Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

BACKGROUND AND AIMS: Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. APPROACH AND RESULTS: To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. CONCLUSIONS: Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

TG68, a Novel Thyroid Hormone Receptor-ß Agonist for the Treatment of NAFLD.

Activation of thyroid hormone receptor ß (THRß) has shown beneficial effects on metabolic alterations, including non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effect of TG68, a novel THRß agonist, on fatty liver accumulation and liver injury in mice fed a high-fat diet (HFD). C57BL/6 mice fed HFD for 17 or 18 weeks, a time when all mice developed massive steatohepatitis, were then given TG68 at a dose of 9.35 or 2.8 mg/kg for 2 or 3 weeks, respectively. As a reference compound, the same treatment was adopted using equimolar doses of MGL-3196, a selective THRß agonist currently in clinical phase III. The results showed that treatment with TG68 led to a reduction in liver weight, hepatic steatosis, serum transaminases, and circulating triglycerides. qRT-PCR analyses demonstrated activation of THRß, as confirmed by increased mRNA levels of Deiodinase-1 and Malic enzyme-1, and changes in lipid metabolism, as revealed by increased expression of Acyl-CoA Oxidase-1 and Carnitine palmitoyltransferase-1. The present results showed that this novel THRß agonist exerts an anti-steatogenic effect coupled with amelioration of liver injury in the absence of extra-hepatic side effects, suggesting that TG68 may represent a useful tool for the treatment of NAFLD.

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

BACKGROUND & AIMS: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. 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 HCCs. METHODS: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. RESULTS: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor ß (TRß). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRß axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. CONCLUSIONS: Collectively, these findings suggest that reactivation of the T3/TRß axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRß, could be useful tools in HCC therapy. LAY SUMMARY: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

Estimation of a significance threshold for epigenome-wide association studies.

Epigenome-wide association studies (EWAS) are designed to characterise population-level epigenetic differences across the genome and link them to disease. Most commonly, they assess DNA-methylation status at cytosine-guanine dinucleotide (CpG) sites, using platforms such as the Illumina 450k array that profile a subset of CpGs genome wide. An important challenge in the context of EWAS is determining a significance threshold for declaring a CpG site as differentially methylated, taking multiple testing into account. We used a permutation method to estimate a significance threshold specifically for the 450k array and a simulation extrapolation approach to estimate a genome-wide threshold. These methods were applied to five different EWAS datasets derived from a variety of populations and tissue types. We obtained an estimate of α=2.4×10-7 for the 450k array, and a genome-wide estimate of α=3.6×10-8. We further demonstrate the importance of these results by showing that previously recommended sample sizes for EWAS should be adjusted upwards, requiring samples between ∼10% and ∼20% larger in order to maintain type-1 errors at the desired level.

High Frequency of ß-Catenin Mutations in Mouse Hepatocellular Carcinomas Induced by a Nongenotoxic Constitutive Androstane Receptor Agonist.

Activation of Wnt/ß-catenin signaling is frequent in human and rodent hepatocarcinogenesis. Although in mice the tumor-promoting activity of agonists of constitutive androstane receptor (CAR) occurs by selection of carcinogen-initiated cells harboring ß-catenin mutations, the molecular alterations leading to hepatocellular carcinoma (HCC) development by the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCP) in the absence of genotoxic injury are unknown. Here, we show that CAR activation per se induced HCC in mice and that 91% of them carried ß-catenin point mutations or large in-frame deletions/exon skipping targeting Ctnnb1 exon 3. Point mutations in HCCs induced by TCP alone displayed different nucleotide substitutions compared with those found in HCCs from mice pretreated with diethylnitrosamine. Moreover, unlike those occurring in HCCs from diethylnitrosamine + TCP mice, they did not result in increased expression of ß-catenin target genes, such as Glul, Lgr5, Rgn, Lect2, Tbx3, Axin2, and Ccnd1, or nuclear translocation of ß-catenin compared with the control liver. Remarkably, in the nontumoral liver tissue, chronic CAR activation led to down-regulation of these genes and to a partial loss of glutamine synthetase-positive hepatocytes. These results show that, although chronic CAR activation per se induces HCCs carrying ß-catenin mutations, it concurrently down-regulates the Wnt/ß-catenin pathway in nontumoral liver. They also indicate that the relationship between CAR and ß-catenin may be profoundly different between normal and neoplastic hepatocytes.

