Mary S. Stack - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The ability to invade host tissues and form distant metastases is a primary distinguishing characteristic of malignant cells as well as the major cause of cancer fatality. Tumor invasion is facilitated by proteolytic degradation of the extracellular matrix (ECM) mediated primarily by plasminogen activators and matrix metalloproteinases. Our long-term goal is to elucidate the molecular mechanisms involved in regulation of tumor cell-associated ECM proteolysis. Our working hypothesis is that tumor cell-mediated basement membrane proteolysis releases fragments of ECM proteins which stimulate proteinase secretion and/or activity and thereby facilitate metastasis. In this study, matrix regulation of the proteinase secretion profiles of metastatic tumor cells and of the biological activity of the secreted proteinases will be determined using in vitro biochemical studies, tumor cells in culture, and an in vivo animal metastasis model. Initially, the effects of specific isolated domains of the ECM proteins type IV collagen (CIV), laminin (LN) and fibronectin (FN) on the biochemical properties of purified tissue plasminogen activator (t-PA) and matrix metalloproteinase-2 (MMP-2, type IV collagenase) will be determined using ligand blotting and kinetic studies. The effect of ECM fragments on the synthesis, secretion and subsequent biological activity of t-PA, MMP-2 and their respective specific proteinase inhibitors will be determined using cultured melanoma cells. Comparison will be made between the proteinase and inhibitor profiles of a highly metastatic melanoma cell line and a parental melanoma line with low metastatic activity. Lastly, the murine melanoma lung colonization model will be used to identify specific ECM fragments, characterized in previous studies, which potentiate or inhibit experimental metastasis in vivo. Together, these studies will increase our understanding of the molecular mechanisms by which fragments of ECM proteins regulate tumor invasion and metastasis.

Periodontal disease is characterized by degradation of the basement membrane between the junctional epithelium and the tooth, resulting in detachment of the epithelium from the tooth surface followed by apical migration and proliferation of epithelial cells. Formation of this long junctional epithelium precludes attachment of periodontal ligament cells to the root surface, thereby preventing successful healing. Stable attachment of epithelia cells to the internal basal lamina prevents this ling junctional epithelium formation and facilitates repair of damaged periodontal tissue. Ultrastructural data indicate that hemidesmosomes are present at the dento-epithelial junction and contribute to maintenance of normal tissue architecture. A newly described laminin isoform, laminin-5, is involved in an unique integrin-mediated interaction with epithelial cells which can induce hemidesmosome assembly, resulting in the formation of a stable cell:matrix attachment and loss of migratory capacity. However, laminin-5 has also been associated with migrating cells, suggesting a role in mediating cellular motility. To address the differential role of laminin-5 in promoting both gingival epithelial cell adhesion and migration, it is the working hypothesis of this proposal that proteolytic processing of laminin-5 by enzymes present in the gingival microenvironment alters laminin-5 structure and thereby modulates its function. To address this hypothesis, experiments are proposed to analyze the normal processing of laminin-5 which accompanies incorporation into the insoluble extracellular matrix; to identify the subunit and site of cleavage of laminin-5 by a variety of proteinases, and to analyze the effect of these proteolytic modifications on cellular functions related to adhesion and migration. A more detailed understanding of the factors which regulate gingival epithelial cell migration may lead to the development of novel therapeutic approaches by which the formation of long junctional epithelium may be inhibited.

DESCRIPTION (provided by applicant): Epithelial ovarian carcinoma is the leading cause of death from gynecologic malignancy, as 75% of women with this disease succumb to complications resulting from metastasis; thus, strategies aimed at prevention of metastasis would generate immediate clinical impact. Metastases are largely confined to the peritoneal cavity, indicating that microenvironmental factors that modulate intraperitoneal adhesion, motility and invasion play a predominant role in ovarian pathobiology. Acquisition of the metastatic phenotype involves disruption of cell-cell contacts and loss of extracellular matrix (ECM) constraints due to upregulation of matrix metalloproteinases (MMPs) and alterations in matrix- and mechano-transduction. While normal ovarian epithelium does not express MMPs, the transmembrane membrane type 1-MMP (MT1-MMP) is significantly elevated in borderline and malignant tumors and in peritoneal metastases. Integrin-mediated interaction of ovarian cancer cells with interstitial collagens, rich in the submesothelial ECM, represents an important early event unique to ovarian cancer metastatic dissemination. Further, our published data demonstrate that engagement of collagen binding integrins increases MT1-MMP expression and support the conclusion that matrix status influences cell surface and peri-cellular matrix degrading potential. Together these data support the hypothesis that a functional link between adhesion and proteolysis regulates ovarian cancer invasive and metastatic behavior. Experiments proposed in Aim 1 will focus on MT1-MMP surface dynamics to elucidate post-translational mechanisms that regulate surface presentation of active MT1-MMP. This will be integrated with experiments in Aim 2 to evaluate the role of matrix interactions and mechanical constraints as epigenetic factors that participate in transcriptional regulation of MT1-MMP gene expression, changes in MT1-MMP surface dynamics and acquisition of the invasive phenotype. The contribution of integrin signaling to loss of junctional E-cadherin, activation of ?-catenin-mediated transcription and E-cadherin ectodomain shedding will be assessed in Aim 3. Together these experiments will provide novel information on mechanisms by which matrix and mechanical cues in the intraperitoneal microenvironment dictate ovarian cancer metastatic potential through proteinase regulation pathways.

