【作者】 李春生；

【导师】 刘铜军；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 结直肠癌是消化道常见的恶性肿瘤之一，循证医学得出，全球范围内结直肠癌的发病率处于恶性肿瘤的第三位，每年约有120万例新增患者。全身化疗临床使用受到较多关注，其目的是通过全身或局部作用，达到消灭或抑制病患体内的残余病灶、微转移灶，提高临床治愈机率，以延长患者中位生存期，最终实现生活质量改善。分子靶向治疗药物的开发及应用，使晚期大肠肿瘤的治疗获益，进一步推进了结直肠癌肿瘤综合治疗的发展。对肿瘤分子靶向治疗的研究已成为当前的热点,针对这些分子事件作为靶点，大量与肿瘤信号传导通路中的激酶相关的拮抗剂正在进行研究中。其中，关于CDK抑制剂的研究已成为新型抗肿瘤药物的研究热点。临床前研究显示出了与细胞周期和CDK相应的生理作用。在不同类型癌症中，cyclin D-CDK4和cyclin-CDK6复合体通过Rb磷酸化驱动细胞周期通过G1-S过渡。因此针对G1期激酶的靶向治疗药物被产生。其中，CDK4、CDK6选择性抑制剂PD-0332991，已经通过抗癌安全测试和I/II期临床测试。目前，没有研究关于这种抑制剂在大肠癌治疗中的治疗潜力，同样，也少有研究关注于细胞周期通路在大肠癌中的应用。临床前和临床研究已经表明PD-0332991的抗癌活性，它作为选择性细胞周期蛋白依赖性激酶4/6抑制剂，在不同肿瘤治疗中具有视网膜细胞瘤蛋白（Rb依赖性。目前还不清楚在结直肠癌，CDK4、CDK6或两者是否都需要Rb磷酸化，因此PD-0332991可用于针对这个CDK-Rb轴的癌症治疗。SUMO蛋白被广泛表达在整个真核生物界，在体内，SUMO化可能会影响靶蛋白在稳定、定位或激活的任何一个方面。SUMO蛋白作用广泛，参与细胞活动的各个阶段，目前已知包括细胞的有丝分裂、生长、分化、衰老及凋亡过程。研究表明，SUMO修饰途径在肿瘤发生、发展中起到重要作用。UBC9水平的增加在许多人类癌症中被发现，并且UBC9过表达能增加癌细胞生长。SUMO的E3蛋白PIAS3（活化的STAT3蛋白抑制剂）在许多不同的癌症类型被上调，SUMO E1酶水平升高与降低与肝细胞癌患者存活率相关。此外，SUMO化能参与并调节重要的肿瘤抑制蛋白。第一部分：PD-0332991通过CDK6-RB轴抑制结直肠癌生长机制研究目的：研究大肠癌组织及细胞系中CDK4、CDK6、Rb等表达情况，明确CDK4、CDK6在大肠癌细胞周期调节中的作用，检验PD-0332991在大肠癌细胞中的抑制作用并研究作用机制。方法：采用酸性磷酸酶法测定细胞生长抑制率，PI染色细胞周期检测，组织总蛋白提取，蛋白电泳及Western Blot检测，慢病毒转染及RNAi技术，shRNA细胞稳定转染等实验技术和方法。结果：本研究表明，大肠癌组织及其匹配的正常结直肠组织比较，G1期CyclinD1，D2，D3，CDK4，CDK6和pRB蛋白在大肠癌组织高表达，CDK4和CDK6控制细胞周期G1-S转变；CDK6敲除，而不是CDK4敲除，明显减少Rb磷酸化，抑制结直肠癌细胞生长。因此，CDK6起着Rb磷酸化和肿瘤生长的关键作用。在结直肠癌细胞中， PD-0332991通过G1期阻滞，诱导阻断Rb磷酸化，抑制细胞的生长。第一次表明CDK6-Rb轴在癌症生长中至关重要，由于可实现CDK6-RB轴的靶向定位，PD-0332991可能被证明是用于治疗结肠直肠癌的新型药物。第二部分：SUMO1参与结直肠癌中CDK6-RB通路的修饰。目的：研究大肠癌细胞系中SUMO1蛋白表达情况，明确并验证SUMO1参与大肠癌细胞周期蛋白的修饰，并探讨及作用机制。方法：采用酸性磷酸酶法细胞活力测定，PI染色细胞周期检测，蛋白电泳及Western Blot检测，慢病毒转染及shRNA细胞稳定转染，免疫沉淀IP等实验技术和方法。结果：研究表明，在大肠癌细胞中SUMO1通路被激活，SUMO1在大肠癌组织过度表达，SUMO1敲除抑制了大肠癌的细胞周期进程和肿瘤生长，细胞周期同步化16小时后，肿瘤细胞进入S期时间明显延长，IP表明CDK6被2种SUMO1单体修饰，CDK6的SUMO化稳定蛋白质，从而维持磷酸化Rb的酶激活。因此，我们在大肠癌细胞研究的前期工作中，初步建立了CDK6-SUMO1调控的细胞周期通路。CDK6-SUMO1-Rb通路的生物学功能使得它可能成为肿瘤分子靶向治疗的一个理想新靶标，并有可能在此基础上进一步发展为高效、特异及低毒的抗肿瘤药物。结论：通过本课题的研究，结直肠癌组织与癌旁正常组织对比，SUMO1，CDK6，CDK4高表达，SUMO1，CDK6，CDK4在结直肠癌细胞系中高表达；PD-0332991通过CDK6-Rb通路的抑制达到结直肠癌中的治疗有效性，PD0332991阻断Rb磷酸化，诱导G1逃逸，进而抑制大肠肿瘤细胞生长；大肠癌细胞COLO320中CDK6被2种SUMO1修饰，SUMO1参与大肠癌细胞COLO320中CDK6-RB通路的修饰调节；SUMO1、CDK6shRNA敲除明显抑制大肠癌COLO320细胞生长，且SUMO1与CDK6分别敲除后细胞生长曲线一致。更多还原

【Abstract】 Colorectal cancer is one of the most common malignant tumor in digestivetract, it’s indicated that the morbidity of colorectal cancer is the third highest inmalignant tumor, there’s1200thousands new patients per year. Systemicchemotherapy is paid close attention in clinical, which aiming at eliminating orrestrain the patients from residual lesions and micrometastasis, improving theprovability of cure, lengthening the survival time and improving their lifequality by systematic and local function.The exploitation and application of the molecular targeted therapy makingcolorectal carcinoma great progress, which also boosting the comprehensivetreatment of the colorectal carcinoma, the related research is the hotspot, aimingat these molecular events, a large amount of researches about kinase relatedantagonists are underway, for example, CDK inhibitor is the hotspot of thenew-style anti-neoplastic drugs whose mechanism is the CDK functioning in thecell cycle.In different types of cancers, Cyclin D-CDK4and cyclin-CDK6complexdrive the cell cycle G1-S transition by Rb phosphorylation. So the related drugsoccur. CDK4and CDK6selective inhibitor PD-0332991has past the anti-cacertests and clinical stage I/II tests. Currently, there’re no related researches aboutapplying potential in colorectal cancer, meanwhile, there’re less people payingattention to the cell cycle passage applied on colorectal cancer. It’s indicated that PD-0332991has anticancer activity, it performs RB dependence in differenttumor treatments considered as selective cell cycle protein dependent kinase. It’sunknown whether CDK4and/or CDK6need Rb phosphorylation, so PD-0332991can be CDK-Rb axis cancer