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.
Abstract
Solid tumors are composed of heterogeneous cell populations that support rapid growth of cancer cells and suppress immune cell responses. Unleashing the body's own immune response to ...eradicate cancer cells is one of the most promising ways to treat cancer patients. However, cancer immunotherapy is not without challenges. Some of these include:
1) Phenotypic plasticity and clonal selection in a highly dynamic, heterogeneous and mutable cell population, which leads to de novo and acquired resistance;
2) Immune editing by cancer cells;
3) Expression of immune check point ligands and receptors to suppress immune response locally within the tumor microenvironment, and systemically using exosomes as immune checkpoint carriers;
4) Cross-talk between cancer stem cells and the immune microenvironment;
5) Feasibility of developing personalized treatment strategies, such as cost vs benefit of CAR-T-cell therapy and time constraint, and humanized antibody production;
6) Systemic toxicity of current immunotherapy strategies;
To address these challenges, we propose to use a recombinant adeno-associated virus (rAAV) platform to simultaneously enhance tumor immunity and target cancer stem cells by intra-tumoral administration. We express programmed cell death ligand-1 (PD-L1), and B7-H3 (CD276), in combination with a Notch1 decoy. The latter was chosen because: 1) Notch is a crucial signaling component of cancer stem cells (CSCs) maintenance and resistance and 2) Studies have shown that within the tumor, regulatory T-cells (Treg) are activated by OX40 and Notch ligand Jagged1. Blocking Notch signaling would have dual advantages: blocking Notch signaling in CSCs, and preventing activation of immunosuppressive Tregs.
Based on current clinical trials and our experimental results, we hypothesized that intra-tumoral injection of rAAV viral particles engineered to express soluble PD-1, B7-H3 and Notch1 decoys in combination with lipopolysaccharide (LPS) is a promising strategy. To test our hypothesis, we used two mouse triple negative breast (TNBC) cancer models: C0321 in the FVB background and M-Wnt in the C57/Bl6 background; both are aggressive, highly metastatic, and similar to human TNBC. Using tumor spheroids in vitro, in the presence of soluble decoys of PD-1, B7-H3 and Notch1 synergistically induced the most effective tumor cell killing, compared to single decoy application. Importantly, intra-tumoral injection of PD-1, B7-H3 and Notch1 decoys in combination with LPS induced necrosis of tumors in vivo in our preliminary findings.
Citation Format: Deniz A. Ucar, Giulia Monticone, Fokhrul Hossain, Samarpan Majumder, Dorota Wyczechowska, Matthew J. Dean, Luis Del Valle, Jovanny Zabaleta, Yong Ran, Sudarvili Shanthalingam, Abraham Bert Chabot, Bridgette M. Collins-Burow, Matthew E. Burrow, Barbara Osborne, Todd Eliot Golde, Lucio Miele. Delivering intra-tumoral immune modulators and targeting cancer stem cells using recombinant- AAVs abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6691.
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.
Dual inhibition of the MEK1/2 and MEK5 signaling pathways suppressed triple‐negative breast cancer cell migration, mesenchymal features, and metastasis. Mechanistically, MEK1/2 and MEK5 function through FRA‐1 activity to exert the observed pro‐migratory and mesenchymal phenotypes.
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.
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.
Abstract
Triple-negative breast cancers (TNBCs) constitute approximately 12% of all breast cancer cases and are approximately twice as prevalent in African-American populations. Louisiana has a high ...proportion of African-American residents (32.3% in 2017), and New Orleans has among the highest incidences of TNBC in the country. Louisiana patients also have a high incidence of co-morbidities that affect breast cancer biology and outcomes, including type 2 diabetes and obesity. TNBCs have an aggressive clinical presentation due to high rates of metastasis, recurrence and chemoresistance. There are currently no clinically approved targeted therapies for TNBC; cytotoxic chemotherapy is the first-line treatment for TNBC, and recurrent, chemoresistant cancers are usually fatal. TNBCs are molecularly heterogeneous, consisting of at least four molecular subgroups, and immunologically heterogeneous. Both molecular and immunologic properties are associated with clinical outcomes and are seriously understudied in patients under-represented in biomedical research. Patient-derived xenografts (PDXs), as well as patient-derived organoids (PDO), are currently the best model for translational oncology therapeutic research because they accurately recapitulate the complex architecture and heterogenous genetic and molecular composition of solid cancers. To date, the majority of TNBC research has been based on Caucasian patients, although incidence rates of TNBC are higher in African-American cohorts. Our collaborative team aims to overcome this obstacle by establishing and characterizing TNBC PDX models that represent this understudied cohort. We currently have ten TNBC PDX models representing different patient ethnicities, responsiveness to chemotherapies, as well as different TNBC molecular subtypes and metastatic behavior. We dissect and evaluate the various individual components (tumor cell biology, stroma, immune, extracellular matrix) of TNBC tumors. We utilize these models in vivo, ex vivo and in vitro to examine how unique kinases and targeted inhibitors affect the distinct tumor characteristics. In addition to in vivo treatment studies, we generated cell lines and PDOs and we utilize novel techniques such as tissue decellularization to examine extracellular matrix components. We also analyze mechanistically relevant transcript (qRT-PCR) and protein (Western blot, immunohistochemistry) expression patterns that are unique to each PDX model to evaluate the effects of targeted therapies. We work with surrounding laboratories in the greater New Orleans area (Tulane, LSU, Xavier) that are also focused on therapeutic discovery of TNBC in a collaborative effort to provide translational models for their projects. Our aim is to leverage novel PDX models from understudied patients with a range of clinical and molecular presentations to guide the selection of therapeutically targetable pathways and therapeutic agents in specific molecular subtypes of TNBC.
