Integrins and Epithelial-Mesenchymal Cooperation in the Tumor Microenvironment of Muscle-Invasive Lethal Cancers

Muscle-invasive lethal carcinomas traverse into and through this specialized biophysical and growth factor enriched microenvironment. We will highlight cancers that originate in organs surrounded by smooth muscle, which presents a barrier to dissemination, including prostate, bladder, esophageal, gastric, and colorectal cancers. We propose that the heterogeneity of cell-cell and cell-ECM adhesion receptors is an important driver of aggressive tumor networks with functional consequences for progression. Phenotype heterogeneity of the tumor provides a biophysical advantage for tumor network invasion through the tensile muscle and survival of the tumor network. We hypothesize that a functional epithelial-mesenchymal cooperation (EMC)exists within the tumor invasive network to facilitate tumor escape from the primary organ, invasion and traversing of muscle, and navigation to metastatic sites. Cooperation between specific epithelial cells within the tumor and stromal (mesenchymal) cells interacting with the tumor is illustrated using the examples of laminin-binding adhesion molecules—especially integrins—and their response to growth and inflammatory factors in the tumor microenvironment. The cooperation between cell-cell (E-cadherin, CDH1) and cell-ECM (α6 integrin, CD49f) expression and growth factor receptors is highlighted within poorly differentiated human tumors associated with aggressive disease. Cancer-associated fibroblasts are examined for their role in the tumor microenvironment in generating and organizing various growth factors. Cellular structural proteins are potential utility markers for future spatial profiling studies. We also examine the special characteristics of the smooth muscle microenvironment and how invasion by a primary tumor can alter this environment and contribute to tumor escape via cooperation between epithelial and stromal cells. This cooperative state allows the heterogenous tumor clusters to be shaped by various growth factors, co-opt or evade immune system response, adapt from hypoxic to normoxic conditions, adjust to varying energy sources, and survive radiation and chemotherapeutic interventions. Understanding the epithelial-mesenchymal cooperation in early tumor invasive networks holds potential for both identifying early biomarkers of the aggressive transition and identification of novel agents to prevent the epithelial-mesenchymal cooperation phenotype. Epithelial-mesenchymal cooperation is likely to unveil new tumor subtypes to aid in selection of appropriate therapeutic strategies. Copyright © 2022 Harryman, Marr, Nagle and Cress.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

New relationships between prostatic intraepithelial neoplasia and prostatic carcinoma

Our group has been studying the progressive molecular changes in prostatic epithelium which precede the invasive phenotype. Initial studies revealed similar alterations in cytoskeletal proteins between high grade prostatic intraepithelial neoplasia (PIN) lesions and invasive carcinoma. Specifically we observed an increased expression of certain cytokeratins and decreased expression of vimentin. We also noted a change in glycosylation as detected by Ulex europaeus staining. Using the latter technique we were able to microdissect and isolated nuclei from areas of low and high grade PIN lesions as well as from invasive carcinoma for morphometric analysis. Similarities in nuclear size, chromatin heterogeneity, and nuclear DNA content between low and high grade PIN and invasive carcinoma in carcinomatous specimens were noted. In contrast, these parameters were significantly different in low grade PIN lesions obtained from benign prostatic transurethral resection (TURP) specimens. In addition, DNA histograms revealed similar proliferative indices between high grade PIN and invasive carcinoma, which differed significantly from low grade PIN. Parameters thought to be relative to the invasive phenotype were also examined, such as the members of the metalloproteinase family; although normal luminal cells fail to express detectable levels of these enzymes, invasive carcinoma and even low grade PIN lesions express both the 72 kDa and 92 kDa type IV collagenase. Taken together, these data indicate that the dysplastic cells of PIN lesions and carcinomas are similar in nuclear and genomic features as well as protease expression. Our current working hypothesis is that these cells are already armed with the necessary proteases to invade the basal lamina but in an inactive form. Tumor progression requires an additional event of protease activation. © 1992 Wiley‐Liss, Inc. Copyright © 1992 Wiley‐Liss, Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Integrin Beta 4E Promotes Endothelial Phenotypic Changes and Attenuates Lung Endothelial Cell Inflammatory Responses