trement.SUMO protein is generally expressed in the whole eucaryotae, in the body,SUMOmay influence the target protein function of stabilization positioning oractivating. SUMO participate in every cell cycle stage including mitosis, growth,differentiation, aging and decay. It’s indicated that SUMO remodeling pathwaymakes difference in tumorgenesis and development. The increase of UBC9isdetected in many cancers, and UBC9overexpression can make tumor cell grow.E3protein PIAS3(activated STAT3protein inhibitor) of SUMO overexpressesin many different kinds of cancer, SUMO E1enzyme level is related to thesurvival rate of the hepatocellular carcinoma. Besides, SUMO can participateand adjust important tumor profiling.Part I: the mechanism research of PD-0332991colorectal cancer inhibitionthrough CDK6-RB axisObjective: Study the expression condition of the CDK4, CDK6, RB etc incolorectal cancer cells, verify the function of CDK4, CDK6in cell cycleadjustment, test the inhibition function of PD-0332991in colorectal cell andresearch the mechanism. Method: application of the acid phosphatase assay tomeasure the cell growth inhibition rate, cell cycle detection through PI dyeing, extract tissue total protein, protein electrophoresis and western Blot examination,lentiviral transfection and RNAi, shRNA cell stabilization transfection etc.Result: It’s indicated through this research that compared by the normalcolorectal tissue, the cancer tissue overexpresses the CyclinD1, D2, D3, CDK4,CDK6and pRB protein in G1stage, CDK4and CDK6control the G1-Stransform, CDK6knockout rather than CDK can obviously reduce Rbphosphorylation. Therefore, CDK6makes key function on RB phosphorylationand tumor growth. In colorectal cancer cell, PD-0332991induces RBphosphorylation blocked and control cells growth through G1stage retardation.Indicate the CDK6-Rb axis make difference in cancer growth for the first time,PD-0332991can be proved as the new type colorectal cancer drugs throughCDK6-RB axis target positioning test.Part II: participation of SUMO1in decoration of CDK6-RB pathway incolorectal cancerObjective: study the SUMO1protein expression condition in colorectalcells, verify the mechanism of the participation of SUMO in cell cycle proteindecoration. Method: cell activity test through acid phosphatase assay, cell cycledetection through PI dyeing, protein electrophoresis and western Blotexamination, lentiviral transfection and RNAi, shRNA cell stabilizationtransfection, Immunoprecipitation etc. Result: it’s indicated that SUMO1pathway is activated in colorectal cells, SUMO1overexpresses, so SUMO1 knockout can depress cell cycle of the cancer cell and tumor growth,16h aftercell cycle synchronization, the time of entering S stage is obviously lengthened.,it’s indicated by IP that CDK6is decorated by two kinds of SUMO1monomer,Sumolation of CDK6can stabilize the protein, thus sustain Rb phosphorylationenzyme activated. Therefore, in the early research, we preliminary establishCDK6-SUMO1cell cycle pathway. The biological function of CDK6-SUMO1-Rb pathway may let it become one of the ideal target, and may becomehigh efficient, specific and low-toxic antitumor drugs based on the current andfollowing research.Conclusion: It’s indicated through this research that compared by thenormal colorectal tissue, the cancer tissue overexpresses the SUMO1, CDK6,CDK4. SUMO1, CDK6and CDK4overexpress in colorectal cell lines;PD-0332991reach treatment effectiveness through inhibition of CDK6-Rbpathway, PD0332991blocks Rb phosphorylation, induce G1escape, and thenblock the growth of tumor cells; COLO320of the colorectal tumor cell isdecorated by two kinds of SUMO1, SUMO1participate in decorative adjust ofCDK6-Rb pathway in COLO320of colorectal tumor cell; SUMO1, CDK6shRNA knockout obviously inhibit COLO320growth, the growth curve ofSUMO1and CDK6separate knockout make consistency.更多还原