Distinct Mechanisms Are Responsible for Nrf2-Keap1 Pathway Activation at Different Stages of Rat Hepatocarcinogenesis

Activation of the Nrf2-Keap1 pathway, the main intracellular defense against environmental stress, has been observed in several human cancers, including hepatocellular carcinoma (HCC). Here, we assessed whether distinct mechanisms of activation may be involved at different stages of hepatocarcinogenesis. We adopted an experimental model consisting of treatment with diethylnitrosamine (DENA) followed by a choline-devoid methionine-deficient (CMD) diet for 4 months. The CMD diet was then replaced with a basal diet, and the animals were killed at 6, 10 or 13 months after DENA injection. Nrf2 activation occurred at early steps of hepatocarcinogenesis and persisted throughout the tumorigenic process. WhileNrf2mutations were extremely frequent at early steps (90%), their incidence diminished with the progression to malignancy (25%). Conversely, while p62 was almost undetectable in early nodules, its accumulation occurred in HCCs, suggesting that Nrf2 pathway activation at late stages is mainly due to Keap1 sequestration by p62. We demonstrate that, in a model of hepatocarcinogenesis resembling human non-alcoholic fatty liver disease,Nrf2mutations are the earliest molecular changes responsible for the activation of the Nrf2-Keap1 pathway. The progressive loss of mutations associated with a concomitant p62 accumulation implies that distinct mechanisms are responsible for Nrf2-Keap1 pathway activation at different stages of hepatocarcinogenesis

Thyroid hormone inhibits hepatocellular carcinoma progression via induction of differentiation and metabolic reprogramming

The limited therapeutic options available for hepatocellular carcinoma (HCC) make mandatory to find alternative effective treatments for this tumor. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of hepatocellular carcinomas (HCCs)

The origin of the Sardinian Blue, Pseudophilotes barbagiae (Lycaenidae): Out-of-Europe or Out-of-Africa?

Trabajo presentado en el XIXth European Congress of Lepidopterology, celebrado en Radebeul (Alemania) del 27 de septiembre al 2 de octubre de 2015.The island of Sardinia is characterized by an extraordinary richness of endemic species, evolved as a result of its geographic position and geological history. Here, we try to reconstruct the evolutionary history of the endemic Sardinian blue butterfly, P. barbagiae de Prins and van der Poorten 1982, using the DNA barcoding region of the COI mitochondrial gene. The species is restricted to a few slopes in the mountainous areas of the island, and has been described as distinct from the continental and Corsican P. baton by characteristics of the male genitalia and wing markings. We analyzed 11 individuals of P. barbagiae and compared them with all Eu- ropean and north African congeneric species: a group of 77 sequences including P. baton (Corsica, Italy, France, Spain), P. vicrama (Romania, Turkey, Kazakhstan, Russia) and P. panoptes (Spain), as well as several BINs containing 20 sequences of P. abencerragus (Spain, Morocco, Tunisia, Portugal), 10 sequences of P. bavius (Romania, Russia) and 4 sequences of P. fatma (Morocco). The Bayesan phylogenetic tree showed that the group with P. baton, P. vicrama and P. panoptes forms a well-supported clade, but DNA barcodes do not distinguish these three species. The largest interspecific distance (4.8%) was found between P. fatma and P. baton ­ P. vicrama ­P. panoptes group, while the smallest (1.2%) was between P.abencerra­ gus and P. barbagiae. The latter also forms a well-supported monophyletic group and has closer affinity to P. abencerragus (1.2%) than to P. baton – P. vicrama – P. pa noptes group (1.8%). Two hypotheses on a European versus an African origin of P. barbagiae will be presented in the light of genetic and biogeographic data. This study will contribute to the emerging picture of butterfly diversification in the Palearctic region, and contribute to our understanding of the evolution of endemism in the European butterfly fauna.N

Thyroid hormone receptor-β agonist induces β-catenin-dependent hepatocyte proliferation in mice: Implications in hepatic regeneration

