2000 — 2005 |
Dipersio, C Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Control of Matrix Proteolysis by Integrin A3b1 in Skin
DESCRIPTION (Adapted from applicant's abstract): The cutaneous basement membrane is a specialized extracellular matrix (ECM) that separates the epidermis from the dermis. During skin development and wound repair, the basement membrane undergoes extensive changes in structure and composition that are associated with changes in cell migration, signal transduction, and gene expression. Integrin alpha3beta1 is an adhesion receptor on epidermal keratinocytes that binds laminin-5 in the basement membrane. Mutation of the alpha3 gene in mouse revealed a critical role for alpha3beta1 in organization of the basement membrane in developing skin. Cell invasive processes such as skin development, wound healing, and carcinoma invasion are facilitated by proteolysis of the pericellular ECM, in which integrins contribute to functional regulation of matrix metalloproteinases (MMPs) or other ECM-degrading proteinases. Alpha3beta1 may have such a role during wound repair, where its high expression on keratinocytes coincides with the activation of ECM-degrading enzymes. However, mechanisms whereby alpha3beta1 controls ECM remodeling in skin development and wound healing are unknown. The proposed research exploits alpha3beta1-deficient mice, and keratinocyte cell lines derived from them, as a unique model system for defining these mechanisms. Preliminary data for these studies show that alpha3beta1 is required in keratinocytes for distinct proteinase function and for expression of genes encoding known ECM proteinases or proteinase inhibitors. Biochemical and molecular methods, with skin and cultured keratinocytes from normal or alpha3beta1-deficient mice, will be used to identify proteinases that are regulated by alpha3beta1. Candidate proteinases will them be tested directly for their abilities to restore alpha3beta1-dependend functions through transfection of alpha3beta1-deficient keratinocytes. To define integrin-mediated signaling pathways that regulate ECM remodeling, signaling proteins that are activated in an alpha3beta1-dependent manner will be identified using immunobiochemical methods. Dominant-negative mutants of these proteins will then be transfected into alpha3beta1-expressing keratinocytes and tested for inhibition of endogenous proteinase activity or expression. This research will provide important insights into the roles of integrins in regulating ECM remodeling at the levels of gene expression and proteinase function, and may contribute to the development of therapeutic approaches to control wound healing or carcinoma invasion.
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1.009 |
2008 |
Dipersio, C Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Mmp-9 Mrna Stability and Tumor Growth by Alpha3 Beta1 Integrin
[unreadable] DESCRIPTION (provided by applicant): Integrin a3b1 is an extracellular matrix receptor that is expressed in many malignant tumors and has been shown to regulate cellular phenotypes associated with epithelial-to-mesenchymal transition (EMT), such as cell proliferation, survival, and invasion. Expression of matrix metalloproteinase MMP-9 is also linked to malignant tumor growth, where it promotes tumor angiogenesis and cell invasion. a3b1 induces MMP-9 in immortalized keratinocytes (MK cells) through post-transcriptional mRNA stability, and this regulation is acquired during cellular immortalization. However, the mechanisms and signaling pathways whereby a3b1 regulates MMP-9 mRNA stability and their roles in tumor growth and progression are unknown. The goal of the proposed research is to answer these questions by exploiting a panel of a3b1-expressing (i.e. a3 wild type) and a3b1- deficient (i.e., a3-null) MK variants that collectively represent different EMT stages: (1) non-immortalized keratinocytes are isolated from neonatal epidermis; (2) immortalized MK cells harbor a p53-null mutation; (2) transformed MK cells additionally express oncogenic RasV12. Loss of p53 and oncogenic Ras activation are common mutations in squamous cell and other carcinomas. Preliminary data from this model indicate that a3b1 and MMP-9 are required for in vivo tumor growth of MK cells, and identify candidate signaling pathways and mechanisms whereby a3b1 may control MMP-9 mRNA stability. The proposed studies will test the hypotheses that MMP-9 mRNA expression is controlled by specific a3b1-mediated signaling pathways that control mRNA stability, and that these mechanisms control tumor growth in vivo. A combination of molecular, genetic, and biochemical approaches will be used to identify mRNA regulatory elements that control a3b1- dependent mRNA stability, and to elucidate specific signaling pathways or integrin functions that are involved in a3b1-mediated MMP-9 mRNA stability. An in vivo tumorigenesis model will be used to investigate the role of a3b1-dependent regulation of MMP-9 for tumor growth. Finally, the human relevance of these observations will be investigated by testing for a3b1-dependent regulation of MMP-9 in several human carcinoma lines, and by assessing expression of a3b1 and MMP-9 in human tumor specimens. PUBLIC HEALTH RELEVANCE: A key to the development of anti-cancer therapies is the identification of molecular targets that are required for tumor growth, progression, and metastasis. The proposed studies will identify novel molecular pathways that are turned on in cancer cells to promote malignant tumor growth and metastasis. These pathways may be exploitable as therapeutic targets. [unreadable] [unreadable] [unreadable] [unreadable]
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1.009 |
2018 — 2020 |
Dipersio, C. Michael |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrin Regulation of Cancer Progression Through Alternative Mrna Splicing and Nonsense-Medidated Decay (Nmd)
