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The organization of cell cytoskeleton is altered in events of epithelial to mesenchymal transition (EMT), promotion of cell motility, and cancer metastases. EMT is associated with decreased cell-cell adhesion, downregulation of epithelial markers like E-Cadherin, cytokeratins, and occludins, and upregulation of mesenchymal markers such as N-cadherin, vimentin, and various transcription factors such as slug and ZEB. Epithelial to mesenchymal transition is also a consequence of drug resistance and is responsible for cancer metastases. Triple negative breast cancer is highly aggressive cancer and patients show poor prognosis and disease-free survival due to the lack of targeted therapy. Mitogen activated protein kinase pathway, including extracellular activated kinase ERK1/2 and ERK5, and phosphoinositide 3-kinase (PI3K) pathway are known to alter the cytoskeleton through the downstream activation of oncogenes such as FRA-1 and loss of focal adhesions. Of these pathways, the MEK5-ERK5 pathway is understudied in triple negative breast cancer TNBC, and there are few research tools available to selectively inhibit this pathway. The diphenylamine analogs were derived from the parent molecule Mekinist, a FDA approved MEK1/2 inhibitor for melanoma, and modified to gain selectivity towards MEK5. SC-1-151, a type-III allosteric inhibitor of MEK5 is a dual MEK1/2 (98.6%) and MEK5 (59%) inhibitor; the molecule inhibits cell viability and colony formation, and attenuates tumor growth. SC-1-151 was serendipitously identified as a mesenchymal to epithelial transition activator in TNBC cell line MDA-MB-231. E-cadherin protein expression and cell morphology were examined to study MET after the treatment of MDA-MB-231 cells with different structural analogs of SC-1-151 after treatment for 5 days. The compound was further found to induce E-cadherin expression and epithelial phenotype in tamoxifen resistant estrogen positive MCF-7 cell line that underwent EMT. The compound is identified to promote this activity by targeting at least the ERK-FRA1-ZEB1 axis. Alkyl or N-Methyl piperazine substituents on the amide of ring 1 produced similar result as SC-1-151, and substituting the amide group with acid or ester also induced MET. In contrast, ortho-fluoro, para-iodo functional groups of the arene ring 2, when replaced with a meta-bromo substituent did not induce MET. We aim to test the compounds on EGF treated MDA-MB-468 cells to observe the attenuation of EGF induced EMT. Future studies will be performed to determine the specific protein interactions of the promising compounds. Citation Format: Bhatt AB, Wright TD, Anna K, Gupta M, Chakrabarty S, Flaherty PT, Hoang V, Burow M, Cavanaugh JE. Study of diphenylamine analogs as inducers of mesenchymal to epithelial transition in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-07.

