INTEGRIN SIGNALING DURING BREAST TUMORIGENESIS

描述(由申请人提供)：我们假设整合素信号在肿瘤的启动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，我们在MMTV-Neu小鼠中引入了？4信号域的靶向性缺失。？4信号的丢失延迟了肿瘤的发生并抑制了侵袭性生长。体外研究表明，β4与ErbB2形成络合物，增强STAT3和c-jun的活性。STAT3有助于破坏上皮细胞的黏附和促进侵袭，而c-jun则是过度增殖所必需的。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小鼠中引入了一种乳腺特异的FAK消融术。值得注意的是，FAK的缺失完全抑制了乳腺肿瘤的发生。此外，沉默FAK可诱导RAS和PI-3K转化的乳腺癌细胞在体外发生生长停滞、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RAS和PI-3K介导的乳腺肿瘤发生所必需的。我们建议：1)检测4和FAK促肿瘤效应的癌基因特异性。正常乳腺上皮细胞将在体外被改变以抑制FAK或？4信号，然后接受各种癌基因的转化试验。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，并接受旨在检查其增殖、抗凋亡、体外侵袭和在NOD/SCID小鼠中形成原位肿瘤的能力的测试。将使用选定的人类乳腺癌细胞系和转基因小鼠模型来证实关键观察结果；2)阐明？4信号扰乱上皮黏附和促进侵袭的机制。将结合生化分析和iRNA介导的抑制，通过突变来研究？4放大ErbB2信号的机制。将通过生化分析、沉默和成像方法研究RAC过度激活导致黏附连接解体的信号通路；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。然后将使用对照和CAS沉默的肿瘤细胞的生化分析来识别CAS依赖的途径。抑制关键的下游效应将被用来确定CAS促进乳腺肿瘤形成的主要信号通路；4)研究？4和FAK协同维持ErbB2启动的乳腺肿瘤形成的机制。将进行生化实验，以确定乳腺肿瘤细胞中由？4和FAK共同激活的信号成分。沉默将被用来测试潜在的关键信号整合子的促肿瘤作用。小鼠遗传学将被用来确定？4和FAK是否在体内协同促进ErbB2启动的乳腺肿瘤的形成。公共卫生报告：乳腺癌细胞在其微环境信号的作用下向恶性程度的增加进化，微环境包括肿瘤相关的成纤维细胞、巨噬细胞、血管生成的内皮细胞以及这些细胞产生的细胞外基质。整合素黏附受体是这个信号网络中的关键角色。然而，它们在乳腺肿瘤发生中的作用还不完全清楚。我们的初步研究表明，4整合素通过放大ErbB2信号来促进乳腺癌的进展，而RAS和PI-3K介导的肿瘤的启动和维持需要粘着斑激酶(FAK)。我们建议研究4和FAK促进乳腺肿瘤发生的机制。这些研究将有助于我们了解乳腺癌的发病机制，并为其治疗寻找新的分子靶点。

DESCRIPTION (provided by applicant): 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 deletions 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 studies 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. PUBLIC HEALTH RELEVANE: Breast carcinoma cells evolve toward increasing malignancy in response to signals from their microenvironment, which include carcinoma-associated fibroblasts, macrophages, angiogenic endothelial cells, and the extracellular matrix that these cells produce. The integrin adhesion receptors are critical players in this signaling network. Yet, their role in breast tumorigenesis is incompletely understood. Our preliminary studies suggest that the ¿4 integrin promote breast carcinoma progression by amplifying ErbB2 signaling, whereas Focal Adhesion Kinase (FAK) is required for Ras- and Pi-3K-mediated tumor initiation and maintenance. We propose to examine the mechanisms through which ¿4 and FAK promote breast tumorigenesis. These studies will contribute to our understanding of the pathogenesis of breast cancer and to the identification of novel molecular targets for its therapy.