G cells (5x106), were bilaterally implanted in the mammary pads of SCID/BEIGE mice and grown for 28 days in the absence of estrogen to allow for tumor formation. Next, treatments with C4 or C5 (1mg/kg/mouse/day, sc,), DMSO (10% in HP-β-CD), tamoxifen (5mg 60-day release pellet implanted), fulvestrant (200mg/kg/week, sc) began and continued for 28 days. Significant tumor inhibition occurred with C4 and C5 vs DMSO- and tamoxifen-treated mice; and similar tumor-inhibiting effects were observed when compared to fulvestrant (% change from baseline: DMSO=211%; tamoxifen=169%; C4=134%; C5=95%; fulvestrant=96%, n=5/group). Analysis of ESR1 levels in tumor and uterine tissue demonstrated increases in levels in tamoxifen-treated mice and no changes in C4-, C5- and fulvestrant-treated mice vs control. ESR1 stability assays were conducted in wildtype MCF7 cells exposed to vehicle (DMSO), and 10μM each of tamoxifen, C4 or C5 for 24h, followed by a withdrawal period (0, 6, 12, 24h) using media containing cycloheximide (100μg/mL). Increases in the half-life (T1/2) of ESR1 were observed in cells exposed to tamoxifen (T1/2=13.2h) or C4 (T1/2=12.6h) while decreases were observed in MCF7 cells exposed to C5 (T1/2=5.1h) compared to DMSO-treated (T1/2=9.6h) and no treatment cells ((T1/2=7.4h). Conclusion: Our findings indicate that tamoxifen-melatonin drug conjugates may be a viable BC treatment option for estrogen-resistant and potentially other resistant cancers while offering uterine protection. Citation Format: Asef Faruk, Sophie Dietrich, Yong Myoung, Afsana Jahan, Mohamed Marzouk, Jane E. Cavanaugh, Simak Ali, Matthew Burow, Darius Zlotos, Paula Witt-Enderby. Anti-tumor actions of novel tamoxifen-melatonin drug conjugates on estrogen-resistant tumors and actions on tumor-and uterine-expressed ESR1 in SCID_BEIGE mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2296.

Glioblastoma (GBM) is the most aggressive and prevalent primary brain tumor in adults, characterized by rapid growth, diffuse infiltration, and a dismal prognosis. Despite advances in conventional therapies, the median survival remains approximately one year, emphasizing the urgent need for novel therapeutic strategies. GD2, a disialoganglioside overexpressed in several malignancies, has been implicated in tumorigenesis and metastasis and has been identified as a cancer stem cell marker. While previous reports have identified high levels of GD2 expression in gliomas compared to normal brain tissue, its role in GBM stemness remains controversial. In this study, we revisited prior findings refuting GD2 ' s involvement in GBM stemness by replicating key tumorigenesis experiments and further explored its impact on stemness properties such as migration and metabolic plasticity. Additionally, a phytochemical screen was used to identify natural compounds as potential inhibitors targeting GD2-mediated tumorigenesis. Our findings aim to clarify GD2 ' s role in GBM and provide insights into novel therapeutic interventions.

Marine cyanobacteria have gained momentum in recent years as a source of novel bioactive small molecules. This paper describes the structure elucidation and pharmacological evaluation of two new (veraguamide O (1) and veraguamide P (2)) and one known (veraguamide C (3)) analogs isolated from a cyanobacterial collection made in the Las Perlas Archipelago of Panama. We hypothesized that these compounds would be cytotoxic in cancer cell lines. The compounds were screened against HEK-293, estrogen receptor positive (MCF-7), and triple-negative breast cancer (MDA-MB-231) cells as well as against a broad panel of membrane-bound receptors. The planar structures were determined based on NMR and MS data along with a comparison to previously isolated veraguamide analogs. Phylogenetic analysis of the collection suggests it to be an Okeania sp., a similar species to the cyanobacterium reported to produce other veraguamides. Veraguamide O shows no cytotoxicity (greater than 100 μM) against ER-positive cells (MCF-7) with 13 μM IC50 against MDA-MB-231 TNBC cells. Interestingly, these compounds show affinity for the sigma2/TMEM-97 receptor, making them potential leads for the development of non-toxic sigma 2 targeting ligands.

