The role of neurofilament-light polipeptide (NEFL) in the onset and progression of hepatocellular carcinoma

Background: Hepatocellular Carcinoma (HCC) is the third cause of cancer-related deaths worldwide and a major health problem. Although many factors, including chromosomal anomalies, genetic polymorphisms, genetic and epigenetic alterations, contribute to HCC onset and progression, the exact mechanisms remain largely unclear. Recently, it has been shown that NEFL gene (Neurofilament-light polypeptide), whose mutations are responsible of motor-neuron disease such as Charcot-Marie-Tooth disease type 2E, is also involved in some types of malignancies. However, no information about NEFL and HCC are available. Aim: Aim of this study was to investigate the role of NEFL in the several stages of HCC development in the Resistant-Hepatocyte (R-H) rat model of hepatocarcinogenesis. Results: microarray analysis and qRT-PCR analysis showed that the expression of NEFL was strongly up-regulated at all stage of the multistep process of hepatocarcinogenesis, including the very early ones, in particular NEFL mRNA was strongly up-regulated in KRT-19 positive preneoplastic lesions and in fully malignant HCCs. In agreement with mRNA levels, immunofluorescence studies identified the neurofilament only in the preneoplastic lesions positive for KRT-19 and in early HCCs. Since the presence of this neurofilament has never been described in the liver, we wished to investigate whether NEFL expression in the carcinogenic process could be the result of a re-acquisition of fetal/neonatal life status or of increased proliferative capacity of the hepatocytes. However, almost undetectable levels of mRNA were observed in the liver of 19 days fetuses or 2 days pups. Moreover, no NEFL up-regulation was observed in actively dividing hepatocytes in liver regeneration occurring following two/third partial hepatectomy (PH), suggesting that NEFL expression in the liver is a specific feature of cancer onset and development. The ectopic expression of NEFL at all stages of rat carcinogenesis implies that NEFL may function as an oncogene in the onset and development of HCC. To assess the clinical significance of NEFL expression in human HCC patients, we determined NEFL mRNA levels in 14 HCC patients. The results showed that, similarly to what observed in the rat model, while NEFL expression was undetectable in normal human liver, it was readily detectable in HCCs and in matched cirrhotic liver. This demonstrates that aberrant expression of NEFL takes place at the onset and progression in human hepatocarcinogenesis, translating our findings to human pathology. Interestingly, although not statistically significant, a clear trend towards an up-regulation of NEFL in HCCs when compared to matched cirrhotic tissue was observed. Notably, when patients were divided into two groups, NEFL low- or high-expressors, based on the median expression level, the results showed that time of recurrence was significantly shortened in high-NEFL expression compared to low-NEFL expression group, thus demonstrating that NEFL expression levels are a predictive factor for HCC prognosis. Searching for possible mechanisms underlying NEFL enhanced expression, and to determine if the up-regulation of NEFL in the carcinogenic process could be related to promoter methylation status, we analyze the CpG island described in UCSC Genome browser in the 5’ flanking genomic region of NEFL gene. Analysis of the methylation status of 10 consecutive CpG dinucleotides in 5 HCC and 3 normal rat livers exhibited no differences in the methylation status in HCC and controls. These results suggest that DNA hypomethylation of the NEFL promoter region is not involved in the regulation of the expression of this gene. Moreover, no change in NEFL-targeting microRNAs could be found in pre- or neoplastic lesions compared to normal liver. Conclusions: These results unveil the yet unknown role of NEFL in the onset and development of HCC and suggest that NEFL could act as an oncogene in the liver. Further studies are required to identify the mechanism(s) involved in regulating the expression of the NEFL gene and their exact role in HCC development. A better knowledge of the role of NEFL might hopefully provide a novel therapeutic target in HCC progression.