Transcription Factor MafB Promotes Hepatocellular Carcinoma Cell Proliferation through Up-Regulation of Cyclin D1.

BACKGROUND/AIMS: MafB, a member of the Maf transcription factor family, plays a key role in the regulation of pancreatic alpha and beta cell differentiation. However, its function in the control of cancer cell proliferation remains unknown. METHODS: The mRNA and protein expression levels of MafB in hepatocellular carcinoma tissues and adjacent non-tumor normal specimens were determined by real-time RT-PCR and Western blot, respectively. Report assay was performed to determine whether the regulation of Cyclin D1 by MafB is at the transcriptional level. The binding of MafB to the Cyclin D1 promoter was determined by Chromatin Immunoprecipitation (ChIP) assays. To determine the potential oncogenic effects of MafB in vivo, HepG2 cells transfected with adenovirus containing empty vector or MafB were injected subcutaneously to the skin under the front legs of the nude mice. RESULTS: In the current study, we showed that MafB was markedly up-regulated in hepatocellular carcinoma (HCC) tissues and cells. Enforced overexpression of MafB enhanced, while its deficiency inhibited HCC cell proliferation. Mechanistically, Cyclin D1, an important regulator of cell cycle progression, was identified as a direct transcriptional target of MafB. Consistently, knockdown of Cyclin D1 largely attenuated the proliferative roles of MafB in HCC cells. Importantly, MafB overexpression significantly promoted cancer cell growth in mice. CONCLUSIONS: Collectively, our results identified a novel HCC regulatory pathway involving MafB and Cyclin D1, the dysfunction of which drives proliferative character in HCC.

DAX-1 inhibits hepatocellular carcinoma proliferation by inhibiting ß-catenin transcriptional activity.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) represents the most common type of liver cancer. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1), an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains, has been known for its fundamental roles in the development, especially in the sex determination and steroidogenesis. Previous studies also showed that DAX-1 played a critical role in endocrine and sex steroid-dependent neoplasms such as adrenocortical, pituitary, endometrial, and ovarian tumors. However, its biological roles in the development of HCC remain largely unexplored. METHODS: Real-time PCR and Western blot were used to detect the expression of DAX-1 in HCC tissues and cell lines. Immunoprecipitation (IP) assay was used to show the interaction between DAX-1 and ß-Catenin. Small interfering RNA (siRNA) was used to silence the expression of DAX-1. BrdU incorporation and Cell-cycle assays were used to detect the role of DAX-1 in HCC cells proliferation. Migration and invasion assays were carried out to test the metastasis ability of DAX-1 in HCC cells. RESULTS: In the present study, we found that mRNA and protein levels of DAX-1 were down-regulated in HCC tissues and cell lines. Furthermore, overexpression of DAX-1 could inhibit while its knockdown using small interfering RNA promoted cell proliferation in several HCC cell lines. At the molecular level, we demonstrated that DAX-1 could interact with ß-Catenin and attenuate its transcriptional activity. CONCLUSION: Therefore, our results suggest a previously unknown DAX-1/ß-Catenin molecular network controlling HCC development.

MicroRNA-195 regulates steroid receptor coactivator-3 protein expression in hepatocellular carcinoma cells.

Multiple studies have shown that steroid receptor coactivator-3 (SRC-3) is upregulated and promotes cell proliferation in several human cancers, including breast, lung, and prostate carcinoma. However, its molecular determinants remain largely unexplored. In the current study, by way of informatics software, we found that MicroRNA-195 (miR-195) could negatively regulate protein levels of SRC-3 through targeting its 3'-untranslated region (3'-UTR) in hepatocellular carcinoma (HCC) cells. As a result, miR-195 mimics inhibited while its antisense enhanced SRC-3 protein levels. Furthermore, miR-195 could modulate cell proliferation and tumor growth in vivo and in vitro. Therefore, our results demonstrate a novel molecular mechanism for the dysregulated expression of SRC-3 in hepatocellular carcinoma.

Celastrol exerts synergistic effects with PHA-665752 and inhibits tumor growth of c-Met-deficient hepatocellular carcinoma in vivo.

PHA665752 (PHA), a selective small molecule c-Met Inhibitor, potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes of a variety of tumor cells including hepatocellular carcinoma cells. However, these effects were impaired in c-Met-deficient cancer cells. In the present study, we investigated the potential anti-human c-Met-deficient hepatocellular carcinoma effects of Celastrol, a novel triterpene, and its combination with PHA. Human hepatocellular carcinoma cells BEL-7402 (c-Met-positive) and Huh7 (c-Met-deficient) were treated with different dose of PHA with or without equal dose of Celastrol, and cell growth, cell cycle and apoptosis were evaluated, respectively, by MTT assay, flow cytometry and Caspase3/7 activity. Nude mice bearing Huh7 xenografts were used to assess the in vivo anti-tumor activity. Our results showed that Celastrol at high concentration (>1.0 µM) induced G2/M arrest and apoptosis with the activation of Caspase3/7 in Huh7 cells whereas at low concentration (<1.0 µM) had no obvious effects. Low concentration Celastrol presented significant combined effects with PHA on Huh7 cells and Huh7 xenografts in terms of growth inhibition, migration inhibition and apoptosis induction. These results suggest that Celastrol and its combination with PHA present the therapeutic potential on c-Met-deficient hepatocellular carcinoma, and deserve further preclinical and clinical studies.