Triple negative breast cancers (TNBCs) are a clinically and biologically aggressive breast cancer (BC) subtype; TNBC tumors have higher rates of metastasis, relapse and acquired/inherent drug resistance. Incidence and mortality rates of TNBC are stratified based on patient ethnicity - patients with African ancestry have higher mortality rates and diagnoses of invasive cancers compared to patients representing other ethnicities. Louisiana has a high proportion of African-American residents (32.7% in 2018), and New Orleans has among the highest incidences of TNBC in the country. Many of our patients present with TNBC tumors that are partially or completely resistant to neoadjuvant chemotherapies. There are currently no clinically approved targeted therapies for TNBC. Current therapeutic discovery focused TNBC research does not aptly address the knowledge gap regarding ethnic disparity in TNBC incidence/mortality rates and TNBC biology. To date, most TNBC-related research and knowledge has been acquired from Caucasian patients, although patients with African and Hispanic ancestries represent the majority of TNBC cases. Patient-derived xenografts (PDXs) are extensively used in BC research, as they mimic complex microanatomy, oncoarchitecture, and cell-cell/cell-stroma interactions of tumors. Here, we demonstrated the unique composition of PDX tumors is not dramatically affected by serial transplantation in mice, based on molecular phenotypes (examined using qRT-PCR and RNA sequencing) and the oncoarchitecture of the extracellular matrix (based on cryogenic scanning electron microscopy). Using these models in basic research facilitates translation of laboratory findings to the clinical setting, and dramatically enhanced drug discovery research. We have established over twelve TNBC PDX models, 90% of which represent patients of African ancestry, and most of which are resistant to neoadjuvant regimens. We focus on dissecting and evaluating kinase inhibitor/targeted drug response to various individual components (tumor cell biology, stroma, immune, extracellular matrix) of chemotherapy resistant TNBC tumors. Histone deacetylase inhibitors (DACi) are a promising therapeutic agent in TNBC systems; they have been shown to suppress tumorigenesis and metastasis in TNBC through suppression of the mesenchymal phenotype in cell line-based studies. In this study we utilized various TNBC PDX models (TU-BcX-2K1, -2O0, 4IC, -4M4, -4QAN, -4QX) to assess these findings in more translational systems. Interestingly, we showed that DACi effect on tumorigenesis and metastasis varied depending on specific TNBC PDXs utilized. These data implicate specific genes/signaling pathways exist in individual patient tumors that can predict tumor responsiveness to DACi. Preliminary data using the NCI oncology drug set implicated the MEK1/2 pathway contributed to sensitization of TNBC cells. Furthermore, we found a disconnect in gene expressions that were previously shown to be affected by DACi therapy (CDH1, VIM, ZEB1, ZEB2) in various derivations of PDX models (cells, PDX-Os, ex vivo, in vivo). These findings demonstrate that testing various derivations of PDX models is crucial to parsing out specific mechanisms of targeted therapies. Our methods presented here to assess targeted drug response and drug resistance using PDX models can be applied to any area of cancer research and is not limited to breast cancer. Citation Format: Margarite Matossian, Steven Elliott, Maryl Wright, Tiffany Chang, Madlin Alzoubi, Henri Wathieu, Rachel Sabol, Alex Alfortish, Hope Burks, Van Hoang, Deniz Ucar, Gabrielle Windsor, Thomas Yan, Jovanny Zabaleta, Fokhrul Hossain, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven Jones, Elizabeth Martin, Lucio Miele, Bridgette Collins-Burow, Matthew Burow. Effect of histone deacetylase inhibitors on patient-derived neoadjuvant chemotherapy resistant triple negative breast cancer xenografts that represent understudied patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-17.

The transcriptional regulator FUBP1 (Far Upstream Element Binding Protein 1) acts as an oncoprotein in hepatocellular carcinoma (HCC) and is important for hematopoietic stem cell (HSC) self-renewal and erythroid maturation in mice. In this study, we investigated the transcriptional network by which FUBP1 controls hematopoiesis and elucidated the relevance of FUBP1 for human erythropoiesis. Furthermore, we shed light on the role of FUBP1 in leukemia initiating cells. Searching for upstream-regulators of FUBP1, we identified E-boxes as potential TAL1 binding sites in the FUBP1 promoter. Indeed, we demonstrated the regulation of FUBP1 expression by TAL1 in human primary CD34+ donor cells. In chromatin immunoprecipitation (ChIP) experiments, the binding of TAL1 to the FUBP1 promoter increased during erythroid differentiation, correlating with up-regulated FUBP1 and TAL1 expression. Activation of the FUBP1 promoter by TAL1 binding was confirmed in luciferase assays. We observed a reduction in erythroid colony-forming units and glycophorin A positive cells derived from erythroid differentiated human CD34+ cells upon knockdown of FUBP1, supporting the hypothesis that FUBP1 is required for efficient erythropoiesis. In the transduction/transplantation leukemia mouse models for BCR-ABL1+ CML and MLL-AF9+ AML, we observed that Fubp1 knockdown resulted in reduced total cell and progenitor cell numbers. In CML, Fubp1 knockdown cells showed lower cell cycle activity and increased apoptosis. Consistently, CML and AML mice transplanted with Fubp1 knockdown cells survived longer than control mice that received transduced bone marrow expressing wildtype FUBP1 levels. Furthermore, pharmacological treatment of AML mice with the FUBP1 inhibitor irinotecan prolonged their survival significantly as a single drug or in combination with Ara-C. Analysis of FUBP1 expression in bone sections derived from CML and AML patients, and from healthy donors by immunohistochemistry showed no increased FUBP1 expression in leukemic samples, but we noticed a shorter overall survival in those AML patients with strong FUBP1 expression. In CML patients, FUBP1 levels correlate with the disease stage. Thus, elevated expression of FUBP1 might be an indicator for the aggressiveness of leukemia. Our data identify TAL1 as an FUBP1 upstream-regulator and confirm the importance of FUBP1 for HSC self-renewal and erythroid maturation, not only in murine but also in human cells. Furthermore, FUBP1 acts as an oncogenic factor in leukemia. Our findings might provide important evidence for the potential use of FUBP1 in clinical settings, e.g. as a molecular target for the treatment of leukemia patients and as a modulator for the production of red cells. Citation Format: Marlene Steiner, Van T. Hoang, Jasmin Yillah, Katharina Gerlach, Jörn Lausen, Hans-Michael Kvasnicka, Thomas Oellerich, Hanibal Bohnenberger, Daniela Krause, Martin Zörnig. The role of FUBP1 in the hematopoietic system and leukemia [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 1500.

Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors and high invasive potential. TNBC cells express high levels of the mesenchymal markers vimentin, fibronectin, N-cadherin, and cadherin11, which are involved in cell invasion and metastasis, and low levels of the epithelial markers E-cadherin, occludins and cytokeratins, which are involved in cellular contact and stability of the cell membrane. Mitogen activated protein kinases, including ERK5 and ERK1/2 are important pro-survival proteins and are known to be upregulated in most cancers. Of these pathways, the MEK5-ERK5 pathway is understudied in triple negative breast cancer, and there are few research tools available to selectively inhibit this pathway. The diphenylamine moiety was derived from the parent molecule Trametinib, a FDA approved MEK1/2 inhibitor for melanoma. Structural modifications were made on the lead molecule to gain selectivity towards MEK. SC-1-151, one of the compounds from this diphenylamine series was identified as a dual MEK1/2 (98.6% inhibition) and MEK5 (59% inhibition) inhibitor. Additionally, we have shown that SC-1-151 attenuates tumor growth in severe combined immunodeficient (SCID) xenograft mice and causes a mesenchymal to epithelial transition (MET) in MDA-MB-231 TNBC cells. The goal of this research is to determine the structural features of diphenylamines that are responsible for MET. E-cadherin and cadherin-11 protein expression and cell morphology were examined to study MET after the treatment of MDA-MB-231 cells with different structural analogs of SC-1-151 for 5 days. Alkyl or N-Methyl piperazine substituents on the amide of ring 1 produced similar result as SC-1-151, and substituting the amide group with acid or ester also induced MET. In contrast, ortho-fluoro, para-iodo functional groups of the arene ring 2, when replaced with a meta-bromo substituent did not induce MET. Therefore, our data suggest the necessary functional groups for inducing MET include the hydrogen atom on the amine linkage, and the iodine atom on the arene ring 2. Future studies will be performed to determine the specific protein interactions of the promising compounds. Citation Format: Akshita B. Bhatt, Thomas D. Wright, Katie Anna, Mohit Gupta, Patrick Flaherty, Van Hoang, Matthew Burow, Jane E. Cavanaugh. Structure-activity relationship of diphenylamine derivatives to target epithelial to mesenchymal transition in triple negative breast cancer [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 4204.

Overall, triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype, which has higher rates of metastasis and recurrence. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2, c-FOS, SNAI1, ZEB1) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes. Citation Format: Matossian M, Burks H, Bowles A, Sabol R, Hoang V, Elliott S, Bunnell B, Zuercher W, Drewry D, Wells C, Alfortish A, Lee S, Hartono A, Jones S, Moroz K, Zea A, Burow M, Collins-Burow B. Patient-derived triple negative breast cancer xenografts as a translational model to screen for novel kinase pathways [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-05.

Triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. The claudin-low molecular subtype has higher rates of metastases and recurrence. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes. Citation Format: Margarite D. Matossian, Hope E. Burks, Annie C. Bowles, Rachel A. Sabol, Van T. Hoang, Bruce Bunnell, William J. Zuercher, David H. Drewry, Carrow Wells, Krzysztof Moroz, Matthew E. Burow, Bridgette Collins-Burow. Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1117. doi:10.1158/1538-7445.AM2017-1117

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with higher rates of metastasis and recurrence than other classified subtypes. The decreased survival rates seen in TNBC can be attributed in part to the lack of targeted therapeutics. HDAC inhibitors (HDACi) have emerged as a potential adjuvant therapy for patients with triple negative disease. Previously, our lab has identified Panobinostat, a pan-DAC inhibitor, as a successful therapy for the reversal of the metastatic phenotype in TNBC. However, this therapy lacks specificity and may have increased side effects as a result. In this study, we aim to dissect the necessary HDACs responsible for driving and maintaining the metastatic phenotype of TNBC, with the goal of informing specific HDAC targeted therapies. Here we show that Romidepsin, an HDAC 1 and 2 specific inhibitor, is capable of recapitulating aspects of Panobinostat treatment in TNBC both in vitro and in a patient derived xenograft model in vivo. To parse the necessity and role of HDAC1 and HDAC2 individually, we created knockdown cell lines and analyzed them for changes in gene expression changes and their respective associated biological processes, including proliferation, migration, mammosphere formation, tumor growth and metastasis. We found that HDAC2 knockdown was able to significantly repress migration and proliferation in TNBC cells and their associated gene expression signatures in vitro. Furthermore, HDAC2 knockdown resulted in decreased tumor growth and metastasis an in vivo mouse model. These results illustrate the efficacy of Romidepsin in inhibition of oncogenic processes and implicate HDAC2 as a potential target for therapeutic intervention in TNBC. Citation Format: Hope E. Burks, Steven Elliott, Van T. Hoang, Margarite D. Matossian, Bridgette Collins Burow, Matthew E. Burow. Inhibition of HDAC2, but not HDAC1, abrogates triple negative breast cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5079. doi:10.1158/1538-7445.AM2017-5079

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 MAPK/extracellular signal-regulated kinases (MEK) pathways has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). Here we proposed to investigate dual inhibition of MEK1/2 and MEK5 as a more efficacious method for intervention to target mesenchymal and highly metastatic breast cancer cells than MEK1/2 or MEK5 alone through the use of a novel pan-MEK inhibitor SC-151. Interestingly, TNBC cells demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET) and exhibited a significant decrease in migration potential following pan-MEK inhibition. Additionally, immuno-compromised mice inoculated with MDA-MB-231 cells and treated with SC-151 demonstrated decreased tumor volumes compared to vehicle-treated animals. To parse the roles of MEK1/2 and MEK5 in EMT and tumorigenesis, we used the CRISPR/Cas9 approach to knock out ERK5 expression in the TNBC cell line MDA-MB-231. Similar to biological changes induced by pan-MEK inhibition, loss of ERK5 promoted epithelial characteristics in TNBC cells at the morphological and molecular level and impaired tumor formation in vivo. Treatment of ERK5-ko cells with SC-151 further enhanced these effects in vitro, suggesting that MEK1/2 and MEK5 play distinct roles in maintaining the mesenchymal phenotype. Further analysis revealed that constitutive activation of MEK5 abrogated the effects of SC-151 on the reversal of EMT, highlighting the requirement for MEK5 inhibition in MET induction. Taken together, these findings show that while the MEK5-ERK5 pathway may be sufficient in EMT regulation, MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Thus, dual MEK inhibition exerts optimal effects in the reversal of EMT. These data present a novel compound and viable therapeutic strategy to target both MEK1/2 and MEK5 in phenotypically mesenchymal and clinically aggressive breast cancer cells, warranting further investigation into mechanisms by which MEK1/2 and MEK5 individually modulate the EMT axis. Additionally, as MEK inhibition has been shown to sensitize resistant cancer cells to targeted therapies, synergistic and sensitizing effects of SC-151 combined with inhibitors of alternative signaling pathways as well as kinases upstream of MEK will be examined. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Hope E. Burks, Margarite Matossian, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Lowry Curley, Muralidharan Anbalagan, Brian G. Rowan, Doug Chrisey, Jane E. Cavanaugh, Patrick T. Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1596.

