Expression of Novel Kinase MAP3K19 in Various Cancers and Survival Correlations.

Mitogen Activated Protein (MAP) kinases are a category of serine/threonine kinases that have been demonstrated to regulate intracellular events including stress responses, developmental processes, and cancer progression Although many MAP kinases have been extensively studied in various disease processes, MAP3K19 is an understudied kinase whose activities have been linked to lung disease and fibroblast development. In this manuscript, we use bioinformatics databases starBase, GEPIA, and KMPlotter, to establish baseline expressions of MAP3K19 in different tissue types and its correlation with patient survival in different cancers.

Liver Kinase B1 Regulates Remodeling of the Tumor Microenvironment in Triple-Negative Breast Cancer.

Liver kinase B1 (LKB1) is a potent tumor suppressor that regulates cellular energy balance and metabolism as an upstream kinase of the AMP-activated protein kinase (AMPK) pathway. LKB1 regulates cancer cell invasion and metastasis in multiple cancer types, including breast cancer. In this study, we evaluated LKB1's role as a regulator of the tumor microenvironment (TME). This was achieved by seeding the MDA-MB-231-LKB1 overexpressing cell line onto adipose and tumor scaffolds, followed by the evaluation of tumor matrix-induced tumorigenesis and metastasis. Results demonstrated that the presence of tumor matrix enhanced tumorigenesis in both MDA-MB-231 and MDA-MB-231-LKB1 cell lines. Metastasis was increased in both MDA-MB-231 and -LKB1 cells seeded on the tumor scaffold. Endpoint analysis of tumor and adipose scaffolds revealed LKB1-mediated tumor microenvironment remodeling as evident through altered matrix protein production. The proteomic analysis determined that LKB1 overexpression preferentially decreased all major and minor fibril collagens (collagens I, III, V, and XI). In addition, proteins observed to be absent in tumor scaffolds in the LKB1 overexpressing cell line included those associated with the adipose matrix (COL6A2) and regulators of adipogenesis (IL17RB and IGFBP4), suggesting a role for LKB1 in tumor-mediated adipogenesis. Histological analysis of MDA-MB-231-LKB1-seeded tumors demonstrated decreased total fibril collagen and indicated decreased stromal cell presence. In accordance with this, in vitro condition medium studies demonstrated that the MDA-MB-231-LKB1 secretome inhibited adipogenesis of adipose-derived stem cells. Taken together, these data demonstrate a role for LKB1 in regulating the tumor microenvironment through fibril matrix remodeling and suppression of adipogenesis.

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

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.

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

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 17ß-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 17ß-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.

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

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.

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

[This corrects the article DOI: 10.1371/journal.pone.0226464.].

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

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.

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.

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.

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

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.

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.

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.

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

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.

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

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.

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

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 (IC50 = 4-8 µM) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 µM) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC50 = 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 κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.

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

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.

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

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 in vitro, ex vivo and in vivo 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.

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

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.

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

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.

Implementing a negative-pressure isolation ward for a surge in airborne infectious patients.

BACKGROUND: During a large-scale airborne infectious disease outbreak, the number of patients needing hospital-based health care services may exceed available negative-pressure isolation room capacity. METHODS: To test one method of increasing hospital surge capacity, a temporary negative-pressure isolation ward was established at a fully functioning hospital. Negative pressure was achieved in a 30-bed hospital ward by adjusting the ventilation system. Differential pressure was continuously measured at 22 locations, and ventilation airflow was characterized throughout the ward. RESULTS: The pressure on the test ward relative to the main hospital hallway was -29 Pa on average, approximately 10 times higher than the Centers for Disease Control and Prevention guidance for airborne infection control. No occurrences of pressure reversal occurred at the entrances to the ward, even when staff entered the ward. Pressures within the ward changed, with some rooms becoming neutrally or slightly positively pressurized. CONCLUSIONS: This study showed that establishing a temporary negative-pressure isolation ward is an effective method to increase surge capacity in a hospital.

Glyceollin, a novel regulator of mTOR/p70S6 in estrogen receptor positive breast cancer.

An estimated 70% of breast cancer tumors utilize estrogen receptor (ER) signaling to maintain tumorigenesis and targeting of the estrogen receptor is a common method of treatment for these tumor types. However, ER-positive (+) breast cancers often acquire drug resistant or altered ER activity in response to anti-estrogens. Here we demonstrate glyceollin, an activated soy compound, has anti-estrogen effects in breast cancers. We demonstrate through estrogen response element luciferase and phosphorylation-ER mutants that the effects of glyceollin arise from mechanisms distinct from conventional endocrine therapies. We show that glyceollin suppresses estrogen response element activity; however, it does not affect ER-alpha (α) phosphorylation levels. Additionally we show that glyceollin suppresses the phosphorylation of proteins known to crosstalk with ER signaling, specifically we demonstrate an inhibition of ribosomal protein S6 kinase, 70 kDa (p70S6) phosphorylation following glyceollin treatment. Our data suggests a mechanism for glyceollin inhibition of ERα through the induced suppression of p70S6 and demonstrates novel mechanisms for ER inhibition.