Aberrant expression of serine/threonine kinase Pim‐3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines

Abstract

Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre-malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre-malignant lesions, we focused on Pim-3, a member of a proto-oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full-length human Pim-3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, Key words: protein serine-threonine kinases; pre-malignant lesions; hepatocellular carcinoma; RNA interference; apoptosis Hepatocellular carcinoma (HCC) is the 8th highest cause of death among human cancers and is endemic in Asia, Africa and southern Europe. Most cases of HCC arise from persistent chronic infection with human hepatitis B virus (HBV) or hepatitis C virus (HCV). 1 Host responses are presumed to be involved in the development of HCC among patients harboring HBV or HCV, because these viruses lack apparent oncogenes and the infected patients develop HCC after suffering from chronic hepatitis-related pathology. 2,3 Hepatitis virus infection induces the generation of virus antigenspecific cytotoxic T lymphocytes, which have been implicated in both the eradication of viruses and liver injury. 4 Cycles of cytotoxic T lymphocyte-mediated liver cell destruction and regeneration are thought to prepare the mitogenic environment. 4,5 To elucidate the molecular and cellular mechanism of HCC initiation and development, Nakamoto et al. 6 has established a mouse model of HCC by using HBV surface antigen (HBsAg) transgenic mice (HBsTg). In this model, bone marrow cells and splenocytes were obtained from syngeneic wild-type mice, which were immunized with HBsAg and were transplanted into HBsTg mice, which were irradiated beforehand. At 15 months after the transplantation, the transgenic mice developed multiple foci of HCC surrounded by non-malignant areas consisting of hepatocytes with atypical nuclear configuration. 6,7 To obtain the molecular insights on hepatocarcinogenesis, we compared the gene expression pattern between non-tumor portion of this model as a pre-malignant lesion and normal tissues by using a fluorescent differential display (FDD) analysis. We observed that several genes are expressed differentially in this pre-malignant lesion, compared to normal liver tissues. Of interest is that the gene expression of Pim-3, originally identified as depolarization-induced gene in a rat pheochromocytoma cell line,...

Accelerated hepatocellular carcinoma development in mice expressing the Pim-3 transgene selectively in the liver

“…Subsequently, Deneen et al (2003) demonstrated that Pim-3 gene transcription was enhanced in the EWS/ETS-induced malignant transformation of NIH 3T3 cells, suggesting the involvement of Pim-3 in tumorigenesis. Consistently, we observed that Pim-3 expression was enhanced in carcinomas but not in normal tissues of human endoderm-derived organs, including the liver (Fujii et al, 2005), pancreas (Li et al, 2006), colon (Popivanova et al, 2007) and stomach (Zheng et al, 2008). Moreover, Pim-3 can inactivate a pro-apoptotic molecule, Bad, and maintain the expression of an anti-apoptotic molecule, Bcl-X L , and prevent apoptosis of human pancreatic cancer and colon cancer cells (Li et al, 2006).…”

“…We previously observed that Pim-3 was expressed selectively in pre-malignant and malignant lesions of the mouse HCC model in transgenic mice expressing hepatitis B virus surface antigen (Fujii et al, 2005). Moreover, Pim-3 protein was detected in a substantial proportion of HCC cells and precancerous lesions in human liver samples, but not normal human liver.…”

“…However, another member of the Pim kinase family, Pim-1, is constitutively active without any further alteration in its conformation because it lacks any regulatory domain . Similarly, Pim-3 lacks any regulatory domain (Fujii et al, 2005) and Pim-3 complementary DNA (cDNA) alone induced phosphorylation of its target protein, Bad, at Ser 112 when it was transfected into human pancreatic cancer cell lines (Li et al, 2006). Consistently, Pim-3 transgenic mice exhibited enhanced phosphorylation of Bad at Ser 112 in the liver.…”

“…We previously identified Pim-3, a proto-oncogene with serine/threonine kinase activity, as the gene selectively expressed in pre-malignant and malignant lesions of the mouse HCC model in transgenic mice expressing hepatitis B virus surface antigen (Fujii et al, 2005). Pim-3 was originally identified as a depolarization-induced gene, KID-1, in PC12 cells, a rat pheochromocytoma cell line (Feldman et al, 1998).…”

“…Moreover, Pim-3 can inactivate a pro-apoptotic molecule, Bad, and maintain the expression of an anti-apoptotic molecule, Bcl-X L , and prevent apoptosis of human pancreatic cancer and colon cancer cells (Li et al, 2006). Similarly, the ablation of endogenous Pim-3 by short interfering RNA reduced the cell growth of human HCC cell lines by inducing their apoptosis (Fujii et al, 2005).…”