Roles for Intracellular pH Dynamics in Cancer

 描述（由申请人提供）：组成性升高的细胞内 pH (pHi) 对于大多数癌症来说都是常见的，无论其组织起源或遗传背景如何。然而，较高的 pHi 如何导致疾病进展以及介导 pHi 依赖性癌细胞行为的分子机制尚未得到充分研究，而且大多不为人所知。我们之前揭示了 pHi 增加如何通过调节控制定向细胞迁移的 pH 传感蛋白来促进转移进展。我们现在将讨论 pHi 的增加如何促进三种额外的癌细胞行为：肿瘤形成、代谢重编程和反复出现的体细胞突变的保留。我们利用我们独特的专业知识在分子、细胞和组织水平上解决 pHi 动力学如何调节细胞功能。我们在连接结构和细胞生物学方面拥有丰富的记录，揭示了 pH 传感器的设计原理和功能意义，pH 传感器定义为在细胞狭窄的 pH 范围内调节活性或配体结合亲和力的蛋白质。为了识别 pH 传感器，我们将信号通路分析、癌症突变数据库以及蛋白质中可电离残基的滴定网络与分子动力学模拟、生物化学和细胞生理学结合起来。我们在使用基因编码生物传感器测量体内实时 pHi 动态方面的专业知识为理解癌细胞生物学带来了一种独特的新方法。在目标 1 中，我们将检验以下假设：pHi 升高对于致瘤行为是必要且充分的。我们将解决小鼠模型中 pHi 依赖性肿瘤发生的机制，以确定我们在果蝇中报道的 H 外流和癌基因表达缺失的综合致死率。我们将确定在没有癌基因的情况下 pHi 增加如何诱导发育不良，并测量肿瘤发生过程中的空间和时间 pHi 动态，作为了解肿瘤细胞异质性的新指标。这些研究包括对 pH 传感器的结构和功能分析，预计可实现致癌行为。在目标 2 中，我们将检验 pHi 增加能够促进癌症代谢重编程的假设。我们将确定 pHi 增加如何促进葡萄糖利用从线粒体氧化磷酸化转变为增加有氧糖酵解的机制。这些研究包括对公认的 pH 敏感糖酵解酶磷酸果糖激酶-1 和乳酸脱氢酶的结构和功能分析。我们还解决了通过磁共振波谱测定增加的 pHi 如何抑制线粒体氧化磷酸化并调节碳命运。在目标 3 中，我们将检验以下假设：pHi 的增加为保留组氨酸残基的体细胞突变提供了选择压力。这些研究得到了 R21 奖项的研究结果的支持，该奖项验证了癌症中反复出现的体细胞突变的 pH 敏感性，并通过生物信息学分析根据氨基酸取代特征识别了癌症亚型，我们将测试其共同的功能特性。我们提案的结果将产生关于癌症发生的分子机制的新见解，从而为针对 pH 传感器的治疗方法提供信息，以限制疾病进展。

 DESCRIPTION (provided by applicant): Constitutively increased intracellular pH (pHi) is common to most cancers regardless of their tissue origin or genetic background. However, how a higher pHi enables disease progression and the molecular mechanisms mediating pHi-dependent cancer cell behaviors are understudied and mostly not understood. We previously revealed how increased pHi can enable metastatic progression by regulating pH-sensing proteins controlling directed cell migration. We now will address how increased pHi enables three additional cancer cell behaviors: tumor formation, metabolic reprograming and retention of recurring somatic mutations. We exploit our unique expertise in resolving at the molecular, cellular and tissue levels how pHi dynamics regulates cell functions. We have a strong track record of bridging structural and cell biology to reveal the design principles and functional significance of pH sensors, defined as proteins with activities or ligand binding affinities regulated within the narrow pH range of the cell. To identify pH sensors we integrate analyses of signaling pathways, cancer mutations databases, and titrating networks of ionizable residues in proteins with molecular dynamics simulations, biochemistry and cell physiology. Our expertise in measuring real-time pHi dynamics in vivo using a genetically encoded biosensor brings a distinct new approach to understanding cancer cell biology. In Aim 1 we will test the hypothesis that increased pHi is necessary and sufficient for tumorigenic behaviors. We will resolve mechanisms for pHi-dependent tumorigenesis in mouse models to determine the synthetic lethality we reported in Drosophila with loss of H+ efflux and oncogene expression. We will determine how increased pHi in the absence of oncogenes induces dysplasia, and measure spatial and temporal pHi dynamics during tumorigenesis as a new metric to inform us about heterogeneity of tumor cells. These studies include a structural and functional analysis of pH sensors predicted to enable oncogenic behaviors. In Aim 2 we will test the hypothesis that increased pHi enables metabolic reprograming in cancer. We will determine mechanisms for how increased pHi can promote a switch in glucose utilization from mitochondrial oxidative phosphorylation to increased aerobic glycolysis. These studies include a structural and functional analysis of the recognized pH-sensitive glycolytic enzymes phosphofructokinase-1 and lactate dehydrogenase. We also resolve how increased pHi suppresses mitochondrial oxidative phosphorylation and regulates carbon fates, determined by magnetic resonance spectroscopy. In Aim 3 we will test the hypothesis that increased pHi provides a selective pressure for the retention of somatic mutations with histidine residues. These studies are supported by findings from an R21 award that verified pH sensitivity of recurring somatic mutations in cancers and with bioinformatics analyses identified cancer subtypes based on amino acid substitution signatures, which we will test for shared functional properties. Outcomes of our proposal will generate new insights on molecular mechanisms enabling cancer that can inform therapeutic approaches targeting pH sensors to limit disease progression.