Downregulation of indolethylamine N-methyltransferase is an early event in the rat hepatocarcinogenesis and is associated with poor prognosis in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide, often preceded by cirrhosis and usually diagnosed at advanced stages; therefore, identifying molecular changes at early stages is an attractive strategy for detection and timely treatment. Here, we investigated the progressive transcriptomic changes during experimental hepatocarcinogenesis to identify novel early tumor markers in an HCC model induced by chronic administration of sublethal doses of diethylnitrosamine. An analysis of differentially expressed genes showed that four processes associated with oxidation-reduction and detoxification were significantly over-represented during hepatocarcinogenesis progression, of which the Nuclear Factor, Erythroid 2 Like 2 pathway showed several dysregulated genes. Interestingly, we also identified 91 genes dysregulated at early HCC stages, but the expression of the indolethylamine N-methyltransferase gene (INMT), as well as the level of its encoding protein, were strongly downregulated. INMT was increased in perivenular hepatocytes of normal livers but decreased in livers of experimental HCC. Furthermore, a gene expression and survival analysis performed using data from the liver hepatocellular carcinoma project of The Cancer Genome Atlas Program revealed that INMT is also significantly downregulated in human HCC and is associated with poor overall survival. In conclusion, by performing a transcriptome analysis of the HCC progression, we identified that INMT is early downregulated in the rat hepatocarcinogenesis and is associated with poor prognosis in human HCC, suggesting that INMT downregulation may be a promising prognostic marker for HCC in high-risk populations.

Comparative subcellular localization of NRF2 and KEAP1 during the hepatocellular carcinoma development in vivo.

The activation of Nuclear Factor, Erythroid 2 Like 2 - Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.

The transcriptome of early GGT/KRT19-positive hepatocellular carcinoma reveals a downregulated gene expression profile associated with fatty acid metabolism.

Hepatocellular carcinoma expressing hepatobiliary progenitor markers, is considered of poor prognosis. By using a hepatocarcinogenesis model, laser capture microdissection, and RNA-Sequencing analysis, we identified an expression profile in GGT/KRT19-positive experimental tumors; 438 differentially expressed genes were found in early and late nodules along with increased collagen deposition. Dysregulated genes were involved in Fatty Acid Metabolism, RXR function, and Hepatic Stellate Cells Activation. Downregulation of Slc27a5, Acsl1, and Cyp2e1, demonstrated that Retinoid X Receptor α (RXRα) function is compromised in GGT/KRT19-positive nodules. Since RXRα controls NRF2 pathway activation, we determined the expression of NRF2 targeted genes; Akr1b8, Akr7a3, Gstp1, Abcc3, Ptgr1, and Txnrd1 were upregulated, indicating NRF2 pathway activation. A comparative analysis in human HCC showed that SLC27A5, ACSL1, CYP2E1, and RXRα gene expression is mutually exclusive with KRT19 gene expression. Our results indicate that the downregulation of Slc27a5, Acsl1, Rxrα, and Cyp2e1 genes is an early event within GGT/KRT19-positive HCC.

Enrichment of progenitor cells by 2-acetylaminofluorene accelerates liver carcinogenesis induced by diethylnitrosamine in vivo.

The potential role of hepatocytes versus hepatic progenitor cells (HPC) on the onset and pathogenesis of hepatocellular carcinoma (HCC) has not been fully clarified. Because the administration of 2-acetylaminofluorene (2AAF) followed by a partial hepatectomy, selectively induces the HPC proliferation, we investigated the effects of chronic 2AAF administration on the HCC development caused by the chronic administration of the carcinogen diethylnitrosamine (DEN) for 16 weeks in the rat. DEN + 2AAF protocol impeded weight gain of animals but promoted prominent hepatomegaly and exacerbated liver alterations compared to DEN protocol alone. The tumor areas detected by γ-glutamyl transferase, prostaglandin reductase-1, and glutathione S-transferase Pi-1 liver cancer markers increased up to 80% as early as 12 weeks of treatment, meaning 6 weeks earlier than DEN alone. This protocol also increased the number of Ki67-positive cells and those of CD90 and CK19, two well-known progenitor cell markers. Interestingly, microarray analysis revealed that DEN + 2AAF protocol differentially modified the global gene expression signature and induced the differential expression of 30 genes identified as HPC markers as early as 6 weeks of treatment. In conclusion, 2AAF induces the early appearance of HPC markers and as a result, accelerates the hepatocarcinogenesis induced by DEN in the rat. Thus, since 2AAF simultaneously administrated with DEN enriches HPC during hepatocarcinogenesis, we propose that DEN + 2AAF protocol might be a useful tool to investigate the cellular origin of HCC with progenitor features.