Abstract Epithelial to mesenchymal transition (EMT) is a biological process through which epithelial cells undergo cytoskeletal changes and transform into a mesenchymal phenotype. This phenotypical transformation leads to enhanced migratory capabilities and increased resistance to apoptosis and drug therapies. The EMT characterized in part by an increase in vimentin, a mesenchymal marker, and a decrease in E-cadherin, an epithelial marker, plays a significant role in cancer metastasis and progression. The MEK5/ERK5 pathway is essential in regulating cell survival, proliferation, and migration. Inhibition of the MEK5/ERK5 pathway by small molecule inhibitors decreases cellular proliferation and impedes migration of cancer cells, including triple negative breast cancer (TNBCs) cells. A type III allosteric inhibitor of MEK5 designed by our collaborators reverses the EMT and induces a mesenchymal to epithelial transition (MET). Using a TNBC cell line, MDA-MB-231, genetically engineered with RFP tagged vimentin, we established a cellular assay to evaluate analogues of our lead type III inhibitor to further investigate the role of the MEK5-ERK pathway in the EMT, design optimal compounds that induce an MET, and explore structure-activity relationships for kinase inhibition. This research will help identify targets for EMT and optimize compound modification to improve potency and efficiency. Citation Format: Asef Faruk, Saloni Patel, Ramez Hallak, Patrick Flaherty, Jane Cavanaugh. Evaluation of natural and synthetic compounds on epithelial to mesenchymal transition in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A008.

Marine cyanobacteria are a rich source of bio-active metabolites that have been utilized as leads for drug discovery and pharmacological tools for basic science research. Here, we describe the re-isolation of a well-known metabolite, barbamide, from Curaçao on three different occasions and the characterization of barbamide’s biological interactions with targets of the mammalian nervous system. Barbamide was originally discovered as a molluscicidal agent from a filamentous marine cyanobacterium. In our hands, we found little evidence of toxicity against mammalian cell cultures. However, barbamide showed several affinities when screened for binding affinity for a panel of 45 receptors and transporters known to be involved in nociception and sensory neuron activity. We found high levels of binding affinity for the dopamine transporter, the kappa opioid receptor, and the sigma receptors (sigma-1 and sigma-2 also known as transmembrane protein 97; TMEM97). We tested barbamide in vitro in isolated sensory neurons from female mice to explore its functional impact on calcium flux in these cells. Barbamide by itself had no observable impact on calcium flux. However, barbamide enhanced the effect of the TRPV1 agonist capsaicin and enhanced store-operated calcium entry (SOCE) responses after depletion of intracellular calcium. Overall, these results demonstrate the biological potential of barbamide at sensory neurons with implications for future drug development projects surrounding this molecule.

Data openly available in a public repository that issues datasets with DOIs.

Substance Use Disorders (SUDs) constitute a national public health crisis resulting in nearly 91,000 overdose related deaths annually. Negative attitudes and stigma within the general population, as well as healthcare professionals, towards individuals with SUDs present a barrier to identification and treatment. SUD related training programs exert a positive influence on the attitudes of health care professionals. To evaluate the impact of a SUDs training seminar on the attitudes and perceptions of occupational therapy, pharmacy, and physician assistant students towards individuals with SUDs, as well as the perceived value of interprofessional education and collaboration. Two standardized assessments were utilized to determine the course impact on knowledge and attitudes regarding interprofessional teams and stigma related to SUDs after completion of a 2-day interprofessional seminar. Analysis of the survey responses revealed statistically significant changes in all factors related to participants’ values and understanding of Interprofessional Education Collaborative (IPEC) compentencies as well as many of the factors regarding attitudes towards SUDs and their treatment. The activities incorporated in the two-day seminar were effective in exerting statistically significant changes in student knowledge of interprofessional teams and attitudes towards SUDs.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be a promising therapeutic molecule for treating Parkinson's disease (PD). However, because of the failure of GDNF infusion in clinical trials, due to its poor bio-distribution, there was a need to develop other molecules with similar properties to GDNF that have improved bioavailability and administration. Dopamine neuron stimulating peptide-11 (DNSP-11) is a synthetic, amidated 11-amino acid neuroactive peptide derived from the human proGDNF domain. DNSP-11 has been shown to protect dopaminergic cells in vitro and restore dopaminergic activity in vivo. Therefore, the objective of our study was to test if DNSP-11 protects human dopaminergic neuroblastoma SH-SY5Y cells against 6-OHDA induced toxicity and to elucidate the protective mechanism of action. DNSP-11 reduced the 6-OHDA-induced increase in caspase-3/7 activity in SH-SY5Y cells at early time points, indicating possible protection against apoptosis. DNSP-11 also activated the cell survival signaling pathways, ERK1, 2, and 5 in SH-SY5Y cells. In addition, a single injection of DNSP-11 in adult rat striatum leads to modulation of these ERK proteins in the substantia nigra. Overall, the results indicate that DNSP-11 protects human dopaminergic neuroblastoma cells against apoptotic cell death and activates neuroprotective ERK signaling pathways, suggesting its potential therapeutic role in Parkinson's disease (PD).

