Marta Anna Kowalik

YAP activation is an early event and a potential therapeutic target in liver cancer development

BACKGROUND & AIMS Although the growth suppressing Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event. METHODS The experimental model used is the resistant-hepatocyte (R-H) rat model. Gene expression was determined by qRT-PCR or immunohistochemistry. Forward genetic experiments were performed in human HCC cells and in murine oval cells. RESULTS All foci of preneoplastic hepatocytes, generated in rats 4weeks after diethylnitrosamine (DENA) treatment, displayed YAP accumulation. This was associated with down-regulation of the β-TRCP ligase, known to mediate YAP degradation, and of microRNA-375, targeting YAP. YAP accumulation was paralleled by the up-regulation of its target genes. Increased YAP expression was also observed in human early dysplastic nodules and adenomas. Animal treatment with verteporfin (VP), which disrupts the formation of the YAP-TEAD complex, significantly reduced preneoplastic foci and oval cell proliferation. In vitro experiments confirmed that VP-mediated YAP inhibition impaired cell growth in HCC and oval cells; notably, oval cell transduction with wild type or active YAP conferred tumorigenic properties in vitro and in vivo. CONCLUSIONS These results suggest that (i) YAP overexpression is an early event in rat and human liver tumourigenesis; (ii) it is critical for the clonal expansion of carcinogen-initiated hepatocytes and oval cells, and (iii) VP-induced disruption of the YAP-TEAD interaction may provide an important approach for the treatment of YAP-overexpressing cancers.

Thyroid hormone (T3) and TRβ agonist GC-1 inhibit/reverse nonalcoholic fatty liver in rats

Nonalcoholic fatty liver disease is the most common noninfectious liver disease in clinical practice, and there is an increasing need for new therapeutic approaches for the treatment of this liver disease. Here, we examined the effect of the thyroid hormone triiodothyronine (T3) and the agonist of the thyroid hormone receptor β isoform (TRβ), GC‐1, on fatty liver and steatohepatitis induced in rodents by a choline‐methionine deficient (CMD) diet. Male Fischer 344 rats fed a CMD diet for 1 wk developed a marked fatty liver and mild hepatitis. Concurrent administration of T3 resulted in a complete prevention of the fatty change associated with increased fatty acid mitochondrial and peroxisomal β‐oxidation. To investigate whether T3 could also reverse fully established fatty liver, rats were fed a CMD diet for 10 wk and then cofed T3 for 1 wk. Coadministration of T3 resulted in a complete regression of liver steatosis associated with a decrease of lipid peroxidation, cyclooxygenase‐2 expression, and activation of phospho‐STAT3 and phospho‐SAPK/JNK. Finally, additional experiments showed that GC‐1, which has no significant side effects on heart rate, prevented and reverted CMD‐induced fat accumulation, and ameliorated steatohepatitis. These results indicate that TR agonists have the potential to inhibit or reverse hepatic steatosis induced by a nutritional model.—Perra, A., Simbula, G., Simbula, M., Pibiri, M., Kowalik, M. A., Sulas, P., Cocco, M. T., Ledda‐Columbano, G. M., Columbano, A. Thyroid hormone (T3) and TRβ agonist GC‐1 inhibit/reverse nonalcoholic fatty liver in rats. FASEB J. 22, 2981–2989 (2008)

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)

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. (Hepatology 2014;58:228–241)

Yes-Associated Protein Regulation of Adaptive Liver Enlargement and Hepatocellular Carcinoma Development in Mice

The Hippo kinase cascade, a growth‐suppressive pathway that ultimately antagonizes the transcriptional coactivator Yes‐associated protein (YAP), has been shown in transgenic animals to orchestrate organ size regulation. The purpose of this study was to determine whether in non–genetically modified mice (1) the Hippo pathway is involved in the regulation of adaptive liver enlargement caused by the mitogen 1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor and (2) a dysregulation of this pathway occurs during the development of chemically induced hepatocellular carcinoma (HCC). We show that liver enlargement caused by TCPOBOP was associated with an increase of YAP protein levels that paralleled the increase in 2‐bromodeoxyuridine incorporation. Interestingly, when a second dose of TCPOBOP was given to mice with enlarged livers, no further increases in liver mass or YAP protein levels were observed, suggesting that the Hippo pathway prevents further growth of the hyperplastic liver. Viral‐mediated exogenous expression of active YAP in mouse livers was able to partially overcome the block of hepatocyte proliferation. We also show that HCCs developed in mice given diethylnitrosamine and then subjected to repeated treatments with TCPOBOP had increased levels of YAP that were associated with down‐regulation of microRNA 375, which is known to control YAP expression, and with enhanced levels of alpha‐fetoprotein and connective tissue growth factor, two target genes of YAP.

