Publications by authors named "Margarite D Matossian"

21 Publications

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ZEB2 regulates endocrine therapy sensitivity and metastasis in luminal a breast cancer cells through a non-canonical mechanism.

Breast Cancer Res Treat 2021 Aug 6;189(1):25-37. Epub 2021 Jul 6.

Department of Medicine, Section of Hematology & Medical Oncology, Tulane University Health Sciences Center, New Orleans, LA, 70112, USA.

Purpose: The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated.

Methods: In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17β-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response.

Results: Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17β-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells.

Conclusion: ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.
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http://dx.doi.org/10.1007/s10549-021-06256-xDOI Listing
August 2021

NEK5 activity regulates the mesenchymal and migratory phenotype in breast cancer cells.

Breast Cancer Res Treat 2021 Aug 1;189(1):49-61. Epub 2021 Jul 1.

Department of Medicine, Tulane University School of Medicine, New Orleans, LA, USA.

Purpose: Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the field of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains understudied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer.

Methods: Stably overexpressing NEK5 cell lines (MCF7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immunofluorescence and quantification of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis.

Results: NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type-specific manner, respectively.

Conclusion: While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating specific steps of metastatic progression.
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http://dx.doi.org/10.1007/s10549-021-06295-4DOI Listing
August 2021

Constitutive activation of MEK5 promotes a mesenchymal and migratory cell phenotype in triple negative breast cancer.

Oncoscience 2021 18;8:64-71. Epub 2021 May 18.

Department of Medicine, Division of Hematology and Oncology, Tulane University, New Orleans, LA 70118, USA.

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.
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http://dx.doi.org/10.18632/oncoscience.535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131078PMC
May 2021

Dual inhibition of MEK1/2 and MEK5 suppresses the EMT/migration axis in triple-negative breast cancer through FRA-1 regulation.

J Cell Biochem 2021 Aug 20;122(8):835-850. Epub 2021 Apr 20.

Department of Medicine, Section of Hematology and Oncology, Tulane University School of Medicine, New Orleans, Louisiana, USA.

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.
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http://dx.doi.org/10.1002/jcb.29916DOI Listing
August 2021

Quantifying Breast Cancer-Driven Fiber Alignment and Collagen Deposition in Primary Human Breast Tissue.

Front Bioeng Biotechnol 2021 15;9:618448. Epub 2021 Mar 15.

Department of Surgery, Louisiana State University Health Sciences Center New Orleans, New Orleans, LA, United States.

Solid tumor progression is significantly influenced by interactions between cancer cells and the surrounding extracellular matrix (ECM). Specifically, the cancer cell-driven changes to ECM fiber alignment and collagen deposition impact tumor growth and metastasis. Current methods of quantifying these processes are incomplete, require simple or artificial matrixes, rely on uncommon imaging techniques, preclude the use of biological and technical replicates, require destruction of the tissue, or are prone to segmentation errors. We present a set of methodological solutions to these shortcomings that were developed to quantify these processes in cultured, human breast tissue under the influence of breast cancer cells and allow for the study of ECM in primary breast tumors. Herein, we describe a method of quantifying fiber alignment that can analyze complex native ECM from scanning electron micrographs that does not preclude the use of replicates and a high-throughput mechanism of quantifying collagen content that is non-destructive. The use of these methods accurately recapitulated cancer cell-driven changes in fiber alignment and collagen deposition observed by visual inspection. Additionally, these methods successfully identified increased fiber alignment in primary human breast tumors when compared to human breast tissue and increased collagen deposition in lobular breast cancer when compared to ductal breast cancer. The successful quantification of fiber alignment and collagen deposition using these methods encourages their use for future studies of ECM dysregulation in human solid tumors.
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http://dx.doi.org/10.3389/fbioe.2021.618448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006399PMC
March 2021

Patient-Derived Xenografts as an Innovative Surrogate Tumor Model for the Investigation of Health Disparities in Triple Negative Breast Cancer.

Womens Health Rep (New Rochelle) 2020 24;1(1):383-392. Epub 2020 Sep 24.

Section of Hematology and Oncology, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.

