In vitro generation of primary cultures of human hyalocytes.

Purpose: A growing number of studies on animal models have demonstrated that some ocular diseases are the result of the interaction between hyalocytes and the ocular inflammatory setting. Endogenous and exogenous substances might alter the structure and behavior of hyalocytes that can contribute to the pathogenesis of some ocular diseases. Obtaining primary cultures of human hyalocytes could help understand the role of these cells in response to different treatments. Methods: Hyalocytes were isolated from eyes of 54 patient volunteers subjected to vitrectomy for different clinical reasons. By testing different matrices and growth media, we reproducibly generated primary cultures of hyalocytes that we characterized morphologically and biologically, basally and upon treatment with several agents (basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF-ß), platelet-derived growth factor subunit-BB (PDGF-BB), ascorbic acid, dexamethasone, and hydrogen peroxide). Results: We were able to generate primary cultures from vitreous human samples, growing the cells on collagen-coated plates in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum; primary cells expressed the hyalocyte markers. Specific cytoskeletal modifications were observed upon treatment with bFGF, TGF-ß, PDGF-BB, ascorbic acid, dexamethasone, and hydrogen peroxide. Only bFGF was able to promote cell growth and hyaluronic acid production. Conclusions: We describe for the first time the generation and the characterization of primary cultures of human hyalocytes from living donors. Although human hyalocytes share some characteristics with animal hyalocytes, human hyalocytes have their own features typical of the species, confirming how important human experimental models are for investigating human pathologies and their treatments.

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.

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.

DNA sequence and taxonomic gap analyses to quantify the coverage of aquatic cyanobacteria and eukaryotic microalgae in reference databases: Results of a survey in the Alpine region.

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

Analysis of 21 X-chromosomal STRs in an Algerian population sample.

Twenty-one X-chromosomal short tandem repeat loci, including the six clusters of linked markers DXS10148-DXS10135-DXS8378 (Xp22), DXS7132-DXS10074-DXS10079 (Xq12), DXS6801-DXS6809-DXS6789 (Xq21), DXS7424-DXS101 (Xq22), DXS10103-HPRTB-DXS10101 (Xq26), DXS8377-DXS10146-DXS10134-DXS7423-DXS10011 (Xq28), and the loci DXS6800 and GATA172D05 were typed in a northwestern Algerian population sample (n = 210; 104 men and 106 women). Allele and haplotype frequencies were calculated. No evidence of linkage disequilibrium was observed between pairs of loci within clusters of linked markers. At locus DXS10148, sequence analysis of a subset of alleles displaying unusual amplicon length (>/= 36 repeat units) and anomalous electrophoretic mobility showed that this marker has a complex molecular structure with different repeat variants within alleles of identical amplicon size.

A Comprehensive PDX Gastric Cancer Collection Captures Cancer Cell-Intrinsic Transcriptional MSI Traits.

Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein-Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/KRAS genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell-intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel "druggable" targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell-intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis. SIGNIFICANCE: This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.

Rituximab Treatment Prevents Lymphoma Onset in Gastric Cancer Patient-Derived Xenografts.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

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 Dual Roles of NRF2 in Cancer.

NRF2 has been traditionally considered as a tumor suppressor because its cytoprotective functions are deemed to be the main cellular defense mechanism against exogenous and endogenous insults, including xenobiotics and oxidative stress. However, several recent studies demonstrate that hyperactivation of the NRF2 pathway creates an environment that favors the survival of normal as well as malignant cells, protecting them against oxidative stress, chemotherapeutic agents, and radiotherapy. In a rapidly advancing field, this review summarizes some of the known mechanisms by which NRF2 can exert its oncogenic functions, and describes the current status of NRF2 inhibitors, providing a clear rationale for the consideration of NRF2 as a powerful putative therapeutic target in cancer treatment.

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.

SCN1B gene variants in Brugada Syndrome: a study of 145 SCN5A-negative patients.

Brugada syndrome is characterised by a typical ECG with ST segment elevation in the right precordial leads. Individuals with this condition are susceptible to ventricular arrhythmias and sudden cardiac death. The principal gene responsible for this syndrome is SCN5A, which encodes the α-subunit of the Nav1.5 voltage-gated sodium channel. Mutations involving other genes have been increasingly reported, but their contribution to Brugada syndrome has been poorly investigated. Here we focused on the SCN1B gene, which encodes the ß1-subunit of the voltage-gated sodium channel and its soluble ß1b isoform. SCN1B mutations have been associated with Brugada syndrome as well as with other cardiac arrhythmias and familial epilepsy. In this study, we have analysed SCN1B exons (including the alternatively-spliced exon 3A) and 3'UTR in 145 unrelated SCN5A-negative patients from a single centre. We took special care to report all identified variants (including polymorphisms), following the current nomenclature guidelines and considering both isoforms. We found two known and two novel (and likely deleterious) SCN1B variants. We also found two novel changes with low evidence of pathogenicity. Our findings contribute more evidence regarding the occurrence of SCN1B variants in Brugada syndrome, albeit with a low prevalence, which is in agreement with previous reports.

Linkage and linkage disequilibrium analysis of X-STRs in Italian families.

Twenty X-chromosomal short tandem repeat (STR) loci were typed in 80 families of Italian descent, composed by mother and two or more sons, for a total of 93 meiosis. The analyzed X-STR panel included six clusters of closely linked markers (each spanning<3cM): DXS10135-DXS10148-DXS8378 (Xp22); DXS7132-DXS10074-DXS10079 (Xq12); DXS6801-DXS6809-DXS6789 (Xq21); DXS7424-DXS101 (Xq22); DXS10103-HPRTB-DXS10101 (Xq26); DXS8377-DXS10134-DXS7423-DXS10146 (Xq28). Recombination fractions between pairs of markers calculated by pedigree analysis were compared with those obtained from the second-generation Rutgers combined linkage-physical map of the human genome. The observed differences confirm that recombination is not homogeneous along the X chromosome and that the conventional subdivision of X-STRs in four groups of completely unlinked markers cannot be regarded as true. Significant linkage disequilibrium was found between markers DXS6801 and DXS6809 (p=0.017). The effect on likelihood calculations of inferring haplotype frequencies from allele distributions rather than haplotype count in the relevant population was evaluated.