【作者】 王晖；

【导师】 黄翼然；

【作者基本信息】 上海交通大学 ， 外科学， 2014， 博士

【摘要】 肾细胞癌(RCC,renal cell carcinoma)是最为常见的泌尿系统恶性肿瘤之一,目前该肿瘤发病率的增长速度为2%。25%的肾癌患者在就诊时肿瘤已经出现局部淋巴结转移或者远处转移。而发生转移的患者死亡率及术后肿瘤的复发率均明显高于早期发现肾癌的患者。肾脏透明细胞癌(cc RCC,clear cell renal cell carcinoma)是肾癌中最为常见的病理类型,约占肾细胞癌总数的80%。肾透明细胞癌快速生长需要有很强的有氧糖酵解、脂肪合成代谢等一系列物质合成过程与之相配合,为肿瘤的快速增殖、侵袭性生长提供物质基础。特别是cc RCC细胞中富含脂滴的病理特点决定了它具备非常强劲的脂肪合成能力。单纯的食物来源已经远远不能满足肿瘤细胞对脂类物质的需求,cc RCC细胞则主要通过从头脂肪酸合成途径来合成获得所需的脂类物质。目前的研究发现cc RCC细胞中脂肪从头合成途径所需的一系列酶表达异常活跃。我的研究课题研究核心是脂肪酸从头合成途径的一个重要的限速酶——硬脂酰辅酶A去饱和酶1(SCD-1),它是催化饱和脂肪酸向单不饱和脂肪酸转化的限速酶,可以催化棕榈酰辅酶A和硬脂酰辅酶A形成棕榈油酰辅酶A和油酰辅酶A。棕榈油酰辅酶A和油酰辅酶A是膜磷脂、三酰甘油酰、胆固醇酯等多种物质生物合成的主要底物。SCD-1是一种定位于内质网上的跨膜蛋白,它对于维持细胞内饱和脂肪酸和不饱和脂肪酸的正常比例有着重要的作用。近来的研究证明SCD-1参与了多种恶性肿瘤的脂质合成代谢,对于肿瘤的生存,增殖和细胞内的信号转导都有重要的作用。目前的研究证实膜受体酪氨酸激酶(RTK)-PI3K-AKT-m TOR通路中包含有多个原癌基因,在恶性肿瘤细胞中,这一通路上的基因表达异常尤为常见。围绕SCD-1表达异常对肾透明细胞癌细胞的PI3K-AKT-m TOR信号通路的影响这一核心问题,课题具体聚焦三个方面:1)肾透明细胞癌组织中和透明细胞细胞系中SCD-1的表达情况及与肿瘤分期分级的相关性;2)抑制SCD-1的表达对于PI3K-AKT-m TOR信号通路的影响;3)靶向m TOR抑制剂对于干扰SCD-1表达的稳转株细胞的影响作用。并根据研究内容展开了三部分的研究工作。第一部分肾透明细胞癌中SCD-1的表达目的:研究SCD-1在肾透明细胞癌细胞株中及肿瘤组织中的表达情况,分析SCD-1的表达与肾透明细胞癌临床分期及细胞分级的相关性。方法:采用Western蛋白印迹方法进行检测透明细胞癌组织和癌膀正常组织中SCD-1的表达及四种透明细胞癌细胞株中SCD-1的表达情况。同时采用免疫组化的方法检测50例肾透明细胞癌石蜡切片中SCD-1蛋白表达情况,并结合患者的临床分期和细胞分级进行分析。结果:SCD-1在肾透明细胞癌组织中的表达量均明显高于癌旁正常组织中的表达量(P<0.05)。SCD-1在四种肾透明细胞癌细胞系中的表达量增高,但不同的细胞株SCD-1的表达量也有所差别。免疫组化结果示:肿瘤组织中SCD-1的表达量也随之增加(P<0.05)。SCD-1的表达率也随着肿瘤细胞分级和淋巴结转移的增加而增加,差异具有统计学意义(p<0.01)。结论:SCD-1在肾透明细胞癌组织中的表达量明显高于邻近的正常组织。SCD-1在786-O、769-P和CAKI肾透明细胞癌细胞株中表达量较高,而在A498细胞株中表达量相对较低。SCD-1的表达量与肾透明细胞癌的分级和临床分期呈正相关关系。SCD-1的表达量与肿瘤的恶性程度有关,组织中的SCD-1表达量可以作为预测肾透明细胞癌恶性程度的一个重要指标。第二部分SCD-1的表达与AKT信号通路的关系目的:研究干扰肾透明细胞癌细胞株SCD-1表达后对PI3K-AKT-mTOR信号转导系统的影响。方法:通过含有SCD-1干扰质粒的病毒干扰786-O和769-P细胞,建立SCD-1稳定干扰细胞株,然后检测PI3K-AKT-m TOR信号通路上重要分子的表达改变。用划痕实验检验SCD-1干扰后对细胞迁移性的影响。结果:SCD-1稳定干扰株细胞AKT的磷酸化水平明显下调,且细胞的侵袭转移能力较未干扰的细胞有明显下降(P<0.05)。稳定干扰SCD-1表达后,细胞内P-AKT(Ser473)和P-AKT(Thr308)两个位点的活化均有下降,Ser473位点的活性降低较Thr308位点的降低更为明显。ERK本底的表达未见明显改变,而P-ERK激活出现明显的下调。AKT的上游的PI3K和PDK1分子的表达并没有发生相应的改变。4E-BP1和P-P70S6K1也出现表达下调的现象。结论:SCD-1稳定干扰细胞株中AKT Ser473磷酸化活化水平明显下降。而Thr308位点磷酸化水平并未受到明显的抑制。SCD-1稳定干扰细胞株中m TOR1下游分子p70S6K1和4E-BP1的表达明显下降,提示肿瘤细胞的蛋白质合成受到抑制SCD-1稳定干扰细胞株中AKT上游分子PDK1和PI3K的活性并未受到影响。AKT对ERK的激活能力受到抑制,ERK的磷酸化激活更依赖于Ser473位点的激活,而非Thr308位点。SCD-1稳定干扰细胞株细胞侵袭能力明显下降。第三部分靶向药物治疗肾透明细胞癌的治疗策略目的:研究PI3K-AKT-m TOR靶向抑制剂对于SCD-1干扰细胞株的抑制效应,探讨增强新的靶向联合治疗方案以提高肾癌的治疗效果。方法:通过应用PI3K/m TOR抑制剂-PI103,AKT抑制剂MK2206,m TOR抑制剂:Rapamycin,AZD8055,RAD001作用于786-O和769-P细胞及其对应的SCD-1稳转株细胞,观察药物对肿瘤生长、增殖及迁移的抑制作用。结果:AZD8055和PI-103两种抑制剂可以增强对786-O和769-P SCD-1稳定干扰细胞株的增殖抑制效应,两种细胞半数抑制浓度均有明显的下降。AZD8055和PI-103两种抑制剂可以进一步抑制786-O和769-P SCD-1稳定干扰细胞株的迁移能力。AZD8055和PI-103可以明显增强对AKT-m TOR通路的抑制作用。结论:mTORC1和mTORC2抑制剂可以有效的增强对SCD-1干扰细胞株的增殖抑制效应,这种抑制效应是通过抑制AKT-m TOR通路产生的。多靶点的m TOR抑制剂和SCD-1的抑制剂的联合应用可以作为一种新的靶向联合治疗策略。更多还原

【Abstract】 Renal cell carcinoma(RCC) is one of the most common malignant tumors of urinary system. Its incidence is increasing at an annual rate of 2%. 