Despite a decline in overall incidence rates for cancer in the past decade, due in part to impressive advancements in both diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer-related deaths in women. BC alone accounts for ∼30% of all new cancer diagnoses in women worldwide. Triple-negative BC (TNBC), defined as having no expression of the estrogen or progesterone receptors and no amplification of the HER2 receptor, is a subtype of BC that does not benefit from the use of estrogen receptor-targeting or HER2-targeting therapies. Differences in socioeconomic factors and cell intrinsic and extrinsic characteristics have been demonstrated in Black and White TNBC patient tumors. The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with TNBC has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established TNBC PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. The primary aim of this review is to emphasize the importance of using clinically relevant translatable tumor models that reflect TNBC human tumor biology and heterogeneity in high-risk patient populations. The focus is to highlight the complexity of BC as it specifically relates to the management of TNBC in Black women. We discuss the importance of utilizing PDX models to study the extracellular matrix (ECM), and the distinct differences in ECM composition and biophysical properties in Black and White women. Finally, we demonstrate the crucial importance of PDX models toward novel drug discovery in this patient population.
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http://dx.doi.org/10.1089/whr.2020.0037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784803PMC
September 2020

Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant-resistant phenotype in breast cancer cells.

Oncol Lett 2021 May 16;21(5):380. Epub 2021 Mar 16.

Department of Medicine, Section of Hematology and Oncology, Tulane University School of Medicine, New Orleans, LA 70112, USA.

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. 'Hits' or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the 'active' inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.
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http://dx.doi.org/10.3892/ol.2021.12641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988660PMC
May 2021

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer.

Transl Oncol 2021 Jun 21;14(6):101046. Epub 2021 Mar 21.

Department of Pharmacology, School of Pharmacy, Duquesne University, Pittsburgh, PA 15219, USA. Electronic address:

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 binding 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 cancers (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 chemotherapeutic 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 markers 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.
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http://dx.doi.org/10.1016/j.tranon.2021.101046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020482PMC
June 2021

Targeting Never-In-Mitosis-A Related Kinase 5 in Cancer: A Review.

Curr Med Chem 2021 Mar 21. Epub 2021 Mar 21.

Tulane University School of Medicine, Department of Pharmacology, New Orleans LA. United States.

Mitotic kinases have integral roles in cell processes responsible for cancer development and progression in all tumor types and are common targets for therapeutics. However, a large subset of the human kinome remains unexplored with respect to functionality in cancer systems. Within the mitotic kinases, the never-in-mitosis kinase (NEK) family, are emerging as novel kinase targets in various cancer types. NEK5 is an understudied member of the NEK family. While there are more recent studies describing physiologic function of NEK5, its role in cancer biology remains widely understudied. However, emerging studies implicate NEK5 has potentially crucial functions in various solid tumors. In this review we discuss current knowledge regarding a role for NEK5 in cancer, and implications of NEK5 expression and activity in tumor development and metastasis. We summarize current studies that examine NEK5 activity in diverse cancer systems and cellular processes. As an understudied kinase, there are currently no selective NEK5-targeting agents to test effects of pharmacologic inhibition in cancer, although there exist recent advancements in this area. Here we also include an update on efforts to develop selective pharmacologic inhibition of NEK5 and we discuss the current direction of NEK5-targeting therapeutic development. Generation of selective NEK5 inhibitors are promising new targeted therapies for cancer growth and metastasis.
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http://dx.doi.org/10.2174/0929867328666210322101749DOI Listing
March 2021

Molecular Mechanisms of Epithelial to Mesenchymal Transition Regulated by ERK5 Signaling.

Biomolecules 2021 01 29;11(2). Epub 2021 Jan 29.

Department of Pharmacology, School of Pharmacy, Duquesne University, Pittsburgh, PA 15282, USA.

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.
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http://dx.doi.org/10.3390/biom11020183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911413PMC
January 2021

Evaluation of deacetylase inhibition in metaplastic breast carcinoma using multiple derivations of preclinical models of a new patient-derived tumor.

PLoS One 2020 9;15(10):e0226464. Epub 2020 Oct 9.

Department of Medicine, Section of Hematology/Oncology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.

