Role of b-Catenin Wingless/Wnt Pathway in Liver Cancer

我们研究了b-catenin基因在肝癌发生中的作用，我们使用了几种由肝脏中c-myc过表达或与TGF-a或TGF-b1联合诱导的肝脏肿瘤转基因小鼠模型。通过b-catenin蛋白的突变和/或核易位判断，b-catenin的激活在c-myc（4/ 17,23.5%）和c-myc/TGF-b1（6/ 18,33.3%）转基因小鼠的肝脏肿瘤中最为常见。然而，在c-myc/TGF-a小鼠中发生的更快生长和组织学上更具侵袭性的肝细胞癌中，这种情况非常罕见（1/ 20,5%）。给c-myc小鼠注射二乙基亚硝胺、苯巴比妥或2-氨基-3,8-二乙基咪唑[4,5-f]喹诺啉（MeIQx）对b-连环蛋白突变的发生没有显著影响。值得注意的是，b-连环蛋白的核积累仅在腺瘤和嗜酸性高分化癌中观察到。此外，嗜酸性表型的癌前病变经常显示局灶性核阳性，与高增殖区域共定位。相反，嗜碱性和透明细胞灶，以及伪腺癌和低分化肝细胞癌，对b-连环蛋白表现出正常或降低的膜免疫反应性。这些研究表明，b-catenin的核易位和Wingless/Wnt信号的激活可能代表了肝癌发生的早期事件，在表型分化程度更高的肝肿瘤亚群中提供了生长优势。这是相当有趣的，因为b-连环蛋白的突变与人类HCC中杂合性损失的减少有关。因此，我们假设肝癌的发展可能通过两条广泛的途径推进。第一种途径将涉及b-连环蛋白激活，而第二种途径将产生基因组不稳定并产生突变表型。我们目前正在研究b-catenin激活与基因组不稳定性之间的关系，通过随机扩增多态性DNA （RAPD）分析，使用22个任意引物分析小鼠肝脏肿瘤的基因组图谱。迄今为止的结果显示，c-myc不存在基因组不稳定性，c-myc/TGF-b1的基因组不稳定性非常低(5.8 ？1.3%)肿瘤显示b-连环蛋白活化。相比之下，在c-myc/TGF-a小鼠中形成的10个hcc中有10个表现出高的基因组不稳定性(25 ？3.0%)。后者与强效肝癌致癌物2-氨基-3,8-二甲基咪唑[4,5-f]喹诺啉治疗后c-myc/ lacZ hcc的基因组不稳定性相当(32.3 ？2.4%)。基因组不稳定的频率与HCC分化程度呈负相关。此外，仅在c-myc/TGF-a（60%）和c-myc/lacZ（80%）低分化hcc中检测到b-catenin和TGF-a RII基因的杂合性缺失，这可能是两种转基因模型中肿瘤进展的标志。综上所述，目前的数据与转基因小鼠模型中存在两种肝癌发生途径是一致的。第一种途径可能意味着c-myc和b-连环蛋白癌基因在稳定的基因组存在下的合作，而第二种途径在基因组不稳定的背景下起作用。

We have investigated the involvement of the b-catenin gene in hepatocarcinogenesis, we used several transgenic mouse models of hepatic tumors induced by over-expression of c-myc in the liver either alone or in combination with TGF-a or TGF-b1. Activation of b-catenin, as judged by the presence of mutations and/or nuclear translocation of the protein, was most frequent in liver tumors from c-myc (4/17, 23.5%) and c-myc/TGF-b1 (6/18, 33.3%) transgenic mice. However, it was very rare in faster growing and histologically more aggressive hepatocellular carcinomas developed in c-myc/TGF-a mice (1/20, 5%). Administration of diethylnitrosamine, phenobarbital or 2-amino-3,8-diethylimidazo[4,5-f]quinoxaline (MeIQx) to the c-myc mouse did not significantly affect the occurrence of b-catenin mutations. Notably, nuclear accumulation of b-catenin was observed only in adenomas and highly-differentiated carcinomas with eosinophilic phenotype. Furthermore, preneoplastic lesions with eosinophilic phenotype frequently displayed focal nuclear positivity, co-localized with areas of high proliferation. In contrast, basophilic and clear-cell foci, as well as pseudo-glandular and poorly-differentiated hepatocellular carcinomas, exhibited a normal or reduced membranous immunoreactivity for b-catenin. These studies suggest that nuclear translocation of b-catenin and activation of Wingless/Wnt signaling may represent an early event in liver carcinogenesis, providing a growth advantage in a subset of hepatic tumors with a more differentiated phenotype. This is of considerable interest since mutations in b-catenin are associated with a decreased loss of heterozygosity in human HCC). We have therefore hypothesized that development of liver cancer may advance via two broad pathways. The first pathway would involve b-catenin activation, while the second one would generate genomic instability and produce a mutator phenotype. We are currently investigating correlation between b-catenin activation and genomic instability by analyzing the genomic profiles of mouse liver tumors by the random amplified polymorphic DNA (RAPD) analysis using 22 arbitrary primers. The results so far have revealed that genomic instability was absent in c-myc and was very low in c-myc/TGF-b1 (5.8 ? 1.3%) tumors which displayed b-catenin activation. In contrast, 10 out of 10 HCCs developed in c-myc/TGF-a mice exhibited a high rate of genomic instability (25 ? 3.0%). The latter was comparable with the rate of genomic instability found in c-myc/ lacZ HCCs after treatment with the potent hepatocarcinogen 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (32.3 ? 2.4%). The frequency of genomic instability was inversely correlated with the degree of HCC differentiation. Furthermore, loss of heterozygosity of b-catenin and TGF-a RII genes was detected only in c-myc/TGF-a (60%) and c-myc/lacZ (80%) poorly-differentiated HCCs, presumably representing a marker of tumor progression in both transgenic models. Taken together, the present data are consistent with the existence of two pathways of hepatocarcinogenesis in the transgenic mouse models. The first pathway would imply the cooperation between c-myc and b-catenin oncogenes in the presence of a stable genome, while the second pathway is operative in a context of genomic instability.