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.
The key to exploiting integrin adhesion receptors as therapeutic targets to inhibit cancer is the identification of the molecular mechanisms whereby they regulate tumor cell functions and gene expression programs that drive malignant progression and metastasis. The goal of the proposed work is to identify novel molecular pathways by which integrins regulate cancer genes, and to determine how to target those pathways.
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