Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation

Background

Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.

Methods

We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.

Results

We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.

Conclusions

HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

     

The Rb family of cell cycle regulatory factors: clinical implications

The retinoblastoma gene family is composed of three members: the product of the retinoblastoma gene (pRb), which is one of the most well studied tumor suppressor genes and two related proteins, pRb2/p130 and p107, which have been shown to be structurally and functionally similar to pRb. The three retinoblastoma family members show growth suppressive properties, although the growth arrest mediated by each of the three pocket regions of the proteins is not identical. This supports the idea that although the three members may complement each other, they are not fully functional or redundant. Among the three family members, the retinoblastoma-related gene product pRb2/p130 is a tumor suppressor gene and an effective candidate target for gene therapy approach. The aim of this review is to examine the role of the Rb family members in growth regulation discussing their putative prognostic and therapeutical impact in human cancer.     

Loss of pRb2/p130 expression is associated with unfavorable clinical outcome in lung cancer.

Altered expression of cell cycle regulators represents a frequent event in both small cell and non-small cell lung cancer (NSCLC). Despite several studies that reported involvement of tumor suppressor genes, such as p53 and pRb, in the development and progression of lung cancer, contrasting opinions exist about the prognostic role of this protein in this neoplasm. We developed an immunohistochemical assay suitable for the detection of pRb2/p130, the last discovered member of the retinoblastoma gene family, on formalin-fixed and paraffin-embedded sections. We evaluated the immunohistochemical expression of pRb2/p130 in 135 lung cancer specimens, and performed Western blot analysis in a subset of 30 corresponding tumor lysates. A high correlation between immunohistochemical data and Western blot results (P = 0.0004) was found. We statistically analyzed the relationship between overall survival (OS) time and pRb2/p130 expression according to the different histological types in 105 patients. We did not find any correlation between pRb2/p130 expression and OS in small cell lung cancers, whereas in NSCLCs a direct relationship between pRb2 and OS was found in both adenocarcinoma (P = 0.0002) and squamous cell carcinoma (P = 0.0002) histotypes. According to univariate analysis, pRb2/p130 was a prognostic factor of which the lost or reduced expression correlated with a shorter OS (P < 0.0000). At multivariate analysis, pRb2/p130 expression was an independent predictor of OS (P = 0.0001) when considered together with histotype. This study demonstrates for the first time the potential independent prognostic value of pRb2/p130 expression on formalin-fixed, paraffin-embedded sections from lung cancer patients. pRb2/p130 immunoreactivity can be used to predict OS in patients with NSCLC and, therefore, may represent a new prognostic marker.     

Overexpression of tumour suppressor retinoblastoma 2 protein (pRb2/p130) in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Southeast Asia. Although inactivation of pRb2/p130 has been reported in a variety of human cancers, its function in HCC has not been established. In this study we report that loss of expression of pRb2/p130 was detected by immunohistochemistry and western blotting in 15.2% (7 of 46) HCCs examined. High levels of pRb2/p130 expression were found in 84.8% (39 of 46) HCCs studied. Western blot analysis revealed that HCC had 3.5-fold higher pRb2/p130 than adjacent benign liver (ABL) tissues. 71.7% (33 of 46) of HCCs examined exhibited both nuclear and cytoplasmic staining for pRb2/p130. Cytoplasmic staining was found in 93.5% (43 of 46) of ABL tissues. Overproduction of pRb2/p130 in HepG2 cells led to growth suppression, cell cycle arrest in G0/G1, altered cell morphology, inhibition of in vitro colony formation and reduction in tumourigenicity in SCID mice. This demonstration suggests a role of pRb2/p130 as a tumour suppressor protein in HCC and the loss of this protein may lead to the development or progression of HCC. Overexpression of pRb2/p130 in HCC was, therefore, suggested to be a programmed protective response of the organism to uncontrolled proliferation.     

The tyrphostin AG1024 accelerates the degradation of phosphorylated forms of retinoblastoma protein (pRb) and restores pRb tumor suppressive function in melanoma cells

Constitutive cell surface receptor kinase signaling and persistent phosphorylation/inactivation of the retinoblastoma (pRb) family of proteins (pRb, p107 and p130, known as pocket proteins) have been implicated in conferring uncontrolled growth to melanoma cells. However, the signals linking receptor kinase activity to neutralization of pocket proteins have not yet been fully elucidated. We therefore used specific chemical inhibitors to examine pRb regulation in melanoma cells. The most efficient agent, AG1024, known as an inhibitor of insulin-like growth factor 1 receptor and insulin receptor, arrested melanoma cell growth in vitro at nanomolar concentrations within 24 h of application. AG1024 inhibited the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and restored pRb tumor suppressive function. The latter was observed by the reduction in the phosphorylated forms of pRb, p107 and p130, and the formation of growth suppressive DNA binding complexes consisting of pRb and E2F1 or E2F3. The loss of phosphorylated forms of pRb at early time points after AG1024 application was not associated with suppression of cyclin-dependent kinases 2 and 4 activity but rather with proteasomal and nonproteasomal degradation. Thus, inhibition of melanoma cell proliferation by AG1024 is mediated by inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 2 signaling and activation of pRb by a mechanism involving protein degradation.     

pRb2/p130 protein expression is correlated with clinicopathologic findings in patients with oral squamous cell carcinoma

