[A little less respite for hepatocellular carcinoma?]

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Pontin and reptin, two related ATPases with multiple roles in cancer.: Pontin and Reptin in cancer

International audienceStudies in model organisms or cultured human cells suggest potential implications in carcinogenesis for the AAA+ ATPases Pontin and Reptin. Both proteins are associated with several chromatin-remodeling complexes and have many functions including transcriptional regulation, DNA damage repair, and telomerase activity. They also interact with major oncogenic actors such as beta-catenin and c-myc and regulate their oncogenic function. We only now begin to get insight into the role of Pontin and Reptin in human cancers. [Cancer Res 2008;68(17):6873-6]

Halofuginone suppresses the lung metastasis of chemically induced hepatocellular carcinoma in rats through MMP inhibition

Halofuginone, an inhibitor of collagen synthesis, appears to be a promising antitumoral drug in preclinical studies. We used a relevant rat model of autochthonous, chemically induced, spontaneously metastasizing hepatocellular carcinoma (HCC) to test the efficacy of halofuginone on tumor progression and matrix metalloproteinase (MMP) expression. Following sequential administration of diethylnitrosamine and N-nitrosomorpholine for 14 weeks, all animals developed HCC and then received halofuginone or its solvent for 10 weeks. The final number of liver tumors was lower in the halofuginone group than in the solvent group (57.2 ± 4.6 vs 68 ± 5.0; P < .01). The percentage of the lung surface infiltrated by metastasis was much smaller in the halofuginone group (0.3 ± 0.2%) than in the solvent group (13.5 ± 10.1%; P < .02). MMP-9 activity was decreased in the halofuginone group by 89% and 63% in non-neoplastic parts of the liver and tumor, respectively. The percentage of active MMP-2 was reduced by 90% in non-neoplastic parts of the liver and by 61% in tumors. This was likely subsequent to a decreased expression of both MMP-14 and tissue inhibitor of matrix metalloproteinase-2, which are required for pro-MMP-2 activation. These results, obtained from a clinically relevant model, further suggest the potential benefit of halofuginone in HCC

In vivo silencing of Reptin blocks the progression of human hepatocellular carcinoma in xenografts and is associated with replicative senescence.: Reptin silencing blocks liver cancer progression

International audienceBACKGROUND & AIMS: We previously showed that Reptin is overexpressed in hepatocellular carcinoma (HCC), and that in vitro depletion of Reptin with siRNAs led to HCC cell growth arrest and apoptosis. Here, we asked whether in vivo targeting of Reptin in established tumours had a therapeutic effect. METHODS: We used lentiviral vectors to construct HuH7 and Hep3B cell lines with doxycycline (Dox)-dependent expression of Reptin (R2) or control shRNA (GL2). Cells were injected subcutaneously into immunodeficient mice, and Dox was given when tumours reached a volume of 250 mm(3). RESULTS: In vitro, the growth of GL2-Dox, GL2+Dox, and R2-Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4 days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid beta-galactosidase and the presence of senescence-associated heterochromatin foci. In xenografted mice, R2+Dox tumour growth stagnated or even regressed with prolonged treatment in contrast with the GL2-Dox, GL2+Dox, and R2-Dox tumours that progressed steadily. The blockage of tumour progression was associated with the induction of senescence and reduced cell proliferation. CONCLUSIONS: In vivo Reptin depletion leads to tumour growth arrest. Reptin may prove a valuable target in HCC

Protease-activated receptor 1 knockout reduces experimentally induced liver fibrosis.

International audienceThrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl(4)) toxicity was similar in wild-type (WT), PAR-1(-/-), and PAR-1(+/-) mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl(4) or its solvent for 6 wk (300 microl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl(4) treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively (P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-beta receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes

Matrix Metalloproteinase 3 Is Present in the Cell Nucleus and Is Involved in Apoptosis

Matrix metalloproteinase (MMP)-3 is a protease involved in cancer progression and tissue remodeling. Using immunofluorescence and immunoelectron microscopy, we identified nuclear localization of MMP-3 in several cultured cell types and in human liver tissue sections. Western blot analysis of nuclear extracts revealed two immunoreactive forms of MMP-3 at 35 and 45 kd, with the 35-kd form exhibiting caseinolytic activity. By transient transfection, we expressed active MMP-3 fused to the enhanced green fluorescent protein (EGFP/aMMP-3) in Chinese hamster ovary cells. We showed that EGFP/aMMP-3 translocates into the nucleus. A functional nuclear localization signal was demonstrated by the loss of nuclear translocation after site-directed mutagenesis of a putative nuclear localization signal and by the ability of the MMP-3 nuclear localization signal to drive a heterologous protein into the nucleus. Finally, expression by Chinese hamster ovary cells of EGFP/aMMP-3 induced a twofold increase of apoptosis rate, compared with EGFP/pro-MMP-3, which does not translocate to the nucleus. Increased apoptosis was abolished by site-directed mutagenesis of the catalytic site of MMP-3 or by using the MMP inhibitor GM6001. This study elucidates for the first time the mechanisms of nuclear localization of a MMP and shows that nuclear MMP-3 can induce apoptosis via its catalytic activity

Adenosine triphosphatase pontin is overexpressed in hepatocellular carcinoma and coregulated with reptin through a new posttranslational mechanism.

International audienceReptin and Pontin are related ATPases associated with stoichiometric amounts in several complexes involved in chromatin remodeling, transcriptional regulation, and telomerase activity. We found that Reptin was up-regulated in hepatocellular carcinoma (HCC) and that down-regulation of Reptin led to growth arrest. We show here that Pontin messenger RNA (mRNA) is also up-regulated in human HCC 3.9-fold as compared to nontumor liver (P = 0.0004). Pontin expression was a strong independent factor of poor prognosis in a multivariate analysis. As for Reptin, depletion of Pontin in HuH7 cells with small interfering RNAs (siRNAs) led to growth arrest. Remarkably, Pontin depletion led to down-regulation of Reptin as shown with western blot, and vice versa. Whereas siRNAs induced a decrease of their cognate mRNA targets, they did not affect the transcripts of the partner protein. Translation of Pontin or Reptin was not altered when the partner protein was silenced. However, pulse-chase experiments demonstrated that newly synthesized Pontin or Reptin stability was reduced in Reptin- or Pontin-depleted cells, respectively. This phenomenon was reversed upon inhibition of proteasome or ubiquitin-activating enzyme (E1). In addition, proteasome inhibition could partly restore Pontin steady-state levels in Reptin-depleted cells, as shown by western blot. This restoration was not observed when cells were also treated with cycloheximide, thus confirming that proteasomal degradation in this setting was restricted to newly synthesized Pontin. CONCLUSION: Reptin and Pontin protein levels are strictly controlled by a posttranslational mechanism involving proteasomal degradation of newly synthesized proteins. These data demonstrate a tight regulatory and reciprocal interaction between Reptin and Pontin, which may in turn lead to the maintenance of their 1:1 stoichiometry