Genetic Analysis of FAK kinase and scaffold functions in breast cancer

描述(由申请人提供)：乳腺癌表现出巨大的细胞、基因组和生物学复杂性，其中多个基因异常作用于肿瘤的发生和癌症的进展。拟议研究的长期目标是了解乳腺癌发展和转移调控的细胞和分子机制。粘着斑激酶(FAK)是一种胞质酪氨酸激酶，在整合素和生长因子受体介导的信号转导中起重要作用，调节多种细胞的黏附、迁移、存活、增殖和分化。FAK与人类乳腺癌和其他恶性肿瘤有关。然而，FAK在体内促进乳腺肿瘤发生的细胞和分子机制仍不清楚。最近，我们在一个特征良好的乳腺癌小鼠模型中显示，乳腺上皮中有条件地敲除FAK通过影响MaCSCs来抑制乳腺肿瘤的发生和发展。为了研究FAK作为酪氨酸激酶和支架在乳腺癌细胞内信号转导中的独特作用，我们最近创造了两个新的FAK基因敲入突变小鼠(FAK/KD小鼠)或P878/881A突变等位基因(FAK/PA小鼠)，分别通过酪氨酸激酶活性或特异的支架功能来干扰其信号转导，以使Src对内嗜酸性蛋白A2的磷酸化和MT1-MMPs的调节。用MMTV-PYMT模型对PA突变敲门小鼠(PA/PA-MT小鼠)的初步分析表明，FAK介导的Src介导的内嗜素A2磷酸化通过促进乳腺肿瘤细胞上皮向间充质转化(EMT)，维持MaCSCs的含量和致瘤性，在乳腺肿瘤的生长和转移中发挥重要作用。此外，我们还鉴定了FAK在维持正常MASCs中的一个功能，发现FAK的不依赖于激酶的功能能够促进MASCs的自我更新。最后，我们还获得了初步的结果，表明FAK在人MaCSCs的调控中发挥了重要作用，这与我们在小鼠模型中更广泛的发现是一致的。基于这些初步研究和以前的研究，我们建议1)。通过对EMT和MaCSCs的调控，探讨FAK支架在乳腺肿瘤生长和转移中的作用机制。研究FAK激酶活性在乳腺癌发生发展中的作用，并对下游依赖和非依赖的信号通路进行剖析。探讨在乳腺癌治疗中靶向FAK支架和蛋白激酶功能的策略。总之，这些研究将为乳腺癌的分子和细胞机制提供重要的见解，可能有助于这种毁灭性疾病的新疗法。

DESCRIPTION (provided by applicant): Breast cancer exhibits enormous cellular, genomic and biological complexity wherein multiple gene aberrations act to drive tumorigenesis and cancer progression. The long term goal of the proposed studies is to understand the cellular and molecular mechanisms in the regulation of breast cancer development and metastasis. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in mediating signal transduction by integrins as well as growth factor receptors in the regulation of cell adhesion, migration, survival, proliferation and differentiation in a variety of cells. FAK has been implicated in human breast cancer as well as other malignancies. However, the cellular and molecular mechanisms by which FAK promotes mammary tumorigenesis in vivo are still not well understood. We showed recently that conditional knockout of FAK in the mammary epithelium suppressed mammary tumorigenesis and progression by affecting MaCSCs in a well characterized breast cancer mouse model. To investigate the unique role of FAK as both a tyrosine kinase and a scaffold in intracellular signaling in breast cancer, we have recently created two novel FAK knock-in mutant mice with the kinase-defective allele (FAK +/KD mice) or the P878/881A mutation allele (FAK +/PA mice) to disrupt its signaling through tyrosine kinase activity or specific scaffolding function for endophilin A2 phosphorylation by Src and regulation of MT1-MMP, respectively. Preliminary analyses of the PA mutant knockin mice using the MMTV-PyMT model (PA/PA-MT mice) revealed that FAK mediated endophilin A2 phosphorylation by Src plays an important role in mammary tumor growth and metastasis by promoting epithelial to mesenchymal transition (EMT) of mammary tumor cells, and by maintaining the content and tumorigenicity of MaCSCs. In addition, we identified a function of FAK in the maintenance of normal MaSCs and found that kinase-independent functions of FAK were able to promote self-renewal of MaSCs. Lastly, we also obtained preliminary results suggesting an important role of FAK in regulation of human MaCSCs, which is consistent with our more extensive findings in mouse models. Based on these preliminary and previous studies, we propose to 1). determine the mechanism of FAK scaffold function in mammary tumor growth and metastasis through regulation of EMT and MaCSCs, 2). examine the role of FAK kinase activity and dissect the downstream kinase-dependent and -independent signaling pathways in breast cancer development and progression, and 3). explore the strategies of targeting FAK scaffold and kinase functions in MaCSCs for breast cancer therapy. Together, these studies will provide significant insights into the molecular and cellular mechanisms of breast cancer that may contribute to novel therapies for this devastating disease.