Roles for Intracellular pH Dynamics in Cancer

 描述(申请人提供)：结构性细胞内pH升高(Phi)在大多数癌症中是常见的，无论其组织来源或遗传背景如何。然而，较高的phi如何使疾病进展，以及调节phi依赖的癌细胞行为的分子机制还没有得到充分的研究，而且大多还不清楚。我们之前揭示了phi增加如何通过调节控制细胞定向迁移的pH敏感蛋白来实现转移进展。我们现在将讨论phi增加如何使癌细胞有三种额外的行为：肿瘤形成、新陈代谢重新编程和保留复发的体细胞突变。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有着良好的记录，以揭示pH传感器的设计原理和功能意义，pH传感器的定义是具有在细胞狭窄的pH范围内调节的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、癌症突变数据库和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相结合。我们在使用基因编码的生物传感器测量体内实时PHI动力学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，我们将检验这一假设，即增加PHI对于肿瘤发生行为是必要的和充分的。我们将在小鼠模型中解析依赖Phi的肿瘤发生的机制，以确定我们报道的在H外流和癌基因表达缺失的果蝇中的合成致死性。我们将确定在没有癌基因的情况下PHI增加如何导致异型增生，并测量肿瘤发生过程中的空间和时间PHI动态，作为一种新的指标来告知我们肿瘤细胞的异质性。这些研究包括对预测能够实现致癌行为的pH传感器的结构和功能分析。在目标2中，我们将检验这一假说，即增加PHI可以使癌症中的代谢重新编程。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对pH敏感的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。我们还解决了增加的phi如何抑制线粒体氧化磷酸化和调节碳的命运，通过磁共振光谱确定。在目标3中，我们将检验这一假设，即增加的phi为保留带有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奖项的发现的支持，该奖项验证了癌症中反复发生的体细胞突变的pH敏感性，并利用基于氨基酸替代签名的生物信息学分析确定了癌症亚型，我们将测试这些亚型是否具有共同的功能特性。我们提案的结果将对癌症的分子机制产生新的见解，这些分子机制可以为针对pH传感器的治疗方法提供信息，以限制疾病的进展。

 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.