【作者】 曹汐汐；

【导师】 许祖德； 刘秀萍；

【作者基本信息】 复旦大学 ， 病理学， 2010， 博士

【摘要】 前言乳腺癌的发病率和死亡率逐年升高,是严重威胁女性身心健康的恶性肿瘤之一。而乳腺癌的复发和转移是影响其疗效的主要因素。因此,迫切需要寻找一个敏感性好且能较早预测乳腺癌转移的分子生物学标记物及能够抑制其增殖、迁移运动和转移的基因治疗靶点,为临床对乳腺癌的复发和转移做出早期预测提供一定的参考依据,从而有助于及时采取更加有效的治疗措施。人RACK1 (receptor of activated C-kinase 1)定位于人的第5号染色体(5q35.3),cDNA全长951 bp,其编码的蛋白质分子量为35kDa。有学者在人黑色素瘤、口腔鳞癌等肿瘤中对RACK1与肿瘤转移的关系进行了研究。但该基因与人乳腺癌增殖、迁移运动和侵袭转移的关系究竟如何,以及RACK1与乳腺癌生物学行为的相关性等问题,目前国内外尚未见系统性的研究报道,更无其促进人乳腺癌增殖、迁移运动和侵袭转移的分子机制的研究报道。本课题试图从人乳腺癌细胞株、裸鼠原位(乳垫下)移植瘤模型和人体乳腺癌组织三方面对RACK1表达与乳腺癌生物学行为的相关关系及其作用的分子机制进行研究,以寻找预测人乳腺癌转移的分子生物学标记物及分子治疗的新靶点。故该课题的研究不仅对了解肿瘤的生物学特性具有明显的理论意义,也具有显著的临床价值和社会效益。第一部分从人乳腺cDNA文库中筛选与PI3K plloa相互作用的候选分子及相互作用的验证目的筛选与PI3K p110α相互作用的新候选分子。方法采用酵母双杂交系统以pGBKT7-PIK3CA质粒为钓饵,在人乳腺cDNA文库中筛选。经酵母重新转化验证后,在哺乳动物细胞中使用免疫共沉淀(immunoprecipitation, IP)和免疫荧光(immunofluorescence, IF)共定位的方法确定候选分子RACK1和PI3Kplloα的相互作用。结果以pGBKT7-PIK3CA质粒为钓饵,在人乳腺cDNA文库中筛选出RACK1作为候选分子。在哺乳动物细胞中免疫共沉淀确定两者相互作用,免疫荧光确定两者共定位于胞质。小结成功构建pGBKT7-PIK3CA质粒并以此为钓饵质粒,利用酵母双杂交系统从人乳腺cDNA文库筛选出RACK1,在酵母和哺乳动物细胞中验证了候选分子RACK1与PI3K p110α存在相互作用,并确定其作为进一步研究的对象。第二部分RACK1对人乳腺癌增殖、迁移和侵袭转移的影响目的明确RACK1对人乳腺癌增殖、迁移和侵袭转移的影响。方法采用脂质体介导法将pEGFP-N3-RACK1转染人乳腺癌细胞MCF7和T-47D,以筛选出稳定高表达RACK1的细胞株。采用生长曲线和流式细胞技术研究RACK1对人乳腺癌细胞体外增殖的影响；使用划痕实验和transwell迁移实验研究RACK1对其体外迁移运动能力的影响；应用transwell侵袭实验研究RACK1对人乳腺癌细胞体外侵袭运动能力的影响；同时建立裸鼠原位移植瘤模型,从体内角度研究高表达RACK1对乳腺癌增殖、侵袭转移的影响。结果获得稳定高表达RACK1的MCF7和T-47D细胞株；体外实验表明,与对照组相比,稳定高表达组的细胞倍增时间明显降低,S期的比例明显增高；体外迁移运动和侵袭能力明显提高。在裸鼠移植瘤实验中发现,稳定高表达RACK1细胞组裸鼠乳垫下移植瘤生长较对照组明显加快,肿瘤的体积和重量明显增加,侵袭性明显提高。小结采用脂质体介导法将pEGFP-N3-RACK1成功转染人乳腺癌细胞MCF7和T-47D,获得稳定高表达RACK1的细胞株。高表达RACK1在体内、外均能促进人乳腺癌增殖、迁移运动和侵袭转移潜能。第三部分RACK1对人乳腺癌增殖、迁移和侵袭转移影响的相关机制研究目的阐明RACK1对人乳腺癌增殖、迁移运动和侵袭转移作用的机制。方法采用Western blot的方法检测稳定高表达RACK1的人乳腺癌细胞和相应抑制剂处理后以及RACK1 siRNA干扰人乳腺癌细胞后,细胞周期相关蛋白表达的变化,及PI3K/Akt和MAPK信号通路中关键分子活性的改变；采用IP和IF检测RACK1和RhoA在细胞中的相互作用；采用细胞体外迁移实验检测Rho激酶抑制剂处理后稳定高表达RACK1的人乳腺癌细胞体外迁移能力的改变；采用GTP-Rho pull-down实验检测稳定高表达RACK1的细胞和Rho激酶抑制剂处理后以及RACK1 siRNA干扰后RhoA活性的改变；采用明胶酶谱实验检测稳定高表达RACK1的人乳腺癌细胞和RACK1 siRNA干扰后细胞MMPs活性的改变;采用免疫组织化学(immnohistochemistry, IHC)的方法在体内研究RACK1促进人乳腺癌增殖、侵袭转移能力的体内机制。结果与空载对照组相比,稳定高表达RACK1的人乳腺癌细胞株p-Akt的表达明显上升,细胞周期素cyclin D1和cyclin D3的表达也明显上升,而p21的表达下降。使用PI3K的抑制剂(LY294002,10μM)12、24h后,cyclin D1和cyclin D3的上调被抑制,而p21的表达上升；IP证实RhoA与RACK1存在相互作用,IF显示两者共定位于MCF7和MCF7/ADR的胞质；与溶剂对照组相比,使用Rho激酶的抑制剂Y-27632(10μM)预处理的稳定高表达细胞,其迁移能力明显被抑制；Rho活性检测实验显示与空载对照组相比,稳定高表达细胞中活化的RhoA显著增多,而在使用了RACK1 siRNA或Y-27632后又明显减少；Western blot和明胶酶谱检测结果显示,与对照相比,稳定高表达RACK1的细胞株CD147的表达和MMP2的活性明显上升；裸鼠IHC结果显示,与空白亲本及空载对照组相比,稳定高表达组裸鼠组织中Ki67和MMP2的表达明显上升。小结RACK1通过调节PIK/Akt信号通路的活性及细胞周期相关蛋白p21的下调、cyclin D1和cyclin D3的升高,参与调控人乳腺癌细胞的增殖能力；RACK1通过与RhoA的相互作用及活化RhoA/Rho激酶通路,促进人乳腺癌细胞的迁移运动；RACK1通过调节CD147的表达及MMP2的活性,促进人乳腺癌的侵袭转移能力。第四部分人乳腺癌组织中RACK1的表达及其与临床参数的关系目的阐明人乳腺癌组织中RACK1的表达及其与临床参数的关系。方法采用IHC的方法在160例人乳腺癌组织标本中研究RACK1的表达及其与临床参数的关系。结果RACK1蛋白主要表达在细胞质中；在160例乳腺癌标本中,40例样本表现为癌细胞RACK1表达评分为2级,有54例评分为1级,有66例评分为O级；RACK1的表达与肿瘤大小(P=0.046)、淋巴结转移(P=0.027)、临床分期(P=0.007)和组织学分级(P=0.004)相关；RACK1的表达与Ki67有很强的相关性,经Pearson’s相关分析,两者的相关系数高达0.930,P<0.001；另外,RACK1与RhoA、HER-2呈正相关；与原位癌百分比、ER, PR呈负相关；Kaplan-Meier生存分析揭示,RACK1的高表达与乳腺癌癌患者较短的生存期之间有显著相关性(P<0.001)；单、多因素生存分析显示RACK1蛋白在乳腺癌患者预后分析中是一个独立的有预后意义的分子指标(P=0.006); RACK1的ROC曲线下的面积为0.833,而其它临床肿瘤分子指标的ROC面积均低于0.80,分别为Ki670.766,ER 0.446,PR0.387和HER-20.689。小结RACK1在乳腺癌患者预后分析中是一个独立的有意义的分子指标；与其他乳腺癌临床常用分子指标相比,RACK1具有更大的优越性和更广阔的应用前景。更多还原

