Preservation of Proteomic Stability and Promotion of Protein Lipidation by HSF1

目的1：探讨HSF1在PI3K/AKT信号驱动下维持组织过度生长中的作用。我们的初步结果表明，PI3K/AKT信号级联是MEFs中热休克（HS）激活HSR/PSR和恶性细胞中构成型HSF1激活所必需的。重要的是，AKT与HSF1发生物理相互作用。此外，AKT磷酸化HSF1的Ser230位点，而组成型活性AKT1的表达或肿瘤抑制因子PTEN的缺失足以激活HSF1。相比之下，AKT抑制剂阻断HSF1 Ser230磷酸化及其与HSP基因启动子的DNA结合。此外，组成型活性PI3K/AKT信号导致小鼠的大脑和肝脏过度生长或增大，情况类似于人类的巨脑畸形和肝脏肿大，导致产后快速死亡。重要的是，在两种组织中同时缺失Hsf1阻碍了过度生长，延长了动物的存活时间。此外，Hsf1缺失也显著阻碍Pten缺失小鼠肝脏过度生长，Pten是一种负调节PI3K活性的肿瘤抑制因子，延长了小鼠的生存期。我们的研究结果进一步表明，组成性活性的PI3K/AKT破坏蛋白质静止并诱导蛋白质毒性应激，Hsf1缺乏显著增强了这种应激。基于这些初步结果，我们计划探究：1)HSF1是否是AKT的新的生理底物；2) HSF1在体内是否以及如何抑制PI3K/AKT信号组成性激活诱导的蛋白毒性应激，从而促进组织过度生长；3)过度生长组织中蛋白质平衡被破坏的分子机制。目的2：探讨HSF1在促进脂质代谢和蛋白脂化中的作用。我们之前的研究表明，HSF1是AMPK的生理底物，AMPK是一个关键的细胞代谢传感器，AMPK介导的Ser121磷酸化负调控HSF1的激活。现在，我们使用缺乏转录活性的HSF1缺失构建体的初步结果表明，与野生型HSF1一样，它们与AMPK相互作用并抑制AMPK Thr172磷酸化，这是其激活的关键修饰，表明HSF1的作用机制与转录无关。相反，Hsf1缺陷引起AMPK激活，AMPK激活被AMPK抑制剂阻断。有趣的是，我们的研究结果表明HSF1可以与AMPK和LKB1共同沉淀，揭示出LKB1-AMPK-HSF1蛋白复合物。此外，在人类肾癌和乳腺癌样本中，较高的HSF1 mRNA水平与AMPK Thr172磷酸化呈负相关，这与我们的机制研究结果一致。我们的初步数据显示，Hsf1缺乏和Hsf1表达增强分别导致细胞脂质含量降低和升高，这表明Hsf1促进脂肪生成，支持恶性肿瘤。引人注目的是，hsf1缺陷小鼠的全身脂肪量明显减少。重要的是，HSF1对细胞脂质含量和体脂量的这些影响可以通过AMPK抑制剂或sirna介导的AMPK敲低显著地恢复，这表明HSF1的脂肪生成作用主要是通过AMPK抑制介导的。在分子水平上，HSF1缺陷导致SREBP1c失活，SREBP1c是控制脂肪生成基因表达的关键转录因子，ACC失活。胆固醇是一种重要的脂质，涉及许多关键的细胞过程，包括膜组成、信号转导和类固醇激素的合成。与细胞脂质含量降低一致，我们的结果显示HSF1缺乏引起的细胞胆固醇水平显著降低，这是由AMPK抑制所挽救的。基于这些初步结果，我们计划研究：1)HSF1抑制AMPK的分子机制；2) HSF1是否促进SHH蛋白的胆固醇化并支持SHH信号传导；3) HSF1是否促进异种移植人黑色素瘤模型中的脂质代谢和SHH胆固醇化。

Aim1: To examine the role of HSF1 in sustaining tissue overgrowth driven by oncogenic PI3K/AKT signaling. Our preliminary results show that the PI3K/AKT signaling cascade is required for activation of the HSR/PSR by heat shock (HS) in MEFs and for constitutive HSF1 activation in malignant cells. Importantly, AKT physically interacts with HSF1. Furthermore, AKT phosphorylates HSF1 at Ser230, and expression of the constitutively active AKT1 or loss of the tumor suppressor PTEN is sufficient to activate HSF1. By contrast, AKT inhibitors block HSF1 Ser230 phosphorylation and its DNA binding to HSP gene promoters. Furthermore, constitutively active PI3K/AKT signaling causes overgrowth or enlargement of both brains and livers in mice, conditions similar to megalencephaly and hepatomegaly in humans respectively, leading to rapid postnatal death. Importantly, simultaneous deletion of Hsf1 in both tissues impedes overgrowth and prolongs animal survival. Moreover, Hsf1 deletion also markedly impedes the liver overgrowth in mice deficient for Pten, a tumor suppressor negatively regulating PI3K activity, prolonging their survival. Our results further show that constitutively active PI3K/AKT disrupts proteostasis and induces proteotoxic stress, which is markedly heightened by Hsf1 deficiency. Based on these preliminary