25% patients suffering from RCC are diagnosed having local lymph node metastasis or distant metastasis. And the reoccurrence rate of postoperative metastasis and mortality are obviously higher than that of patients with local renal cell carcinoma. Clear cell renal cell carcinoma(cc RCC) is the most common pathological type in renal cell carcinoma, accounting for about 80% of the total renal cell carcinoma. Rapid growth of cc RCC needs strong aerobic glycolysis, lipid metabolism and protein synthesis. It is reported cc RCC cell is rich in lipid droplets which reflects it has a very strong ability of fat synthesis. De novo lipid synthesis is a major pathway to obtain lipid required for cc RCC cells. It is found that de novo lipid synthesis in cc RCC cells required a series of active enzyme expression. Stearoyl-coenzyme A desaturase 1(SCD-1) is one of the most important enzymes. It is the rate limiting enzyme which can catalyze palmitoyl coenzyme A and stearoyl- coenzyme A into palmitoyl oleoyl coenzyme A and oleoyl coenzyme A respectively. Palm-oil-acyl-co A and oleoyl-co A are the main substrate of membrane phospholipids, triglycerides and cholesterol ester. SCD-1 is a transmembrane protein in the surface of endoplasmic reticulum. It plays an important role in maintaining the normal proportion of the saturated fatty acid and unsaturated fatty acid in cells. Recent studies have proved that SCD-1 is involved in the synthesis of lipids metabolism and signal transduction in many malignant tumors. Membrane receptor tyrosine kinase(RTK)-PI3K-AKT-m TOR is a important signal transduction pathway in cells. A series of proto oncogene are involved in this pathway which are particularly important in tumorigenesis due to their abnormal expression. Our research work mainly focus the effect of abnormal SCD-1 expression onPI3K-AKT-m TOR signal pathway in renal clear cell carcinoma cells. We accomplish our research work from three aspects: 1) the expression of SCD-1 in cc RCC cells and tumor tissue; the correlation between SCD-1 expression in tumor tissues and tumor clinically pathological feature; 2) the effect of SCD-1 inhibition on PI3K-AKT-m TOR signaling pathway in cc RCC; 3) effect of m TOR inhibitor on stably SCD-1 interference cell linesPart I Expression of SCD-1 in renal clear cell carcinomaObjective: To investigate the expression of SCD-1 in clear cell renal cell carcinoma and its association with clinically pathological feature of the tumorMethods: The expression of SCD-1 protein in clear cell carcinoma tissues and cell lines were detected by Western blotting. We detected SCD-1expreesion in 50 cases of renal clear cell carcinoma specimen by immunohistochemistry and investigated its association with tumor clinical stage and cell classification.Results: The expression of SCD-1 in renal clear cell carcinoma tissues was significantly higher than that in the adjacent normal tissues. The expression of SCD-1 was higher in 786-O, 769-P, CAKI cell lines, and relatively low in A498 cells. The results of immunohistochemistry showed that SCD-1 expression was correlated with the tumor grade and stage(P<0.05 and p<0.01 respectively).Conclusion: The expression of SCD-1 in renal clear cell carcinoma tissues was significantly higher than that in the adjacent normal tissues. There is a increase in the expression of SCD-1 in four clear cell renal carcinoma cell lines. There is a positive correlation between SCD-1expression in renal cell carcinoma and tumor stage and grade. The expression of