【作者】 焦旸；

【导师】 童建； 樊赛军；

【作者基本信息】 苏州大学 ， 卫生毒理学， 2007， 博士

【摘要】 乳癌是女性最常见的恶性肿瘤,具有发病率高、侵袭性强、发病早期即可发生远处转移等特点,是危害女性健康的主要恶性疾病之一。乳癌的发生机制错综复杂,涉及大量基因或基因以外的变化和众多相互交通的细胞信号通路,这为人们寻找安全理想的乳癌诊疗方法带来极大挑战。因此,对乳癌发生、发展机制的研究一直是近年来乳癌研究中的主要方向之一。肿瘤抑制基因主要的生物学功能是通过抑制细胞的恶性转化从而抑制肿瘤的发生,其编码蛋白产物主要参与对细胞周期进程的调控并能诱导细胞凋亡,此外还可参与对细胞间粘附力、蛋白酶活性等与肿瘤侵袭性或转移性相关的功能调节;肿瘤抑制基因的突变或其正常功能的改变往往引起细胞恶性转化,最终导致癌变。近年来对肿瘤抑制基因广泛、深入的研究为乳癌的临床诊治提供了重要的理论依据和实验基础,肿瘤抑制基因也因此成为乳癌发生机制中的一个研究热点。Tob1基因(transducer of ERBB2, 1)属于TOB/BTG抗增殖家族,已知该家族所有成员都具有抗细胞增殖活性和细胞周期负性调控作用。众多研究报道Tob1基因可通过对cyclin D1的表达调控参与对细胞周期进程的调控,并在多种人类肿瘤中均具有肿瘤抑制基因特征。然而Tob1在恶性肿瘤发生中的具体作用机制至今尚不完全清楚,其在乳癌发生、发展中的相关功能研究也未见报道。本研究首先通过Western Blot法、Northern Blot法以及免疫组化染色法对多种人类乳癌细胞及乳癌组织中Tob1的表达情况进行了检测,结果发现在多种乳腺肿瘤细胞及多数乳癌组织中Tob1的表达水平均有不同程度的下降或缺失。采用MTT生存分析法发现由重组腺病毒介导的外源性Tob1能显著抑制乳癌细胞的生长;而在实验动物模型中,Tob1的高水平表达还可以显著抑制乳腺肿瘤的生长以及肿瘤新生血管的形成。综合体内、体外以及动物实验结果,首次证实Tob1在乳癌的发生、发展过程中发挥肿瘤抑制因子作用。随后,为阐明Tob1发挥乳癌抑制功能的具体作用机制,采用流式细胞术分析了Tob1的高水平表达对细胞周期进程及细胞凋亡的影响,结果显示外源性Tob1可引起乳癌细胞中细胞周期G0/G1期以及G2/M期阻滞,并能显著增加细胞凋亡的发生,这一结果与采用Western Blot法和RT-PCR法检测到的细胞周期G1期调控关键因子cyclin D1的表达下降以及凋亡相关因子Bax的表达水平的下降相一致。雌激素(estrogen)/雌激素受体(estrogen receptor, ER)信号通路的异常激活是乳癌发生的重要机制之一,本实验采用GST捕获法、免疫共沉淀法等检测了Tob1与ER间的相互作用。实验中采用自行构建的一系列可表达Tob1全长、各片段缺失体以及“LXXLL”模序突变体的重组表达质粒载体进行Tob1的功能研究,结果发现Tob1可以和ER发生相互结合,而且这种结合作用与Tob1的氨基末端有关,但并不完全依赖于LXXLL模序。此外,荧光素酶活性测定结果显示Tob1可显著抑制ER的转录活性。上述结果共同提示Tob1可能通过雌激素/ER信号通路在乳癌的发生过程中发挥重要生物学作用。采用相同实验方法,还检测到Tob1不仅与ER信号通路下游关键调控因子cyclin D1存在体内外相互作用,并能显著抑制cyclin D1的转录活性。综合Tob1对cyclin D1不同表达水平的调控作用,初步阐明了Tob1对乳癌的细胞周期进程及细胞生长产生影响的具体分子机制。最后通过集落形成实验、TUNEL法、宿主细胞修复实验以及Western Blot法,首次证实重组腺病毒介导的Tob1高水平表达可通过对受照射细胞中凋亡相关基因的表达调控,增强辐射诱导的细胞凋亡;外源性Tob1通过对DNA损伤修复通路中关键因子的表达调控,抑制受照射细胞的DNA损伤修复能力;Tob1可能分别通过上述机制增加人类乳癌细胞对电离辐射的敏感性。总之,本研究首次证实Tob1可分别通过对细胞周期进程的调控以及对细胞周期凋亡的诱导发挥乳癌抑制因子作用,同时Tob1可通过对乳癌细胞中DNA损伤修复的调控作用而增强乳癌细胞对放射治疗的敏感性。这些研究结果为乳癌的发生机制提供了新的理论基础和实验依据,结合后续动物模型实验和临床前实验,将为人类征服这一恶性肿瘤性疾病提供新的研究手段和工具。更多还原

