Identification and experimental validation of druggable epigenetic targets in hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.

Prioritizing Popular Proteins in Liver Cancer: Remodelling One-Carbon Metabolism.

Primary liver cancer (HCC) is recognized as the fifth most common neoplasm and the second leading cause of cancer death worldwide. Most risk factors are known, and the molecular pathogenesis has been widely studied in the past decade; however, the underlying molecular mechanisms remain to be unveiled, as they will facilitate the definition of novel biomarkers and clinical targets for more effective patient management. We utilize the B/D-HPP popular protein strategy. We report a list of popular proteins that have been highly cocited with the expression "liver cancer". Several enzymes highlight the known metabolic remodeling of liver cancer cells, four of which participate in one-carbon metabolism. This pathway is central to the maintenance of differentiated hepatocytes, as it is considered the connection between intermediate metabolism and epigenetic regulation. We designed a targeted selective reaction monitoring (SRM) method to follow up one-carbon metabolism adaptation in mouse HCC and in regenerating liver following exposure to CCl4. This method allows systematic monitoring of one-carbon metabolism and could prove useful in the follow-up of HCC and of chronically liver-diseased patients (cirrhosis) at risk of HCC. The SRM data are available via ProteomeXchange in PASSEL (PASS01060).

Lamin A is involved in the development of vascular calcification induced by chronic kidney failure and phosphorus load.

Vascular calcification remains one of the main factors associated to morbidity and mortality in both ageing and chronic kidney disease. Both hyperphosphataemia, a well-known promoter of vascular calcification, and abnormal processing defects of lamin A/C have been associated to ageing. The main aim of this study was to analyse the effect of phosphorus load in the differential expression pattern of genes and proteins, particularly of lamin A/C, which are involved in phenotypic change of the vascular smooth muscle cells to osteoblast-like cells. The in vivo study of the calcified abdominal aortas from nephrectomized rats receiving a high phosphorus diet showed among others, a repression of muscle related proteins and overexpression of lamin A/C. Similar results were observed in vitro, where primary vascular smooth muscle cells cultured in calcifying medium showed increased expression of prelamin A and lamin A and abnormalities in the nuclear morphology. Co-immunoprecipitation assays showed novel and important physical interactions between lamin A and RUNX2 during the process of calcification. In fact, the knockdown of prelamin A and lamin A inhibited the increase of Runx2, osteocalcin and osteopontin gene expression, calcium deposition, nuclear abnormalities and the RUNX2 protein translocation into the nucleus of the cell. These in vivo and in vitro results highlight the important role played by lamin A in the process of vascular calcification.

Vitamin D-binding protein as a biomarker of active disease in acute intermittent porphyria.

UNLABELLED: Acute intermittent porphyria (AIP) is an autosomal dominant metabolic disorder caused by a deficiency of hepatic porphobilinogen deaminase (PBGD). The disease is characterized by life threatening acute neurovisceral attacks. The aim of this study was to identify metabolites secreted by the hepatocytes that reflect differential metabolic status in the liver and that may predict response to the acute attack treatment. Plasma vitamin D binding protein (VDBP) from a mouse model of AIP displayed an abnormal migration in 2D-electrophoresis that is efficiently recovered upon gene therapy leading to liver specific over-expression of the PBGD protein. The change in VDBP mobility results from a differential isoelectric point suggesting a post-translational modification that takes place preferably in the liver. Liquid chromatography-mass spectrometry (LC-MS) analysis of human samples before and after glycosidase treatment revealed glycosylated plasma VDBP specifically in patients with recurrent attacks of AIP. Glycosylated VDBP recovered normal values in three severely afflicted AIP patients submitted to therapeutic liver transplantation. Our findings suggest that post-translational modification of VDBP might be considered as a promising biomarker to study and monitor the liver metabolic status in patients with AIP. SIGNIFICANCE: We describe an increased glycosylation of VDBP in porphyric livers. Normal glycosylation was recovered upon liver gene therapy in a mouse model of porphyria or after liver transplantation in severely afflicted patients with AIP. Moreover, quantification of glycosylated VDBP by our ELISA immunoassay or LC-MS protocol in patients undergoing PBGD-gene therapy (www.aipgene.org) may be used as a marker indicating improvement or normalization of the patient's hepatic metabolism. This article is part of a Special Issue entitled: HUPO 2014.

Characterization of herpes simplex virus 1 strains as platforms for the development of oncolytic viruses against liver cancer.

BACKGROUND: Diverse oncolytic viruses (OV) are being designed for the treatment of cancer. The characteristics of the parental virus strains may influence the properties of these agents. AIMS: To characterize two herpes simplex virus 1 strains (HSV-1 17syn(+) and HFEM) as platforms for virotherapy against liver cancer. METHODS: The luciferase reporter gene was introduced in the intergenic region 20 locus of both HSV-1 strains, giving rise to the Cgal-Luc and H6-Luc viruses. Their properties were studied in hepatocellular carcinoma (HCC) cells in vitro. Biodistribution was monitored by bioluminescence imaging (BLI) in athymic mice and immune-competent Balb/c mice. Immunogenicity was studied by MHC-tetramer staining, in vivo killing assays and determination of specific antibody production. Intratumoural transgene expression and oncolytic effect were studied in HuH-7 xenografts. RESULTS: The H6-Luc virus displayed a syncytial phenotype and showed higher cytolytic effect on some HCC cells. Upon intravenous or intrahepatic injection in mice, both viruses showed a transient transduction of the liver with rapid relocalization of bioluminescence in adrenal glands, spinal cord, uterus and ovaries. No significant differences were observed in the immunogenicity of these viruses. Local intratumoural administration caused progressive increase in transgene expression during the first 5 days and persisted for at least 2 weeks. H6-Luc achieved faster amplification of transgene expression and stronger inhibition of tumour growth than Cgal-Luc, although toxicity of these non-attenuated viruses should be reduced to obtain a therapeutic effect. CONCLUSIONS: The syncytial H6-Luc virus has a strong oncolytic potential on human HCC xenografts and could be the basis for potent OV.