Triiodothyronine (T3) induces hepatocyte proliferation in rodents. Recent work has shown molecular mechanism for T3's mitogenic effect to be through activation of β-catenin signaling. Since systemic side effects of T3 may preclude its clinical use, and hepatocytes mostly express T3 hormone receptor β (TRβ), we investigated if selective TRβ agonists like GC-1 may also have β-catenin-dependent hepatocyte mitogenic effects. Here we studied the effect of GC-1 and T3 in conditional knockouts of various Wnt pathway components. We also assessed any regenerative advantage of T3 or GC-1 when given prior to partial hepatectomy in mice. Mice administered GC-1 showed increased pSer675-β-catenin, cyclin D1, BrdU incorporation, and PCNA. No abnormalities in liver function tests were noted. GC-1-injected liver-specific β-catenin knockouts (β-catenin LKO) showed decreased proliferation when compared to wild-type littermates. To address if Wnt signaling was required for T3- or GC-1-mediated hepatocyte proliferation, we used LRP5-6-LKO, which lacks the two redundant Wnt coreceptors. Surprisingly, decreased hepatocyte proliferation was also evident in LRP5-6-LKO in response to T3 and GC-1, despite increased pSer675-β-catenin. Further, increased levels of active b-catenin (hypophosphorylated at Ser33, Ser37, and Thr41) were evident after T3 and GC-1 treatment. Finally, mice pretreated with T3 or GC-1 for 7 days followed by partial hepatectomy showed a significant increase in hepatocyte proliferation both at the time (T0) and 24 h after surgery. In conclusion, like T3, TRβ-selective agonists induce hepatocyte proliferation through β-catenin activation via both PKA- and Wnt-dependent mechanisms and confer a regenerative advantage following surgical resection. Hence, these agents may be useful regenerative therapies in liver transplantation or other surgical settings

The thyromimetic KB2115 (Eprotirome) induces rat hepatocyte proliferation

Although the hepatomitogenic activity of T3 is well established, the wide range of harmful effects exerted by this hormone precludes its use in regenerative therapy. The aim of this study was to investigate whether an agonist of TR, KB2115 (Eprotirome) could exert a mitogenic effect in the liver, without most of the adverse T3/TR-dependent side effects. F-344 rats treated with KB2115 for one week displayed a massive increase in bromodeoxyuridine incorporation (from 20 to 40% vs. 5% of controls), which was associated with increased mitotic activity in the absence of significant signs of liver toxicity. Noteworthy, while cardiac hypertrophy typical of T3 was not observed, beneficial effects, such as lowering blood cholesterol levels, were associated to KB2115 administration. Following a single dose of KB2115, hepatocyte proliferation was evident as early as 18 hours demonstrating its direct mitogenic effect. No increase of serum transaminase levels or apoptosis was observed prior to- or concomitantly with S phase. While KB2115-induced mitogenesis was not associated to enhanced expression of c-fos, c-jun and c-myc, cyclin D1 levels rapidly increased. In conclusion, KB2115 induces hepatocyte proliferation without overt toxicity. Hence this agent may be useful for regenerative therapies in liver transplantation or other surgical settings

mRNA-miRNA networks identify metabolic pathways associated to the anti-tumorigenic effect of thyroid hormone on preneoplastic nodules and hepatocellular carcinoma

Background: Thyroid hormones (THs) inhibit hepatocellular carcinoma (HCC) through different mechanisms. However, whether microRNAs play a role in the antitumorigenic effect of THs remains unknown. Methods: By next generation sequencing (NGS) we performed a comprehensive comparative miRNomic and transcriptomic analysis of rat hepatic preneoplastic lesions exposed or not to a short-term treatment with triiodothyronine (T3). The expression of the most deregulated miRs was also investigated in rat HCCs, and in human hepatoma cell lines, treated or not with T3. Results: Among miRs down-regulated in preneoplastic nodules following T3, co-expression networks revealed those targeting thyroid hormone receptor-β (Thrβ) and deiodinase1, and Oxidative Phosphorylation. On the other hand, miRs targeting members of the Nrf2 Oxidative Pathway, Glycolysis, Pentose Phosphate Pathway and Proline biosynthesis – all involved in the metabolic reprogramming displayed by preneoplastic lesions– were up-regulated. Notably, while the expression of most miRs deregulated in preneoplastic lesions was not altered in HCC or in hepatoma cells, miR-182, a miR known to target Dio1 and mitochondrial complexes, was down-deregulated by T3 treatment at all stages of hepatocarcinogenesis and in hepatocarcinoma cell lines. In support to the possible critical role of miR-182 in hepatocarcinogenesis, exogenous expression of this miR significantly impaired the inhibitory effect of T3 on the clonogenic growth capacity of human HCC cells. Conclusions: This work identified several miRNAs, so far never associated to T3. In addition, the precise definition of the miRNA-mRNA networks elicited by T3 treatment gained in this study may provide a better understanding of the key regulatory events underlying the inhibitory effect of T3 on HCC development. In this context, T3-induced down-regulation of miR-182 appears as a promising tool

