Integrins in Cancer Invasion

描述(申请人提供)：这个项目的长期目标是确定由微环境信号触发并由整合素？3？1和整合素？6？4驱动的癌症侵袭机制。这些整合素协商细胞与上皮基底膜的主要成分层粘连蛋白-5(Ln-5)的相互作用。？6？4和Ln-5的表达改变与肿瘤进展有关。然而，对可能的因果机制的定义一直具有挑战性，因为有报道称？6？4/Ln-5功能的丧失和获得都促进了肿瘤的发生。在这项提案中，我们计划评估综合观点协调这些对比数据的有效性。在目标1中，我们将测试这一假设，即侵袭的癌细胞为自分泌的细胞外基质，特别是Ln-5铺平了道路。这是对传统癌细胞迁移观点的挑战，这一假说是基于发生在重塑或愈合的上皮细胞中从静态黏附到迁移的生理转换，并得到了我们的初步实验的支持，在这些实验中，Ln-5或？3？1抗体抑制了癌细胞的散射，但？6抗体不抑制？6抗体的散射。为了验证这一假设，我们将确定 Ln-5对运动原LPA和HGF诱导的细胞散射是必不可少的，以及这些运动刺激是否协调Ln-5的上调和/或分泌。在目标2中，我们将验证Ln-5/？6？4/半桥粒轴调节细胞-细胞黏附和肿瘤进展的假设。？6？4与Ln-5结合可拮抗？3？1介导的迁移，并通过ErbB2增强细胞间的黏附。提示？6？4对肿瘤进展的负调控作用，与ErbB2基因扩增有关的肿瘤预后不良。最近来自我们和其他人的体内数据表明，分泌的Ln-5异源三聚体具有肿瘤抑制作用。我们的假设是基于这样的假设，即这些明显相互矛盾的数据反映了癌细胞通过半桥粒(HD)锚定到Ln-5的状态，在细胞从迁移到静止以及从静止到迁移的过程中，癌细胞受到？6？4的调节。为了验证这一假设，我们将研究？6？4/HD轴在细胞-细胞黏附和ErbB2定位中的作用，这些细胞系通过基因操作表达正常或致癌的ErbB2，以及HD组分的siRNA被击倒。读数分析包括体外细胞-细胞黏附测量和体内异种肿瘤模型。在目标3中，我们将检验Ln-5的2DIII结构域通过EGFR促进肿瘤进展的假设。许多报道和我们自己的初步微阵列分析表明，单体Ln-5？2链的表达与侵袭性一致。？2DIII蛋白降解片段与EGFR结合，我们的初步数据表明，它在体内上调了对凋亡和肿瘤发生的抵抗力。我们将通过siRNA敲除和挽救？2链或？2DIII片段变体来测试？2DIII对体外形态和存活以及体内肿瘤生长和侵袭的影响，并评估其与其他ErbB家族成员的结合。总之，这3个AIMS将检验分泌型Ln-5异源三聚体(阴性)和单体？2(阳性)对肿瘤进展的相反影响的综合观点的有效性和局限性。了解癌症侵袭的这些机制可能为控制癌症侵袭和转移的新的分子靶点指明方向，癌症侵袭和转移是癌症死亡的主要原因。

DESCRIPTION (provided by applicant): The long-term goal of this project is to define mechanisms of cancer invasion triggered by microenvironment cues and driven by integrins ?3?1 and ?6?4. These integrins negotiate interactions of cells with laminin-5 (Ln-5), a major component of epithelial basement membranes. Altered expression of both ?6?4 and Ln-5 is associated with cancer progression. Definition of possible causal mechanisms, though, has been challenging because both loss and acquisition of ?6?4/Ln-5 functions were reported to promote tumorigenesis. In this proposal, we plan to assess the validity of an integrated view reconciling these contrasting data. In Aim 1, we will test the hypothesis that invading cancer cells "pave their way" with autocrine-secreted extracellular matrix, particularly Ln-5. A challenge of conventional views of cancer cell migration, this hypothesis is based on the physiological switch from static adhesion to migration that occurs in remodeling or healing epithelia, and is supported by our preliminary experiments in which cancer cell scattering was inhibited by Ln-5 or ?3?1, but not ?6 antibodies. To test this hypothesis, we will determine whether secreted Ln-5 is essential for cell scattering induced by the motogens LPA and HGF, and whether these motogenic stimuli coordinate upregulation and/or secretion of Ln-5. In Aim 2, we will test the hypothesis that a Ln- 5/?6?4/hemidesmosome axis regulates cell-cell adhesion and tumor progression. Engagement of ?6?4 with Ln-5 antagonizes ?3?1-mediated migration and enhances cell-cell adhesion via ErbB2. This suggests negative regulation of tumor progression by ?6?4, vis-¿-vis cancer poor prognosis associated with ErbB2 gene amplification. Recent in vivo data, from ourselves and others, suggest a tumor suppressive role for secreted Ln-5 heterotrimers. Our hypothesis is based on the premise that these apparently contradictory data reflect the status of cancer cell anchoring to Ln-5 via hemidesmosomes (HD), regulated by ?6?4 in cells switching from migratory to stationary and vice versa. To test this hypothesis, we will investigate the role of the ?6?4/HD axis in cell-cell adhesion and ErbB2 localization in cell lines genetically manipulated to express normal or oncogenic ErbB2, and siRNA knocked-down of HD components. Readout assays include cell-cell adhesion measurements in vitro and xenogeneic tumor models in vivo. In Aim 3, we will test the hypothesis that the ?2DIII domain of Ln-5 promotes tumor progression through EGFR. Expression of monomeric Ln-5 ?2 chain is consistently associated with invasion by many reports and our own preliminary microarray analyses. The ?2DIII proteolytic fragment binds EGFR and our preliminary data indicate it upregulates resistance to apoptosis and tumorigenesis in vivo. We will test the ?2DIII effects on in vitro morphology and survival and in vivo tumor growth and invasion, by siRNA knockdown and rescue with ?2 chain or ?2DIII fragment variants, and will assess its binding to additional ErbB family members. In summary, the 3 aims will test validity and limitations of an integrated view of the opposing effects on tumor progression by secreted Ln-5 heterotrimers (negative) and monomeric ?2 (positive). Understanding these mechanisms of cancer invasion may point to new molecular targets for controlling cancer invasion and metastasis, the major cause of mortality in cancer.