We previously reported integrin beta 4 (ITGB4) is an important mediator of lung vascular protection by simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitor. In this study, we report increased endothelial cell (EC) expression specifically of ITGB4E, an ITGB4 mRNA splice variant, by simvastatin with effects on EC protein expression and inflammatory responses. In initial experiments, human pulmonary artery ECs were treated using simvastatin (5 μM, 24 h) prior to immunoprecipitation of integrin alpha 6 (ITGA6), which associates with ITGB4, and Western blotting for full-length ITGB4 and ITGB4E, uniquely characterized by a truncated 114 amino acid cytoplasmic domain. These experiments confirmed a significant increase in both full-length ITGB4 and ITGB4E. To investigate the effects of increased ITGB4E expression alone, ECs were transfected with ITGB4E or control vector, and cells were seeded in wells containing Matrigel to assess effects on angiogenesis or used for scratch assay to assess migration. Decreased angiogenesis and migration were observed in ITGB4E transfected ECs compared with controls. In separate experiments, PCR and Western blots from transfected cells demonstrated significant changes in EC protein expression associated with increased ITGB4E, including marked decreases in platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin (VE-cadherin) as well as increased expression of E-cadherin and N-cadherin along with increased expression of the Slug and Snail transcription factors that promote endothelial-to-mesenchymal transition (EndMT). We, then, investigated the functional effects of ITGB4E overexpression on EC inflammatory responses and observed a significant attenuation of lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, including decreased phosphorylation of both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as reduced inflammatory cytokines (IL-6 and IL-8), expressed in the media of EC after either LPS or excessive cyclic stretch (CS). Finally, EC expression-increased ITGB4E demonstrated decreased barrier disruption induced by thrombin as measured by transendothelial electrical resistance. Our data support distinct EC phenotypic changes induced by ITGB4E that are also associated with an attenuation of cellular inflammatory responses. These findings implicate ITGB4E upregulation as an important mediator of lung EC protection by statins and may lead to novel therapeutic strategies for patients with or at risk for acute lung injury (ALI). Copyright © 2022 Chen, Gard, Epshtein, Camp, Garcia, Jacobson and Cress.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A Humanized Monoclonal Antibody Targeting Extracellular Nicotinamide Phosphoribosyltransferase Prevents Aggressive Prostate Cancer Progression

Prostate cancer (PCa) is the major cause of cancer-related death in males; however, effective treatments to prevent aggressive progression remain an unmet need. We have previously demonstrated that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator that promotes PCa invasion. In the current study, we further investigate the therapeutic effects of an eNAMPT-neutralizing humanized monoclonal antibody (ALT-100 mAb) in preclinical PCa orthotopic xenograft models. We utilized human aggressive PCa cells (DU145 or PC3) for prostate implantation in SCID mice receiving weekly intraperitoneal injections of either ALT-100 mAb or IgG/PBS (control) for 12 weeks. Prostatic tumors and solid organs were examined for tumor growth, invasion, and metastasis and for biochemical and immuno-histochemistry evidence of NFκB activation. ALT-100 mAb treatment significantly improved overall survival of SCID mice implanted with human PCa orthotopic prostate xenografts while inducing tumor necrosis, decreasing PCa proliferation and reducing local invasion and distal metastases. The ALT-100 mAb inhibits NFκB phosphorylation and signaling in PCa cells both in vitro and in vivo. This study demonstrates that eNAMPT neutralization effectively prevents human PCa aggressive progression in preclinical models, indicating its high potential to directly address the unmet need for an effective targeted therapy for patients with aggressive PCa. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A method to reuse archived H&E stained histology slides for a multiplex protein biomarker analysis

Archived Hematoxylin and Eosin (H&E) stained pathology slides are routinely stored to index formalin-fixed paraffin-embedded (FFPE) sample tissue blocks. FFPE blocks are clinically annotated human tumor specimens that can be valuable in studies decades after the tissue is collected. If stored properly, they have the potential to yield a valuable number of serial sectioned slides for diagnostic or research purposes. However, some retrospective studies are limited in scope because the tissue samples have been depleted or not enough material is available in stored blocks for serial sections. The goal of these studies was to determine if archived H&E-stained slides can be directly reutilized by optimizing methods to de-stain and then re-stain the H&E stained slides to allow the detection of several biomarkers of interest using a conjugated antibody with chromogen multiplex immunohistochemistry procedure. This simple but innovative procedure, combined with image analysis techniques, demonstrates the ability to perform precise detection of relevant markers correlated to disease progression in initially identified tumor regions in tissue. This may add clinical value in retaining H&E slides for further use. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

An Actin-, Cortactin- and Ena-VASP-Linked Complex Contributes to Endothelial Cell Focal Adhesion and Vascular Barrier Regulation