Triple negative breast cancer (TNBC) is associated with poor survival, metastatic recurrence, increased mortality, and has few viable therapeutic options. Given the high mortality associated with metastasis of this disease, discovery of therapeutics targeting the metastasis axis is imperative. Kinases have been demonstrated to have an essential role in regulation of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Elucidating specific signaling pathways that promote EMT has the potential to provide novel targeting strategies in the treatment of TNBC. Here we demonstrate a partially non-selective polo-like kinase 1 (PLK1) inhibitor, GSK346294, is capable of EMT reversal in phenotypically mesenchymal TNBC cell lines, as evidenced by enhanced E-cadherin expression and loss of cellular migration potential. Structurally analogous but highly specific PLK inhibitors did not exhibit similar EMT changes, which led us to investigate off-target kinases inhibited by GSK346294. Follow-up kinase profiling studies revealed members of the NEver-in-mitosis-related Kinase family (NEK5 and NEK9) as additional kinases inhibited by GSK346294, leading us to investigate their individual roles in the progression of EMT. NEK5 overexpression in a non-invasive breast cancer cell line increased both migration and invasion in trans-well assays. Additionally, enhanced NEK5 expression altered the TNBC gene expression profile, enhancing expression of genes associated with growth factor signaling (MET, EGFR) and the mesenchymal gene signature (SLUG, VIM). To date, no studies exist which delineate the regulatory roles of NEK5 and NEK9 in EMT or cellular invasion/motility. Further investigation into the function of NEK5 and/or NEK9 in the metastatic progression may provide novel therapeutic targets for the treatment of TNBC, the implication of which could impact the treatment and management of neoplastic disease and metastasis beyond breast cancer. Citation Format: Margarite D. Matossian, Steven Elliott, Van T. Hoang, Hope E. Burks, Theresa B. Phamudy, Doug Chrisey, William J. Zuercher, David H. Drewry, Carrow Wells, Bridgette Collins-Burow, Matthew E. Burow. Targeting an unconventional kinase-invasion axis in breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 684.

The mitogen-activated protein kinase (MAPK) pathway has well-established roles in cellular processes including proliferation, differentiation, and regulation of cell fate, namely survival and apoptosis. In breast cancer, constitutive activation of the MAPK/extracellular signal-regulated kinases (ERK) pathways have been linked to chemoresistance and metastatic progression through distinct mechanisms including the activation of epithelial-to-mesenchymal transition (EMT). Our previous studies have shown that overexpression of MEK5 promotes EMT markers and induces the progression to a mesenchymal phenotype. Here, we tested the effects of a novel MEK1/2 and MEK5 inhibitor, SC-1-151, and other known MAPK signaling inhibitors (PD184,352 (MEK1/2), AZD6244 (MEK1/2), BIRB796 (p38)) on a panel of mesenchymal and highly metastatic breast cancer cell lines. While the MEK1/2 and p38 inhibitors decreased cell viability across cell lines, only the dual inhibition of MEK1/2 and MEK5 though the use of SC-1-151 demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET). Furthermore, the cells exhibited a significant decrease in migration potential following SC-1-151 treatment. Further analysis of the effects of SC-1-151 in the triple-negative breast cancer cell lines revealed an alteration of the genes associated with EMT, notably a decrease in expression of Fra-1, a transcription factor downstream of MAPK. Immuno-compromised mice inoculated with the MDA-MD-231 cell line and treated with SC-1-151 demonstrated decreased tumor volumes compared to vehicle-treated animals at day 30 post cell injection, implicating the role of MEK inhibition on tumorigenesis. These data demonstrate the need for a better understanding of the dual role of MEK1/2 and MEK5 signaling in breast cancer, and suggest that inhibition of the MEK1/2 and MEK5 signaling pathways leads to a decrease in EMT and cell migration. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Henry C. Segar, Hope Burks, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Doug Chrisey, Jane E. Cavanaugh, Patrick Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Dual role of MEK1/2 and MEK5 in the reversal of epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2014-1052

