Alterations of E-cadherin and Proteasome Function in Progression of Lung Cancer

University Of Colorado Denver, Aurora CO

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Abstract

Lung cancer is the leading cause of cancer deaths in the United States and develops progressively from genetically damaged bronchial epithelial cells. Despite surgery and radiation, most lung cancer patients die from metastatic disease and recurrent disease within the lung. The ability of the cancer cell to migrate, cross boundaries, and flourish in other tissues contributes substantially to treatment failure. Based on observations that 3p loss was a nearly universal event in lung cancer, Project 1 was initiated to characterize 3p genetic alterations and discover candidate tumor suppressor genes. We constructed physical contigs across the entire chromosome and used these reagents to investigate 3p loss. Because LOH was extensive, we searched for and identified small homozygous deletions. From 3p21.3 we cloned SEMA3F, a member of the semaphorin gene family. Based on data from our group and others, SEMA3F correlates with disease stage, is a competitive antagonist of VEGF, is lost early in lung tumor development, inhibits epithelial cell spreading, and is pro-apoptotic when overexpressed. A second discovery we made involved a distinct 3p21.3 homozygous deletion confined to the beta-catenin gene. Beta-catenin provides both an essential link between E-cadherin and the actin cytoskeleton in adherens junctions, and is also a critical component of the WNT signalling pathway. Using a human tissue microarray, we demonstrated that downregulation of beta-catenin and E-cadherin negatively affects lung cancer progression and survival. We also found that a 3p-encoded WNT ligand, WNT7a, was downregulated in most lung cancers. We have now shown that E-cadherin, H-cadherin, beta-catenin, WNT7a and SEMA3F can be upregulated by pharamacologic inhibitors of GSK3beta, HDACs, or combined agents, and that this treatment changes migratory behavior. In Aim 1, NSCLC lines expressing low endogenous cadherins will be treated with agents that inhibit HDACs, cytosine methylation or GSK3beta and examined for anti-tumor effects in vitro and in vivo. Transcriptional repressors will be blocked using RNA inhibition or anti-sense oligos. Changes in critical cadherin-regulating genes will be assessed in pre-neoplastic lesions and tumor microarrays. Aim 2 is based on knowledge that tyrosine phosphorylation disrupts beta-catenin / E-cadherin interactions and leads to endocytosis of E-cadherin. We hypothesize that the function of upregulated E-cadherin in Aim 1 will be compromised in some cases. An analysis of beta-catenin/E-cadherin tyrosine phosphorylation will be performed in the presence or absence of EGFR kinase inhibitors and these results incorporated into the treatment strategies of Aim 1. A similar analysis will be performed in resected primary tumors for evidence of prognosis and clinical/biologic correlates. In Aim 3, we will test the hypothesis that SEMA3F is an inhibitor of metastasis acting, at least in part, by competing with VEGF and VEGF-C receptors. This will include an analysis of tumorigenicity and metastatic capacity of SEMA3F overexpressing cell lines, and the effects of SEMA3F on lymphatic and other vascular proliferations in vivo. Based on our understanding of Semaphorin-VEGF interactions affecting dual signalling systems, we hypothesize that VEGF receptor inhibitors (e.g., PTK787 ) combined with exogenous SEMA3F will have additive or synergistic negative effects on cell migration. Lastly, selected components (VEGF-C/VEGFR3, SEMA3F, NRP1 and 2) will be examined by real-time RT-PCR in pre-neoplastic lesions, including those with angiogenic squamous dysplasia, and by immunohistochemistry in tissue microarrays from high-grade neuroendocrine and NSCLC tumors.