? DESCRIPTION (provided by applicant): Integrins hold promise as therapeutic targets to inhibit malignant progression and metastasis. However, barriers remain that must be overcome before integrins can be fully exploited as clinical targets. The work proposed in this application will address some of these barriers in the context of investigating novel mechanisms whereby the laminin-binding integrin, ?3?1, regulates tumor cell function and the expression of cancer-associated genes. Published studies from our group and others have established clear roles for ?3?1 on breast cancer cells and other tumor cells in a number of functions that promote tumor growth, progression, and metastasis. Our recent work using RNAi-mediated suppression of ?3?1 in human breast cancer cells has identified ?3?1-dependent expression of cyclooxygenase-2 (COX-2/PTGS2) as a major pro-tumorigenic function of ?3?1 function. Indeed, we recently validated correlated expression of ?3 and COX-2 in human clinical samples of invasive ductal carcinoma. We have now determined through exon microarray analysis and follow-up PCR-based studies that ?3?1 regulates alternative exon usage (AEU) of a variety of genes, including alternative splicing of the COX-2 mRNA. Further studies revealed that ?3?1-deficient cells generate a COX-2 mRNA splice variant that retains an intron harboring premature termination codons (PTCs), which targets the mRNA for nonsense-mediated decay (NMD). These findings are significant, as post-transcriptional mRNA processing and stability are emerging as major modes of gene regulation in cancer, yet little is known about how microenvironmental signals are transduced into tumor cells to control mRNA processing or target mRNAs for degradation. In the current work, we will test the hypotheses that suppression of ?3?1 in breast cancer cells reduces COX-2 mRNA stability through synergistic mechanisms of (1) mRNA splicing/intron retention that targets the transcript for NMD, and (2) enhancement of the NMD pathway. We will also test importance of ?3?1 binding to laminins or the tetraspanin protein CD151 in the maintenance of normal COX-2 mRNA splicing and NMD suppression. This work will be completed using state-of-the-art minigene splice reporters and high- throughput cDNA and RNAi screens to identify ?3?1-dependent trans-regulators of COX-2 mRNA spicing, combined with cell culture and xenograft models that we have established to investigate ?3?1 functions in tumor progression and metastasis. Results from these experiments should identify novel mechanisms of ?3?1-dependent COX-2 mRNA splicing and NMD suppression, and determine ?3?1 binding functions that control this regulation, thereby revealing ?3?1-dependent vulnerabilities of breast cancer cells that can be exploited as therapeutic targets.
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1.009 |
2019 — 2020 |
Dipersio, C. Michael Van De Water, Livingston (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Keratinocyte Integrin Crosstalk During Wound Healing.
PROJECT SUMMARY Epidermal keratinocytes are vital to normal wound healing by restoring the epidermal barrier and secreting paracrine factors that govern diverse processes including wound angiogenesis and myofibroblast function. In pathogenic settings, impaired epidermal function results in chronically insufficient (e.g., diabetic ulcers) or over- exuberant healing (e.g., hypertrophic scars). Our long-term goal is to develop therapeutic paradigms through which integrins can be manipulated to modulate pathogenic keratinocyte function. While it is well established that integrins regulate proliferation, migration and growth factor signaling, their roles in orchestrating wound keratinocyte functions remain enigmatic. Moreover, while normal and wound keratinocytes express integrin ?9?1, in vivo, upon explanation integrin ?9?1 is lost, confounding observations made in previous studies, in vitro. Using genetically defined, virally transduced keratinocytes that express integrins ?3?1 and/or ?9?1 in different combinations, we discovered in the last project period that ?9?1 exerts a cross-suppressive effect on wound cell function and gene expression that is governed by ?3?1, including paracrine signals that promote endothelial cell function and autocrine signals that regulate basement membrane assembly. Using genetically defined mice that we have derived expressing different combinations of ?3?1 and/or ?9?1 in the epidermis, we also found that deletion of ?9?1 from epidermis promoted wound angiogenesis and enhanced laminin ?2 processing in the regenerating, epidermal basement membrane after injury. Based on our recently published studies and new foundation data, we now hypothesize that ?9?1 cross-suppresses ?3?1-dependent keratinocyte functions through inhibition of a novel ?3?1-FAK-YAP/TAZ signaling axis. We further hypothesize that this signaling axis controls a gene expression program that promotes keratinocyte wound functions, including paracrine stimulation of endothelial cells and fibroblasts and autocrine regulation of basement membrane assembly. This hypothesis will be tested in three Aims using a combination of co-culture models, qPCR arrays, proteomics, PAC-seq mRNA analysis, cell biology, and defined genetic mouse models. At the end of this project period, we will have built on the foundation developed in the first project period to elucidate the complex signaling network downstream of integrin signaling in keratinocytes that governs paracrine and autocrine signaling in normal wounds. We will also have determined how these integrin signaling pathways are altered in epidermal tumors in which angiogenesis and other wound processes persist. In doing so, we will have developed the basis for novel integrin targeting therapeutics to modulate keratinocyte function and wound outcome.
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1.009 |