Metaplastic breast carcinoma (MBC) is a clinically aggressive and rare subtype of breast cancer, with similar features to basal-like breast cancers. Due to rapid growth rates and characteristic heterogeneity, MBC is often unresponsive to standard chemotherapies; and novel targeted therapeutic discovery is urgently needed. Histone deacetylase inhibitors (DACi) suppress tumor growth and metastasis through regulation of the epithelial-to-mesenchymal transition axis in various cancers, including basal-like breast cancers. We utilized a new MBC patient-derived xenograft (PDX) to examine the effect of DACi therapy on MBC. Cell morphology, cell cycle-associated gene expressions, transwell migration, and metastasis were evaluated in patient-derived cells and tumors after treatment with romidepsin and panobinostat. Derivations of our PDX model, including cells, spheres, organoids, explants, and in vivo implanted tumors were treated. Finally, we tested the effects of combining DACi with approved chemotherapeutics on relative cell biomass. DACi significantly suppressed the total number of lung metastasis in vivo using our PDX model, suggesting a role for DACi in preventing circulating tumor cells from seeding distal tissue sites. These data were supported by our findings that DACi reduced cell migration, populations, and expression of mesenchymal-associated genes. While DACi treatment did affect cell cycle-regulating genes in vitro, tumor growth was not affected compared to controls. Importantly, gene expression results varied depending on the cellular or tumor system used, emphasizing the importance of using multiple derivations of cancer models in preclinical therapeutic discovery research. Furthermore, DACi sensitized and produced a synergistic effect with approved oncology therapeutics on inherently resistant MBC. This study introduced a role for DACi in suppressing the migratory and mesenchymal phenotype of MBC cells through regulation of the epithelial-mesenchymal transition axis and suppression of the CTC population. Preliminary evidence that DACi treatment in combination with MEK1/2 inhibitors exerts a synergistic effect on MBC cells was also demonstrated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226464PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546483PMC
November 2020

ERK5 Is Required for Tumor Growth and Maintenance Through Regulation of the Extracellular Matrix in Triple Negative Breast Cancer.

Front Oncol 2020 3;10:1164. Epub 2020 Aug 3.

Section of Hematology & Medical Oncology, Department of Medicine, Tulane University School of Medicine, New Orleans, LA, United States.

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. 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.
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http://dx.doi.org/10.3389/fonc.2020.01164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416559PMC
August 2020

A novel screening approach comparing kinase activity of small molecule inhibitors with similar molecular structures and distinct biologic effects in triple-negative breast cancer to identify targetable signaling pathways.

Anticancer Drugs 2020 09;31(8):759-775

Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana.

Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.
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http://dx.doi.org/10.1097/CAD.0000000000000962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092417PMC
September 2020

Pharmacological, Mechanistic, and Pharmacokinetic Assessment of Novel Melatonin-Tamoxifen Drug Conjugates as Breast Cancer Drugs.

Mol Pharmacol 2019 08 20;96(2):272-296. Epub 2019 Jun 20.

Division of Pharmaceutical, Administrative and Social Sciences, Duquesne University, Pittsburgh, Pennsylvania (M.H., T.D.W., B.P.M., P.A.W.-E.); Department of Pharmaceutical Chemistry, German University in Cairo, New Cairo City, Cairo, Egypt (M.A.M., D.P.Z.); Purdue University, West Lafayette, Indiana (S.A.); Section of Hematology and Medical Oncology, Department of Medicine, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana (M.D.M., S.E., M.W., M.A., B.M.C.-B., M.E.B.); Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, Wuerzburg, Germany (U.H.); Indiana University School of Medicine, Indianapolis, Indiana (R.E.S.); and Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (P.A.W.-E.)

Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC = 4-8 M) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC = 80-211 M) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC = 181-304 mM; efficacy ∼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor B. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.
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http://dx.doi.org/10.1124/mol.119.116202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666385PMC
August 2019

Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models.

Breast Cancer Res 2019 05 22;21(1):67. Epub 2019 May 22.

Center for Stem Cell Research and Regenerative Medicine, Tulane University, 1430 Tulane Ave, #8699, New Orleans, LA, 70112, USA.

Background: Breast cancer is the second leading cause of cancer deaths in the USA. Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer with high rates of metastasis, tumor recurrence, and resistance to therapeutics. Obesity, defined by a high body mass index (BMI), is an established risk factor for breast cancer. Women with a high BMI have increased incidence and mortality of breast cancer; however, the mechanisms(s) by which obesity promotes tumor progression are not well understood.

Methods: In this study, obesity-altered adipose stem cells (obASCs) were used to evaluate obesity-mediated effects of TNBC. Both in vitro and in vivo analyses of TNBC cell lines were co-cultured with six pooled donors of obASCs (BMI > 30) or ASCs isolated from lean women (lnASCs) (BMI < 25).

Results: We found that obASCs promote a pro-metastatic phenotype by upregulating genes associated with epithelial-to-mesenchymal transition and promoting migration in vitro. We confirmed our findings using a TNBC patient-derived xenograft (PDX) model. PDX tumors grown in the presence of obASCS in SCID/beige mice had increased circulating HLA1 human cells as well as increased numbers of CD44CD24 cancer stem cells in the peripheral blood. Exposure of the TNBC PDX to obASCs also increased the formation of metastases. The knockdown of leptin expression in obASCs suppressed the pro-metastatic effects of obASCs.