SCD-1 is related to tumor malignancy and SCD-1 expression can be an important predictor of malignancy of clear cell renal cell carcinoma.Part II The relationship between the expression of SCD-1 and AKT signal pathwayObjective: To study the effect of inhibition of the expression of SCD-1 on AKT signal transduction system in clear cell renal cell line.Methods: SCD-1 expression was stably inhibited by viruses in 786-O and 769-P cells. We detected expression changes of PI3K-AKT-m TOR signal pathway using Western Blot. Wound healing assay was performed in transfected cells to test the effect on cell migration.Results: AKT phosphorylation were significantly inhibited in SCD-1 interfered cells. The invasion and metastasis ability of SCD-1 interfered cells decreased significantly compared to control cells(P<0.05). AKT(Ser473) and AKT(Thr308) activation decreased in SCD-1 interfered cells, while the inhibition of Ser473 phosphorylation is more obvious than Thr308 sites. There is a significant activation decreasing of P-ERK whereas expression of ERK has no change. Expression of PI3 K and PDK1 which is upstream molecular of the AKT has not changed. Expression of 4E-BP1 and P-P70S6K1 had the same trend as AKT.Conclusion: AKT Ser473 phosphorylation decreased significantly in SCD-1 interfered cell lines while Thr308 phosphorylation was not fully inhibited. Expression of p70S6K1 and 4E-BP1 decreased significantly in the interfered cell lines which suggested that protein synthesis in tumor cells was inhibited. PDK1 and PI3 K activity were not interfered by SCD-1 interference which meant AKT upstream molecules was not affected. ERK activation was inhibited, which meant the phosphorylation of ERK was more dependent on the Ser473 site instead of Thr308 site. Cell invasion ability of SCD-1 interfered cells decreased significantly.Part III Strategy of target drugs application for the treatment of clear cell renal cell carcinomaObjective: To study the inhibitory effect of PI3K-AKT-mTOR target inhibitors on SCD-1 interfered cell lines, and to improve the therapeutic result of combination targeted therapy on renal cell carcinoma.Method: We applied PI3K/mTOR inhibitor-PI103, AKT inhibitor MK2206, mTOR inhibitor: Rapamycin, AZD8055 and RAD001 to 786-O, 769-P cells and the corresponding SCD-1 interfered cell lines. Then we detected the inhibition effect of drugs on tumor growth and migration.Results: AZD8055 and PI-103 could enhance the effect of proliferation inhibition caused by SCD-1 interference in 786-O and 769-P cells, which could decrease half inhibitory concentration significantly. AZD8055 and PI-103 could further inhibit the migration ability of SCD-1 interfered 786-O and 769-P cells which proved AZD8055 and PI-103 could significantly enhance the inhibitory effect on AKT-m TOR pathway.Conclusion: mTORC1 and mTORC2 inhibitors can enhance the inhibitory effect on cell proliferation significantly in SCD-1 interfered cells, which indicates the inhibitory effect is produced by inhibiting the AKT-m TOR pathway. The combined application of multi-target m TOR inhibitors and SCD-1 inhibitors can be used as a new therapy strategy to cure clear cell renal cell carcinoma.更多还原