Thyroid Hormone Receptor Ligands Induce Regression of Rat Preneoplastic Liver Lesions Causing Their Reversion to a Differentiated Phenotype

Triiodothyronine (T3), through interaction with its intracellular thyroid hormone receptors (TRs), influences various physiological functions, including metabolism, development, and growth. We investigated the effect of T3 and the selective TR‐β agonist GC‐1 in two models of hepatocarcinogenesis. Preneoplastic lesions were induced in F‐344 rats via a single dose of diethylnitrosamine, followed by a choline‐deficient (CD) diet for 10 weeks. Rat subgroups were then fed the CD diet or a CD diet containing either 4 mg/kg T3 or 5 mg/kg GC‐1 for another week. Rats fed a CD diet alone showed a large number (65/cm2) of preneoplastic lesions positive for the placental form of glutathione S‐transferase (GSTP). Coadministration of T3 for the last week caused an almost complete disappearance of the foci (3/cm2). A reduction of GSTP‐positive foci was also observed in rats fed a CD + GC‐1 diet (28/cm2 versus 75/cm2 of rats fed a CD diet alone) in the absence of significant differences in labeling or apoptotic index of preneoplastic hepatocytes between the two groups. An antitumoral effect of GC‐1 was also observed with the resistant hepatocyte model of hepatocarcinogenesis. Nodule regression was associated with a return to a fully differentiated phenotype, indicated by the loss of the fetal markers GSTP and gamma glutamyl transpeptidase, and reacquisition of the activity of glucose 6‐phosphatase and adenosine triphosphatase, two enzymes expressed in normal hepatocytes. Conclusion: Our results indicate that activated TRs negatively influence the carcinogenic process through induction of a differentiation program of preneoplastic hepatocytes. The results also suggest that TRs could be a meaningful target in liver cancer therapy. (HEPATOLOGY 2009.)

Nrf2, but not β‐catenin, mutation represents an early event in rat hepatocarcinogenesis

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. (Hepatology 2015;62:851‐862)

Gender-Specific Interplay of Signaling through β-Catenin and CAR in the Regulation of Xenobiotic-Induced Hepatocyte Proliferation

Aberrant signaling through the Wnt/β-catenin pathway is a critical determinant in human and rodent liver carcinogenesis and generally accepted to be a potent driver of proliferation. Xenobiotic agonists of the constitutive androstane receptor (CAR) induce massive acute hyperplasia of mouse liver and facilitate the outgrowth of hepatocellular carcinomas with activated β-catenin. In the present study, the interplay of β-catenin-dependent and CAR-dependent signaling in the liver and its effect on hepatocyte proliferation were analyzed in transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) following treatment with two CAR agonists, 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) and phenobarbital. Hepatocyte-specific knockout of β-catenin inhibited CAR agonists-induced hepatocyte proliferation in male mice. By contrast, the proliferative effect of CAR agonists was strongly augmented in female β-catenin knockout animals. This was due to prolonged proliferation of the knockout hepatocytes. CAR-mediated hepatocyte proliferation was, at least in part, dependent on estrogen signaling and was associated with enhanced expression of FoxM1 and elevated activity of the PDK1/p90RSK pathway. In conclusion, our study shows that gender-specific factors determine whether β-catenin signaling plays a pro- or an antiproliferative role in the regulation of mouse hepatocyte proliferation induced by CAR agonists.

Metabolic reprogramming identifies the most aggressive lesions at early phases of hepatic carcinogenesis.

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions.

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

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 TRβ1 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, TRβ1 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. TRβ1 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 TRβ1 and of its target genes in Krt‐19+ preneoplastic rat lesions and was associated with nodule regression. In HCC, TRβ1 down‐regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRβ1‐targeting microRNAs ([miR]‐27a, ‐181a, and ‐204). An inverse correlation between TRβ1 and miR‐181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. Conclusion: Down‐regulation of TRs, especially TRβ1, 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. (Hepatology 2015;61:249–259)

Emerging Role of the Pentose Phosphate Pathway in Hepatocellular Carcinoma

In recent years, there has been a revival of interest in metabolic changes of cancer cells as it has been noticed that malignant transformation and metabolic reprogramming are closely intertwined. The pentose phosphate pathway (PPP) is one of the fundamental components of cellular metabolism crucial for cancer cells. This review will discuss recent findings regarding the involvement of PPP enzymes in several types of cancer, with a focus on hepatocellular carcinoma (HCC). We will pay considerable attention to the involvement of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Subsequently, we discuss the inhibition of the PPP as a potential therapeutic strategy against cancer, in particular, HCC.