Conclusions: Leptin signaling is a potential mechanism through which obASCs promote metastasis of TNBC in both in vitro and in vivo analyses.
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http://dx.doi.org/10.1186/s13058-019-1153-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530039PMC
May 2019

Obesity-Altered Adipose Stem Cells Promote ER⁺ Breast Cancer Metastasis through Estrogen Independent Pathways.

Int J Mol Sci 2019 Mar 20;20(6). Epub 2019 Mar 20.

Center for Stem Cell Research, Tulane University School of Medicine, New Orleans, LA 70112, USA.

Adipose stem cells (ASCs) play an essential role in tumor microenvironments. These cells are altered by obesity (obASCs) and previous studies have shown that obASCs secrete higher levels of leptin. Increased leptin, which upregulates estrogen receptor alpha (ERα) and aromatase, enhances estrogen bioavailability and signaling in estrogen receptor positive (ER⁺) breast cancer (BC) tumor growth and metastasis. In this study, we evaluate the effect of obASCs on ER⁺BC outside of the ERα signaling axis using breast cancer models with constitutively active ERα resulting from clinically relevant mutations (Y537S and D538G). We found that while obASCs promote tumor growth and proliferation, it occurs mostly through abrogated estrogen signaling when BC has constitutive ER activity. However, obASCs have a similar promotion of metastasis irrespective of ER status, demonstrating that obASC promotion of metastasis may not be completely estrogen dependent. We found that obASCs upregulate two genes in both ER wild type (WT) and ER mutant (MUT) BC: and . This study demonstrates that obASCs promote metastasis in ER WT and MUT xenografts and an ER MUT patient derived xenograft (PDX) model. However, obASCs promote tumor growth only in ER WT xenografts.
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http://dx.doi.org/10.3390/ijms20061419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470828PMC
March 2019

Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model.

BMC Cancer 2019 Mar 7;19(1):205. Epub 2019 Mar 7.

Department of Medicine, Section of Hematology & Medical Oncology, Tulane University School of Medicine, New Orleans, LA, USA.

Background: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking.

Methods: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining.

Results: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres.

Conclusions: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.
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http://dx.doi.org/10.1186/s12885-019-5401-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407287PMC
March 2019

Panobinostat suppresses the mesenchymal phenotype in a novel claudin-low triple negative patient-derived breast cancer model.

Oncoscience 2018 Mar 29;5(3-4):99-108. Epub 2018 Apr 29.

Tulane University School of Medicine, Department of Medicine, Section of Hematology & Medical Oncology, New Orleans LA, USA.

Claudin-low triple negative breast cancer (CL-TNBC) is a clinically aggressive molecular TNBC subtype characterized by a propensity to metastasize, recur and acquire chemoresistance. CL-TNBC has a diverse intra- and extracellular composition and microenvironment, and currently there are no clinically approved targeted therapies. Histone deacetylase inhibitors (HDACi) have been investigated as therapeutic agents targeting invasive TNBC phenotypes. However, further studies are required to evaluate HDAC inhibition in CL-TNBC. Here, we utilize a novel CL- TNBC patient-derived xenograft model to study the various and diverse therapeutic potential targets within CL-TNBC tumors. To evaluate effects of the pan-HDACi panobinostat on metastasis and the mesenchymal phenotype of CL-TNBC, we utilize immunohistochemistry staining and qRT-PCR in , and studies. Further, we evaluate pan-HDAC inhibition on stem-like subpopulations using 3D mammosphere culture techniques and quantification. Finally, we show that pan- HDACi suppresses collagen expression in CL-TNBC. In this study, we provide evidence that pan-HDAC inhibition has effects on various components of the CL-TNBC subtype, and we demonstrate the potential of our novel CL-TNBC PDX model in therapeutic discovery research.
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http://dx.doi.org/10.18632/oncoscience.412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978446PMC
March 2018

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

Breast Cancer Res Treat 2018 Jun 1;169(2):381-390. Epub 2018 Feb 1.

Department of Medicine, Section of Hematology & Medical Oncology, Tulane University School of Medicine, New Orleans, LA, USA.

Background: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods.

Methods: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0.

Results: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix.

Conclusions: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.
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http://dx.doi.org/10.1007/s10549-018-4685-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948145PMC
June 2018

Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes.

PLoS One 2017 3;12(8):e0177802. Epub 2017 Aug 3.

Department of Medicine: Section of Hematology and Oncology, Tulane University, New Orleans, Louisiana, United States of America.

Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds' effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177802PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542472PMC
September 2017
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