【作者】 黄淑红；

【导师】 张红卫； 谢经武；

【作者基本信息】 山东大学 ， 发育生物学， 2007， 博士

【摘要】 癌症是人类生命的最大威胁之一，研究癌症致病机理是当代生物学和医学研究的热门和前沿领域。肺癌是世界癌症相关死亡重要的类型，我国主要城市中肺癌发病率居各种恶性肿瘤的首位，死亡率已经超过世界平均水平。在美国，肺癌平均每年夺去150，000的生命(超过乳腺癌，前列腺癌和结直肠癌死亡人数的总合)。新发现的肺癌病人中，75％已经是肺癌晚期，医学认为存活期仅为10个月。肝癌是人类消化系统主要的肿瘤类型，而肝细胞肝癌(Hepatocellular carcinomas，HCC)是其中最主要的类型，也是世界上重要的恶性肿瘤之一。肝癌在中国的恶性肿瘤死亡序位已高居第二位，年死亡率为20-40/10万人。在美国，每年因HCC死亡的人数为14000人，占肿瘤相关死亡人数的第八位。大量患者(70-85％)由于年老或者其它疾病，预后非常差，而且化疗在HCC的治疗方面也作用甚微。Sonic Hedgehog(SHH)通路在胚胎发育、器官极性和细胞增殖过程中都起重要作用。作为胚胎发育过程中起到重要作用的信号通路，SHH信号通路已经被很清楚地证明，在成体组织中通过突变或者其他机制而被激活，从而在肿瘤发生中起到至关重要的作用。包括皮肤基底细胞癌，肺癌，胃癌，前列腺癌，结直肠癌，胰腺癌等。特异性抑制SHH通路活性可以抑制这些肿瘤细胞的增殖。本论文主要关注中国高发性肿瘤肝细胞肝癌和肺癌中SHH通路的情况。主要研究思路是：首先检查SHH通路靶基因以及主要作用分子的表达；然后寻找这种异常的通路活性是如何产生的；最后研究SHH通路在细胞功能，增殖等方面的作用，本研究的成果可为阐明肿瘤发病的机理提供新资料。对于肺癌中SHH通路的研究开始于1997年，并已有大量证据显示SHH通路在不同肺癌类型中存在不同类型的激活。但是关于其激活的机率还没有报道。本文第二部分通过原位杂交和免疫组化等技术，检测了肺癌中是否存在存在SHH通路的激活，发现在肺癌中仅有不超过10％的病例存在SHH靶基因的表达上调。在部分存在SHH异常激活的病例中，发现了通路负调控基因Su(fu)的表达缺失。结果表明，SHH的异常激活在肺癌中仅存在于一小部分病例中，并且这种激活可能由于SHH配体的过表达或者Su(fu)的功能缺失引起。本文首次报道了SHH通路中其他一些重要成员在肺癌中的表达情况。如通路受体之一Smoothened(SMO)，负调控基因Rab23，下游靶基因platelet=derived growth factor receptor alpha(PDGFRα)，hedgehog interacting protein(HIP)以及SHH启动子调控子hepatocyte nuclear factor 3-beta(HNF3β)。并且检测和分析了在皮肤基底细胞癌中存在获得功能型突变的SMO的第十外显子是否在肺癌中也存在突变，而产生了配体非依赖的通路活性。研究结果显示，虽然SHH通路在肺癌中的激活并不普遍，但是一些通路重要成员却存在高频率的过表达，包括SMO，Rab23以及PDGFRα，而通路抑制基因之一的HIP则在一些病例中表达下调。另外，本实验中，首次报道Rab23基因的核定位。结果暗示了SHH通路的激活方式可能在不同肿瘤类型中存在差异。并且，虽然通过PCR-SSCP检测发现较为普遍的SMO第十外显子出现异常条带，但是测序发现仅有一个病例存在杂和突变，显示出SMO的第十外显子突变在肺癌中可能不是引起通路激活的主要原因之一，但是在肿瘤病例中出现的异常SSCP条带仍然给了我们有趣的线索。综上所述，研究表明，虽然肺癌中SHH激活并非广泛存在，但许多SHH通路的分子在肺癌中广泛表达。文章第三部分首次报道SHH信号通路在HCC中高频率的激活。在115例癌症组织和44例癌旁组织中检测了SHH的表达，以及靶基因PTC1、Gli1的表达。在超过60％的HCC组织中存在SHH的表达，与之一致的是，超过50％的病例存在靶基因PTC1和Gli1的表达，显示了SHH活性在HCC中广泛存在。同时检测了HCC细胞系Hep3B中存在SHH激活，并且通路抑制剂KAAD-cyclopamine处理细胞，可以引起细胞凋亡以及生长抑制，而这种作用在转染了pCS2-Gli1表达载体的细胞中则被减弱。采用RNAi技术抑制Gli1活性，同样可以引起细胞凋亡，而在没有通路活性的HepG2细胞中，这些处理则没有影响。实验揭示了SHH通路在HCC的生长、增殖中起重要作用。SHH信号通路是很多的分子形成一个复杂的网络。作者也检测了其他通路分子的表达情况。结果除了显示出SMO的过表达，以及部分肿瘤也存在Rab23表达的上调，这种上调暗示虽然作为通路的负调控分子，Rab23可能同PTC1一样，也是通路的靶基因之一。与肺癌的情况相似，在HCC中也发现了Rab23的核定位。原位杂交结果还显示，HCC中没有PDGFRα的表达，这一结果暗示HCC中的SHH通路下游靶基因网络可能不同于其他肿瘤。通过PCR-SSCP检测同样也发现较为普遍的第十外显子出现异常条带。突变的结果把一个脯氨酸Pro变成了丝氨酸Ser，这是一个从非极性氨基酸，变成极性氨基酸的突变，这个突变有可能改变胞内区的结构域，从而引起蛋白功能的改变。但是在检测的十例组织中，仅有一例存在这种突变，也说明SMO第十外显子的突变可能不是引起HCC中SHH通路激活的原因。研究创新点：本论文的工作通过对大量肺癌病例的检测，首次研究了肺癌中SHH信号通路异常激活的机率。首次系统研究了SHH通路关键分子在肺癌中表达的情况。首次报道了在中国高发性肝细胞肝癌中有大量病例存在SHH信号通路异常激活的情况。并且，在细胞水平上，检测了SHH信号通路活性对HCC-Hep3B细胞的生长、增殖的作用，系统研究了SHH通路关键分子在肝癌中表达的情况。这一系列创新性研究成果必将有助于揭示肿瘤发生的分子机理。更多还原

【Abstract】 Cancer is one of the biggest threat facing human life, study on the mechanism of cancer development is a hotspot and foreland.Lung cancer is the leading cause of cancer-related death, claiming more than 150,000 lives every year in the US alone (which exceeds the combined mortality from breast, prostate, and Colorectal cancers). The incidence of lung cancer has run the first rank of the all the malignancy in the main cities of the China, and the mortality rate has gone beyond the average rate of the world. Patients with advanced stage of lung cancer, which represents 75% of all new