INTEGRIN SIGNALING DURING BREAST TUMORIGENESIS

我们假设整合素信号在肿瘤的发生和发展过程中起着关键作用。研究 在ErbB 2介导的乳腺肿瘤发生过程中，我们引入了一种靶向缺失的整合素4， MMTV-Neu小鼠中的信号传导结构域。<$4信号的丢失延迟了肿瘤的发生并抑制了侵袭性 增长离体研究表明，<$4与ErbB 2形成复合物，并增强STAT 3和STAT 4的活化。 STAT 3有助于破坏上皮粘附并促进侵袭，而c-Jun是细胞增殖所必需的。 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 在MMTV-PyMT小鼠中引入了FAK的乳腺特异性消融。值得注意的是，FAK的缺失 完全抑制了乳腺肿瘤的发生。此外，FAK的沉默诱导Ras-和PI-3 K-的表达。 转化的乳腺癌细胞在体外经历生长停滞和衰老或凋亡。这些 结果表明，4通过放大ErbB 2信号促进肿瘤进展，而FAK是必需的。 Ras和PI-3 K介导的乳腺肿瘤发生。我们建议：1）检查癌基因特异性 4和FAK的促肿瘤发生作用。正常的乳腺上皮细胞将在体外被改变， 抑制FAK或p4信号传导，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遗传操作， 抑制FAK或FA 4信号传导，并进行旨在检查其增殖、抵抗 在NOD/SCID小鼠中诱导细胞凋亡、体外侵袭和形成原位肿瘤。主要观察结果将是 使用选择的人乳腺癌细胞系和转基因小鼠模型证实; 2）为了阐明 通过哪一机制4信号中断上皮粘附和促进入侵。机制 将通过使用诱变结合生物化学方法来检查Δ 4扩增ErbB 2信号传导的方法。 分析和iRNA介导的抑制。Rac过度激活导致的信号通路 将通过生物化学分析、沉默和成像来研究粘附连接的分解 方法：3）研究FAK促进乳腺肿瘤发生的分子机制。 沉默将用于确认CAS介导FAK的促肿瘤发生作用。的生化分析 然后将对照和CAS沉默的肿瘤细胞用于鉴定CAS依赖性途径。键的抑制 下游效应器将被用来确定主要的信号通路，通过它CAS促进 乳腺肿瘤发生; 4）检查通过哪些机制？4和FAK合作，以维持 ErbB 2启动的乳腺肿瘤发生。将进行生化实验以识别信号传导 在乳腺肿瘤细胞中由<$4和FAK共同激活的组分。沉默将被用来测试亲- 潜在的关键信号整合子的致瘤作用。小鼠遗传学将用于确定是否有4和FAK 在体内协同促进ErbB 2引发的乳腺肿瘤发生。

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.