A Large Set of miRNAs Is Dysregulated from the Earliest Steps of Human Hepatocellular Carcinoma Development.

Hepatocellular carcinoma (HCC) typically results from a stepwise process characterized by the development of premalignant lesions, such as low- or high-grade dysplastic nodules (LGDNs and HGDNs, respectively), in a cirrhotic setting. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional regulation of gene expression that can act as oncogenes or tumor suppressors. Whether and which miRNAs are involved in the early stages of HCC development remains elusive. Here, small-RNA sequencing was applied to profile miRNA expression in 55 samples (cirrhotic nodules; CNs), LGDNs, HGDNs, early HCCs, and small progressed HCCs, obtained from 17 patients bearing HCCs of different etiologies. An miRNA expression signature of 62 miRNAs distinguishing small progressed HCCs from matched CNs was identified. Interestingly, 52 of these miRNAs discriminated CNs from LGDNs/HGDNs, regardless of etiology, and remained modified along the tumorigenic process. Functional analysis of the predicted mRNA targets of deregulated miRNAs identified common modifications between the early and late stages of HCC development likely involved in the stepwise process of HCC development. Our results demonstrate that miRNA deregulation happens very early in HCC in humans, implying their crucial role in the tumorigenic process. The identification of miRNAs discriminating CNs from neoplastic nodules may have relevant translational implications in early diagnosis.

Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment, tracing a biomarker signature specific for this tumor.

Colorectal cancer (CRC) develops through the accumulation of both genetic and epigenetic alterations. However, while the former are already used as prognostic and predictive biomarkers, the latter are less well characterized. Here, performing global methylation analysis on both CRCs and adenomas by Illumina Infinium HumanMethylation450 Bead Chips, we identified a panel of 74 altered CpG islands, demonstrating that the earliest methylation alterations affect genes coding for proteins involved in the crosstalk between cell and surrounding environment. The panel discriminates CRCs and adenomas from peritumoral and normal mucosa with very high specificity (100%) and sensitivity (99.9%). Interestingly, over 70% of the hypermethylated islands resulted in downregulation of gene expression. To establish the possible usefulness of these non-invasive markers for detection of colon cancer, we selected three biomarkers and identified the presence of altered methylation in stool DNA and plasma cell-free circulating DNA from CRC patients.

Genetic inactivation of Nrf2 prevents clonal expansion of initiated cells in a nutritional model of rat hepatocarcinogenesis.

BACKGROUND & AIMS: Dysregulation of the Keap1-Nrf2 pathway has been observed in experimental and human tumors, suggesting possible roles of the pathway in cancer development. Herein, we examined whether Nrf2 (Nfe2l2) activation occurs at early steps of rat hepatocarcinogenesis, to assess critical contributions of Nrf2 to the onset of hepatocellular carcinoma (HCC). METHODS: We used wild-type (WT) and Nrf2 knockout (Nrf2KO) rats treated with a single injection of diethylnitrosamine (DENA) followed by choline-devoid methionine-deficient (CMD) diet. This experimental model causes massive fatty liver and steatohepatitis with fibrosis and enables identification of early stages of hepatocarcinogenesis. RESULTS: We found that Nrf2 activation takes place in early preneoplastic lesions identified by the marker glutathione S-transferase placental form (GSTP). Nrf2 missense mutations, known to disrupt the Keap1-Nrf2 binding, were present in 65.7% of GSTP-positive foci. Nrf2KO rats were used to directly investigate whether Nrf2 is critical for initiation and/or clonal expansion of DENA-damaged hepatocytes. While Nrf2 genetic inactivation did not alter DENA-induced initiation, it led to increased liver injury and chronic compensatory hepatocyte regeneration when rats were fed a CMD diet. However, in spite of such a permissive environment, the livers of Nrf2KO rats did not display any preneoplastic lesion unlike those of WT rats. CONCLUSIONS: These results demonstrate that, in a model of hepatocarcinogenesis resembling human non-alcoholic fatty liver disease: i) Nrf2 is activated at early steps of the tumorigenic process and ii) Nrf2 is mandatory for the clonal expansion of initiated cells, indicating that Nrf2 is critical in the onset of HCC. LAY SUMMARY: Dysregulation of the Keap1-Nrf2 molecular pathway has been observed in human tumors. In a nutritional model of hepatocarcinogenesis, the protein Nrf2 is frequently mutated/activated at early steps of the tumorigenic process. Herein, we show that Nrf2 is mandatory for the development of preneoplastic lesions. These results suggest that Nrf2 has a critical role in the onset of hepatocellular carcinoma.

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

The thyromimetic agent GC-1 induces hepatocyte proliferation via Wnt/ß-catenin signaling and may promote regeneration in both acute and chronic liver insufficiencies. However, ß-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 ß-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-ß-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 ß-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 ß-catenin and its downstream targets. GC-1 exerts a notable antitumoral effect on hMet-S45Y-ß-catenin HCC by inactivating Met signaling. GC-1 does not promote ß-catenin activation in HCC. Thus, GC-1 may be safe for use in inducing regeneration during chronic hepatic insufficiency.

GC-1: A Thyromimetic With Multiple Therapeutic Applications in Liver Disease.

Thyroid hormones (THs), namely, 3,5,3'-triiodo-l-thyronine (T3) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine or T4), influence a variety of physiological processes that have important implications in fetal development, metabolism, cell growth, and proliferation. While THs elicit several beneficial effects on lipid metabolism and improve myocardial contractility, these therapeutically desirable effects are associated to a thyrotoxic state that severely limits the possible use of THs as therapeutic agents. Therefore, several efforts have been made to develop T3 analogs that could retain the beneficial actions (triglyceride, cholesterol, obesity, and body mass lowering) without the adverse TH-dependent side effects. This goal was achieved by the synthesis of TRß-selective agonists. In this review, we summarize the current knowledge on the effects of one of the best characterized TH analogs, the TRß1-selective thyromimetic, GC-1. In particular, we review some of the effects of GC-1 on different liver disorders, with reference to its possible clinical application. A brief comment on the possible therapeutic use of GC-1 in extrahepatic disorders is also included.

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 TRß, KB2115 (Eprotirome), could exert a mitogenic effect in the liver, without most of the adverse T3/TRα-dependent side effects. F-344 rats treated with KB2115 for 1 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 h, demonstrating its direct mitogenic effect. No increase in serum transaminase levels or apoptosis was observed prior to or concomitantly with the S phase. While KB2115-induced mitogenesis was not associated to enhance 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.

Constitutive androstane receptor (Car)-driven regeneration protects liver from failure following tissue loss.

BACKGROUND & AIMS: Liver can recover following resection. If tissue loss is too excessive, however, liver failure will develop as is known from the small-for-size-syndrome (SFSS). The molecular processes underlying liver failure are ill-understood. Here, we explored the role and the clinical potential of Nr1i3 (constitutive androstane receptor, Car) in liver failure following hepatectomy. METHODS: Activators of Car, various hepatectomies, Car(-/-) mice, humanized CAR mice, human tissue and ex vivo liver slice cultures were used to study Car in the SFSS. Pathways downstream of Car were investigated by in vivo siRNA knockdown. RESULTS: Excessive tissue loss causing liver failure is associated with deficient induction of Car. Reactivation of Car by an agonist normalizes all features associated with experimental SFSS. The beneficial effects of Car activation are relayed through Foxm1, an essential promoter of the hepatocyte cell cycle. Deficiency in the CAR-FOXM1 axis likewise is evident in human SFSS. Activation of human CAR mitigates SFSS in humanized CAR mice and improves the culture of human liver slices. CONCLUSIONS: Impaired hepatic Car-Foxm1 signaling provides a first molecular characterization of liver that fails to recover after tissue loss. Our findings place deficient regeneration as a principal cause behind the SFSS and suggest CAR agonists may bear clinical potential against liver failure. LAY SUMMARY: The unique regenerative capacity of liver has its natural limits. Following tissue loss that is too excessive, such as through extended resection in the clinic, liver failure may develop. This is known as small-for-size-syndrome (SFSS) and represents the most frequent cause of death due to liver surgery. Here we show that deficient induction of the protein Car, a central regulator of liver function and growth, is a cause of liver failure following extended resection; reactivation of Car through pharmacological means is sufficient to prevent or rescue the SFSS.

