Strong antineoplastic effects of metformin in preclinical models of liver carcinogenesis.

Studies suggest that metformin, widely used for treating Type 2 diabetes, possesses innate antineoplastic properties. For metabolic syndrome patients with hepatocellular carcinoma (HCC), metformin may provide antitumoral effects. We evaluated the impact of metformin on tumour growth and visceral fat composition using relevant preclinical models of metabolic syndrome. Studies were performed in three hepatoma cell lines, in HepG2 xenograft mice fed with standard chow (SC) diet, 60% high-fat diet (HFD) or 30% fructose diet (FR), and an ex vivo model of human cultured HCC slices. Visceral fatty acid composition was analysed by magnetic resonance imaging (MRI). Metformin had a dose-dependent inhibitory effect on cell proliferation and apoptosis in vitro through the deregulation of mTOR/AMPK, AKT and extracellular signal regulated kinase (ERK) signalling pathways. Tumour engraftment rates were higher in HFD mice than SC mice (hepatic: 79% compared with 25%, P=0.02) and FR mice (subcutaneous: 86% compared with 50%, P=0.04). Subcutaneous tumour volume was increased in HFD mice (+64% compared with FR and SC, P=0.03). Metformin significantly decreased subcutaneous tumour growth via cell-cycle block and mammalian target of rapamycin (mTOR) pathway inhibition, and also induced hypoxia and decreased angiogenesis. In ex vivo tumour slices, metformin treatment led to increased necrosis, decreased cyclin D1 and increased carbonic anhydrase-9 (CA-9). Metformin caused qualitative changes in visceral fat composition of HFD mice, with decreased proportions of polyunsaturated fatty acids (14.6% ± 2.3% compared with 17.9% ± 3.0%, P=0.04). The potent antitumoral effects of metformin in multiple preclinical models implicating several molecular mechanisms provide a strong rationale for clinical trials including combination studies in HCC patients.

Progression from cirrhosis to cancer is associated with early ubiquitin post-translational modifications: identification of new biomarkers of cirrhosis at risk of malignancy.

Cirrhosis is a lesion at risk of hepatocellular carcinoma (HCC). Identifying mechanisms associated with the transition from cirrhosis to HCC and characterizing biomarkers of cirrhosis at high risk of developing into cancer are crucial for improving early diagnosis and prognosis of HCC. We used MALDI imaging to compare mass spectra obtained from tissue sections of cirrhosis without HCC, cirrhosis with HCC, and HCC, and a top-down proteomics approach to characterize differential biomarkers. We identified a truncated form of monomeric ubiquitin lacking the two C-terminal glycine residues, Ubi(1-74), the level of which increased progressively, from cirrhosis without HCC to cirrhosis with HCC to HCC. We showed that kallikrein-related peptidase 6 (KLK6) catalysed the production of Ubi(1-74) from monomeric ubiquitin. Furthermore, we demonstrated that KLK6 was induced de novo in cirrhosis and increased in HCC in parallel with accumulation of Ubi(1-74). We investigated in vitro the possible consequences of Ubi(1-74) accumulation and demonstrated that Ubi(1-74) interferes with the normal ubiquitination machinery in what is likely to be a kinetic process. Our data suggest that de novo KLK6 expression during early liver carcinogenesis may induce production of Ubi(1-74) by post-translational modification of ubiquitin. Given the deleterious effect of Ubi(1-74) on protein ubiquitination and the major role of ubiquitin machinery in maintenance of cell homeostasis, Ubi(1-74) might severely impact a number of critical cellular functions during transition from cirrhosis to cancer. Ubi(1-74) and KLK6 may serve as markers of cancer risk in patients with cirrhosis.

Hepatocellular carcinoma protein carbonylation in virus C and metabolic syndrome patients.

Metabolic syndrome (MS) is becoming the leading cause of chronic liver diseases worldwide. Hepatocellular carcinoma (HCC) development in MS is peculiar compared to other chronic liver diseases. Carbohydrate and lipid metabolic imbalance in MS increase reactive oxygen species damaging proteins. In the present work we study the difference in protein oxidative damage (carbonylation) in human HCC derived from virus C infection (VHC) and from MS (MS_HCC) as the only subjacent cause. We selected a patient cohort containing of 10 non-tumoral and 10 tumoral liver resections in each study group (virus C and MS HCC) based on clinical patient history and histological parameters. Protein samples were labeled to saturation using CF 647-hydrazide™ dye. This approach allows us to perform carbonyl detection alongside with a DIGE experiment. We detected a total of 1184 spots with 36 differentially expressed proteins and 47 spots differentially carbonylated between VHC and MS_HCC (fold change >1.5, p<0.05). VHC up-regulated proteins are involved in signaling pathways related to cancer development such as signaling by EGFR, Wnt, Cdc20 and cell cycle. Further, up-regulated proteins in MS HCC, are implicated in metabolism of carbohydrates and amino acids. Differential carbonylation analysis between VHC and MS_HCC showed protein damage in proteins such as glucose phosphate isomerase, isocitrate dehydrogenase, and 3-ketoacyl-CoA thiolase. Higher protein carbonylation in MS_HCC samples was observed in proteins involved in redox response and lipid metabolism. In conclusion, the observed difference in protein oxidative damage between MS and Virus C derived carcinoma could account for the different cancer development pathway.