cases, have a median survival time of only 10 months.Liver cancer, with hepatocellular carcinoma (HCC) as the major tumor type, is a malignancy of worldwide significance. The mortality rate of lung cancer has run the second rank of the all the malignancy in China, and the mortality rate for year is 20-40/100,000. HCC ranks as the eighth cause of cancer-related death in American men with 14 000 deaths yearly and is the most rapidly increasing type of cancer in the United States. The medical oncology community is largely unprepared for this looming epidemic of HCC. A majority (70-85%) of patients present with advanced or unresectable disease, making the prognosis of HCC dismal, and systemic chemotherapy is quite ineffective in HCC treatment.Sonic Hedgehog signaling pathway play important role in embryo development, tissue polarity and cell proliferation. Also, it has been proved that this important signaling pathway is activated in adult tissue via mutation or some other mechanism, including basal cell carcinoma, lung cancer, gastric cancer, prostate cancer, Colorectal adenocarcimas and pancreas cancer etc. Special inhibit of the pathway would suppress the cancer cell proliferation.This article mainly focus on the role of Sonic Hedgehog signaling pathway in the popular cancers in China, hepatocellular carcinoma and lung cancer.The work is divided into three parts:1. Target gene expression- Gli1, HIP and PTC1transcripts (in situ hybridization and RT-PCR)proteins (Immunohistochemistry and Western blotting)2. Specific alterations (over-expression or gene mutation)SHH, Su(Fu), HIP, PTC1 or Rab233. Requirement of the Hedgehog pathway for cell functionsCell growth, cell invasion or tumor progressionThe study on the involvement of SHH pathway in lung cancer began at 1997, and since then, a mass of evidences showed that it is involved in different kinds of lung cancers via different mechanism. But its frequency remains unknown. The second part of article, the author examined activation of this pathway in lung cancers by in-situ hybridization and immunohistochemistry, and find that less than 10% of the tumors have elevated Hedgehog target gene expression. Some primary tumors were found with no detectable Su(Fu), a negative regulator of the pathway. These data indicate that activation of the Hedgehog pathway is activated in only subsets of lung cancers. And these activity may caused by overexpression of Sonic Hedgehog or lose expression of Su(Fu).After that, the author checked the expression of some important players of SHH pathway in lung cancers, including Smoothened (SMO), Rab23, the downstream target platelet-derived growth factor receptor alpha (PDGFRα), hedgehog interacting protein (HIP) and hepatocyte nuclear factor 3-beta (HNF3β). Also the author detected whether there is any mutation in the 10 exon of SMO, some gain of function mutation of which could cause ligand independent activity.The result indicate that, some (such as SMO, Rab23 and PDGFRα) but not all players of the Hedgehog pathway express in many lung cancer specimens although other Hedgehog target