The metabolic gene HAO2 is downregulated in hepatocellular carcinoma and predicts metastasis and poor survival.

BACKGROUND & AIMS: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis. METHODS: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice. RESULTS: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability. CONCLUSION: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC.

Local hypothyroidism favors the progression of preneoplastic lesions to hepatocellular carcinoma in rats.

UNLABELLED: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3'-L-triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant-hepatocyte rat model (R-H model), we found down-regulation of TRß1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRß1 and, to a lesser extent, TRα1 down-regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin-19 (Krt-19) and characterized by a higher proliferative activity, compared to the Krt-19 negative ones. TRß1 down-regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up-regulation of TRß1 and of its target genes in Krt-19(+) preneoplastic rat lesions and was associated with nodule regression. In HCC, TRß1 down-regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRß1-targeting microRNAs ([miR]-27a, -181a, and -204). An inverse correlation between TRß1 and miR-181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. CONCLUSION: Down-regulation of TRs, especially TRß1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor.

Nrf2, but not ß-catenin, mutation represents an early event in rat hepatocarcinogenesis.

UNLABELLED: Hepatocellular carcinoma (HCC) develops through a multistage process, but the nature of the molecular changes associated with the different steps, the very early ones in particular, is largely unknown. Recently, dysregulation of the NRF2/KEAP1 pathway and mutations of these genes have been observed in experimental and human tumors, suggesting their possible role in cancer development. To assess whether Nrf2/Keap1 mutations are early or late events in HCC development, we investigated their frequency in the rat Resistant Hepatocyte model, consisting of the administration of diethylnitrosamine followed by a brief exposure to 2-acetylaminofluorene. This model enables the dissection of all stages of hepatocarcinogenesis. We found that Nrf2/Keap1 mutations were present in 71% of early preneoplastic lesions and in 78.6% and 59.3% of early and advanced HCCs, respectively. Mutations of Nrf2 were more frequent, missense, and located in the Nrf2-Keap1 binding region. Mutations of Keap1 occurred at a much lower frequency in both preneoplastic lesions and HCCs and were mutually exclusive with those of Nrf2. Functional in vitro and in vivo studies showed that Nrf2 silencing inhibited the ability of tumorigenic rat cells to grow in soft agar and to form tumors. Unlike Nrf2 mutations, those of Ctnnb1, which are frequent in human HCC, were a later event as they appeared only in fully advanced HCCs (18.5%). CONCLUSION: In the Resistant Hepatocyte model of hepatocarcinogenesis the onset of Nrf2 mutations is a very early event, likely essential for the clonal expansion of preneoplastic hepatocytes to HCC, while Ctnnb1 mutations occur only at very late stages. Moreover, functional experiments demonstrate that Nrf2 is an oncogene critical for HCC progression and development.

Thyroid Hormone Receptor ß Agonist Induces ß-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 ß-catenin signaling. Since systemic side effects of T3 may preclude its clinical use, and hepatocytes mostly express T3 hormone receptor ß (TRß), we investigated if selective TRß agonists like GC-1 may also have ß-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-ß-catenin, cyclin D1, BrdU incorporation, and PCNA. No abnormalities in liver function tests were noted. GC-1-injected liver-specific ß-catenin knockouts (ß-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-ß-catenin. Further, increased levels of active ß-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, TRß-selective agonists induce hepatocyte proliferation through ß-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.

Tri-iodothyronine induces hepatocyte proliferation by protein kinase A-dependent ß-catenin activation in rodents.