Thyroid Hormone Receptor-β Agonist GC-1 Inhibits Met-β-Catenin-Driven Hepatocellular Cancer

The thyromimetic agent GC-1 induces hepatocyte proliferation via Wnt/β-catenin signaling and may promote regeneration in both acute and chronic liver insufficiencies. However, β-catenin activation due to mutations in CTNNB1 is seen in a subset of hepatocellular carcinomas (HCC). Thus, it is critical to address any effect of GC-1 on HCC growth and development before its use can be advocated to stimulate regeneration in chronic liver diseases. In this study, we first examined the effect of GC-1 on β-catenin-T cell factor 4 activity in HCC cell lines harboring wild-type or mutated-CTNNB1. Next, we assessed the effect of GC-1 on HCC in FVB mice generated by hydrodynamic tail vein injection of hMet-S45Y-β-catenin, using the sleeping beauty transposon-transposase. Four weeks following injection, mice were fed 5 mg/kg GC-1 or basal diet for 10 or 21 days. GC-1 treatment showed no effect on β-catenin-T cell factor 4 activity in HCC cells, irrespective of CTNNB1 mutations. Treatment with GC-1 for 10 or 21 days led to a significant reduction in tumor burden, associated with decreased tumor cell proliferation and dramatic decreases in phospho-(p-)Met (Y1234/1235), p-extracellular signal-related kinase, and p-STAT3 without affecting β-catenin and its downstream targets. GC-1 exerts a notable antitumoral effect on hMet-S45Y-β-catenin HCC by inactivating Met signaling. GC-1 does not promote β-catenin activation in HCC. Thus, GC-1 may be safe for use in inducing regeneration during chronic hepatic insufficiency

DataSheet_1_mRNA-miRNA networks identify metabolic pathways associated to the anti-tumorigenic effect of thyroid hormone on preneoplastic nodules and hepatocellular carcinoma.zip

BackgroundThyroid hormones (THs) inhibit hepatocellular carcinoma (HCC) through different mechanisms. However, whether microRNAs play a role in the antitumorigenic effect of THs remains unknown.MethodsBy next generation sequencing (NGS) we performed a comprehensive comparative miRNomic and transcriptomic analysis of rat hepatic preneoplastic lesions exposed or not to a short-term treatment with triiodothyronine (T3). The expression of the most deregulated miRs was also investigated in rat HCCs, and in human hepatoma cell lines, treated or not with T3.ResultsAmong miRs down-regulated in preneoplastic nodules following T3, co-expression networks revealed those targeting thyroid hormone receptor-β (Thrβ) and deiodinase1, and Oxidative Phosphorylation. On the other hand, miRs targeting members of the Nrf2 Oxidative Pathway, Glycolysis, Pentose Phosphate Pathway and Proline biosynthesis – all involved in the metabolic reprogramming displayed by preneoplastic lesions– were up-regulated. Notably, while the expression of most miRs deregulated in preneoplastic lesions was not altered in HCC or in hepatoma cells, miR-182, a miR known to target Dio1 and mitochondrial complexes, was down-deregulated by T3 treatment at all stages of hepatocarcinogenesis and in hepatocarcinoma cell lines. In support to the possible critical role of miR-182 in hepatocarcinogenesis, exogenous expression of this miR significantly impaired the inhibitory effect of T3 on the clonogenic growth capacity of human HCC cells.ConclusionsThis work identified several miRNAs, so far never associated to T3. In addition, the precise definition of the miRNA-mRNA networks elicited by T3 treatment gained in this study may provide a better understanding of the key regulatory events underlying the inhibitory effect of T3 on HCC development. In this context, T3-induced down-regulation of miR-182 appears as a promising tool.</p