Search

Studies 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 β-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]

Thrombin 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 (CCl4) 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 CCl4or its solvent for 6 wk (300 μl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl4treatment 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-β 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.

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 M 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%a; P < .02). MM P-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 metal loproteinase-2, which are required for proMMP-2 activation. These results, obtained from a clinically relevant model, further suggest the potential benefit of halofuginone in HCC.

> Malgre d’importants progres recents, les mecanismes moleculaires de la carcinogenese hepatique restent tres imparfaitement connus. Plus encore, les perspectives therapeutiques sont extremement minces. C’est pour cette raison que notre equipe a decide de rechercher de nouveaux acteurs de la carcinogenese hepatique, et de nouvelles cibles therapeutiques potentielles, a l’aide d’une etude comparative du proteome de quatre carcinomes hepatocellulaires (CHC) et du foie peri-tumoral correspondant [1]. Ce travail a permis de mettre en evidence de nombreuses proteines dont l’expression est deregulee dans les CHC, et parmi elles, la reptine (encore appelee RUVBL2, TIP48 ou TIP49b). Jusqu’alors, la reptine avait ete etudiee essentiellement dans des organismes modeles ou elle avait ete detectee dans des complexes de haut poids moleculaire impliques dans le remodelage de la chromatine, la regulation transcriptionnelle, ou la reparation de l’ADN, comme INO80 [2] ou TIP60 [3]. La reptine a une importante homologie avec la pontine ; ces deux proteines sont proches de l’helicase bacterienne RuvB et appartiennent a la famille des AAA+ ATPases. Elles interagissent l’une avec l’autre en formant des dodecameres contenant un hexamere de chaque proteine [4]. Notre attention avait ete attiree par l’interaction de ces proteines avec deux oncogenes importants pour le CHC, la β-catenine [5] et c-myc [6]. La plupart des travaux montraient que la reptine se comportait comme un antagoniste de l’activite transcriptionnelle du complexe β-catenine/TCF-LEF alors que la pontine avait une activite agoniste ; cependant, recemment d’autres travaux ont revele que la reptine etait necessaire a l’effet represseur de la β−catenine sur l’expression du gene anti-metastatique KAI-1/CD82 [7].

Two lines of mice, one resistant (Car-R) and one susceptible (Car-S) to skin carcinogenesis, were produced by bi-directional selective breeding. To see whether the characteristics of susceptibility or resistance to tumorigenesis were also expressed in the liver and lung, the two lines were submitted comparatively to treatment with 5,9-dimethyl dibenzo[ c , g ]carbazole (DiMeDBC), a potent hepatocarcinogenic derivative of the ubiquitous heterocyclic carcinogenic pollutant, 7 H -dibenzo[ c , g ]carbazole (DBC). An inversion of genetic predisposition to carcinogenesis in liver was observed. Car-R animals displayed rapid tumorigenesis in 100% of cases while Car-S mice were remarkably less sensitive, showing a 4-fold lower mean tumor multiplicity and a 4-month longer latency time. In parallel adduct formation by DiMeDBC and DBC in liver DNA was analyzed by the 32 P-postlabeling method, showing a remarkably higher level in Car-R mice than in Car-S animals. These data indicate that tissue-specific sensibility in carcinogenesis may involve gene expression at various levels.