UNLABELLED: Thyroid hormone (T3), like many other ligands of the steroid/thyroid hormone nuclear receptor superfamily, is a strong inducer of liver cell proliferation in rats and mice. However, the molecular basis of its mitogenic activity, which is currently unknown, must be elucidated if its use in hepatic regenerative medicine is to be considered. F-344 rats or C57BL/6 mice were fed a diet containing T3 for 2-7 days. In rats, administration of T3 led to an increased cytoplasmic stabilization and nuclear translocation of ß-catenin in pericentral hepatocytes with a concomitant increase in cyclin-D1 expression. T3 administration to wild-type (WT) mice resulted in increased hepatocyte proliferation; however, no mitogenic response in hepatocytes to T3 was evident in the hepatocyte-specific ß-catenin knockout mice (KO). In fact, T3 induced ß-catenin-TCF4 reporter activity both in vitro and in vivo. Livers from T3-treated mice demonstrated no changes in Ctnnb1 expression, activity of glycogen synthase kinase-3ß, known to phosphorylate and eventually promote ß-catenin degradation, or E-cadherin-ß-catenin association. However, T3 treatment increased ß-catenin phosphorylation at Ser675, an event downstream of protein kinase A (PKA). Administration of PKA inhibitor during T3 treatment of mice and rats as well as in cell culture abrogated Ser675-ß-catenin and simultaneously decreased cyclin-D1 expression to block hepatocyte proliferation. CONCLUSION: We have identified T3-induced hepatocyte mitogenic response to be mediated by PKA-dependent ß-catenin activation. Thus, T3 may be of therapeutic relevance to stimulate ß-catenin signaling to in turn induce regeneration in selected cases of hepatic insufficiency.

MicroRNA/gene profiling unveils early molecular changes and nuclear factor erythroid related factor 2 (NRF2) activation in a rat model recapitulating human hepatocellular carcinoma (HCC).

UNLABELLED: Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then used to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the resistant-hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the nuclear factor erythroid related factor 2 (NRF2) pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19-positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated with human HCC as well underlines the translational value of our results. CONCLUSION: This study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target.

Timed regulation of P-element-induced wimpy testis-interacting RNA expression during rat liver regeneration.

UNLABELLED: Small noncoding RNAs comprise a growing family of molecules that regulate key cellular processes, including messenger RNA (mRNA) degradation, translational repression, and transcriptional gene silencing. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) represent a class of small RNAs initially identified in the germline of a variety of species, where they contribute to maintenance of genome stability, and recently found expressed also in stem and somatic cells, where their role and responsiveness to physiopathological signals remain elusive. Here, we investigated piRNA expression in rat liver and its response to the stimuli exerted by regenerative proliferation of this organ. Quantitative polymerase chain reaction analysis identify in the liver the RNAs encoding PIWIL2/HILI, PIWIL4/HIWI2, and other components of the piRNA biogenesis pathways, suggesting that this is indeed functional. RNA sequencing before, during, and after the wave of cell proliferation that follows partial hepatectomy (PH) identified ∼1,400 mammalian germline piRNAs expressed in rat liver, including 72 showing timed changes in expression 24-48 hours post-PH, a timing that corresponds to cell transition through the S phase, returning to basal levels by 168 hours, when organ regeneration is completed and hepatocytes reach quiescence. CONCLUSION: The piRNA pathway is active in somatic cells of the liver and is subject to regulation during the pathophysiological process of organ regeneration, when these molecules are available to exert their regulatory functions on the cell genome and transcriptome, as demonstrated by the identification of several liver mRNAs representing candidate targets of these regulatory RNAs.

Met as a therapeutic target in HCC: facts and hopes.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and its burden is expected to increase further in the next years. In spite of the advances of classical therapies, such as surgery, transplantation, use of radiofrequency and transarterial embolization, the prognosis of this neoplasm has not considerably improved over the past few years. The advent of targeted therapies and the approval of the systemic treatment of advanced HCC with the kinase inhibitor sorafenib have provided some hope for the future. Even if the molecular mechanisms responsible for the onset and progression of HCC are still largely unknown, new therapeutic targets have recently come to the spotlight. One of these targets is the tyrosine kinase receptor for the Hepatocyte Growth Factor, encoded by the MET gene, known to promote tumor growth and metastasis in many human organs. In this review we will summarize the contrasting results obtained in vitro (in HCC cell lines) and in animal experimental models and we will also try to analyze the reasons for the opposite findings, suggesting that the HGF/MET axis can have either a promoting or a suppressive role in the development of HCC. We will also reconsider the evidence of activation of this pathway in human HCCs and discuss the results of the clinical trials performed with MET inhibitors. The final purpose is to better clarify which can be the role of MET as a therapeutic target in HCC.