Understanding the role of SKP2 in small cell lung cancer progression

描述(申请人提供)：背景：小细胞肺癌(SCLC)每年影响约50,000人。由于缺乏有效的治疗选择，这些患者中的许多人在确诊后五年的存活率不到5%。显然，找到杀死小细胞肺癌肿瘤细胞的方法是当务之急。像许多肿瘤一样，小细胞肺癌在基因组的特定部分表现出经常性的拷贝数改变(CNA)。对其他肿瘤类型的研究表明，发现是什么反复出现的基因组异常导致肿瘤存活，可以导致更有效的治疗方案和患者生存。在这一假象下，小细胞肺癌的两个重复的基因组改变是独立的，视网膜母细胞瘤易感基因(RB1)的一个几乎普遍的突变(~95%)和Skp2基因的基因组扩增(~65%)。以往的工作表明，在Rb-/-小鼠的垂体和甲状腺肿瘤中，Skp2的缺失是人工合成的致死作用。因此，导致小细胞肺癌发生的突变组合可能被用来促进小细胞肺癌细胞的死亡。目的/假设：我假设很大一部分小细胞肺癌细胞对Skp2靶向治疗敏感。我的初步研究支持这一观点。我建议定义Skp2敏感的SCLC细胞的分子图谱，并确定Skp2缺失启动这些细胞凋亡的机制。具体目标：(1)以确定哪些小细胞肺癌细胞系需要Skp2来进行增殖和存活。(2)目的：探讨肿瘤细胞中pRb和Skp2缺失的综合致死机制。(3)目的：阐明在Skp2缺失后，小细胞肺癌细胞的代偿性生存途径。研究设计：在慢病毒shRNA敲除Skp2或干扰shRNA对照后，将在MGH癌症中心分子治疗中心的58个小细胞肺癌细胞系中进行细胞活性分析。基因表达谱将被用来识别区分Skp2依赖株和Skp2非依赖SCLC细胞的特征。在Skp2基因敲除后，将编制对Skp2丢失敏感和抗性的品系的基因表达谱。以前的工作表明Skp2的缺失通过涉及p27功能的机制诱导细胞凋亡；或者，其他工作表明Skp2的缺失促进了内质网应激。众所周知，高水平的内质网应激会导致细胞凋亡。因此，p27和内质网应激反应的作用将在SCLC细胞中Skp2丢失后的合成致死性的背景下进行评估。基因表达谱将为理解为什么某些细胞而不是其他细胞对Skp2的丢失敏感提供基础。对于单独耐受Skp2缺失的细胞，对细胞应激反应的评估将在Skp2存在和不存在的情况下确定。这将导致发现与Skp2基因敲除一起导致小细胞肺癌细胞死亡的联合途径。 与公共卫生相关：小细胞肺癌(SCLC)是最致命的癌症之一，确诊5年后，仍有不到10%的人仍然活着。这一建议将进一步确定Skp2蛋白在促进小细胞肺癌发展中所起的作用。此外，这项建议旨在巩固Skp2抑制作为小细胞肺癌患者潜在靶向抗肿瘤治疗的使用。

DESCRIPTION (provided by applicant): Background: Small cell lung cancer (SCLC) affects ~50,000 people a year. A lack of effective treatment options offers many of these patients a less than 5% survival rate five years after diagnosis. Identifying ways to kill SCLC tumor cells is clearly an imperative. Like many tumors, SCLC tumors display recurrent copy number alterations (CNA) in specific parts of the genome. Studies of other tumor types have shown that discovering what recurrent genomic abnormalities drive tumor survival can lead to more effective treatment protocols and patient survival. Under this pretense two recurrent genomic changes in SCLC stand alone, a near universal mutation (~95%) of the retinoblastoma susceptibility gene (RB1) and genomic amplification of the Skp2 locus (~65%). Previous work demonstrated that in Rb-/- pituitary and thyroid tumors in mice, loss of Skp2 was synthetic lethal. Therefore, the combinations of mutations that contribute to SCLC development may be exploited to promote SCLC cell death. Objective/Hypothesis: I hypothesize that a significant portion of SCLC cells will be sensitive to Skp2 targeted therapy. My preliminary studies support this notion. I propose to define the molecular profile of a Skp2- sensitive SCLC cell and to identify the mechanism by which loss of Skp2 initiates apoptosis in these cells. Specific Aims: (1.) To determine which SCLC cell lines require Skp2 for proliferation and survival. (2.) To identify the mechanism of synthetic lethality between loss of pRB and Skp2 in tumor cells. (3.) To elucidate the compensatory survival pathways in SCLC cells that promote viability following loss of Skp2. Study Design: Cell viability assays will be done in a panel of 58 SCLC cell lines housed at Center for Molecular Therapeutics at the MGH Cancer Center following lentiviral shRNA knockdown of Skp2 or a Scramble shRNA control. Gene expression profiles will be used to identify signatures that distinguish Skp2- dependent lines, from Skp2-independent SCLC cells. Gene expression profiles will be compiled following Skp2 knockdown in lines both sensitive and resistant to loss of Skp2. Previous work has suggested that loss of Skp2 induces apoptosis through a mechanism involving p27 function; alternatively, other work has shown that previous work demonstrated that loss of Skp2 promotes ER-stress. High levels of ER-stress are known to lead to apoptosis. Therefore the role of p27 and the ER-stress response will be assessed in the context of the synthetic lethality following loss of Skp2 in SCLC cells. The gene expression profiles will provide a basis for understanding why some cells and not others are sensitive to loss of Skp2. For cells tolerant of Skp2 loss alone, assessment of response to cellular stress will be determined in the presence and absence of Skp2. This will lead to identification of combined approaches that together with Skp2 knockdown will lead to SCLC cell death. PUBLIC HEALTH RELEVANCE: With fewer than 10% of people still alive, 5 years after diagnosis, small cell lung cancer (SCLC) is one of the most deadly forms of cancer. This proposal will further determine the role that the protein Skp2 plays in promoting SCLC development. Furthermore, this proposal aims to solidify the use of Skp2 inhibition as a potential targeted antitumor therapy for patients with SCLC.