Molecular and Genetic Studies of Bladder Tumorigenesis

膀胱癌非常常见，治疗成本高，但研究非常少。膀胱癌的发病率 癌症在退伍军人中的发病率高于普通人群，因为这种疾病主要是 折磨着有吸烟史的老年男性患膀胱癌的风险更高 脊髓损伤的患者--这种情况在退伍军人中非常普遍。有许多挑战， 膀胱癌的临床管理，包括复发和进展的不可预测性， 低级别非侵袭性肿瘤，肌肉侵袭性肿瘤的侵袭性及其 尽管进行了根治性化疗，但仍有转移，顺铂耐药率高，缺乏有效的化疗方案， 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 痛苦和折磨，高昂的医疗费用和低质量的生活。尽管其流行，临床挑战 和社会经济后果，膀胱癌已收到的关注水平远远低于许多少 常见和致命的疾病。直到最近，对膀胱癌的研究相对较少， FDA批准的治疗晚期膀胱癌的新药。而整个基因组外显子组 最近，人类膀胱癌的转录组分析为研究竞技场注入了新的活力，许多 这些新发现并没有从根本上改变临床实践。主 因此，该项目的目标是深入了解基因和分子事件， 主要作用，膀胱癌的形成和发展，然后利用这些信息，以开发新的 诊断和治疗策略。为了实现这一总体目标，在2000年期间进行了一系列研究。 上一个资助期的重点是丙酮酸激酶（PKM 2）的亚型2，这是一种关键的限速酶， 有氧糖酵解或瓦尔堡效应。主要的发现包括：（i）PKM 2在绝大多数人中上调， 低级别乳头状和高级别浸润性膀胱癌;（ii）PKM 2的RNAi抑制显著降低了细胞增殖， （iii）紫草素结合并抑制PKM 2介导的肿瘤细胞增殖并增加凋亡和自噬; 生长;（iv）紫草素和顺铂一起在体外和体内对膀胱癌生长的抑制作用更强。 体内;（v）获得性顺铂耐药性与PKM 2上调密切相关;和（vi）组成性 PKM 2的尿路上皮特异性敲除可减少膀胱癌患者低级别乳头状膀胱肿瘤的形成 基因工程小鼠这些和其他数据表明PKM 2在膀胱肿瘤中的关键重要性。 肿瘤发生，并为本次更新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PKM 2在不同疾病中的作用和作用机制的理解， 膀胱癌的发病途径和分期及其治疗潜力。第一个系列将定义 肿瘤发生阶段，在此期间PKM 2是必不可少的。先进的小鼠基因工程方法 将被用来烧蚀PKM 2在一个时间控制的方式之前或之后形成的低等级 乳头状或高度浸润性膀胱癌。第二个系列将研究不同的突变是否 RTK-RAS-PI 3 K是膀胱癌中最常见的改变途径，其会聚于上调PKM 2。 培养细胞中特定突变的过表达和敲低将随后进行表达分析 PKM 2和RNA剪接因子。第三个系列将确定PKM 2的功能意义 顺铂耐药膀胱癌细胞中PKM 2的上调以及是否通过RNAi或紫草素重新抑制PKM 2， 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 应该提供新的见解PKM 2过表达的作用和潜在的机制， 膀胱癌的发生、发展及抑制PKM 2对顺铂减量的治疗价值 耐药性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.