【Abstract】 IntroductionThe incidence and mortality of breast carcinoma are increasing each year, which is one of the major causes of death in women. Among various prognostic indicators of breast carcinoma, disease recurrence and metastasis are key factors affecting the outcome of primary breast carcinoma therapy. Identification of sensitive and specific biomarkers for metastatic potential and their regulatory genes in breast carcinoma cells, therefore, is important for both early clinical detection and effective prophylactic therapy.Human RACK1 (receptor of activated C kinase 1) is located in 5q35.3, with a cDNA length of 951 bp that codes for a 35 kDa protein. Several published reports have focused on the effects of RACK1 expression on melanoma and oral squamous cell carcinoma metastasis. The exact role RACK1 plays in breast carcinoma proliferation, migration and invasion/metastasis is, however, still largely unclear. Specifically, there has been no report on the relation and mechanisms underlying RACK1 expression and the biological behavior of breast carcinoma in either Chinese literature or elsewhere.Using three experimental models - respected clinical specimens of human breast carcinoma, established breast carcinoma cell lines stably transfected with RACK1, and xenograft in nude mouse mammary pad - this study investigated the impact of RACK1 on the metastatic potential of breast carcinoma in an effort to identify RACK1 as a biomarker predicting early metastasis and a potential target for therapy. Part I Screening a Human Mammary cDNA Library with PI3K p110αas Bait and the Interaction ConfirmationPurpose To identify novel PI3K p110α-interacting proteinsMethods A yeast two hybrid system was utilized (pGBKT7-PIK3CA as a bait) to screen a human mammary cDNA librasy. After verification in yeast cells, we performed IP and IF to confirm the interation between PI3K p110αand RACK1Results We screened a human mammary cDNA library with PI3K p110αas bait. Of the several newly implicated proteins, RACK1 was chosen for successive detailed analysis. The immunoprecipitation and immunofluorescence analyses sufficiently identified RACK1 as a novel binding partner of PI3K p110α.Conclusions RACK1 is a novel binding partner of PI3K p110α.Part II Promotion of Proliferation, Migration and Invasion/Metastasis by RACK1 in Human Breast CarcinomaPurpose To investigate the effect of RACK1 on proliferation, migration and invasion/metastasis of human breast carcinomaMethods We established breast carcinoma cell lines stably-transfected with RACK1. MTT assay and flow cytometry was used to evaluate the effects of RACK1 on proliferation. Migration assaywas used to analyze the effects of RACK1 on migration in vitro. Transwell invasion assay was utilized to evaluate the effects of RACK1 on invasion. Furthermore, an orthotopic nude mouse model was established to investigate the effects of RACK1 on tumor growth and metastasis in