Understanding the role of SKP2 in small cell lung cancer progression

背景：小细胞肺癌（SCLC）每年影响约5万人。由于缺乏有效的治疗方案，许多患者在确诊后5年的生存率低于5%。确定杀死SCLC肿瘤细胞的方法显然是必要的。像许多肿瘤一样，SCLC肿瘤在基因组的特定部分表现出复发性拷贝数改变（CNA）。对其他肿瘤类型的研究表明，发现复发性基因组异常驱动肿瘤存活可以带来更有效的治疗方案和患者生存。在这种伪装下，SCLC中有两种复发性基因组变化，视网膜母细胞瘤易感基因（RB1）的近普遍突变（~95%）和Skp2位点的基因组扩增（~65%）。先前的研究表明，在小鼠Rb-/-垂体和甲状腺肿瘤中，Skp2的缺失是合成致死的。因此，促进SCLC发展的突变组合可能被利用来促进SCLC细胞死亡。目的/假设：我假设很大一部分SCLC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细胞的分子谱，并确定Skp2缺失引发这些细胞凋亡的机制。具体目标：(1)；确定哪些SCLC细胞系需要Skp2来增殖和存活。(2)。探讨肿瘤细胞中pRB和Skp2缺失之间的合成致死性机制。(3)。阐明SCLC细胞在Skp2缺失后促进生存的代偿性生存途径。研究设计：在慢病毒shRNA敲低Skp2或Scramble shRNA对照后，将在MGH癌症中心分子治疗中心的58个SCLC细胞系中进行细胞活力测定。基因表达谱将用于识别区分Skp2依赖性细胞系和Skp2非依赖性SCLC细胞的特征。基因表达谱将在Skp2基因缺失敏感和抗性细胞系中被敲除。先前的研究表明，Skp2的缺失通过涉及p27功能的机制诱导细胞凋亡；另外，其他研究表明，先前的研究表明Skp2的缺失会促进er应激。高水平的内质网应激可导致细胞凋亡。因此，p27的作用和内质网应激反应将在SCLC细胞中Skp2缺失后的合成致死率的背景下进行评估。基因表达谱将为理解为什么一些细胞对Skp2的缺失敏感而另一些细胞不敏感提供基础。对于仅耐受Skp2缺失的细胞，将在Skp2存在和缺失的情况下确定对细胞应激反应的评估。这将导致鉴定与Skp2敲低一起导致SCLC细胞死亡的联合方法。

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.