Retention of somatic mutations in cancers by changes in pH sensing

描述(申请人提供)：癌症中的体细胞基因突变是随机发生的，但一个重要的悬而未决的问题是如何保留选择性突变。保留在一定程度上是由健康特征决定的，例如克服增殖障碍或免疫监视，使其适应肿瘤微环境。我们的建议测试了一种关于健康特征和适应的新想法，这些特征和适应有助于保留癌症中的基因突变。这一新想法是基于大多数癌症的保守特征，即细胞内pH(phi 7.5-7.6)高于正常细胞(phi 7.1-7.2)。我们预测，选择性体细胞突变会导致pH感知的增强或丧失，这为PHI较高的癌症提供了适应优势。为了限制测试这一新想法的范围和风险，我们将重点研究Arg&gt；在癌症中的突变，并预测这些突变编码了PH值感知的收益。我们的特定假设是，癌症中phi的增加为选择性精氨酸对组氨酸突变的保留提供了一种适应性优势。我们的初步分析显示，由于密码子偏向，Arg&Gt；His突变的发生率高于预期，包括CpG突变效应。我们对Arg&GT的pH感测增益的预测；他的突变是基于组氨酸在溶液中具有近中性的PKA，因此可以在正常细胞的phi处质子化，在癌细胞的较高phi处不带电。相反，因为Arg的pKa~12很可能保持在正常细胞和癌细胞的phi。在目标1中，我们使用生物信息学方法测试保留组氨酸突变的进化力量。我们将开发新的生物信息学技术来对Arg&gt；的体细胞突变进行排序，并表征在这些位置上运行的历史和当代进化力量，以量化突变偏向，并确定相对于整个进化时间尺度的预期，改变他的突变的PKA在癌症中的保留程度。在目标2中，我们对重复出现的候选突变进行了实验测试，以获得酸碱度感知。我们将在体外以及在正常和转化的克隆细胞中确定野生型和突变型蛋白的pH依赖功能。我们还将通过计算预测突变的组氨酸和精氨酸的PKA，并使用分子动力学模拟测试蛋白质结构中依赖于pH的构象变化。据我们所知，与pH敏感有关的癌症中的突变，如我们将测试的最常见的突变之一P53-R273H，还没有报道，体细胞突变相对于更高的 癌症的PHI。如果正确，我们的假设将产生一个实质性的新观点，为什么癌症中的一些体细胞突变会被保留下来。此外，成功证实我们的假设将使未来的研究有说服力，以测试His&gt；Arg突变是否导致pH敏感丧失，以及涉及酪氨酸、丝氨酸和苏氨酸残基的突变，这些突变在磷酸化时具有接近中性的PKA。因此，我们的发现可以广泛应用，并对针对导致癌症的突变的pH依赖的治疗策略产生重大影响。

DESCRIPTION (provided by applicant): Somatic gene mutations in cancers occur randomly but a significant unresolved question is how selective mutations are retained. Retention is in part determined by fitness traits such as overcoming barriers to proliferation or immune surveillance that confer an adaptation to the tumor microenvironment. Our proposal tests a new idea on fitness traits and adaptation contributing to retention of gene mutations in cancers. This new idea is based on the conserved trait of most cancers having a higher intracellular pH (pHi 7.5-7.6) than normal cells (pHi 7.1-7.2). We predict that selective somatic mutations confer gain or loss of pH sensing that provides an adaptive advantage to the higher pHi of cancers. To limit the scope and risk of testing this new idea we will focus on Arg>His mutations in cancers with the prediction that these mutations encode a gain of pH sensing. Our specific hypothesis is that increased pHi in cancers confers an adaptive advantage for retention of selective arginine to histidine mutations. Our preliminary analysis reveals a higher incidence of Arg>His mutations than expected from codon bias, including CpG mutational effects. Our prediction on gain of pH sensing by Arg>His mutations is based on histidine having a pKa near neutral in solution and hence can be protonated at the pHi of normal cells and uncharged at the higher pHi of cancer cells. In contrast, because Arg has a pKa ~12 it likely remains charged at the pHi of normal and cancer cells. In Aim 1 we test evolutionary forces for retention of histidine mutations by using bioinformatics approaches. We will develop novel bioinformatics techniques for ranking Arg>His somatic mutations, and characterize the historical and contemporaneous evolutionary forces operating at these positions to quantify mutational biases and determine the extent to which pKa altering His mutations are retained in cancers relative to what would be expected across evolutionary timescales. In Aim 2 we experimentally test recurring Arg>His candidate mutations for gain of pH sensing. We will determine pH-dependent functions of wild type and mutant proteins in vitro and in normal and transformed clonal cells. We also will computationally predict pKa's of mutated histidines and arginines and test pH-dependent conformational changes in protein structure using molecular dynamics simulations. To our knowledge mutations in cancers conferring pH sensing, such as p53-R273H, one of the most commonly occurring mutations we will test, has not been reported, nor has the retention of somatic mutations relative to the higher pHi of cancers. If correct, our hypothesis would generate a substantial new view on why some somatic mutations in cancers are retained. Additionally, successfully confirming our hypothesis would be compelling for future studies to test His>Arg mutations for loss of pH-sensing, and mutations involving tyrosine, serine and threonine residues that have pKa's near neutral when phosphorylated. Hence, our findings could be applied broadly and have substantial impact on therapeutic strategies to target pH dependence of mutations driving cancers.