Mice of the XVIInc/Z and DBA/2N strains, which are responsive and nonresponsive, respectively, to the aryl hydrocarbon (Ah) receptor, were treated with the hepatocarcinogen 5,9-dimethyldibenzo[c,g]carbazole and their livers were examined by nuclease P1-enhanced 32P-postlabeling for the levels of DNA adducts formed. Pretreatment at the doses usually reported in the literature with the cytochrome P4501A (CYP1A) inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), β-naphthoflavone (BNF), and isosafrole modulated DNA adduction. In XVIInc/Z mice, DNA adduction was totally inhibited by TCDD (a CYP1A1/1A2 inducer), BNF (a CYP1A1/1A2 inducer), and isosafrole (a CYP1A2 inducer). In DBA/2N mice, in which DNA adduction was also inhibited by TCDD, about 25% of the DNA adduct levels persisted after pretreatment with BNF (not a CYP1A1/1A2 inducer in this strain) or isosafrole (a CYP1A2 inducer in this strain). The increase (in all cases less than twofold) in the levels of the phase-II drug-metabolizing enzymes glutathione S-transferase and uridine diphospho-glucuronyltransferase after treatment with inducers cannot explain the total disappearance of DNA adducts. Assays of 5-bromo-2′-deoxyuridine incorporation did not show any induction of DNA synthesis which could explain the decrease in adducts. These results suggest that in vivo 1) increases in CYP1A enzymes by inducers are not correlated with enhanced levels of certain DNA adducts; and 2) phase-II drug metabolizing enzymes are not the main cellular protection pathway for detoxification. An additional mechanism, perhaps also induced by the Ah receptor but highly dependent on the dose of inducer, could be involved in parallel to multidrug resistance (mdr); further experiments are needed to identify this process used by the cell to enhance its protection against toxic or genotoxic effects. Environ. Mol. Mutagen. 32: 314–324, 1998 © 1998 Wiley-Liss, Inc.

International audience; Reptin 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.

Background & 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 250mm 3 . Results In vitro , the growth of GL2−Dox, GL2+Dox, and R2−Dox cells was undistinguishable whereas that of R2+Dox cells stopped 4days after Dox treatment. The growth decrease was associated with increased apoptosis, and evidence of replicative senescence, as shown by staining for acid β-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.

International audience; n.a

International audience; 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.

Statins have beneficial effects in early pre-clinical models of hepatocellular carcinoma (HCC). Our aim was to test the efficacy of pravastatin on the progression of established HCC in rat, and to study its mechanisms.HCC was induced with diethylnitrosamine and N-nitrosomorpholine. After 14 weeks, all rats developed HCC and then received pravastatin or its solvent for 10 weeks (10 rats/group).Liver tumor mass was lower in pravastatin group (PG) than control group (CG), as estimated from the number of liver tumors (p0.004) and the liver weight/body weight ratio (p0.04). Every CG rat surviving at 24 weeks (4/4) had lung metastasis, against only 5/8 in PG. Moreover, the percentage of lung surface occupied by metastasis was 10-fold smaller in PG than CG (p0.016). Pravastatin decreased liver matrix metalloproteinase (MMP)-9 activity and mostly suppressed MMP-2 activation (p0.004), likely because it decreased expression of MMP-14 and tissue inhibitor of matrix metalloproteinases-2 (p0.01), required for MMP-2 activation.Pravastatin reduces progression and limits metastatic diffusion of established HCC. This could be linked to the decreased MMP activity. These results, obtained in a very aggressive HCC model, further suggest the potential benefit of statins in human HCC.

The potent multitissue carcinogen 7H-dibenzo-[c,g]carbazole and nine methylated derivatives, synthesized on the basis of the positions in the parent compound that are involved in metabolism, were tested for their ability to induce sarcomas and hepatic tumors in XVllnc/Z mice. In addition, the capacity of these compounds to induce DNA adducts in skin and liver was investigated by 32 P-postlabeling analysis after their topical administration. Induction by these compounds of cytochromes P450 of the 1A family in liver and skin was investigated and correlated to their carcinogenic potential. A clear correlation was found between the tissue specificity of DNA binding and the capacity of each compound to induce skin or liver tumors. In contrast, no direct relationship was observed between the capacity of the compounds to induce cytochromes 1 A 1/1A2 and the tissue specificity of carcinogenesis or DNA binding in liver or skin. The results are discussed with respect to the positions of methyl groups in the 7H-dibenzo[c,g]carbazole molecule.

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.