Mechanisms of FIP200 regulation of breast cancer through its autophagy and non-autophagy functions

乳腺癌是美国女性最常见的恶性肿瘤，仍然是对健康的主要威胁。 发病率高，致命性强。尽管最近在破译基因方面取得了显著进展 与乳腺癌相关的突变，我们对转移和转移机制基础的理解 乳腺癌的复发仍然非常有限。拟议研究的长期目标是了解 参与乳腺癌细胞这些特性的分子和细胞机制最终是 对病人的致命性负责。FIP200(200 kDa的FAK家族相互作用蛋白)是在我们的 实验室，随后被证明是ULK1/Atg13/FIP200复合体的组成部分，对 诱导自噬。我们最近已经证明，FIP200消融抑制了发育和 乳腺癌的进展，为动物自噬的促肿瘤作用提供了第一个证据 具有完整免疫功能的模型。我们还开发了一种在肿瘤后删除FIP200的诱导系统 体内发育，并证明FIP200缺失导致的自噬急性中断在 肿瘤阻碍了它们的生长。在初步研究中，我们确定了两种不同的乳腺癌人群。 干细胞样细胞(BCSCs)的实验结果表明，FIP200对两者的维持和致瘤性均有损害 BCSCs。我们的初步数据表明转化生长因子-β/SMAD和EGFR/STAT3信号通路参与了信号转导 FIP200分别对这两种BCSCs进行调控。在另一组初步研究中，我们发现 FIP200中的582-585残基与Atg13结合，并产生了一种新的FIP200敲入突变小鼠 这些残基突变为ALA(命名为KI等位基因，编码不与Atg13结合的FIP200-4A)。分析 KI/KI小鼠和来自KI/KI胚胎的MEF显示出自噬和非自噬功能 FIP200在调节不同生物过程中的作用。这些研究进一步揭示了一种新的非自噬 FIP200保护正常和转化的MEF免受肿瘤坏死因子α诱导的细胞凋亡的作用因此这是独一无二的 突变的Ki等位基因以及在初步研究中准备的额外的小鼠模型为我们提供了强大的 FIP200自噬和FIP200调控乳腺癌机制的遗传学分析工具 非自噬功能在体内发挥作用。基于这些强有力的初步数据，并使用我们独特的新型鼠标 FIP200对CD29hiCD61+和ALDH+BCSCs的调控机制 在PYMT和BRCA1缺陷的乳腺癌小鼠模型中；2)检查自噬和非自噬 FIP200在BCSCs和乳腺癌发生发展中的调控作用 靶向FIP200自噬和非自噬功能治疗乳腺癌的策略探讨 心理治疗。综上所述，这些研究将为深入了解糖尿病的分子和细胞机制提供重要的见解。 乳腺癌的转移和复发可能有助于这种毁灭性疾病的新疗法。

Breast cancer is the most common malignancy among US women and remains a major health threat with high incidence and lethality. Despite the remarkable progress made recently in deciphering the genetic mutations associated with breast cancer, our understanding of the mechanistic basis for metastasis and relapse of breast cancer is still very limited. The long-term goal of the proposed studies is to understand the molecular and cellular mechanisms involved in these properties of breast cancer cells that are ultimately responsible for patient lethality. FIP200 (FAK-family Interacting Protein of 200 kDa) was initially identified in our laboratory and subsequently shown to be a component of the ULK1/Atg13/FIP200 complex essential for the induction of autophagy. We have shown recently that FIP200 ablation inhibited the development and progression of breast cancer, providing the first evidence of a pro-tumorigenic role for autophagy in animal models with intact immune functions. We also developed an inducible system to delete FIP200 after tumor development in vivo and demonstrated that acute disruption of autophagy by FIP200 deletion in established tumors blocked their growth. In preliminary studies, we identified two distinct populations of breast cancer stem-like cells (BCSCs) and showed that FIP200 ablation impaired the maintenance and tumorigenicity of both BCSCs. Our preliminary data implicated TGF-β/Smad and EGFR/Stat3 signaling pathways in mediating FIP200 regulation of these two BCSCs respectively. In another set of preliminary studies, we identified residues 582-585 in FIP200 for binding to Atg13 and generated a new FIP200 knock-in mutant mouse with these residues mutated to Ala (designated as KI allele, encoding FIP200-4A lacking binding to Atg13). Analysis of KI/KI mice and MEFs derived from KI/KI embryos showed both autophagy and non-autophagy functions of FIP200 in regulating different biological processes. These studies further revealed a new non-autophagy function of FIP200 to protect normal and transformed MEFs from TNFα-induced apoptosis. Thus this unique mutant KI allele as well as additional mouse models prepared in preliminary studies provides us with powerful tools for the genetic analysis of mechanisms of FIP200 regulation of breast cancer through its autophagy and non-autophagy functions in vivo. Based on these strong preliminary data and using our unique novel mouse models, we propose to 1) determine the mechanisms of FIP200 regulation of CD29hiCD61+ and ALDH+ BCSCs in PyMT and BRCA1-deficient mouse models of breast cancer; 2) examine autophagy and non-autophagy functions of FIP200 in the regulation of BCSCs and breast cancer development and progression; and 3) explore the strategies of targeting FIP200 autophagy and non-autophagy functions in BCSCs for breast cancer therapy. Together, these studies will provide significant insights into the molecular and cellular mechanisms of breast cancer metastasis and relapse that may contribute to novel therapies for this devastating disease.