DESCRIPTION: (Adapted from the investigator's abstract) The invasive behavior of oral carcinoma requires coordinated cellular events including basement membrane attachment and detachment, extracellular matrix (ECM) proteolysis, and acquisition of motility. Altered expression of matrix binding integrins is associated with oral carcinoma progression. Integrins can promote an hierarchy of cellular responses dictated by the physical nature of the integrin engagement, thereby transducing distinct signals from the ECM. Production of two distinct classes of ECM-degrading proteinases, plasminogen activators (PA) and matrix metalloproteinases (MMP) is also in early event in malignant progression. The correlation between enhanced expression of the serine proteinase urinary-type PA (uPA or urokinase), MMP-9 (gelatinase B) and tumor progression is well described. Binding of urinary type-PA (uPA) o its cellular receptor (uPAR) leads to enhanced pericellular plasmin formation, which in turn directly degrades ECIV giycoproteins and activates selected MMPs such as MMP-9. Moreover, uPAJR may also regulate invasive behavior via novel, proteinase-independent mechanisms, by modifying integrin adhesive functions and modulating integrin signaling pathways. Our data demonstrate that a3b1 integrin aggregation alters expression of both uPA and MMP-9. Further, a3bl integrin aggregation induces uPA/R/a3b 1 integrin association and MAP kinase (MAPK) activation, resulting in enhance( proteinase transcription Based on these results, it is the working hypothesis of this proposal that a functional link between adhesion and proteolysis regulates oral carcinoma invasive behavior. Specifically we propose a multi-functional interaction of the uPA/R system with carcinoma cell integrins, such that integrin-in edited adhesion modulates cellular, iPA expression, while subsequent uPA/uPAR/integrin interactions in turn regulate downstream adhesive events that control proteinase expression, proliferation, adhesion and motility. To test this hypothesis, we will assess the specific physical parameters of a3b1 integrin engagement that control proteinase induction. The ability of uPAIR to modulate a3b1 signaling and modify proteinase expression and proliferation will then be analyzed. Immunohistochemical and biochemical analysis of normal and tumor tissues will be employed to evaluate integrin, proteinase, and MAPK expression and activity. The functional contribution of induced proteinases to the cellular invasive phenotype will then be evaluated. The long term goal of the proposed research is to provide a more detailed understanding of the functional link between adhesion and proteolysis and the con tribution of this in terplay to regulation of metastasis.

DESCRIPTION: (provided by applicant) The invasive behavior of oral squamous cell carcinoma (OSCC) requires coordinated cellular events including dissociation of cell-cell contacts, integrin-mediated basement membrane attachment and detachment, extracellular matrix (ECM) proteolysis, changes in cytoskeletal dynamics and the acquisition of a migratory phenotype. In the previous funding period, we have focused on the contribution of proteolytic processing of the ECM component laminin-5 (LN5) to regulation of motility. LN5 engages both the alpha 3 beta 1 and alpha 6 beta 4 integrins and, in contrast to the majority of ECM components, staining for LN5 persists in OSCC lesions. Production of ECM-degrading matrix metalloproteinases (MMPs) is an early event in malignant progression. ProMMP-2 is a soluble proteinase that is activated post-translationally at the cell/matrix interface by membrane type 1 MMP (MT1-MMP). Inactive proMT1-MMP is processed intracellularly by furin and the mature enzyme plays a dual role by cleaving both matrix proteins and proMMP-2. Relative to the normal oral mucosa, expression of both MMP-2 and MT1-MMP is enhanced in OSCC lesions. Further, we have previously demonstrated that alpha 3 beta 1 integrin aggregation promotes proMMP-2 processing via an MT1-MMP-dependent mechanism. However, cellular regulation of furin-mediated proMT1-MMP zymogen activation in OSCC and the effect of alpha 6 beta 4 integrin aggregation have not been addressed. Altered expression of E-cadherin, a transmembrane protein that promotes cell-cell contact, is also associated with OSCC progression. Our preliminary data suggest that E-cadherin engagement can differentially regulate both the expression and activation of proMMP-2. Together these preliminary results support the hypothesis that proteinase expression and subsequent tumor cell behavior may be regulated on a cellular level via the engagement of cell-matrix and cell-cell adhesion molecules. Adhesion molecules also regulate changes in cytoskeletal dynamics that accompany motility; however the effect of direct alteration of cytoskeletal structure using mechanical force and the impact on proteinase expression in OSCC is unknown. The overall goal of Project 2 is to assess the mechanistic link between acquisition of proteolytic potential and induction of migratory and invasive behavior. To achieve this goal, we will evaluate the effect of integrin aggregation on membrane proteinase expression and trafficking, examine the mechanism of E-cadherin-mediated regulation of proteolytic potential, assess the potential correlation between proteinase expression and cytoskeletal dynamics, and elucidate the contribution of proteolytic activity to motility and invasion.

