Role of CPT1A as a lysine succinyltransferase in tumorigenesis

项目摘要 致癌性或肿瘤抑制性信号传导通常导致翻译后修饰（PTM）的不平衡 在人类癌症中。受体酪氨酸激酶HER 2是一种癌基因，在细胞中扩增或过表达。 约25%的乳腺癌患者患有乳腺癌，并且与肿瘤侵袭性和预后不良相关。一 最近的研究表明，HER 2以激酶活性依赖性方式易位至线粒体， 改变乳腺癌细胞的代谢并促进其耐药性。与这些发现一致，我们最近的 研究表明，致癌成纤维细胞生长因子受体（FGFR）1，也位于 线粒体，直接酪氨酸磷酸化线粒体丙酮酸脱氢酶激酶（PDHK）1， 增强其酶活性以促进小鼠肿瘤生长。综合来看，这些结果强烈表明 包括HER 2在内的致癌酪氨酸激酶的线粒体定位调节线粒体代谢， 促进肿瘤生长的途径。然而，哪些线粒体代谢酶受 乳腺癌中的HER 2完全未知。因此，我们对线粒体进行了磷酸化蛋白质组学分析， 从HER 2 + BT474乳腺癌细胞中富集组分，发现线粒体肉毒碱 棕榈酰转移酶（CPT）1A在Y514处被酪氨酸磷酸化。进一步的生化分析表明， CPT 1A Y514通过HER 2磷酸化促进其肉毒碱棕榈酰转移酶（CPT酶）活性。 有趣的是，我们最近发现CPT 1A作为赖氨酸琥珀酰转移酶具有额外的酶活性 在体外和体内，CPT 1A是LSTase的一种酶，它能使底物蛋白的赖氨酸残基琥珀酰化。 哺乳动物细胞中的第一个也是唯一的LSTase。重要的是，CPT 1A Gly 710（G710 E）的突变选择性地 灭活的CPT酶活性而不是LST酶活性。与CPT 1A WT相似，CPT 1A G710 E增加了总 赖氨酸琥珀酰化，而不影响细胞内琥珀酰辅酶A水平。这些发现表明 CPT 1A作为LSTase具有前所未有的作用，可调节赖氨酸琥珀酰化依赖性信号转导 在细胞中独立于其促进线粒体脂肪酸氧化的典型CPT酶活性（FAO） 通路然而，值得注意的是，CPT 1A的LSTase活性是如何调节的，以及LSTase活性是否被调节。 对于肿瘤发生的重要性尚不清楚。以前的研究报告说，多种类型的癌症，包括 黑色素瘤、前列腺癌、乳腺癌和卵巢癌显示CPT 1A表达升高， CPT 1A与si/shRNAs降低癌细胞增殖。我们还发现CPT 1A mRNA的高表达， 在HER 2+乳腺癌患者中， 在其他类型的乳腺肿瘤中观察到mRNA表达和患者生存率降低。另外我们 发现CPT 1A的敲低以及拉帕替尼对HER 2的抑制，减少了CPT 1A-底物基序， LVxxK琥珀酰化和抑制BT474细胞增殖。这些结果表明，CPT 1A LSTase活性和HER 2信号在乳腺癌细胞增殖调节中的作用因此，我们假设， HER 2介导的CPT 1A在Y514的磷酸化可能促进其LSTase活性， 典型的CPTase活性，以重新编程代谢，从而增加肿瘤增殖。为了解决这个问题， 我们在目标1中提出，研究HER 2依赖性CPT 1A Y514磷酸化如何影响其双重作用， 酶活性在目标2中，我们将研究新的LSTase和典型的LSTase中的一个或两个是否都是有效的。 CPT 1A的CPTase活性改变乳腺癌代谢。在目标3中，我们将研究LSTase是否 CPT 1A的活性通过CPT 1A Y514磷酸化促进乳腺癌细胞增殖和肿瘤生长。 总的来说，拟议的研究将对双重酶活性的作用产生新的机理见解 （LSTase和CPTase活性），并提供了全面的 了解两种不同的PTM（酪氨酸磷酸化和赖氨酸琥珀酰化）如何协调， 通过信号中枢蛋白CPT 1A改变代谢以促进肿瘤生长。

Project Summary Oncogenic or tumor suppressive signaling often causes imbalances in posttranslational modifications (PTMs) in human cancer. The receptor tyrosine kinase HER2 is an oncogene amplified or overexpressed in approximately 25% of breast cancers and is associated with tumor aggressiveness and poor prognosis. A recent study demonstrated that HER2 is translocated to mitochondria in a kinase activity-dependent manner to alter metabolism and promote drug resistance in breast cancer cells. Consistent with these findings, our recent study showed that oncogenic fibroblast growth factor receptor (FGFR) 1, which is also localized in mitochondria, directly tyrosine phosphorylates mitochondrial pyruvate dehydrogenase kinase (PDHK) 1, enhancing its enzyme activity to promote tumor growth in mice. Taken together, these results strongly suggest that mitochondrial localization of oncogenic tyrosine kinases, including HER2, regulate mitochondrial metabolic pathways that contribute to tumor growth. However, which mitochondrial metabolic enzymes are regulated by HER2 in breast cancer is totally unknown. Thus, we performed phospho-proteomic analyses of mitochondria- enriched fractions from HER2+ BT474 breast cancer cells and found that mitochondrial carnitine palmitoyltransferase (CPT) 1A is tyrosine phosphorylated at Y514. Further