Caffeine regulates splicing of cancer-related genes: dissecting the mechanism

描述(申请人提供)：Pre-mRNA的选择性剪接显著增加了人类蛋白质组的复杂性和功能多样性，在发育决策、基因表达调控以及当异常时在人类疾病的发病中发挥关键作用。重要的是，超过1000个剪接变异体与癌症表型相关。虽然对这些变异体的功能分析及其在癌症中的可能作用仍处于起步阶段，但它们作为癌症生物标记物/治疗靶点的潜在重要性已经引起了人们的极大兴趣。我们现在提供大量的初步数据表明，咖啡因，一种在人类饮食中大量消耗的刺激剂，可以诱导包括肿瘤抑制基因KLF6在内的一大批癌症相关基因的选择性剪接。以KLF6为原型，我们已经证明了这种诱导是快速和可逆的，并且发生在剪接水平上，而不是差异稳定水平上。我们还证明了四个经典的咖啡因信号通路在咖啡因诱导的选择性剪接中几乎没有作用，表明一个新的分子机制是可行的。重要的是，自从上一次提交以来，我们发现KLF6的选择性剪接受到SR蛋白SC35的调控，并且该蛋白是由咖啡因诱导的。我们现在假设，咖啡因至少部分地通过诱导SC35来调节选择性剪接，并可能模仿在癌细胞中异常激活的内源性途径。为了进一步剖析咖啡因对癌症相关基因剪接的影响，我们现在提议：1)以KLF6为原型，研究SC35在咖啡因介导的选择性剪接中的作用；2)研究剪接因子与KLF6 ESS-1和ISE-1在体内外的相互作用，并询问SC35调控KLF6剪接的模型：3)确定咖啡因上调SC35的机制；4)确定咖啡因是否在体内正常组织中诱导“癌症特异性”剪接变体。我们观察到咖啡因可以调节与癌症表型有关的各种基因的选择性剪接，这一观察具有非常重要的意义和及时的意义。我们期望这些研究将导致识别对该癌症基因亚集的剪接调节有共同作用的元件/因子/途径。我们还希望确定高咖啡因摄入量是否可以在体内正常细胞中诱导与癌症相关的剪接变体，导致瞬时表达，可能会混淆生物标记物分析和靶向治疗。

DESCRIPTION (provided by applicant): Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity and functional diversity, playing a key role in developmental decisions, gene expression regulation and, when aberrant, in human disease onset. Importantly, over 1000 splicing variants have been associated with the cancer phenotype. Although the functional analysis of these variants and their possible role in cancer is still in its infancy, their potential importance as cancer biomarkers/therapeutic targets has garnered much interest. We now present a substantial body of preliminary data showing that caffeine, a highly consumed stimulant in the human diet, induces alternative splicing of a large subset of cancer-related genes including the tumor suppressor KLF6. Using KLF6 as a prototype, we have shown that this induction is rapid and reversible and occurs at the level of splicing rather than differential stability. We also demonstrate that the four classic caffeine signaling pathways have little if any role in caffeine-induced alternative splicing, indicating that a novel molecular mechanism is operative. Importantly, since the previous submission we have found that alternative splicing of KLF6 is regulated by the SR protein SC35, and that this protein is induced by caffeine. We now hypothesize that caffeine regulates alternative splicing, at least in part, by the induction of SC35, and may mimic an endogenous pathway aberrantly activated in cancer cells. To further dissect the impact of caffeine on the splicing of cancer-related genes, we now propose to: 1) Investigate the role of SC35 in caffeine-mediated alternative splicing, using KLF6 as a prototype; 2) Investigate the interaction of splicing factors with KLF6 ESS-1 and ISE-1 in vitro and in vivo, and interrogate a model for regulation of KLF6 splicing by SC35: 3) Determine the mechanism by which caffeine elevates SC35 and 4) Determine whether caffeine induces "cancer-specific" splice variants in normal tissues in vivo. Our observation that caffeine can regulate alternative splicing of a variety of genes involved in the cancer phenotype is both highly significant and timely. We expect that these studies will result in the identification of elements/factors/pathways that are common to the splicing regulation of this cancer gene subset. We also expect to determine whether high caffeine consumption can induce cancer-related splice variants in normal cells in vivo, resulting in transient expression that may confound both biomarker analysis and targeted therapy.