Novel insights into the molecular mechanisms of α-fetoprotein expression and malignant phenotypes of hepatocellular carcinoma

MicroRNA122 (miR-122) is highly liver-specific and constitutes more than 70% of microRNAs (miRNAs) in the normal adult liver. α-fetoprotein (AFP) is the most conventional tumor marker used for detecting and surveying hepatocellular carcinoma (HCC), one of the more common and lethal cancers worldwide. In a recent issue of Nature Communications, Kojima et al. have revealed a strong association between the two biomarkers,¹ providing novel insights into the roles of miR-122 in AFP expression and malignant phenotypes of HCC.

miRNAs are a class of short (∼20–23 nucleotides), single-stranded and endogenous non-coding RNAs that regulate post-transcriptional gene expression. An imperfect base-pairing between a miRNA and the 3'-untranslated region of the target mRNA results in mRNA degradation or attenuated translation. An mRNA may be targeted by multiple miRNAs, and a miRNA may target multiple mRNAs.² As the most abundant and specific miRNA in liver, miR-122 is a crucial regulator for ensuring liver maintains normal functions, and altered miR-122 expression has been observed in various hepatic diseases, such as HCC, viral hepatitis and hepatic fibrosis.³

AFP is a major plasma protein produced by the fetal liver and yolk sac in large quantities, but its expression decreases after birth. AFP is thought to be the fetal form of serum albumin because it binds to copper, nickel, estradiol, fatty acids and bilirubin, and shares homologous regions with albumin.⁴ The concentration of serum AFP is extremely low in adults, except in patients with hepatocarcinogenesis, hetatocyte regeneration and embryonic carcinomas. Thereby, serum AFP is used as a biomarker for the diagnosis and surveillance of patients with a subset of tumors (HCC, germ cell tumors and metastatic cancers of the liver) in non-pregnant women, men and children.⁵ In general, excessive production of AFP is related to the more malignant tumor behaviors of HCC, such as vascular invasion and intrahepatic metastasis.

However, it has so far remained unclear how AFP expression and malignant phenotypes of HCC are regulated, although some molecules, such as miR-122, CUX1 (cut-like homeobox 1), ZBTB20 (zinc finger and BTB domain-containing protein 20) and RhoA (Ras homolog gene family member A), have been reported to be associated with AFP expression and/or the malignancy of HCC. Now Kojima et al. have identified miR-122/CUX1/miR-214/ZBTB20/AFP as a pathway for AFP expression and miR-122/CUX1/RhoA as a regulatory paradigm for the aggressive characteristics of HCC (Figure 1). Through three in vitro and in vivo approaches, they have demonstrated that decreased miR-122 expression in HCC is closely linked with elevated AFP expression and aggressive tumor behavior.

Figure 1.

Research approaches and proposed regulatory pathways for AFP expression and malignant phenotypes of HCC.

In the first approach, the authors established two miR-122-silenced HCC cell lines. Compared to the parent HCC cells, the silence of miR-122 resulted in increased AFP expression and a more invasive phenotype in the culture system. The invasive phenotypes of the miR-122-silenced HCC cells were also confirmed in vivo by transplantation of the cells into the livers of nude mice (orthotopic xenograft). The transcription factor CUX1 regulates gene expression, morphogenesis, differentiation and cell cycle progression.⁶ In this study, the authors have proved that as a direct target of miR-122, CUX1 plays a central role in the pathways. Decreased miR-122 expression in HCC results in increased CUX1 production, which in turn promotes the activity of RhoA, a small GTPase that is closely associated with cell migration and invasion.⁷ Furthermore, CUX1 represses ZBTB20 expression via miR-214, whereas ZBTB20 is a key repressor of AFP gene transcription in liver.⁸

In the second approach, the authors generated transgenic mice that expressed antisense miR-122. In agreement with their in vitro results using miR-122-silenced HCC cells, the miR-122/CUX1/miR-214/ZBTB20/AFP pathway was further verified in vivo using the liver tissues from the transgenic mice in which miR-122 was functionally silenced. Nevertheless, the transgenic mice did not display abnormal morphological development in their livers, suggesting that decreased miR-122 expression itself does not cause liver cells to become transformed.

In the third approach, the authors investigated the correlations among miR-122 expression, AFP expression and HCC grade using the clinical HCC samples. They confirmed that AFP expression and HCC grade were inversely correlated with miR-122 expression in human HCC samples.

These findings provide new insights into the precise mechanisms by which AFP expression and malignant phenotypes of HCC are regulated, and highlight the potential for miR-122 and associated molecules to be used as diagnostic biomarkers or therapeutic targets against HCC. However, complexities and discrepancies with AFP expression and malignant phenotypes of HCC have been uncovered in a number of studies.

First, AFP serum levels are raised in several clinical conditions, including in some normal healthy subjects and in patients with hereditary persistence of AFP (a rare benign disorder with elevated AFP levels),⁹ hepatic inflammation, cirrhosis and several kinds of malignancies besides HCC. Notably, a large proportion of HCC samples do not express elevated levels of AFP.¹⁰ It is difficult to explain the above clinical phenotypes using the miR-122/CUX1/miR-214/ZBTB20/AFP and miR-122/CUX1/RhoA pathways alone; other pathways must be present. Second, analysis of RNAs from 20 human HCC samples showed that miR-122 was significantly downregulated in only 50% of the tumors compared to non-malignant liver tissue from the same individuals.¹¹ Therefore, it is reasonable to argue the importance of miR-122 in HCC and to question why the miR-122 expression levels in the other 50% of HCC are not affected.

The above contradictory phenomena indicate that regulatory mechanisms for AFP expression and malignant phenotypes of HCC are extremely complex. To develop new therapeutics and diagnostics based on miR-122 and associated signaling molecules, it will be essential to address such questions and identify the possible mechanisms underlying these discrepancies. In addition, it may be necessary to analyze the whole profiles of altered gene expression in HCC versus normal liver tissue after miR-122 is silenced because each miRNA may target multiple mRNAs and each mRNA may be targeted by multiple miRNAs.