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.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options. A defining feature of TNBC is the propensity to metastasize and acquire resistance to cytotoxic agents. Mitogen activated protein kinase (MAPK) and extracellular regulated kinase (ERK) signaling pathways have integral roles in cancer development and progression. While MEK5/ERK5 signaling drives mesenchymal and migratory cell phenotypes in breast cancer, the specific mechanisms underlying these actions remain under-characterized. To elucidate the mechanisms through which MEK5 regulates the mesenchymal and migratory phenotype, we generated stably transfected constitutively active MEK5 (MEK5-ca) TNBC cells. Downstream signaling pathways and candidate targets of MEK5-ca cells were based on RNA sequencing and confirmed using qPCR and Western blot analyses. MEK5 activation drove a mesenchymal cell phenotype independent of cell proliferation effects. Transwell migration assays demonstrated MEK5 activation significantly increased breast cancer cell migration. In this study, we provide supporting evidence that MEK5 functions through FRA-1 to regulate the mesenchymal and migratory phenotype in TNBC.

Extracellular signal-regulated kinase (ERK5) is an essential regulator of cancer progression, tumor relapse, and poor patient survival. Epithelial to mesenchymal transition (EMT) is a complex oncogenic process, which drives cell invasion, stemness, and metastases. Activators of ERK5, including mitogen-activated protein kinase 5 (MEK5), tumor necrosis factor α (TNF-α), and transforming growth factor-β (TGF-β), are known to induce EMT and metastases in breast, lung, colorectal, and other cancers. Several downstream targets of the ERK5 pathway, such as myocyte-specific enhancer factor 2c (MEF2C), activator protein-1 (AP-1), focal adhesion kinase (FAK), and c-Myc, play a critical role in the regulation of EMT transcription factors SNAIL, SLUG, and β-catenin. Moreover, ERK5 activation increases the release of extracellular matrix metalloproteinases (MMPs), facilitating breakdown of the extracellular matrix (ECM) and local tumor invasion. Targeting the ERK5 signaling pathway using small molecule inhibitors, microRNAs, and knockdown approaches decreases EMT, cell invasion, and metastases via several mechanisms. The focus of the current review is to highlight the mechanisms which are known to mediate cancer EMT via ERK5 signaling. Several therapeutic approaches that can be undertaken to target the ERK5 pathway and inhibit or reverse EMT and metastases are discussed.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

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.

Conventional mitogen-activated protein kinase (MAPK) family members regulate diverse cellular processes involved in tumor initiation and progression, yet the role of ERK5 in cancer biology is not fully understood. Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. ERK5 signaling contributes to drug resistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). More recently a role for ERK5 in regulation of the extracellular matrix (ECM) has been proposed, and here we investigated the necessity of ERK5 in TNBC tumor formation. Depletion of ERK5 expression using the CRISPR/Cas9 system in MDA-MB-231 and Hs-578T cells resulted in loss of mesenchymal features, as observed through gene expression profile and cell morphology, and suppressed TNBC cell migration. In vivo xenograft experiments revealed ERK5 knockout disrupted tumor growth kinetics, which was restored using high concentration Matrigel™ and ERK5-ko reduced expression of the angiogenesis marker CD31. These findings implicated a role for ERK5 in the extracellular matrix (ECM) and matrix integrity. RNA-sequencing analyses demonstrated downregulation of matrix-associated genes, integrins, and pro-angiogenic factors in ERK5-ko cells. Tissue decellularization combined with cryo-SEM and interrogation of biomechanical properties revealed that ERK5-ko resulted in loss of key ECM fiber alignment and mechanosensing capabilities in breast cancer xenografts compared to parental wild-type cells. In this study, we identified a novel role for ERK5 in tumor growth kinetics through modulation of the ECM and angiogenesis axis in breast cancer.

