Signaling of Integrin Alpha 7

描述（申请人提供）：肝脏和乳腺的研究表明，细胞外基质在限制上皮细胞生长和促进上皮细胞分化方面起着关键作用。然而，导致抑制上皮细胞过度生长的信号机制尚不清楚。最近，我们对ITGA7基因（ITGA7）进行了突变分析。我们在前列腺癌、肝细胞癌、平滑肌肉瘤和多形性胶质母细胞瘤中发现ITGA7突变，频率从25%到83%不等。许多这些突变引起了蛋白质的截短、微缺失或移码。有趣的是，在前列腺癌和肝细胞癌中，ITGA7突变患者的临床复发率更高。恢复ITGA7的表达后，PC3和Du145异种前列腺肿瘤小鼠模型的肿瘤体积、转移率和死亡率均显著降低。ITGA7诱导细胞周期蛋白依赖性激酶抑制剂3 （CDKN3）和RACGAP1的表达。siRNA敲低CDKN3和RACGAP1完全逆转了IGGA7的生长抑制作用，而仅部分逆转了IGGA7的迁移抑制活性。最近，Annexin V染色和TUNEL实验表明，ITGA7过表达诱导PC3细胞凋亡。通过酵母双杂交系统，我们发现ITGA7的c端与细胞死亡蛋白HtrA2相互作用。在ITGA7的c端发现了一个30个氨基酸的基序，对其与HtrA2的相互作用至关重要。表达ITGA7诱导HtrA2蛋白酶活性。siRNA敲低HtrA2可消除ITGA7诱导的细胞死亡。另外，ITGA7的表达诱导了整合素连接激酶（ILK）的激酶活性。有趣的是，我们还发现ILK，一种丝氨酸/苏氨酸激酶和与整合素和细胞骨架相关的结构蛋白，结合并磷酸化了一种称为myopodin的肿瘤抑制基因，该基因调节细胞生长和细胞运动。基于这些初步数据，我们假设ITGA7激活HtrA2和ILK信号通路，达到诱导细胞凋亡、抑制细胞生长和减缓迁移的目的。在本研究中，我们提出：1)通过分析PC3和DU145细胞中与HtrA2结合缺陷的突变体ITGA7分子，阐明ITGA7/HtrA2相互作用在介导细胞死亡和抑制肿瘤侵袭中的作用；2)通过分析myopodin缺陷细胞系统，探讨ILK/myopodin相互作用在ITGA7调节细胞运动、细胞生长和抑制侵袭性中的作用；3)研究ITGA7敲除对TRAMP小鼠癌变的影响，探讨ITGA7在纯ITGA7敲除小鼠前列腺组织发育、细胞分化和肿瘤发生中的生物学作用。

DESCRIPTION (provided by applicant): Studies in liver and breast have indicated that extracellular matrix plays a critical role in limiting the growth and promoting the differentiation of epithelial cells. However, the signaling mechanism that leads to inhibition of over-growth of epithelial cells is not well understood. Recently, we performed a mutational analysis on ITGA7 gene (ITGA7). We found mutations on ITGA7 in prostate cancer, hepatocellular carcinoma, leiomyosarcoma and glioblastoma multiforms with frequencies ranging from 25% to 83%. Many of these mutations caused truncation, micro-deletion or frameshift of the protein. Interestingly, patients with ITGA7 mutations had higher rate of clinical relapse in both prostate cancer and hepatocellular carcinoma. Mouse model of PC3 and Du145 xenografted prostate tumors showed a dramatic reduction in tumor volume, rate of metastasis and rate of mortality when expression of ITGA7 was restored in these cell lines. ITGA7 induced the expression of cyclin dependent kinase inhibitor 3 (CDKN3) and RACGAP1. siRNA knocking down of CDKN3 and RACGAP1 completely reversed the growth inhibition effect of IGGA7, while only partially reversed the migration inhibition activity. Recently, Annexin V staining and TUNEL assays indicated that over-expression of ITGA7 induced apoptosis in PC3 cells. Through a Yeast two-hybrid system, we identified that the C-terminus of ITGA7 interacted with HtrA2, a cell death protein. A 30 amino acid motif located in the C-terminus of ITGA7 was identified as critical for its interaction with HtrA2. Expression of ITGA7 induced protease activity of HtrA2. siRNA knocking down of HtrA2 abolished the cell death induction by ITGA7. Separately, expression of ITGA7 induced the kinase activity of integrin link kinease (ILK). Interestingly, we also found that ILK, a serine/threonine kinase and structural protein associated with integrins and the cytoskeleton, bound and phosphorlyated a tumor suppressor gene called myopodin, which regulates cell growth and cell motility. Based on these preliminary data, we hypothesize that ITGA7 activates HtrA2 and ILK signaling pathways to achieve induction of apoptosis, cell growth inhibition and migration deceleration. In this study, we propose: 1) To elucidate the role of ITGA7/HtrA2 interaction in mediating cell death and inhibition of cancer invasion by analyzing the mutant ITGA7 molecule defective of binding with HtrA2 in PC3 and DU145 cells; 2) To investigate the role of ILK/myopodin interaction in ITGA7 regulated cell motility, cell growth and inhibition of invasiveness by analyzing myopodin defective cell system; 3) To study the impact of ITGA7 knock-out on the carciongenesis of TRAMP mice, and to investigate the biological role of ITGA7 in prostate gland tissue development, cell differentiation and tumorigenesis in pure ITGA7 knockout mice.