Imaging mass spectrometry reveals modified forms of histone H4 as new biomarkers of microvascular invasion in hepatocellular carcinomas.

UNLABELLED: Microvascular invasion (MiVI) is a major risk factor in postoperative tumor recurrence and mortality in hepatocellular carcinoma (HCC). Unfortunately, this histological feature is usually missed by liver biopsy because of limited sampling, and MiVI is commonly detected only after surgery and examination of the full resected specimen. To date, there exists no reliable tool for identifying MiVI prior to surgical procedures. This study aimed to compare the proteome of HCC with and without MiVI in order to identify surrogate biomarkers of MiVI. A training cohort comprising surgically resected primary HCC with MiVI (n = 30) and without MiVI (n = 26) was subjected to matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS). Comparative analysis of acquired mass spectra of the two groups yielded 30 differential protein peaks, among which 28 were more strongly expressed in HCC with MiVI. Among these, two peaks were identified as N-term acetylated histone H4 dimethylated at lysine (K) 20, and N-term acetylated histone H4 dimethylated at K20 and acetylated at K16. Both peaks were validated in the training cohort and in an independent validation cohort (n = 23) by immunohistochemistry and western blot. CONCLUSION: These results demonstrate the potential of MALDI IMS for uncovering new relevant biomarkers of MiVI in HCC, and highlight the role of epigenetic modifications in the prognosis of HCC. Preoperative detection of modified forms of histone H4 expression in tumor biopsies would be helpful in management of patients with HCC.

Cullin7: a new gene involved in liver carcinogenesis related to metabolic syndrome.

BACKGROUND: Metabolic syndrome (MS) is an emerging risk factor in hepatocellular carcinoma (HCC). HCC related to MS may occur either in advanced fibrosis or before the development of cirrhosis, suggesting involvement of different molecular pathways according to the features of background liver. OBJECTIVE: To investigate genomic aberrations in HCC related to MS in order to identify new target genes involved in liver carcinogenesis. METHODS: Chromosomal aberrations of HCC obtained from 20 patients with MS (HCC/MS) were studied by comparative genomic hybridisation and compared with HCC related to hepatitis C virus (HCV) infection (HCC/HCV, n=10) and, within the group of HCC with MS, according to the condition of the background liver (presence or absence of significant fibrosis). RESULTS: Among the most frequent chromosomal alterations observed in HCC, 6p21.1 amplification had a higher incidence in HCC/MS than in HCC/HCV (60% vs 20%, p<0.01). Advanced fibrosis/cirrhosis in the peritumoral liver was the only clinicopathological factor associated with the 6p21.1 amplicon in HCC/MS. Increased expression of cullin7 (CUL7), a gene located at the 6p21.1 locus, was demonstrated in HCC with the 6p21.1 amplicon, in parallel with a decrease in cyclin D1 expression. CUL7 downregulation using siRNA transfection in hepatoma cell lines induced significant cyclin D1 expression (by promoting its degradation), decreased cell proliferation and increased apoptosis. CONCLUSIONS: This study demonstrates specific genomic alterations in HCC/MS and points to CUL7 as a novel gene potentially involved in liver carcinogenesis associated with MS, the amplification of which might influence cell proliferation.

The expression of the hypoxia markers CA9 and CXCR4 is correlated with survival in patients with neuroendocrine tumours of the ileum.

BACKGROUND/AIM: The hypoxia-inducible factor pathway regulates the expression of a diverse group of molecules such as CA9 and CXCR4. Our aim was to investigate the expression of these markers in a series of patients with an ileal neuroendocrine tumour (IET) at various stages of tumorigenesis. METHODS: The immunohistochemical expression of CA9 and CXCR4 was examined in 51 patients with a resected IET. A 'hypoxic score' was calculated, integrating the expression of both CA9 and CXCR4 (hypoxic score 0: absence of expression of both molecules; hypoxic score 1: expression of CXCR4 and/or CA9). Results were compared to histoprognostic factors (including tumour size, stage and grade, WHO and TNM classifications, presence of vascular or perineural invasion, presence of a fibrotic stroma and microvascular density) and to survival. RESULTS: All tumours were well differentiated. 69% of tumours were less than 25 mm. 46% of tumours largely infiltrated the intestinal wall (≥T3, subserosa and serosa) and 90% were classified as N1 and/or 63% as M1. 57% of tumours were of grade G1, 43% of grade G2. Grade G2 (p=0.004) and larger tumour infiltration (≥T4; p=0.03) correlated with lower survival. Hypoxic score 1 correlated with a greater tumour size (p=0.034), larger tumour infiltration (T3 or T4; p=0.001), grade G2 (p=0.046), presence of lymph node metastasis (p=0.0066) and with lower survival of patients (p=0.03). CONCLUSION: The hypoxia-inducible factors CA9 and CXCR4 were found associated to the malignant progression of neuroendocrine tumours of the ileum. Their expression may reflect higher tumour aggressivity.