DESCRIPTION (provided by applicant): Receptor Cross-Talk in Metastatic Dissemination Epithelial ovarian carcinoma (EOC) will affect 1 out of every 69 women born in the US today. Currently, 80% of women newly diagnosed with EOC already have metastatic disease, indicating that intervention in the metastatic process will improve long-term survival of women with EOC. Metastasis occurs through a unique mechanism involving shedding of non-adherent cells as multi-cellular aggregates (MCAs) into the peritoneal cavity followed by intra-peritoneal (IP) implantation, and is often associated with peritoneal ascites. The factors that regulate the terminal transition from free-floating MCA to life-threatening peritoneally anchored metastatic lesion are currently unknown. Studies in the previous funding period highlighted the role of the soluble microenvironmental regulators EGF and lysophosphatidic acid (LPA) in regulation of epithelial (E)-cadherin (Ecad) expression and function. These mechanistic studies generated exciting new data on metalloproteinase-catalyzed Ecad ectodomain shedding, the role of the soluble Ecad ectodomain in human EOC ascites, and on changes in ?-catenin dynamics resulting from altered junctional integrity. Further, we performed a detailed IHC analysis of Ecad expression in primary human EOC, developed a panel of assays with which to evaluate MCA dynamics and metastatic success, and identified a set of gene products regulated by altering the mechanical environment of the cell. These results form the basis of the current hypothesis that cadherin switching and IP mechanobiology actively contribute to metastatic success. Studies in Aim 1 will focus on cadherin switching and MCA dynamics using a panel of cadherin-modified cell lines and a suite of in vitro assays designed to mechanistically evaluate the effect of cadherin composition on key cellular events in EOC metastasis. Experiments in Aim 2 will evaluate the effect of altered peritoneal mechanobiology on the suite of measures of MCA metastatic success and will determine whether IP mechanical forces affect mesothelial receptivity to metastatic implantation. Aim 3 will combine analysis of human tumors and murine IP metastasis models for a direct examination of altered cadherin profiles, IP mechanobiology, and metastatic dissemination in vivo. The long-term goal of these studies is to cultivate a molecular level understanding of EOC metastasis, necessary for the development of EOC-specific therapies that effectively target metastatic disease.

