Maria Maddalena Angioni

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.

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)

Neonatal exposure to estradiol in rats influences neuroactive steroid concentrations, GABAA receptor expression, and behavioral sensitivity to anxiolytic drugs.

J. Neurochem. (2010) 113, 1285–1295.

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.

A long term, non-tumorigenic rat hepatocyte cell line and its malignant counterpart, as tools to study hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death. Search for genes/proteins whose expression can discriminate between normal and neoplastic liver is fundamental for diagnostic, prognostic and therapeutic purposes. Currently, the most used in vitro hepatocyte models to study molecular alterations underlying transformation include primary hepatocytes and transformed cell lines. However, each of these models presents limitations. Here we describe the isolation and characterization of two rat hepatocyte cell lines as tools to study liver carcinogenesis. Long-term stable cell lines were obtained from a HCC-bearing rat exposed to the Resistant-Hepatocyte protocol (RH cells) and from a rat subjected to the same model in the absence of carcinogenic treatment, thus not developing HCCs (RNT cells). The presence of several markers identified the hepatocytic origin of both cell lines and confirmed their purity. Although morphologically similar to normal primary hepatocytes, RNT cells were able to survive and grow in monolayer culture for months and were not tumorigenic in vivo. On the contrary, RH cells displayed tumor-initiating cell markers, formed numerous colonies in soft agar and spheroids when grown in 3D and were highly tumorigenic and metastatic after injection into syngeneic rats and immunocompromised mice. Moreover, RNT gene expression profile was similar to normal liver, while that of RH resembled HCC. In conclusion, the two cell lines here described represent a useful tool to investigate the molecular changes underlying hepatocyte transformation and to experimentally demonstrate their role in HCC development.

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.

Expression analysis of HLA-E and NKG2A and NKG2C receptors points at a role for natural killer function in ankylosing spondylitis

Background Ankylosing spondylitis (AS) is a complex chronic inflammatory disease strongly associated with the majority of human leucocyte antigen (HLA)-B27 alleles. HLA-E molecules are non-classical major histocompatibility complex (MHC) class I molecules that specifically interact with the natural killer receptors NKG2A (inhibitory) and NKG2C (activating), and have been recently proposed to be involved in AS pathogenesis.‘’ Objective To analyse the expression of HLA-E and the CD94/NKG2 pair of receptors in HLA-B27-positive patients with AS and healthy controls (HC) bearing the AS-associated B*2705 and the non-AS-associated B*2709 alleles. Methods The level of surface expression of HLA-E molecules on CD14+ peripheral blood mononuclear cell was evaluated in 21 HLA-B*2705 patients with AS, 12 HLA-B*2705 HC, 12 HLA-B*2709 HC and 6 HLA-B27-negative HC using the monoclonal antibody MEM-E/08 by quantitative cytofluorimetric analysis. The percentage and density of expression of HLA-E ligands NKG2A and NKG2C were also measured on CD3−CD56+ NK cells. Results HLA-E expression in CD14+ cells was significantly higher in patients with AS (587.0, IQR 424–830) compared with B*2705 HC (389, IQR 251.3–440.5; p=0.0007), B*2709 HC (294.5, IQR 209.5–422; p=0.0004) and HLA-B27-negative HC (380, IQR 197.3–515.0; p=0.01). A higher number of NK cells expressing NKG2A compared with NKG2C were found in all cohorts analysed, as well as a higher cell surface density. Conclusion The higher surface level of HLA-E molecules in patients with AS compared with HC, concurrently with a prevalent expression of NKG2A, suggests that the crosstalk between these two molecules might play a role in AS pathogenesis, accounting for the previously reported association between HLA-E and AS.

AIF-1 gene does not confer susceptibility to Behçet’s disease: Analysis of extended haplotypes in Sardinian population

Background Behçet’s disease (BD) is a polygenic immune-mediated disorder characterized by a close association with the HLA-B*51 allele. The HLA region has a strong linkage disequilibrium (LD) and carries several genetic variants (e.g. MIC-A, TNF-α genes) identified as associated to BD because of their LD with HLA-B*51. In fact, the HLA-B*51 is inherited as part of extended HLA haplotypes which are well preserved in patients with BD. Sardinian population is highly differentiated from other Mediterranean populations because of a distinctive genetic structure with very highly preserved HLA haplotypes. Patients and methods In order to identify other genes of susceptibility to BD within the HLA region we investigated the distribution of human Allograft Inflammatory Factor-1 (AIF-1) gene variants among BD patients and healthy controls from Sardinia. Six (rs2736182; rs2259571; rs2269475; rs2857597; rs13195276; rs4711274) AIF-1 single nucleotide polymorphisms (SNPs) and related extended haplotypes have been investigated as well as their LD within the HLA region and with HLA-B*51. Overall, 64 BD patients, 43 HLA-B*51 positive healthy controls (HC) and 70 random HC were enrolled in the study. Results HLA-B*51 was the only allele with significantly higher frequency (pc = 0.0021) in BD patients (40.6%) than in HC (9.8%). The rs2259571T AIF-1 variant had a significantly reduced phenotypic, but not allelic frequency in BD patients (72.1%; pc = 0.014) compared to healthy population (91.3%). That was likely due to the LD between HLA-B*51 and rs2259571G (pc = 9E-5), even though the rs2259571G distribution did not significantly differ between BD patients and HC. Conclusion No significant difference in distribution of AIF-1 SNPs haplotypes was observed between BD patients and HC and between HLA-B*51 positive BD patients and HLA-B*51 positive HC. Taken together, these results suggest that AIF-1 gene is not associated with susceptibility to BD in Sardinia.

