Molecular Analysis of Signal Transduction in Cancer Development and Progression

总结/摘要 拟议研究的长期目标是了解信号 癌症发展和进展的转导机制。以前的资金 期间集中在粘着斑激酶（FAK）和其他几种蛋白质在 调节细胞迁移和乳腺癌的发展和转移。我们有 阐明了FAK介导的Src磷酸化的机制， 抑制MT 1-MMP的内吞作用以刺激癌细胞侵袭。使用条件 KO方法，我们发现乳腺上皮特异性FAK缺失导致 小鼠乳腺重度小叶-肺泡发育不全和分泌性不成熟 腺。此外，我们证明了乳腺上皮细胞中FAK的失活， 细胞抑制乳腺肿瘤的发生和发展， 癌症干细胞/祖细胞使用充分表征的MMTV-PyMT小鼠模型， 人类乳腺癌我们还创建了定义的纤连蛋白梯度， 微流体方法，并分析了FAK和许多其他信号转导的作用， 分子在定向细胞迁移的调节。确定的其他研究a N-WASP和hnRNPK之间的新相互作用，并表明hnRNPK可能 作为N-WASP的负调节剂，抑制丝状伪足的形成和细胞 蔓延这项资助也支持了我们对核定位肌动蛋白作用的研究 调节蛋白N-WASP和Arp 2/3复合物在RNA聚合酶II- 依赖转录在初步研究中，我们有了意外的发现， FIP 200的失活，FIP 200是一种假定的乳腺肿瘤抑制因子， 癌细胞系，抑制（而不是促进）乳腺肿瘤发生， 在体内乳腺癌的MMTV-PyMT小鼠模型中的进展。我们进一步 显示FIP 200缺失乳腺肿瘤细胞的固有增殖缺陷， 以及肿瘤微环境中免疫细胞浸润的增加可能 有助于抑制MMTV中的乳腺肿瘤发生和进展- 具有FIP 200的条件性KO的PyMT小鼠。根据这些初步研究，我们 建议确定FIP 200及其相关信令 通路调节乳腺癌的发展和进展， 分子、细胞、免疫学和小鼠遗传学方法。我们将（1）。分析 FIP 200调控乳腺癌细胞增殖的分子机制 分离的肿瘤细胞的原代培养，2）。研究提升的作用和机制 FIP 200抑制乳腺肿瘤发展的免疫细胞浸润 消融; 3）。探讨FIP 200在乳腺肿瘤中的潜在作用 上维护

Summary/Abstract The long term goal of the proposed studies is to understand the signal transduction mechanisms in cancer development and progression. The previous funding period focused on the role of focal adhesion kinase (FAK) and several other proteins in the regulation of cell migration and breast cancer development and metastasis. We have illustrated the mechanisms by which FAK-mediated Src phosphorylation of endophilin A2 inhibits endocytosis of MT1-MMP to stimulate cancer cell invasion. Using a conditional KO approach, we showed that mammary epithelial-specific deletion of FAK leads to severe lobulo-alveolar hypoplasia and secretory immaturity of the murine mammary gland. Furthermore, we demonstrated that inactivation of FAK in mammary epithelial cells suppresses mammary tumorigenesis and progression by affecting mammary cancer stem/progenitor cells using the well-characterized MMTV-PyMT mouse model of human breast cancer. We also created the defined fibronectin gradients using microfluidics approaches and analyzed the role of FAK and a number of other signaling molecules in the regulation of directional cell migration. Additional studies identified a novel interaction between N-WASP and hnRNPK and showed that hnRNPK may function as a negative regulator of N-WASP to inhibit filopodia formation and cell spreading. This grant also supported our work on the role of nuclear localized actin regulatory proteins N-WASP and Arp2/3 complex in the regulation of RNA polymerase II- dependent transcription. In preliminary studies, we made the unexpected finding that inactivation of FIP200, a putative breast tumor suppressor based on initial studies in cancer cell lines, inhibits (rather than promotes) mammary tumorigenesis and progression in the MMTV-PyMT mouse model of breast cancer in vivo. We further showed that both intrinsic proliferative defects of FIP200-null mammary tumor cells as well as increased infiltration of immune cells in the tumor microenvironment may contribute to the suppression of mammary tumorigenesis and progression in MMTV- PyMT mice with conditional KO of FIP200. Based on these preliminary studies, we propose to determine the mechanisms by which FIP200 and its associated signaling pathways regulate breast cancer development and progression using a combination of molecular, cellular, immunological and mouse genetic approaches. We will 1). analyze molecular mechanisms of FIP200 regulation of mammary tumor cell proliferation using primary culture of isolated tumor cells, 2). examine the role and mechanisms of elevated immune cell infiltration in the suppression of mammary tumor development upon FIP200 ablation, and 3). investigate the potential role of FIP200 in mammary tumor maintenance.