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

乳腺癌是美国妇女中最常见的恶性肿瘤， 高发病率和致命性。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 乳腺癌细胞的这些特性所涉及的分子和细胞机制， 导致病人死亡FIP 200（200 kDa的FAK家族相互作用蛋白）最初在我们的研究中被鉴定。 实验室，随后被证明是ULK 1/Atg 13/FIP 200复合物的一个组成部分， 诱导自噬。我们最近已经表明，FIP 200消融抑制了发展， 乳腺癌的进展，提供了动物自噬的促肿瘤发生作用的第一个证据 具有完整免疫功能的模型。我们还开发了一个诱导系统，在肿瘤发生后删除FIP 200。 在体内的发展，并证明了急性破坏自噬的FIP 200删除在建立 肿瘤阻碍了它们的生长。在初步研究中，我们确定了两个不同的乳腺癌人群， 干细胞样细胞（BCSC），并显示FIP 200消融损害了两者的维持和致瘤性。 BCSC。我们的初步数据表明TGF-β/Smad和EGFR/Stat 3信号通路介导了 FIP 200分别调节这两种BCSC。在另一组初步研究中，我们发现 FIP 200中582-585位残基用于与Atg 13结合，并产生了新的FIP 200敲入突变小鼠， 这些残基突变为Ala（命名为KI等位基因，编码缺乏与Atg 13结合的FIP 200 -4A）。分析 的KI/KI小鼠和来自KI/KI胚胎的MEF显示出自噬和非自噬功能， FIP 200调节不同的生物过程。这些研究进一步揭示了一种新的非自噬 FIP 200保护正常和转化的MEFs免受TNFα诱导的凋亡的功能。这一独特的 突变KI等位基因以及在初步研究中制备的其他小鼠模型为我们提供了强有力的 FIP 200通过自噬调节乳腺癌机制的遗传分析工具， 体内非自噬功能。基于这些强有力的初步数据，并使用我们独特的新型小鼠 模型，我们建议1）确定FIP 200调节CD 29 hiCD 61+和ALDH+ BCSCs的机制 在乳腺癌的PyMT和BRCA 1缺陷小鼠模型中; 2）检查自噬和非自噬 FIP 200在调节BCSC和乳腺癌发展和进展中的功能;以及3） 探索靶向乳腺癌BCSCs中FIP 200自噬和非自噬功能的策略 疗法总之，这些研究将提供重要的见解的分子和细胞机制， 乳腺癌转移和复发，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.