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

我们已经研究了b-catenin基因在肝癌发生中的作用，我们使用了几种c-myc单独或联合转化生长因子-α或转化生长因子-β1在肝脏过度表达诱发肝癌的转基因小鼠模型。在c-myc(4/17，23.5%)和c-myc/转化生长因子-b1(6/18，33.3%)转基因小鼠的肝肿瘤中，b-catenin的激活(根据蛋白的突变和/或核转位判断)最为常见。然而，在c-myc/TGF-a小鼠中生长更快、组织学上更具侵袭性的肝细胞癌中，它是非常罕见的(1/20，5%)。给c-myc小鼠注射二乙基亚硝胺、苯巴比妥或2-氨基-3，8-二乙基咪唑并[4，5-f]喹恶啉(MeIQx)对b-catenin突变的发生无明显影响。值得注意的是，只有在腺瘤和具有嗜酸性表型的高分化癌中才能观察到b-连环蛋白的核聚集。此外，嗜酸粒细胞表型的癌前病变通常表现为局灶性核阳性，与高增殖区共存。相反，嗜碱性和透明细胞病灶，以及假腺性和低分化肝细胞癌，对b-连环蛋白的膜免疫反应正常或降低。这些研究表明，b-catenin的核转位和Wingless/Wnt信号的激活可能是肝癌发生的早期事件，为分化程度更高的一类肝肿瘤提供了生长优势。这是相当有意义的，因为b-连环蛋白的突变与人类肝细胞癌杂合性丢失的减少有关)。因此，我们假设肝癌的发展可能通过两条广泛的途径进行。第一种途径将涉及b-连环蛋白的激活，而第二种途径将导致基因组不稳定并产生突变表型。我们目前正在研究b-连环蛋白的激活与基因组不稳定性之间的关系，方法是使用22个随机引物对小鼠肝肿瘤的基因组图谱进行随机扩增多态性DNA(RAPD)分析。结果表明，c-myc中不存在基因组不稳定性，而表现为b-catenin活性的c-myc/TGF-b1肿瘤中基因组不稳定性很低(5.8±1.3%)。相比之下，在c-myc/转化生长因子-a小鼠中，10/10的肝癌细胞表现出很高的基因组不稳定性(25？3.0%)。后者与c-myc/lacz肝癌细胞经强致癌物2-amino-3，8-dimethyl-imidazo[4，5-f]quinoxaline处理后的基因组不稳定性(32.3？2.4%)相当。基因组不稳定性频率与肝细胞癌分化程度呈负相关。此外，仅在c-myc/转化生长因子-α(60%)和c-myc/lacZ(80%)低分化肝癌中检测到b-catenin和转化生长因子-αRII基因杂合性缺失，这可能代表了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。第一个途径意味着c-myc和b-catenin癌基因在稳定的基因组存在的情况下合作，而第二个途径在基因组不稳定的情况下起作用。

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.