Citation Format: Margarite D. Matossian, Steven Elliott, Hope E. Burks, Maryl Wright, Rachel A. Sabol, Van T. Hoang, Deniz A. Ucar, Alex Alfortish, Jovanny Zabaleta, Fokhrul Hossain, Tiffany Chang, Henri Wathieu, Nicholas Pashos, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven D. Jones, Elizabeth C. Martin, Lucio Miele, Bridgette M. Collins-Burow, Matthew E. Burow. Applications of patient-derived triple-negative breast cancer xenografts that represent understudied patients in Louisiana in targeted therapeutic research abstract. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C110.
Abstract 967: Targeting notch one notch above Ucar-Bilyeu, Deniz A.; MATOSSIAN, Margarite D.; Barnes, VAN Hoang ...
Cancer research (Chicago, Ill.),
07/2018, Letnik:
78, Številka:
13_Supplement
Journal Article
Recenzirano
Abstract
Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard ...chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem cell-like (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling, particularly Notch1, is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch directly have been impaired by the systemic toxicity of the Gamma Secretase Inhibitors (GSI) used, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that target Notch signaling in breast cancer cells indirectly and selectively are a potentially attractive strategy. However, no such target has been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening method, we have identified a small molecule compound that targets MAP2K5 (MEK5) and decreases phosphorylation of MAPK7 (ERK5). Expression of ERK5 is associated with poor prognosis in TNBC. Consistent with ERK5KO cells, suppression of ERK5 phosphorylation decreased the amount of Notch1 and Jagged1 protein and mRNAs. More importantly, a selective MEK5 inhibitor, SC-181, reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells without suppressing T-cell proliferation. Treatment with nanomolar concentration of this compound decreased the number and size of mammospheres in a dose- dependent manner. Our preliminary results suggest that targeting the MEK5-ERK5 pathway is a promising strategy to selectively target Notch signaling in TNBC CSC without systemic Notch inhibition.
Citation Format: Deniz A. Ucar-Bilyeu, Margarite D. MATOSSIAN, VAN Hoang Barnes, Fokhrul M. Hossain, Mohit Gupta, HOPE E. BURKS, THOMAS D. WRIGHT, Jane Cavanaugh, Patrick Flaherty, Matthew E. Burow, Lucio Miele. Targeting notch one notch above abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 967.
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.
Annual bluegrass (ABG) (Poa annua L.) is a prolific seed producer in the spring on golf courses that in turn decreases aesthetic quality and trueness of ball roll on cool‐season putting greens. Proxy ...(ethephon) applied twice in the spring after green‐up is the current industry standard after the loss of Embark (mefluidide) from the turf and ornamental market. However, plant growth regulators including Proxy have been used for years to help suppress ABG seedheads with inconsistent success. The primary objective of this study was to determine if ABG seedhead suppression is improved by adding a late fall application of Proxy to the two traditional spring applications of plant growth regulators at nine locations with diverse environments. A second objective was to determine the importance of including Primo Maxx (trinexapac‐ethyl) in fall and spring applications. Adding a late fall application of Proxy prior to the two spring applications (F+S+S) improved control of ABG seedheads over the traditional two spring applications (S+S), but the magnitude of improvement varied among locations. When treatments were applied F+S+S, the industry standard tank mixture of Proxy + Primo Maxx provided consistent ABG seedhead control and turf quality, similar to Proxy + Fiata (phosphonate + proprietary pigment) and equal to or better than Proxy alone.
PDF
