Molecular and Genetic Studies of Bladder Tumorigenesis

膀胱癌非常常见，治疗成本很高，但研究极少。膀胱癌的发病率 退伍军人的癌症比普通人群高得不成比例，因为这种疾病主要是 有吸烟史的老年男性会感到痛苦。患膀胱癌的风险更高 脊髓损伤的患者--一种在退伍军人中非常普遍的情况。在这方面有很多挑战 膀胱癌的临床处理，包括复发和进展的不可预见性 低级别非侵袭性肿瘤、肌肉侵袭性肿瘤的侵袭性及其倾向 尽管进行了根治性膀胱切除术，但顺铂耐药的频率很高，缺乏有效的转移 转移性疾病的治疗。因此，患有这种疾病的患者不可避免地会经历大量的 痛苦和苦难、高昂的医疗费用和低生活质量。尽管它很流行，但临床挑战 以及社会经济分支，膀胱癌受到的关注程度远远低于其他许多人 常见且致命的疾病。直到最近，致力于膀胱癌的研究相对较少，如果 FDA批准了任何治疗晚期膀胱癌的新药。而整个基因组，外显子组 最近对人类膀胱癌的转录组分析为研究领域注入了新的活力，许多 这些新发现并没有转化为从根本上改变临床实践。初级阶段 因此，这个项目的目标是深入了解基因和分子事件 在膀胱癌形成和发展中的主要作用，然后利用这些信息开发新的 诊断和治疗策略。为实现这一总体目标，在 上一个资助期专注于关键限速酶--丙酮酸激酶(PKM2)的异构体2 有氧糖酵解或Warburg效应。主要发现包括：(1)PKM2在绝大多数细胞中上调 低级别乳头状癌和高级别浸润性膀胱癌；(Ii)抑制PKM2的RNAi显著减少细胞 紫草素结合并抑制PKM2介导的肿瘤细胞 (4)紫草素和顺铂联合应用对膀胱癌的体外和体外生长抑制作用明显增强。 体内；(V)对顺铂的获得性耐药性与PKM2上调密切相关；以及(Vi)结构性 尿路上皮特异性PKM2基因敲除减少低级别乳头状膀胱癌的形成 转基因小鼠。这些数据和其他数据证明了PKM2在膀胱中的重要作用 肿瘤的发生，并为这项续展申请中提出的工作奠定了基础。三个系列的研究是 旨在扩大和加深我们对PKM2在不同环境中的作用和作用机制的理解 膀胱癌的途径、分期及其治疗潜力。第一个系列将定义 肿瘤形成阶段(S)，在此阶段，PKM2是不可或缺的。复杂的小鼠基因工程方法 将被用来在低品位形成之前或之后以时间控制的方式烧蚀PKM2 乳头状癌或高级别浸润性膀胱癌。第二个系列将研究发散突变 在RTK-RAS-PI3K中，膀胱癌中最常见的改变途径汇聚为上调PKM2。 在培养细胞中过度表达和敲除特定突变之后将进行表达分析 PKM2和RNA剪接因子的表达。第三系列将确定PKM2的功能意义 RNAi或紫草素Re对顺铂耐药膀胱癌细胞的上调及对PKM2的抑制作用 使用体外和体内方法使这些细胞对顺铂敏感。这三个系列的研究加在一起 应对PKM2过表达的作用(S)和潜在机制(S)提供新的见解 膀胱癌的发生发展及抑制PKM2降低顺铂的治疗价值 耐药和治疗各种形式的膀胱癌。

Bladder cancer is very common, costly to manage but extremely poorly studied. The incidence of bladder cancer is disproportionally higher in the Veterans than in the general population because the disease mainly afflicts the aging males with a history of cigarette smoke. The risk of developing bladder cancer is even higher in patients with spinal cord injuries – a condition very prevalent in the Veterans. There are many challenges in the clinical management of bladder cancer including the unpredictable nature of recurrence and progression of low-grade non-invasive tumors, the aggressiveness of muscle-invasive tumors and their tendency to metastasize despite radical cystectomy, the high frequency of cisplatin resistance and the lack of effective therapy for metastatic disease. Patients with this disease therefore inevitably experience tremendous amount of pain and suffering, high medical expenses and low quality of life. Despite its prevalence, clinical challenges and socioeconomic ramifications, bladder cancer has received a level of attention far below many less common and deadly diseases. Until recently, relatively little research was devoted to bladder cancer and few if any new drugs were approved by the FDA to treat advanced bladder cancer. While the whole genome, exome and transcriptome profiling of human bladder cancer of late has injected new life into the research arena, many of the new findings have not been translated into fundamentally changing the clinical practice. The primary goals of this project are therefore to acquire a deep understanding the genetic and molecular events that play principal roles in bladder cancer formation and progression and then to utilize such information to develop new diagnostic and therapeutic strategies. Toward this overall goal, a range of studies were carried out during the last funding period that focused on isoform 2 of pyruvate kinase (PKM2), a key rate-limiting enzyme controlling aerobic glycolysis or Warburg effect. Major findings include that (i) PKM2 is upregulated in a vast majority of low-grade papillary and high-grade invasive bladder cancer; (ii) RNAi inhibition of PKM2 markedly reduces cell proliferation and increases apoptosis and autophagy; (iii) shikonin binds and inhibits PKM2-mediated tumor cell growth; (iv) shikonin and cisplatin together are much more inhibitory of bladder cancer growth in vitro and in vivo; (v) acquired resistance to cisplatin is strongly associated with PKM2 upregulation; and (vi) constitutive urothelium-specific knockout of PKM2 reduces the formation of low-grade papillary bladder tumors in genetically engineered mice. These and other data demonstrate the critical importance of PKM2 in bladder tumorigenesis and set a stage for work proposed in this renewal application. Three series of studies are designed to broaden and deepen our understanding of the role and mechanisms of action of PKM2 in different pathways and stages of bladder cancer and its therapeutic potentials. The first series will define the tumorigenic stage(s) during which PKM2 is indispensable. Sophisticated mouse genetic engineering approach will be employed to ablate PKM2 in a time-controlled manner before or after the formation of low-grade papillary or high-grade invasive bladder cancer. The second series will examine whether divergent mutations of the RTK-RAS-PI3K, the most prevalently altered pathway in bladder cancer, converge to upregulate PKM2. Overexpression and knockdown of specific mutations in cultured cells will be followed by expression analyses of PKM2 and RNA splicing factors. The third series will determine the functional significance of PKM2 upregulation in cisplatin-resistant bladder cancer cells and whether inhibiting PKM2 by RNAi or shikonin re- sensitize these cells to cisplatin using in vitro and in vivo approaches. Together, these three series of studies should offer novel insights into the role(s) and underlying mechanism(s) of PKM2 overexpression during bladder cancer formation and progression and the therapeutic value of inhibiting PKM2 in reducing cisplatin resistance and in treating various forms of bladder cancer.