results, we plan to interrogate: 1) whether HSF1 is a new physiological substrate for AKT; 2) whether and how HSF1 suppresses proteotoxic stress induced by constitutive activation of PI3K/AKT signaling and thereby promotes tissue overgrowth in vivo; and 3) the molecular mechanisms underlying disrupted proteostasis in overgrown tissues. Aim 2: To examine the role of HSF1 in promoting lipid metabolism and protein lipidation. Our previous studies revealed that HSF1 is a physiological substrate for AMPK, a key cellular metabolic sensor, and that the AMPK-mediated Ser121 phosphorylation negatively regulates HSF1 activation. Now, our preliminary results using HSF1 deletion constructs deficient for transcriptional activity show that, just like the wild-type HSF1, they interact with AMPK and suppress AMPK Thr172 phosphorylation, a modification key to its activation, indicating a transcription-independent mechanism of action of HSF1. Conversely, Hsf1 deficiency causes AMPK activation, which is blocked by the AMPK inhibitor. Interestingly, our results show that HSF1 can be co-precipitated with both AMPK and LKB1, revealing a LKB1-AMPK-HSF1 protein complex. Furthermore, in human kidney and breast cancer samples higher HSF1 mRNA levels are inversely correlated with AMPK Thr172 phosphorylation, congruent with the results of our mechanistic studies. Our preliminary data show that Hsf1 deficiency and enhanced HSF1 expression result in diminished and heightened cellular lipid content, respectively, suggesting that HSF1 promotes lipogenesis to support malignancy. Strikingly, Hsf1-deficient mice display markedly reduced whole-body fat mass. Importantly, these effects of HSF1 on cellular lipid content and body fat mass can be markedly rescued by either AMPK inhibitors or siRNA-mediated AMPK knockdown, suggesting that the lipogenic effect of HSF1 is largely mediated via AMPK suppression. At the molecular level, HSF1 deficiency causes inactivation of SREBP1c, a key transcription factor controlling lipogenic gene expression, in addition to inactivation of ACC. Cholesterol is an important lipid implicated in many key cellular processes, including membrane composition, signaling transduction, and synthesis of steroid hormones. Congruent with diminished cellular lipid content, our results reveal a markedly reduced cellular cholesterol level caused by HSF1 deficiency, which is rescued by AMPK inhibition. Based on these preliminary results, we plan to investigate: 1) the molecular mechanisms underlying AMPK suppression by HSF1; 2) whether HSF1 promotes cholesteroylation of sonic hedgehog (SHH) proteins and supports SHH signaling; and 3) whether HSF1 promotes lipid metabolism and SHH cholesteroylation in xenografted human melanoma models.