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，其中多种基因畸变驱动肿瘤发生和癌症进展。这些研究的长期目标是了解乳腺癌发展和转移的细胞和分子调控机制。Focal adhesion kinase （FAK）是一种细胞质酪氨酸激酶，主要通过整合素和生长因子受体介导信号转导，调控多种细胞的粘附、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尚不清楚。我们最近发现，在一个特征明确的乳腺癌小鼠模型中，有条件地敲除乳腺上皮中的FAK通过影响MaCSCs来抑制乳腺肿瘤的发生和进展。为了研究FAK作为酪氨酸激酶和支架在乳腺癌细胞内信号传导中的独特作用，我们最近创建了两个具有激酶缺陷等位基因（FAK +/KD小鼠）或P878/881A突变等位基因（FAK +/PA小鼠）的新型FAK敲入突变小鼠，分别通过酪氨酸激酶活性或特异性支架功能通过Src和MT1-MMP调节来破坏其信号传导。利用MMTV-PyMT模型（PA/PA- mt小鼠）对PA突变敲入小鼠进行的初步分析显示，FAK介导的Src介导的亲内啡肽A2磷酸化通过促进乳腺肿瘤细胞上皮向间质转化（EMT）以及维持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，我们确定了FAK在维持正常MaSCs中的功能，并发现FAK的激酶无关功能能够促进MaSCs的自我更新。最后，我们还获得了初步结果，表明FAK在调节人MaCSCs中起重要作用，这与我们在小鼠模型中更广泛的发现是一致的。基于这些前期和前期的研究，我们提出：1)通过调控EMT和MaCSCs来确定FAK支架功能在乳腺肿瘤生长和转移中的作用机制；2)研究FAK激酶活性在乳腺癌发生和进展中的作用，并解析下游激酶依赖和非依赖的信号通路；3)探索靶向FAK支架和激酶功能在MaCSCs中的治疗策略。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.