vivo.Results Experiments in breast carcinoma cell lines stably-transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration and invasion. Conclusions RACK1 promotes proliferation, migration and invasion/metastasis in human breast carcinoma in vitro and in vivo.PartⅢThe Mechanisms Underlying the Promotion of Proliferation, Migration and Invasion/Metastasis Induced by RACK1 in Human Breast CarcinomaPurpose To clarify possible mechanisms underlying the promotion of proliferation, migration and invasion/metastasis induced by RACK1 in human breast CarcinomaMethods We used Western blot to evaluate the cell cycle related protein expression level and activations of PI3K/Akt and MAPK pathway in breast carcinoma cells stably transfected with RACK1 treated with Rho kinase inhibitor and siRNA specifically targeting RACK1, compared with control cells. We used IP and IF to investigate the interaction of RACK1 and Rho A. Trans well migration assay was also applied to investigate the changes in migration of breast carcinoma cell treated with Rho kinase inhibitor. Besides, gelatin zymographic analysis, GTP-Rho pull-down assay and immnohistochemistry were utilized in this section as well.Results Experiments in breast carcinoma cell lines stably-transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration and invasion. In cell lines stably-transfected with RACK1, p-Akt, cyclin D1, cyclin D3 and CD 147 expression, as well as MMP2 activity, were elevated. RACK1-induced migration could be inhibited by the addition of Rho-kinase inhibitor.Conclusions RACK1 upregulates proliferation via PI3K/Akt pathway, targeting p21, cyclin D1 and cyclin D3; migration via interaction with RhoA and activation of RhoA/Rho kinase pathway; invasion via CD 147 and MMPs.PartⅣRACK1 Expression and its Correlation with Clinicopathological Indicators in 160 Breast Carcinoma Patients Purpose To clarify RACK1 Expression and its correlation with clinicopathological indicators in 160 breast carcinoma patientsMethods IHC was performed in 160 breast carcinoma samples to investigate RACK1 Expression and its correlation with clinicopathological indicators.Results RACK1 immunoreactivity was readily detected in the cytoplasm and occasionally in the nucleus. Cases scoring 0,1 and 2 were 66,54 and 40, respectively. We did observe significant correlations of RACK1 with tumor size (P= 0.046), lymph node metastasis (P = 0.027), clinical stage (P = 0.007) and histological grading (P = 0.004). Pearson’s correlation coefficient between RACK1 and Ki67 was 0.930, P<0.001. Kaplan-Meier survival analysis of 160 cases revealed a correlation between higher RACK1 expression levels and shorter disease-specific survival times (P<0.001). Factors that affected survival by univariate analyses included tumor size, lymph node metastasis, RACK1, Ki67 and HER-2 (all P<0.05). The ROC areas for RACK1, Ki67, ER, PR and HER-2 were 0.833,0.766,0.446,0.387 and 0.689, respectively.Conclusions RACK1 is a superior independent biomarker for diagnosis and prognosis comparing with currently widely-used diagnostic index in breast carcinoma.更多还原