biochemical analyses showed that CPT1A Y514 phosphorylation by HER2 promotes its carnitine palmitoyltransferase (CPTase) activity. Interestingly, we recently found that CPT1A has an additional enzymatic activity as a lysine succinyltransferase (LSTase) to succinylate lysine residues of substrate proteins in vitro and in vivo .To our knowledge, CPT1A is a first and only LSTase in mammalian cells. Importantly, mutation of CPT1A Gly710 (G710E) selectively inactivated CPTase activity but not LSTase activity. Similar to CPT1A WT, CPT1A G710E increased total lysine succinylation in cells without affecting intracellular succinyl-CoA levels. These findings suggest the unprecedented role of CPT1A as LSTase that can regulate lysine succinylation-dependent signal transduction in cells independent of its canonical CPTase activity that facilitates mitochondrial fatty acid oxidation (FAO) pathway. Notably, however, how the LSTase activity of CPT1A is regulated and whether LSTase activity is important for tumorigenesis is not known. Previous studies reported that multiple types of cancers including melanoma, prostate, breast, and ovarian cancers showed elevated expression of CPT1A and knockdown of CPT1A with si/shRNAs decreased cancer cell proliferation. We also found that high CPT1A mRNA expression in HER2+ breast cancer patients strongly correlates with reduced survival while no correlation between CPT1A mRNA expression and reduced patient survival was observed in other types of breast tumors. In addition, we found that knockdown of CPT1A, as well as inhibition of HER2 by lapatinib, decreased CPT1A-subsrate motif LVxxK succinylation and suppressed BT474 cell proliferation. These results suggest a link between CPT1A LSTase activity and HER2 signaling in regulation of breast cancer cell proliferation. We thus hypothesize that HER2-mediated phosphorylation of CPT1A at Y514 may promote its LSTase activity in addition to the canonical CPTase activity to reprogram metabolism, thereby increasing tumor proliferation. To address this, we propose in Aim 1 to examine how HER2-dependent CPT1A Y514 phosphorylation affects its dual enzymatic activities. In Aim 2, we will examine whether either or both of the novel LSTase and canonical CPTase activities of CPT1A alter breast cancer metabolism. In Aim 3, we will examine whether the LSTase activity of CPT1A promotes breast cancer cell proliferation and tumor growth via CPT1A Y514 phosphorylation. Overall, the proposed studies will shed new mechanistic insight into the role of the dual enzymatic activities (LSTase and CPTase activities) of CPT1A in HER2+ breast tumorigenesis and provide the comprehensive understanding of how two distinct PTMs (tyrosine phosphorylation and lysine succinylation) are coordinated to alter metabolism through the signaling hub protein CPT1A to promote tumor growth.