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

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

Epithelial to mesenchymal transition (EMT) is a cellular program that converts non-motile epithelial cells into invasive mesenchymal cells. EMT is implicated in cancer metastasis, chemo-resistance, cancer progression, and generation of cancer stem cells (CSCs). Inducing mesenchymal to epithelial transition (MET), the reverse phenomenon of EMT, is proposed as a novel strategy to target triple negative and tamoxifen-resistant breast cancer. Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors, a highly invasive mesenchymal phenotype, and a lack of targeted therapy. Estrogen receptor-positive breast cancer can be targeted by tamoxifen, an ER antagonist. However, these cells undergo EMT over the course of treatment and develop resistance. Thus, there is an urgent need to develop therapeutic interventions to target these aggressive cancers. In this study, we examined the role of novel diphenylamine analogs in converting the mesenchymal phenotype of MDA-MB-231 TNBC cells to a lesser aggressive epithelial phenotype. Using analog-based drug design, a series of diphenylamine analogs were synthesized and initially evaluated for their effect on E-cadherin protein expression and changes incell morphology, which was quantified by measuring the spindle index (SI) value. Selected compound 1 from this series increases the expression of E-cadherin, a primary marker for epithelial cells, and decreases the mesenchymal markers SOX2, ZEB1, Snail, and vimentin. The increase in epithelial markers and the decrease in mesenchymal markers are consistent with a phenotypic switch from spindle-like morphology to cobblestone-like morphology. Furthermore, Compound 1 decreases spheroid viability, cell migration, and cell proliferation in triple negative BT-549 and tamoxifen-resistant MCF-7 breast cancer cells.

Epithelial to mesenchymal transition is an important cellular adaptation that helps cancer cells acquire a spindle-like phenotype from a cuboidal phenotype, degrade the extracellular matrix, invade the neighboring tissues, and metastasize to other organs and form secondary tumor. Cellular plasticity is governed by growth factors that act in a paracrine manner to activate downstream oncogenes and regulate the activity of epigenetic factors, which facilitate phenotypic switch from epithelial to mesenchymal, ultimately leading to increased cell migration and invasion. The intracellular phosphorylation cascade that is downstream of growth factor receptors plays an important role in transmitting the signal from the extracellular environment into the nucleus, thereby completing the loop required to elicit a cellular response. Triple negative breast cancer (TNBC), characterized by loss of hormone receptors is a highly aggressive form of cancer and patients show poor prognosis and disease-free survival due to lack of targeted therapy. In contrast, estrogen positive breast cancer can be targeted by estrogen receptor antagonists or CDK4/6 inhibitors, but drug resistance and relapse is often associated with epithelial to mesenchymal transition and poor patient outcome. Hence, targeting the mesenchymal phenotype with small molecule inhibitors is an emerging strategy to attenuate the invasive and aggressive nature of cancer cells. The MEK5-ERK5 pathway is understudied in triple negative breast cancer and there are few research tools available to selectively inhibit this pathway. Diphenylamine derivatives, synthesized as putative MEK5 inhibitors from parent MEK1/2 inhibitor trametinib, are effective in inducing mesenchymal to epithelial transition in MDA-MB-231 triple negative breast cancer cells, as indicated by an increase in E-cadherin expression, which is a marker of epithelial phenotype, and a decrease in spindle index, an important cell shape determinant. Compound 1, a dual MEK1/2 (98%) and MEK5 (59%) inhibitor was further characterized in functional assays; Compound 1 was found to significantly inhibit cell viability, proliferation, migration, spheroid viability, and colony formation in MDA-MB-231 cells. Compound 1 is effective in reversing the mesenchymal phenotype of MDA-MB-231, BT-549, and tamoxifen resistant-MCF-7 breast cancer cells. Signaling crosstalk and drug resistance in cancer limits the applicability of monotherapy. Our current work is focused on treating diverse breast cancer cells with combination of novel compound 1 and paclitaxel, ipatasertib, JQ-1, and LBH589 to induce synthetic lethality at lower concentration, restore drug sensitivity, and enhance selectivity. Citation Format: Akshita B. Bhatt, Thomas D. Wright, Katie Anna, Mohit Gupta, Suravi Chakrabarty, Van Hoang, Matthew Burow, Patrick T. Flaherty, Jane E. Cavanaugh. Novel diphenylamine analogs induce mesenchymal to epithelial transition and enhance the sensitivity of breast cancer cells to conventional chemotherapeutic agents [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 1879.

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.