INTEGRIN SIGNALING DURING BREAST TUMORIGENESIS

我们假设整合素信号在肿瘤的发生和发展中起关键作用。为了研究 4整合素在ErbB2介导的乳腺肿瘤发生中的作用，我们引入了一种靶向缺失的 MMTV-Neu小鼠的？4信号域。？4信号的丢失延迟了肿瘤的发生并抑制了侵袭力 成长。体外研究表明，β4与ErbB2形成络合物，增强STAT3和 C-Jun.STAT3有助于破坏上皮细胞的黏附和促进侵袭，而c-jun则是必需的 过度增殖。为了研究粘着斑激酶(FAK)在乳腺肿瘤发生中的作用，我们 介绍了一种乳腺特异性FAK在MMTV-PyMT小鼠中的消融方法。值得注意的是，FAK的删除 完全抑制了乳腺肿瘤的发生。此外，沉默FAK还可诱导RAS-和PI-3K- 转化乳腺癌细胞，使其在体外经历生长停滞、衰老或凋亡。这些 结果表明，β4通过放大ErbB2信号促进肿瘤进展，而FAK是必需的 用于RAS和PI-3K介导的乳腺肿瘤发生。我们建议：1)检测癌基因的特异性 4和FAK的促肿瘤作用。正常乳腺上皮细胞将在体外改变为 抑制FAK或？4信号，然后用各种癌基因进行转化试验。 相反，携带不同癌基因的乳腺肿瘤细胞将在体外进行基因操作，以 抑制FAK或？4信号，并接受旨在检测其增殖、抵抗 细胞凋亡，体外侵袭，在NOD/SCID小鼠体内形成原位肿瘤。主要观察结果将是 利用选定的人乳腺癌细胞系和转基因小鼠模型进行确认；2)阐明 信号转导扰乱上皮细胞黏附和促进侵袭的机制。这一机制 通过哪一种方式放大ErbB2信号将通过突变结合生化来检测 分析和iRNA介导的抑制。RAC过度激活引起的信号转导途径 将通过生化分析、沉默和成像来研究粘连连接的解体 方法：3)研究FAK促进乳腺肿瘤发生的分子机制。 沉默将被用来证实CAS介导FAK的促肿瘤作用。生物化学分析 对照和CAS沉默的肿瘤细胞将被用来识别CAS依赖的通路。键的抑制 下游效应器将被用来识别CAS促进的主要信号通路 乳腺肿瘤的发生；4)研究？4和FAK协同维持的机制 ERBB2启动的乳腺肿瘤发生。将进行生化实验以识别信号 乳腺肿瘤细胞中由？4和FAK联合激活的成分。将使用静音来测试PRO- 潜在的关键信号整合子的致癌作用。小鼠的遗传学将被用来确定？4和FAK 体内合作促进ErbB2启动的乳腺肿瘤形成。

We hypothesize that integrin signaling plays key roles during tumor initiation and progression. To study the role of the ¿4 integrin during ErbB2-mediated mammary tumorigenesis, we introduced a targeted deletion of the ¿4 signaling domain in MMTV-Neu mice. Loss of ¿4 signaling delayed tumor onset and inhibited invasive growth. Ex vivo studies indicated that ¿4 forms a complex with ErbB2 and enhances activation of STAT3 and c-Jun. STAT3 contributes to disrupt epithelial adhesion and promote invasion, whereas c-Jun is required for hyperproliferation. To examine the role of Focal Adhesion Kinase (FAK) in mammary tumorigenesis, we introduced a mammary gland-specific ablation of FAK in MMTV-PyMT mice. Notably, deletion of FAK completely suppressed mammary tumorigenesis. In addition, silencing of FAK induced Ras- and PI-3K- transformed mammary carcinoma cells to undergo growth arrest and senescence or apoptosis in vitro. These results suggest that ¿4 promotes tumor progression by amplifying ErbB2 signaling, whereas FAK is required for Ras- and PI-3K-mediated mammary tumorigenesis. We propose: 1) To Examine the Oncogene Specificity of the Pro-tumorigenic Effects of ¿4 and FAK. Normal mammary epithelial cells will be altered in vitro to suppress FAK or ¿4 signaling and then subjected to transformation assays with various oncogenes. Conversely, mammary tumor cells carrying distinct oncogenes will be genetically manipulated in vitro to suppress FAK or ¿4 signaling and subjected to assays designed to examine their ability to proliferate, to resist apoptosis, to invade in vitro, and to form orthotopic tumors in NOD/SCID mice. Key observations will be confirmed using selected human breast cancer cell lines and transgenic mouse models; 2) To Elucidate the Mechanisms through which ¿4 Signaling Disrupts Epithelial Adhesion and Promotes Invasion. The mechanism by which ¿4 amplifies ErbB2 signaling will be examined by using mutagenesis in combination with biochemical analysis and iRNA-mediated inhibition. The signaling pathways through which hyperactivation of Rac causes disassembly of adherens junctions will be studied by using biochemical analysis, silencing, and imaging methods; 3) To Study the Molecular Mechanisms through which FAK Promotes Mammary Tumorigenesis. Silencing will be used to confirm that CAS mediates the pro-tumorigenic effect of FAK. Biochemical analysis of control and CAS-silenced tumor cells will then be used to identify CAS-dependent pathways. Inhibition of key downstream effectors will be used to identify the major signaling pathways through which CAS promotes mammary tumorigenesis; 4) To Examine the Mechanisms through which ¿4 and FAK Cooperate to Sustain ErbB2-initiated Mammary Tumorigenesis. Biochemical experiments will be conducted to identify signaling components jointly activated by ¿4 and FAK in mammary tumor cells. Silencing will be used to test the pro- tumorigenic role of potential key signaling integrators. Mouse genetics will be used to determine if ¿4 and FAK cooperate in vivo to promote ErbB2-initiated mammary tumorigenesis.