Abstract A02: OXPHOS inhibition and pentose phosphate pathway induction are early events priming preneoplastic lesions toward HCC development

Introduction: A shift towards a Warburg metabolism in which aerobic glycolysis is increased has long been associated to cancer cell transformation. However, whether the switch from oxidative phosphorylation to glycolysis can occur at early stages of cancer development, particularly in hepatocellular carcinoma (HCC), remains elusive. Materials and Methods: Preneoplastic hepatic lesions and Hepatocellular carcinomas were induced in rats subjected to the Resistant-Hepatocyte (RH) model, consisting of a single dose of dietthylnitrosamine (DENA) and a 2-week feeding a diet supplemented with 2-acetylaminoaminofluorene (2-AAF). In vitro experiments were performed in HCC cells obtained by perfusion of HCC-bearing rats or immortalized rat hepatocytes. Results and discussion: Using the Resistant-Hepatocyte (R-H) model, we show that the acquisition of the Warburg phenotype is a very early event in rat HCC development as demonstrated by concomitant MCT4 expression and oxidation/inhibition of pyruvate kinase M2 (PKM2). In keeping with this, we also observed inhibition of succinate dehydrogenase (SDH) by the chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) and an increase in the expression and activity of citrate synthase (CS). In these preneoplastic lesions, metabolic reprogramming towards the Pentose Phosphate Pathway (PPP) was indicated by a strong increase in the expression and activity of glucose-6-phosphate dehydrogenase (G6PD). G6PD increased expression was observed exclusively in the highly proliferating KRT-19 positive preneoplastic lesions, considered the HCC precursor lesions in the R-H model, and was associated with low levels of miR-1, a miRNA known to target G6PD. Accordingly, forced expression of miR-1 down-regulated G6PD expression in HCC cells. PPP induction has been suggested to be one of the mechanisms by which deregulated NRF2-KEAP1 signaling promotes cellular proliferation and tumorigenesis. Since in the R-H rat model a sustained activation of the NRF2/KEAP1 pathway occurs in KRT-19+ nodules, we investigated the effect of impairing NRF2 in cells derived from R-H rat HCC. Notably, NRF2 silencing decreases G6PD and increases miR-1 expression, consequently inhibiting PPP and PKM2 oxidation. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients. Conclusion: Our results demonstrate that Warburg metabolic deregulation and PPP induction are early events in HCC development. Crucially, TRAP1 and NRF2 are key regulators of this metabolic reprogramming in preneoplastic hepatocytes Citation Format: Marta A. Kowalik, Giulia Guzzo, Andrea Morandi, Andrea Perra, Silvia Menegon, Maria M. Angioni, Silvia Giordano, Paola Chiarugi, Andrea Rasola, Amedeo Columbano. OXPHOS inhibition and pentose phosphate pathway induction are early events priming preneoplastic lesions toward HCC development. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A02.

Abstract 1009: Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development

Introduction and aim: Among the several changes underlying metabolic reprogramming of cancer cells, increased glucose utilization and its uncoupling from oxygen availability is a well-established phenomenon and has been recognized as a hallmark of cancer. To what extent these metabolic changes are important for the progression of slow growing tumors and whether a metabolic rewiring occurs in the very early stages of neoplastic progression represent key questions on the significance of these metabolic alterations in cancer. Here, we compared the metabolic features of preneoplastic hepatic lesions with those of advanced hepatocellular carcinomas (HCCs) and of proliferating liver, following partial hepatectomy (PH). Materials and Methods: Expression levels, activity and modulation of several enzymes with key roles in glycolysis, pentose phosphate pathway (PPP) and oxidative phosphorylation (OXPHOS) were assessed in preneoplastic hepatic lesions and HCC, induced in rats exposed to the Resistant-Hepatocyte (R-H) model. In vitro experiments were performed on HCC cells obtained by perfusion of HCC-bearing rats. Expression of metabolic genes was also investigated in two different cohorts of human patients carrying HCC. Results and discussion: A switch from OXPHOS to PPP was observed in very early preneoplastic lesions generated 10 weeks after the treatment with DENA. Notably, this metabolic reprogramming was observed only in the most aggressive preneoplastic lesions, characterized by positivity for cytokeratin 19 (CK-19+). PPP induction, shown by a strong increase in the expression and activity of glucose 6-phosphate dehydrogenase (G6PD) was supported both by inhibition of pyruvate kinase activity and by TP53-inducible glycolysis and apoptosis regulator (TIGAR) induction. Importantly, such metabolic rewiring was not observed in normal hepatocytes, undergoing proliferation following 2/3 partial hepatectomy (PH). Activation of the NRF2/KEAP1 pathway and down-regulation of miR-1 accompanied the metabolic reprogramming in CK-19+ preneoplastic lesions. Accordingly, NRF2 silencing decreased G6PD and increased miR-1 expression, consequently inhibiting PPP, while forced expression of miR-1 downregulated G6PD expression in HCC cells. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients. Conclusion: These results demonstrate that metabolic reprogramming takes place at early stages of hepatocarcinogenesis and is likely the consequence of the concomitant activation of the NRF2-KEAP1 pathway. Citation Format: Marta A. Kowalik, Giulia Guzzo, Andrea Morandi, Andrea Perra, Silvia Menegon, Ionica Masgras, Elena Trevisan, Maria M. Angioni, Francesca Fornari, Luca Quagliata, Giovanna M. Ledda-Columbano, Laura Gramantieri, Luigi Terracciano, Silvia Giordano, Paola Chiarugi, Andrea Rasola, Amedeo Columbano. Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1009.