genes are not frequently detected in lung cancer. Loss of HIP expression was found in several cases of lung cancers. In addition, the article report the nucleus location of Rab23 in lung cancer cells. From these data, we suggest that the regulation of SHH pathway may differ from tumor type to tumor type. By PCR-SSCP, nearly all the tumor samples showed a abnormal line, but only one of them have a mutation of SMO exon 10 were found by DNA sequencing. It indicate that the mutation of exon 10 of SMO may not be the main reason of SHH pathway activity in lung cancers. But the abnormal line in tumor samples is still an interesting clue for us.The third part of this article provides evidence to indicate that Hedgehog signaling activation occurs frequently in HCC. The author detected expression of SHH, PTC1 and Gli1 in 115 cases of HCC and in 44 liver tissues adjacent to the tumor. Expression of SHH is detectable in about 60% of HCCs examined. Consistent with this, Hedgehog target genes PTC1 and Gli1 are expressed in over 50% of the tumors, suggesting that the Hedgehog pathway is frequently activated in HCCs. Of five cell lines screened, the author found Hep3B cells with detectable Hedgehog target genes. Specific inhibition of Hedgehog signaling in this cell lines by smoothened (SMO) antagonist, KAAD-cyclopamine, inhibits cell growth and results in apoptosis. In contrast, no effects are observed after these treatments in HCC36 and HepG2 cells, which do not have detectable Hedgehog signaling. Thus, our data indicate that Hedgehog signaling activation is an important event for development of human HCCs.Also the author checked the expression of some important players of SHH pathway in HCC, including Smoothened (SMO), Rab23, the downstream target platelet-derived growth factor receptor alpha (PDGFRα), hedgehog interacting protein (HIP) and hepatocyte nuclear factor 3-beta (HNF3β), and whether there is any mutation in the 10 exon of SMO.The result indicate that, some (such as SMO, Rab23) players of the Hedgehog pathway express in many HCC specimens, but no expression of PDGFRαis detected by in situ hybridization. Loss of HIP expression was found in several cases of lung cancers. Also, Nucleus location of Rab23 is found in HCC cells. From these data, we suggest that the regulation of SHH pathway may differ from lung cancer. By PCR-SSCP, nearly all the tumor samples detected (10) showed an abnormal line, but only one of them have a mutation of SMO exon 10 were found by DNA sequencing.InnovationBy detecting a mass of lung cancer samples, this article gives the first report about the frequency of abnormal activity in lung cancers. For the first time, the author checked the expression of some important players of SHH pathway in lung cancers. For the first time, this article provides evidence to indicate that Hedgehog signaling activation occurs frequently in HCC. Also the article detected the effect of Hedgehog signaling activation on the growth and proliferation of HCC - Hep3B cell line, checked the expression of some important players of SHH pathway in lung cancers.更多还原