BACKGROUND: pRb2/p130 is one of the retinoblastoma (Rb) gene family and a suppressor oncogene. Immunohistochemically, the expression of pRb2/p130 was reported to be correlated inversely with the degree of malignancy in lung carcinoma and endometrial carcinoma. In the current study, the correlation between expression of pRb2/p130 and clinicopathologic factors in oral squamous cell carcinoma was investigated. METHODS: One hundred twenty-two specimens from patients with oral squamous cell carcinoma were investigated by staining with a polyclonal antibody against pRb2/p130. The correlation between the expression of pRb2/p130 and various clinicopathologic factors was studied. RESULTS: Positive staining for pRb2/p130 was observed in 61 of 122 cases (50.0%). pRb2/p130 expression was found to be correlated significantly with clinical stage (P = 0.050), cervical lymph node metastasis (P = 0.035), and tumor differentiation (P = 0.050). In the entire group a significantly reduced 5-year cumulative survival rate was observed in patients with pRb2/p130-negative tumors compared with patients whose tumors positively expressed pRb2/p130 (P = 0.0004). When tested with Cox proportional hazards regression analysis, the most significant independent prognostic factor for the entire group of 122 patients was found to be pRb2/p130 expression. CONCLUSIONS: Expression of pRb2/p130 may be a good prognostic indicator in patients with oral squamous cell carcinoma and also may be utilized for the subclassification of tumors with the Grade 3 mode of carcinoma invasion.     

In-vivo analysis of hpv e7 protein association with prb, p107 and p130

The two-hybrid system was used to detect interactions in vivo between HPV E7 and three 'Rb-like proteins', pRb, p107 and p130. The association between pE7 and pRb parallel the oncogenic potential of the specific HPV types. In contrast, the interaction between pE7 and p107 or p130 differ. While the HPV 16 E7 protein associates with the 'Rb-like' proteins strongly, both HPV 18 and 6b E7 proteins bind more weakly. We tested several HPV 6 E7 mutants carrying single amino acid mutations. Substitution of the glycine at position 22 with an aspartate was the only mutation capable of increasing the ability of HPV 6 E7 protein to bind pRb. However, association with p107 and p130 by the HPV 6 E7 protein was also increased by mutation of the arginine at position 4 with an aspartate. These data suggest that pRb, p107 and p130 interact with similar but non-identical domains of pE7. In addition, we used amphotropic retroviruses encoding the HPV 18 E6 and the different E7 genes to analyze their immortalizing activity. The wild-type HPV Is and 16 E7 genes complemented the HPV 18 E6 gene to immortalize human keratinocytes. In comparison, none of the cells infected with HPV 6 E7, wildtype or mutant- encoding retroviruses, became immortal. Thus, our data suggest that HPV 6 E7 lacks a property independent of pRb-association which is required for immortalization of human keratinocytes.     

Universal inactivation of both p16 and p15 but not downstream components is an essential event in the pathogenesis of T-cell acute lymphoblastic leukemia.

p16/p15 regulate the cell cycle pathway by inhibiting the cyclin Ds-CDK4/6 mediated phosphorylation of pRb. We reported previously that in T-cell acute lymphoblastic leukemia (T-ALL), p16 and p15 were frequently (approximately 70%) inactivated at the DNA level by deletion, mutation, or hypermethylation. Therefore, we hypothesize that inactivation of the cell cycle regulatory pathway may be essential in the pathogenesis of T-ALL, and that the remaining T-ALL with a wild-type p16/p15 gene likely harbor inactivation of these genes at RNA or protein levels. Alternatively, the downstream components of the pathway including CDK4/6, cyclin Ds, and pRb may be deregulated. In 124 primary T-ALLs, we found inactivation of the p16 and p15 genes at the DNA level in 79 (64%) and 64 (52%) samples, respectively. Only 9 of the 45 samples with wild-type p16 expressed p16 protein, whereas the remaining 36 lacked p16 expression at the RNA or protein level. In the 60 samples with an intact p15 gene, only 2 expressed p15 mRNA, and the only one analyzed lacked p15 protein. Overall, the abrogation rates for p16 and p15 at DNA/RNA/protein levels were 93% (115 of 124) and 99% (123 of 124), respectively. Although no alterations were evident in cyclin Ds or CDK4/6, pRb was hyperphosphorylated in the majority of samples investigated. These findings strongly support that both p16 and p15 are specific targets in the deregulation of the cell cycle pathway in T-ALL and that the inactivation of these genes is most likely essential in the pathogenesis of this disease.     

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.     

Retinoblastoma gene family expression in lymphoid tissues

It appears more and more clear that retinoblastoma (RB) family of proteins represents key molecules in tumour suppression. This family consists of pRb/p105, p107 and pRb2/p130, which participate in a gene regulatory network that governs the cellular response to antimitogenic signals, and whose deregulation constitutes one of the hallmarks of cancer. Irrespective of their structural and biochemical similarities, RB proteins carry out different functional tasks. The expression of RB gene family in the reactive lymphoid tissues again confirms the different role of each member in cell cycle control and differentiation of normal cells. These different functional properties appear to be maintained in tumours lymphoid tissues, where alterations of the RB/p105 gene appear to be relatively rare. In this review, we will summarize the current knowledge about the role of the RB proteins in reactive and neoplastic lymphoid tissue.