Polo-like kinase 1 and sphingosine-1-phosphate circuitry enhances TSC-mutant cell survival

肾脏疾病(囊肿、血管平滑肌脂肪瘤(AML)、肾功能障碍和肾癌)是结节性硬化症(TSC)发病率和死亡率的主要原因。结节性硬化症是一种罕见的疾病，由TSC1或TSC2基因功能缺失突变导致PI3K/mTOR信号通路异常激活引起。使用mTORC1抑制剂(MTORi)治疗可部分减少TSC中血管肌脂肪瘤的体积，但停止治疗后肿瘤会重新生长。由于mTORi的这些主要的细胞抑制作用，需要终身治疗，但副作用的风险增加。鞘磷脂是细胞功能的重要介体，包括增殖、坏死和凋亡。与我们的建议相关的是，神经酰胺和鞘氨醇-1-磷酸(S1P)这两种中央生物活性鞘磷脂在调节细胞死亡和存活方面表现出相反的作用。鞘磷脂信号与有丝分裂事件和Polo-like kinase1(PLK1)的激活有关。PLK1与TSC1相互作用，并使其磷酸化，从而调节mTORC1。TSC缺陷的细胞有异常的有丝分裂，PLK1水平升高，并且对PLK1抑制剂敏感。在初步的数据中，我们证明了在TSC2缺失的AML来源的细胞中，负责合成和作用主要生物活性的鞘氨醇、神经酰胺和鞘氨醇-1-磷酸(S1P)的三种关键酶，即二氢神经酰胺脱饱和酶1(DEGS1)、酸性神经酰胺酶(ASAH1)和鞘氨醇激酶1(SPHK1)显著激活。我们的中心假设是TSC突变，通过增加PLK1和S1P受体1和3(S1PR1和S1PR3)的表达，调节生物活性鞘脂的产生，促进细胞生存。成功完成拟议的AIM 1研究将建立稳定的AML来源细胞，其PLK1、S1PR1和S1PR3基因缺失，并使用CRISPR/Cas9基因编辑技术进行非靶向控制。成功完成拟议的AIM 2研究将证明CRISPR/Cas9基因敲除PLK1、S1PR1和S1PR3基因对鞘磷脂作用和细胞体外存活的生物学影响。这个项目的意义在于，它将成功地开发出关键的AML来源的PLK1、S1PR1或S1PR3基因敲除细胞，为进一步研究PLK1和鞘磷脂通路在TSC肾血管肌脂瘤中的作用奠定基础。这些未来的研究预计将产生积极的影响，因为对鞘磷脂代谢和作用的基于机制的了解将导致改善TSC和其他肾脏疾病的治疗策略。

Renal disease (cysts, angiomyolipomas (AML), renal dysfunction, and renal cell carcinoma) is a major cause of morbidity and mortality in Tuberous Sclerosis Complex (TSC), a rare disease caused by loss-of-function mutations in TSC1 or TSC2 gene that lead to aberrant activation of the PI3K/mTOR signaling pathway. Treatment with mTORC1 inhibitors (mTORi) partially decreases the volume of angiomyolipomas in TSC, but tumors regrow when treatment is discontinued. Because of these primarily cytostatic effects of mTORi, life-long therapy is required with increased risk for side effects. Sphingolipids play key roles as mediators of cellular functions, including proliferation, necrosis, and apoptosis. Relevant to our proposal, the two central bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), exhibit opposing roles in regulating cell death and survival. Sphingolipid signaling is associated with mitotic events and activation of polo-like kinase 1 (PLK1). PLK1 interacts with and phosphorylates TSC1 to regulate mTORC1. TSC-deficient cells have aberrant mitosis, increased PLK1 levels and are sensitive to PLK1 inhibitors. In preliminary data we demonstrate that three key enzymes responsible for the synthesis and action of the primary bioactive sphingolipids, ceramide, and sphingosine-1-phosphate (S1P), namely dihydroceramide desaturase 1 (DEGS1), acid ceramidase (ASAH1), and sphingosine kinase 1 (SPHK1), are markedly activated in TSC2-null AML-derived cells. Our central hypothesis is that TSC mutations, by increasing the expression of PLK1 and S1P receptor 1 and 3 (S1PR1 and S1PR3), regulate the production of bioactive sphingolipids that facilitate cell survival. Successful completion of the proposed Aim 1 studies will establish stable AML-derived cells with depleted PLK1, S1PR1, and S1PR3 genes and non-targeting control using CRISPR/Cas9 gene editing technology. Successful completion of the proposed Aim 2 studies will demonstrate the biological consequences of CRISPR/Cas9 knock-out PLK1, S1PR1, and S1PR3 genes on sphingolipid actions and cell survival in vitro. The significance of this project is that it will successfully develop critical AML-derived PLK1, S1PR1, or S1PR3 gene knock-out cells with validated biological functions for future studies to investigate PLK1 and sphingolipid circuitry in TSC renal angiomyolipomas. These future studies are expected to have a positive impact because a mechanism-based understanding of sphingolipid metabolism and actions will lead to improved strategies for the treatment of TSC and other renal diseases.