【Abstract】 Breast cancer is one of the most common malignancies of women in China and the world,which results from genetic or epigenetic changes of both malignant cells and the host cells interacting with the tumor, involving numerous cross-talking pathways. The complex underlying mechanisms lead to poor understanding of breast carcinogenesis, so as to difficulties in finding efficient, safe and selective therapeutics of breast cancer.Tumor suppress genes can reduce and inhibit the alteration of normal cells’turning into tumor cells, and the functions of their protein products fall into several categories including repression of genes essential for the cell cycle, coupling the cell cycle to DNA damage, inducing apoptosis, blocking loss of contact inhibition, and inhibiting metastasis etc. Many studies suggest that any mutation of one or more tumor suppressor genes will increase the probability of carcinogenesis. Over the last decade, the growing knowledge of tumor suppressor genes and proteins associated with the development and progression of breast cancer has provided us the foundation on mechanisms contributing to breast carcinogenesis, as well as the better opportunities for therapy of this disease.Tob1 (transducer of ERBB2, 1) is a member of the TOB/BTG antiproliferative protein family characterized by its anti-proliferative activity with the abilities to regulate cell cycle negatively in the periphery. Some previous studies suggest that Tob1 plays a role in the control of G1-S progression by suppressing cyclin D1 expression, and therefore may act as a tumor suppressor gene. However, it remains unknown whether and how Tob1functions in breast carcinogenesis ,and it is the purpose of this study to elucidate these issues.By using Western Blot, Northern Blot and immunohistochemistry staining assays, it first demonstrated a lower or un-detectable level of Tob1 expression in human breast cancer cell lines and human breast cancer tissues compared to normal controls in this study. Enhanced expression of Tob1 notably inhibited in vitro growth of breast cancer cells. By using animal models with nude mice, it was found that Tob1 over-expression significantly suppressed tumor growth and angiogenesis. Taken together with in vitro, in vivo experiments and clinic data, all these results suggest that Tob1 may function as a tumor suppressor in breast cancer development and progression.In order to study the specific pathways and underlying mechanism(s) contributing to Tob1 anti-tumor activity in breast cancer, the effects of Tob1 on cell cycle regulation and apoptosis was examined by flow-cytometry assay, and the result showed an increased expression of Tob1 which caused a cell cycle G0/G1 and G2/M arrest ,and programmed cell death (apoptosis) induction associated with a down-regulation of cyclin D1, a key regulator of G1-to-S phase progression of the cell cycle, and a up-regulation of Bax, a pro-apoptotic Bcl-2 family protein.Abnormal activation of Estrogen/ER singling plays an important role in breast cancer development, progression and therapy. By using Co-immunoprecipitation, Western blot and GST pull-down assays, a series of recombinant vectors expressing wild type, truncation and“LXXLL”mutation Tob1 were constructed to analyze in vitro and in vivo association of Tob1 with ER. It was found that Tob1’s interacting with ER required the N-terminus of Tob1 but not the LXXLL motif within the N-terminus. Luciferase promoter assays revealed that Tob1 significantly inhibited the estrogen-driven ER transcription activation, proposing Tob1 as an important mediator in the estrogen/ER signaling pathway in breast carcinogenesis.Tob1 was also found to be associated with cyclin D1, one of the important downstream regulators of estrogen/ER signaling pathway. Demonstrated by in vitro and in vivo binding assays, Tob1 also suppressed the activity of cyclin D1 promoter. Together, these results provided a potential pathway for Tob1’s mediating cell cycle progression and cell growth.Finally, the results revealed for the first time that Tob1 increased sensitivity of breast cancer cells to ionizing radiation, by ways of reduction or inhibition of expression of critical proteins in DNA repair process such as DNA-dependent protein kinase (DNA-PK), Ku70, Ku80 and X-ray-sensitive complementation group 4 (XRCC4) as well as enhancement of radiation-mediated Bax expression.In summary, it has been demonstrated that Tob1 either functions as a tumor suppressor gene in breast cancer progression by mediating cell cycle progression and apoptosis induction or acts as a radiosensitizor in breast cancer therapy by regulating DNA damage repair. Therefore, it will put forward our understanding in promotion of diagnosis and radiotherapy for breast cancer.更多还原