The basal sub-type, which shares features with triple-negative breast cancer (TNBC), is among the most lethal breast cancer subtype, characterized by a highly aggressive and metastatic phenotype. Although pathways that may represent targets for novel therapeutic intervention for basal like breast cancer (BLBC) have begun to be elucidated, the ability to define and selectively target the invasive and metastatic phenotype of basal-type/TNBC remains a major challenge facing the breast cancer field. Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is a known tumor suppressor in many cancers including breast. Low LKB1expression has been observed in breast cancer patients and we report a significant association between loss of LKB1 expression and poor prognosis specifically in the basal sub-type of breast cancer. Induction of LKB1 expression in BLBC cell lines inhibited invasiveness in vitro as well as lung and brain metastatic burden in an orthotopic xenograft tumor model. Further analysis of BLBC cell lines overexpressing LKB1 by next generation sequencing (RNA-seq) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extra cellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). We further demonstrated marked inhibition of matrix metalloproteinase 1 (MMP-1) expression and activity via regulation AP-1 family member cJun. Additionally, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating a reversal of the EMT phenotype in a triple-negative breast cancer cell line MDA-MB-231. We have demonstrated a role for LKB1 expression in the regulation of cell invasion and metastasis in addition to tumorigenesis. Taken together these data support future development of therapeutic agents to induce the LKB1 signaling pathway in BLCB/triple-negative breast cancer. Citation Format: Lyndsay V. Rhodes, Chandra R. Tate, Hope E. Burks, Van T. Hoang, Diari Gilliam, Elizabeth C. Martin, Steven Elliott, David FB Miller, Aaron Buechlein, Douglas Rusch, Haixu Tang, Kenneth P. Nephew, Matthew E. Burow, Bridgette M. Collins-Burow. The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2014-1571

While the precise molecular mechanisms underlying metastasis remain unclear, epithelial-to-mesenchymal transition (EMT), the loss of an epithelial cell phenotype and acquisition of a mesenchymal cell phenotype, has been implicated in cancer cell invasion and dissemination. The ZEB family of transcription factors, which includes ZEB1 and ZEB2, has been demonstrated to mediate this transition by downregulating the expression of genes associated with an epithelial phenotype. We sought to investigate the effects of direct ZEB family overexpression on EMT in estrogen receptor-positive (ER+) breast cancer cell systems. We overexpressed ZEB1 or ZEB2 in the epithelial, ER+, luminal A breast cancer cell lines MCF-7 and ZR75. Overexpression of individual ZEB1 and ZEB2 levels were confirmed and localization of the ZEB factors to the nucleus was confirmed by confocal microscopy in both cell lines. ZEB2 overexpressing cells, but not ZEB1 overexpressing cells, showed increased migration and invasion in vitro compared to the vector control in both MCF-7 and ZR75 cell lines, suggesting differential function of the two ZEB family members. Additionally, MCF-7-ZEB2 xenografts exhibited increased lung metastasis compared to MCF-7-vector cells. To elucidate the effects of ZEB on our ER+ cell line we performed next generation deep sequencing on MCF-7 -vector, ZEB1 and ZEB2 overexpressing cells. Analysis of total gene regulation using the NCI Pathway Interaction Database demonstrated an increase in genes associated with RhoA activity, an important mediator of cell motility, in ZEB2 overexpressing cells. However, the ZEB overexpressing cells show no change in morphology and canonical EMT markers remained unchanged between cell lines, suggesting a potential post-translational modification affecting ZEB1 and ZEB2 function. Together these results indicate that ZEB factors drive motility in breast cancer cells but are incapable of promoting a complete EMT in ER+ cells, warranting further investigation into the mechanisms involved in ZEB action. Elucidating the pathways involved in ZEB family function is an important step in understanding the mechanisms underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay Rhodes, Elizabeth Martin, Theresa Phamduy, Steven Elliot, Van Hoang, Henry Segar, Aaron Buechlein, Douglas Rusch, Dave Miller, Melody Baddoo, Erik Flemington, Kenneth Nephew, Douglas Chrisey, Bridgette Collins-Burow, Matthew Burow. ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2014-1034