Roles of autophagy-mediated pathways in the pathogenesis and treatment of TSC

描述(申请人提供)：TSC患者发展为多系统疾病，包括脑部、心脏、皮肤和肾脏的良性肿瘤。到10岁时，80%的TSC儿童患有肾血管平滑肌脂肪瘤和/或肾囊肿。哺乳动物雷帕霉素靶标复合体1(TORC1)在结节性硬化症复合体(TSC)中被激活，是细胞生长、细胞代谢和自噬的主要调节因子。TORC1抑制剂的治疗部分缩小了与TSC相关的脑和肾脏病变的大小，但当治疗停止时，它们会重新出现。我们的中心假设是自噬和细胞代谢的失调在TSC的发病机制和TSC相关的肾脏病变对TORC1靶向治疗的反应中起着关键作用。在目标1和2中，我们将检验这样的假设，即在TSC2缺陷细胞中低水平的自噬会导致“代谢饥饿”表型，使TSC2缺陷细胞对进一步的自噬抑制高度敏感。与这一假设一致，我们 已经发现抑制自噬会导致代谢失调，并降低TSC2缺陷细胞在体内的生长。在目标3中，我们将解决这样的假设，即依赖p62/seecestosome1的信号网络促进TSC2缺陷细胞的生长和存活。与这一假设一致，我们发现p62在人血管肌脂瘤和其他TSC2缺陷细胞中积聚是低自噬的结果，并且p62的shRNA下调抑制了TSC2缺陷细胞的体内生长。我们的体内策略(目标2和3)包括TSC2缺陷的血管肌脂肪瘤来源细胞和TSC2+/-小鼠的肾囊/囊腺瘤定量。在整个提案中，我们将利用创新、最先进的技术，包括使用“海马”系统实时监测生物能量参数、与布罗德研究所合作进行代谢组谱分析、高通量合成致命性药物筛选，以及血管肌脂肪瘤细胞的体内生物发光检测。将产生新的试剂，包括2个新的小鼠模型(TSC2+/-ATG5+/-和TSC2+/-p62-/-)。和血管肌脂肪瘤来源的细胞，其shRNA下调关键分子。这个项目的意义在于，它将首次揭示自噬依赖的细胞网络如何在TSC的肾脏疾病发病机制中发挥作用。我们预计这个项目将产生很高的影响，因为TSC相关的损害对儿童和成人都有破坏性的后果，而且TORC1信号网络在包括多囊肾病在内的其他人类疾病中调节失调。 与公共卫生相关：对于患有TSC的儿童和成人，迫切需要新的治疗范例，对他们来说，唯一的非手术治疗选择是持续的，也许是终生的，使用TORC1抑制剂治疗。我们将确定自噬依赖的信号网络影响TSC的发病机制和靶向治疗的中心机制，从而为TSC和许多其他人类疾病带来新的治疗范式，在这些疾病中，TORC1信号轴被失调。

DESCRIPTION (provided by applicant): TSC patients develop multi-system disease including benign tumors of the brain, heart, skin and kidney. By age 10, 80% of children with TSC have renal angiomyolipomas and/or renal cysts. Mammalian target of rapamycin (mTOR) complex 1 (TORC1), which is activated in tuberous sclerosis complex (TSC), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment with TORC1 inhibitors partially decreases the size of TSC-associated brain and kidney lesions, but they regrow when treatment is stopped. Our central hypothesis is that dysregulation of autophagy and cellular metabolism plays a critical role in the pathogenesis of TSC and in the response of TSC-associated renal lesions to TORC1-targeted therapy. In Aims 1 and 2, we will test the hypothesis that low levels of autophagy in TSC2-deficient cells lead to a "metabolic starvation" phenotype, making TSC2-deficient cells hypersensitive to further autophagy inhibition. Consistent with this hypothesis, we have found that inhibiting autophagy induces metabolic dysregulation and decreases the in vivo growth of TSC2-deficient cells. In Aim 3, we will address the hypothesis that p62/sequestosome1-dependent signaling networks promote the growth and survival of TSC2-deficient cells. Consistent with this hypothesis, we have found that p62 accumulates in human angiomyolipomas and other TSC2-deficient cells as a consequence of low autophagy, and that shRNA down regulation of p62 inhibits the in vivo growth of TSC2-deficient cells. Our in vivo strategy (Aims 2 and 3) includes TSC2-deficient angiomyolipoma-derived cells and renal cyst/cystadenoma quantitation in Tsc2+/- mice. Throughout the proposal, we will utilize innovative, state-of-the-art technology including real-time monitoring of bioenergetic parameters using the "Seahorse" system, metabolomic profiling in collaboration with the Broad Institute, high throughput synthetic-lethality drug screening, and in vivo bioluminescent detection of angiomyolipoma cells. Novel reagents will be generated, including 2 new mouse models (Tsc2+/-Atg5+/- and Tsc2+/-p62-/-.) and angiomyolipoma-derived cells with shRNA down regulation of key molecules. The significance of this project is that it will reveal for the firsttime how autophagy-dependent cellular networks contribute to the pathogenesis of renal disease in TSC. We expect this project to have high impact because TSC-associated lesions have devastating consequences in both children and adults and because the TORC1 signaling network is dysregulated in other human diseases including polycystic kidney disease. PUBLIC HEALTH RELEVANCE: Novel therapeutic paradigms are urgently needed for children and adults with TSC, for whom the only non- surgical treatment option is continuous, perhaps life-long, treatment with TORC1 inhibitors. We will identify the central mechanisms through which autophagy-dependent signaling networks impact the pathogenesis and targeted therapy of TSC, leading to new therapeutic paradigms for TSC and the many other human diseases in which the TORC1 signaling axis is dysregulated.