Background : Embryonic gene Cripto-1 (CR-1) is not detected or expressed at low levels in normal adult tissues, however, is re-expressed in numerous tumors, including hepatocellular carcinoma (HCC) and CR-1 transgene overexpression led to mammary epithelial hyperplasia and adenocarcinoma, which prompted us to investigate whether hepatocyte-specific overexpression of CR-1 in transgenic mice can initiate hepatocarcinogenesis.
Methods : Semi-quantitative RT-PCR, qRT-PCR, Western blotting or immunohistochemical staining were employed to detected CR-1 mRNA transcript or CR-1 expression in HCC tissue specimens. Combined use of CR-1 transgenic mice, microarray, 2/3 partial hepatectomy (PHx), and TCGA and GEO database examined the functions of CR-1 in initiate hepatocelluar carcinogenesis.
Results: Here we firstly provided the direct evidence that a truncated 1.7-kb mRNA transcript from CR-1 geneis dominantly expressed in HCC specimens, suggesting the importance of short CR-1 mRNA form in HCC progression.We found that CR-1 is frequently upregulated in HCC specimens. Hepatocyte-specific overexpression of CR-1 in transgenic mice promoted hepatocyte proliferation after 2/3 PHx, CR-1 positively regulated HCC cell proliferation and invasion in vitro, and CR-1 overexpression promoted tumour growth of HCC cells in nude mice. In this study, we demonstrated that CR-1 transgenic overexpression in transgenic liver abnormally activated downstream pathways (i.e., AKT, Wnt/b-catenin, Stat3, MAPK/ERK, JNK, TGF-b and Notch) and led to the deregulated expression profile of genes, including up-regulated genes (i.e., CD5L, S100A8, S100A9, Timd4, Orm2, Orm3, Saa1, Saa3, Itih3, Itih4, Ly6e, IGHG1, IGHG2B and Vnn3) and down-regulated genes (i.e.,PDK4, DMBT1, G0S2, Plk2, Plk3, Gsta1 and Gsta2), all of which are key regulators of cellular proliferation, inflammation response, cellular malignant transformation, hepatocarcinogenesis or HCC progression, however, there were no histological signs of precancerous lesions, hepatocyte dysplasia and HCC formation in livers samples from 3-, 6- or 8-month-old RCLG/Alb-Cre transgenic mice, suggesting that the constitutive expression of CR-1 alone is not sufficient to promote hepatocarcinogenesis, unless other factors, i.e., a second hit, is present.
Conclusions: Taken together, we provide the first in vivo genetic evidence that CR-1 overexpression in transgenic mouse liver promotes hepatocyte proliferation after 2/3 PHx, and causes the deregulated molecular alterations involved in HCC oncogenesis, however, these abnormally molecular alterations are not sufficient to initiate hepatocarcinogenesis in mice. Keywords : Cripto-1 (CR-1), hepatocellular carcinoma (HCC), transgenic mice, hepatocarcinogenesis