Integrin Regulation of Cancer Progression Through Alternative mRNA Splicing and Nonsense-Medidated Decay (NMD)

 描述(由申请人提供)：整合素有望成为抑制恶性进展和转移的治疗靶点。然而，在整合素作为临床靶点被充分利用之前，仍然存在必须克服的障碍。本申请中提出的工作将在研究层粘连蛋白结合整合素α3β1调节肿瘤细胞功能和癌症相关基因表达的新机制的背景下解决其中一些障碍。我们小组和其他人发表的研究已经确定了α3β1在乳腺癌细胞和其他肿瘤细胞上的明确作用，在促进肿瘤生长、进展和转移的许多功能中。我们最近利用RNA干扰抑制α3β1在人乳腺癌细胞中的表达，发现α3β1依赖的环氧合酶2(COX-2/PTGS2)的表达是α3β1功能的主要促肿瘤功能。事实上，我们最近证实了α-3和COX-2在人类浸润性导管癌临床样本中的相关表达。我们现在已经通过外显子微阵列分析和后续的基于聚合酶链式反应的研究确定，α3β1调节多种基因的替代外显子使用，包括COX-2mRNA的选择性剪接。进一步的研究表明，α3β1缺陷细胞产生COX-2mRNA剪接变异体，该剪接变异体保留含有提前终止密码子的内含子，该内含子针对该基因的无义介导的衰变。这些发现意义重大，因为转录后mRNA处理和稳定性正在成为癌症基因调控的主要模式，但关于微环境信号如何被转导到肿瘤细胞来控制mRNA处理或靶向降解mRNAs，人们知之甚少。在目前的工作中，我们将验证这样的假设，即在乳腺癌细胞中抑制α3β1降低了COX-2mRNA的稳定性，这是通过(1)以NMD转录物为靶点的MRNA剪接/内含子保留和(2)增强NMD途径的协同机制来实现的。我们还将测试α3β1与层粘连蛋白或Tetraspanin蛋白CD151结合在维持正常COX-2mRNA剪接和抑制NMD方面的重要性。这项工作将使用最先进的微型基因剪接报告和高通量cDNAi和RNA干扰筛选来鉴定α3β1依赖的COX-2mRNA调味的反式调节因子，结合我们建立的细胞培养和异种移植模型来研究α3β1在肿瘤进展和转移中的作用。这些实验的结果应该确定α3β1依赖的COX-2mR NA剪接和NMD抑制的新机制，并确定控制这一调节的α3β1结合功能，从而揭示α3β1依赖 可被用作治疗靶点的乳腺癌细胞的脆弱性。

 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, α3β1, regulates tumor cell function and the expression of cancer-associated genes. Published studies from our group and others have established clear roles for α3β1 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 α3β1 in human breast cancer cells has identified α3β1-dependent expression of cyclooxygenase-2 (COX-2/PTGS2) as a major pro-tumorigenic function of α3β1 function. Indeed, we recently validated correlated expression of α3 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 α3β1 regulates alternative exon usage (AEU) of a variety of genes, including alternative splicing of the COX-2 mRNA. Further studies revealed that α3β1-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 α3β1 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 α3β1 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 α3β1-dependent trans-regulators of COX-2 mRNA spicing, combined with cell culture and xenograft models that we have established to investigate α3β1 functions in tumor progression and metastasis. Results from these experiments should identify novel mechanisms of α3β1-dependent COX-2 mRNA splicing and NMD suppression, and determine α3β1 binding functions that control this regulation, thereby revealing α3β1-dependent vulnerabilities of breast cancer cells that can be exploited as therapeutic targets.