INTEGRIN SIGNALING DURING BREAST TUMORIGENESIS

描述（由申请人提供）：我们假设整合素信号传导在肿瘤发生和进展过程中起关键作用。 为了研究整合素<$4在ErbB 2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信号结构域的靶向缺失。 4信号的丢失延迟了肿瘤的发生并抑制了侵袭性生长。 离体研究表明，Δ 4与ErbB 2形成复合物，并增强STAT 3和c-Jun的活化。STAT 3有助于破坏上皮粘附并促进侵袭，而c-Jun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，沉默FAK诱导Ras和PI-3 K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Ras和PI-3 K介导的乳腺肿瘤发生所必需的。 我们提出：（1）检测<$4和FAK的促肿瘤作用的癌基因特异性。 将在体外改变正常乳腺上皮细胞以抑制FAK或FA 4信号传导，然后用各种癌基因进行转化测定。 相反，将在体外对携带不同癌基因的乳腺肿瘤细胞进行遗传操作以抑制FAK或FAK 4信号传导，并进行旨在检查其增殖、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定。 关键观察结果将使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4信号传导破坏上皮粘附并促进侵袭的机制。 这一机制，4扩增ErbB 2信号传导将通过使用诱变结合生物化学分析和iRNA介导的抑制来检查。 通过生化分析、沉默和影像学方法研究Rac过度激活导致粘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。 沉默将用于确认CAS介导FAK的促肿瘤发生作用。 然后将使用对照和CAS沉默的肿瘤细胞的生化分析来鉴定CAS依赖性途径。 关键下游效应物的抑制将用于鉴定CAS促进乳腺肿瘤发生的主要信号传导途径; 4）检查Δ 4和FAK合作维持ErbB 2启动的乳腺肿瘤发生的机制。 将进行生物化学实验以鉴定乳腺肿瘤细胞中由ε 4和FAK联合激活的信号传导组分。 沉默将用于测试潜在的关键信号传导整合剂的促肿瘤发生作用。 将使用小鼠遗传学来确定Δ 4和FAK是否在体内合作以促进ErbB 2引发的乳腺肿瘤发生。 公共卫生救济金：乳腺癌细胞响应来自其微环境的信号而朝着恶性程度增加的方向进化，所述微环境包括癌相关成纤维细胞、巨噬细胞、血管生成内皮细胞和这些细胞产生的细胞外基质。 整合素粘附受体是该信号网络中的关键参与者。 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ErbB 2信号促进乳腺癌进展，而粘着斑激酶（FAK）是Ras和Pi-3 K介导的肿瘤发生和维持所必需的。 我们建议检查通过哪些机制？4和FAK促进乳腺肿瘤的发生。 这些研究将有助于我们了解乳腺癌的发病机制，并确定其治疗的新的分子靶点。

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.