BACKGROUND/AIMS: Increase in vascular permeability is a cardinal feature of all inflammatory diseases and represents an imbalance in vascular contractile forces and barrier-restorative forces, both of which are highly dependent on actin cytoskeletal dynamics. In addition to the involvement of key vascular barrier-regulatory, actin-binding proteins, such as nmMLCK and cortactin, we recently demonstrated a role for a member of the Ena-VASP family known as Ena-VASP-like (EVL) in promoting vascular focal adhesion (FA) remodeling and endothelial cell (EC) barrier restoration/preservation. METHODS: To further understand the role of EVL in EC barrier-regulatory processes, we examined EVL-cytoskeletal protein interactions in FA dynamics in vitro utilizing lung EC and in vivo murine models of acute inflammatory lung injury. Deletion mapping studies and immunoprecipitation assays were performed to detail the interaction between EVL and cortactin, and further evaluated by assessment of changes in vascular EC permeability following disruption of EVL-cortactin interaction. RESULTS: Initial studies focusing on the actin-binding proteins, nmMLCK and cortactin, utilized deletion mapping of the cortactin gene (CTTN) to identify cortactin domains critical for EVL-cortactin interaction and verified the role of actin in promoting EVL-cortactin interaction. A role for profilins, actin-binding proteins that regulate actin polymerization, was established in facilitating EVL-FA binding. CONCLUSION: In summary, these studies further substantiate EVL participation in regulation of vascular barrier integrity and in the highly choreographed cytoskeletal interactions between key FA and cytoskeletal partners. © Copyright by the Author(s). Published by Cell Physiol Biochem Press.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors

The microenvironment of solid tumors is dynamic and frequently contains pockets of low oxygen levels (hypoxia) surrounded by oxygenated tissue. Indeed, a compromised vasculature is a hallmark of the tumor microenvironment, creating both spatial gradients and temporal variability in oxygen availability. Notably, hypoxia associates with increased metastasis and poor survival in patients. Therefore, to aid therapeutic decisions and better understand hypoxia’s role in cancer progression, it is critical to identify endogenous biomarkers of hypoxia to spatially phenotype oncogenic lesions in human tissue, whether precancerous, benign, or malignant. Here, we characterize the glucose transporter GLUT3/SLC2A3 as a biomarker of hypoxic prostate epithelial cells and prostate tumors. Transcriptomic analyses of non-tumorigenic, immortalized prostate epithelial cells revealed a highly significant increase in GLUT3 expression under hypoxia. Additionally, GLUT3 protein increased 2.4-fold in cultured hypoxic prostate cell lines and was upregulated within hypoxic regions of xenograft tumors, including two patient-derived xenografts (PDX). Finally, GLUT3 out-performs other established hypoxia markers; GLUT3 staining in PDX specimens detects 2.6–8.3 times more tumor area compared to a mixture of GLUT1 and CA9 antibodies. Therefore, given the heterogeneous nature of tumors, we propose adding GLUT3 to immunostaining panels when trying to detect hypoxic regions in prostate samples. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

EVL is a novel focal adhesion protein involved in the regulation of cytoskeletal dynamics and vascular permeability

Increases in lung vascular permeability is a cardinal feature of inflammatory disease and represents an imbalance in vascular contractile forces and barrier-restorative forces, with both forces highly dependent upon the actin cytoskeleton. The current study investigates the role of Ena-VASP-like (EVL), a member of the Ena-VASP family known to regulate the actin cytoskeleton, in regulating vascular permeability responses and lung endothelial cell barrier integrity. Utilizing changes in transendothelial electricial resistance (TEER) to measure endothelial cell barrier responses, we demonstrate that EVL expression regulates endothelial cell responses to both sphingosine-1-phospate (S1P), a vascular barrier-enhancing agonist, and to thrombin, a barrier-disrupting stimulus. Total internal reflection fluorescence demonstrates that EVL is present in endothelial cell focal adhesions and impacts focal adhesion size, distribution, and the number of focal adhesions generated in response to S1P and thrombin challenge, with the focal adhesion kinase (FAK) a key contributor in S1P-stimulated EVL-transduced endothelial cell but a limited role in thrombin-induced focal adhesion rearrangements. In summary, these data indicate that EVL is a focal adhesion protein intimately involved in regulation of cytoskeletal responses to endothelial cell barrier-altering stimuli. Keywords: cytoskeleton, vascular barrier, sphingosine-1-phosphate, thrombin, focal adhesion kinase (FAK), Ena-VASP like protein (EVL), cytoskeletal regulatory protein © The Author(s) 2021.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]