[unreadable] DESCRIPTION (provided by applicant): Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. The 5-year survival rate has remained at a low 50% for the past 20 years, highlighting our limited understanding of the molecular events that govern OSCC initiation, progression and metastasis. As the high mortality from OSCC is attributed to metastasis, a more detailed analysis of the molecular events that potentiate OSCC dissemination are a necessary prerequisite to the development of novel early detection and treatment strategies. Analysis of epigenetic changes associated with OSCC metastasis has identified loss of E-cadherin along with enhanced expression of urinary type plasminogen activator (uPA) and ?3 integrin as key candidate biomarkers for prediction of poor disease outcome. The proteinase uPA binds to a glycosylphosphatidyl inositol (GPI)-linked receptor, uPAR, whereupon it initiates zymogen activation cascades leading to proteolytic modification of basement membrane proteins, potentiating invasion and metastasis. Furthermore, results obtained during the previous funding period have identified a matrix- induced physical interaction between uPA/R and ?3?1 integrin that initiates a Src/MEK/ERK-dependent signaling pathway culminating in activation of the uPA promoter. In contrast to integrin signaling, activation of E-cadherin through formation of de novo junctions represses proteinase activity and invasion. Cross-talk between ?3?1 integrin and E-cadherin is evident, as integrin clustering downregulates surface E-cadherin, thereby destabilizing cell-cell junctional contacts. These data support the hypothesis that a functional link between adhesion and proteolysis regulates OSCC invasive behavior. Proposed experiments will elucidate mechanisms by which uPAR, as a lateral ?3?1 integrin ligand, can modulate ?3?1 signaling and thereby regulate OSCC metastatic behavior. Experiments proposed in Aim 1 will focus on matrix regulation of uPAR/?3?1 membrane dynamics to elucidate mechanisms whereby lipid raft partitioning of uPAR/?3?1 can regulate signal transduction and alter gene expression. Aim 2 will evaluate the mechanisms by which uPAR modulates integrin-regulated changes in E-cadherin junctional integrity and activation of ?-catenin-mediated transcription in response to junction dissolution. These mechanistic data will be integrated in Aim 3 to assess epigenetic factors in OSCC progression using in vitro and in vivo model systems that more accurately reflect the in vivo microenvironment. Together these experiments will provide novel data on mechanisms whereby lateral interactions of uPAR with ?3?1 integrin contribute to OSCC tumor dissemination and may provide the rationale for long-term studies that target this interaction as a novel therapeutic strategy. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): The Gordon Research Conference on Matrix Metalloproteinases (MMP GRC) and the integrated Gordon- Kenan Research Seminar (MMP GRS) for young scientists constitute the premier forum for intellectual exchange on this family of enzymes crucial in cancer progression and metastasis, connective tissue disorders such as arthritis, inflammatory conditions and developmental biology. The multi-disciplinary scope of the conference includes the MMP, ADAM, and ADAMTS proteinase families as well as their natural and synthetic inhibitors. From inception, the MMP field has been inherently trans-disciplinary, combining tools and approaches from structural biology and biochemistry, genomics and proteomics, cell and developmental biology, medicine and pathology to address key questions of basic and translational importance. This application is in support of funding to enable the 2015 MMP Gordon Research Conference (GRC) to showcase innovative and integrative research on MMPs and related proteins. The MMP GRC will be held in partnership with an affiliated Gordon Research Seminar (GRS) designed by and for junior investigators and trainees, held immediately preceding the GRC. The conferences will be held August 1-7 at Sunday River Resort, Newry, ME. The residential nature of these conferences generates a retreat-like format that promotes close interactions, fosters collaborations and engenders a sense of community among participants who are drawn from academia, pharma, and government sectors worldwide. The 2015 MMP GRC/GRS have a focus on MMP regulation in disease and development and include a new emphasis on mechanobiology and imaging integrated throughout sessions on cancer, inflammation, cellular/biochemical regulation and molecular biophysics. The conference comprises 9 sessions that emphasize presentation of unpublished data and offer abundant time for discussion with additional free time for informal interactions. Sessions are chaired by experienced scientists and new investigators. The international slate of speakers achieves a strong gender balance and a representative cross-section of ethnic/racial diversity, geographic distribution, and early career investigators. Community renewal is well represented by ensuring that the majority of invited speakers are new to the conference and/or were not on the 2013 program and >30% of speakers will be selected from submitted abstracts. Two poster sessions will provide additional presentation opportunities and enhance opportunities for lively discussion. The MMP GRC/GRS provides an unparalleled opportunity to stimulate and inspire, to advance scientific knowledge with free exchange of ideas, to facilitate collaborations, and to nurture and promote development of the future biomedical workforce, with the ultimate goal of promoting human health.

ABSTRACT: This project proposes to develop a new Partnership for Aging and Cancer Research by bringing together NIH extra-mural and intra-mural investigators to collaboratively evaluate aging and the ovarian cancer (OvCa) microenvironment. Advanced age is a significant risk factor for OvCa incidence and negatively affects survival. We have demonstrated that immune system dysregulation is associated with aging, reflected by both increased myelopoiesis and decreased lymphopoiesis, such that immune cells exhibiting exhausted and over activated phenotypes are enhanced in aging. Additionally age-related changes in plasma exosome concentration and content differentially alter B cell activation. Furthermore, our pre-clinical studies have shown that aged mice develop consistently greater peritoneal tumor burden relative to young cohorts with concomitant changes in tumor immune cell composition and that pro-inflammatory signaling supports a pro-metastatic phenotype. Proposed experiments will test the hypothesis that host aging promotes OvCa metastatic progression through changes in the peritoneal proteome and through dysregulation of the peritoneal immune landscape. To address this hypothesis, Aim 1 will characterize age-related changes in the proteome (secreted, cell surface and intracellular) of tumor-naïve primary peritoneal mesothelial cells, examine age- associated alterations in the ascites proteome of tumor-bearing young vs aged mice, and assess the functional consequences of ascites-derived exosome-mediated information transfer to tumor cells and host peritoneal mesothelium. Complementary experiments in Aim 2 will characterize changes in the immune landscape of both host and tumor tissues in young vs aged mice, examine the contribution of aging peritoneal B1a lymphocytes to OvCa growth and assess the role of ascites-derived exosome-mediated information transfer to alterations in immune cell populations in the aged host. With successful completion of the studies proposed herein through this collaborative partnership, we will provide an unprecedented portrait of the aged ovarian tumor microenvironment (both tumor- and host-derived) to identify critical determinants of metastatic success for future mechanistic evaluation and therapeutic intervention.