Aldo-Keto Reductases as Early Biomarkers of Hepatocellular Carcinoma: A Comparison Between Animal Models and Human HCC.

BACKGROUND: The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC. AIMS: To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis. METHODS: Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4-17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat. RESULTS: The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues. CONCLUSIONS: Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.

Ptgr1 expression is regulated by NRF2 in rat hepatocarcinogenesis and promotes cell proliferation and resistance to oxidative stress.

Prostaglandin reductase-1 (Ptgr1) is an alkenal/one oxidoreductase that is involved in the catabolism of eicosanoids and lipid peroxidation such as 4-hydroxynonenal (4-HNE). Recently, we reported that Ptgr1 is overexpressed in human clinical and experimentally induced samples of hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated and its role in carcinogenesis are not yet known. Here, we studied parameters associated with antioxidant responses and the mechanisms underlying the induction of Ptgr1 expression by the activation of Nuclear Factor (erythroid-derived-2)-like-2 (NRF2). For these experiments, we used two protocols of induced hepatocarcinogenesis in rats. Furthermore, we determined the effect of PTGR1 on cell proliferation and resistance to oxidative stress in cell cultures of the epithelial liver cell line, C9. Ptgr1 was overexpressed during the early phase in altered hepatocyte foci, and this high level of expression was maintained in persistent nodules until tumors developed. Ptgr1 expression was regulated by NRF2, which bound to an antioxidant response element at -653bp in the rat Ptgr1 gene. The activation of NRF2 induced the activation of an antioxidant response that included effects on proteins such as glutamate-cysteine ligase, catalytic subunit, NAD(P)H dehydrogenase quinone-1 (NQO1) and glutathione-S-transferase-P (GSTP1). These effects may have produced a reduced status that was associated with a high proliferation rate in experimental tumors. Indeed, when Ptgr1 was stably expressed, we observed a reduction in the time required for proliferation and a protective effect against hydrogen peroxide- and 4-HNE-induced cell death. These data were consistent with data showing colocalization between PTGR1 and 4-HNE protein adducts in liver nodules. These findings suggest that Ptgr1 and antioxidant responses act as a metabolic adaptation and could contribute to proliferation and cell-death evasion in liver tumor cells. Furthermore, these data indicate that Ptgr1 could be used to design early diagnostic tools or targeted therapies for HCC.

Increased expression of prostaglandin reductase 1 in hepatocellular carcinomas from clinical cases and experimental tumors in rats.

To identify novel tumor-associated proteins, we analyzed the protein expression patterns from experimental hepatocellular carcinoma (HCC) that were induced using hepatocarcinogenesis models in rats. Rats were subjected to two previously described protocols of hepatocarcinogenesis using diethylnitrosamine as a carcinogen: the alternative Solt-Farber (aS&F) protocol, which induces HCC within 9 months, and Schiffer's model, which induces cirrhosis and multifocal HCC within 18 weeks. The patterns of protein expression from tumors and normal liver tissue were examined by SDS-PAGE and the bands identified at 33-34 kDa were analyzed by mass spectrometry. The prostaglandin reductase 1 (PTGR1) showed the highest number of peptides, with a confidence of level >99%. The increased expression of PTGR1 in tumors was confirmed in these two models by Western blotting and by increase in alkenal/one oxidoreductase activity (25-fold higher than normal liver). In addition, the gene expression level of Ptgr1, as measured by qRT-PCR, was increased during cancer development in a time-dependent manner (200-fold higher than normal liver). Furthermore, PTGR1 was detected in the cytoplasm of neoplastic cells in rat tumors and in 12 human HCC cases by immunohistochemistry. These analyses were performed by comparing the expression of PTGR1 to that of two well-known markers of hepatocarcinoma, Glutathione S-transferase pi 1 (GSTP1) in rats and glypican-3 in humans. The increased expression and activity of PTGR1 in liver carcinogenesis encourage further research aimed at understanding the metabolic role of PTGR1 in HCC and its potential application for human cancer diagnosis and treatment.

Laser capture microdissection after γ-glutamyl transferase histochemistry: an optimization for gene expression analysis.

γ-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 µg of total RNA from 15 to 18 mm² of microdissected tissue (20 µm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip-capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM.