Researchers from the Beijing Institute of Biotechnology this week published research suggesting that the suppression of a specific microRNA by the hepatitis B virus is responsible for the cancer growth and metastasis that is sometimes caused by the infection.
Although the data were generated in cell culture and in a mouse xenograft model, the findings could point to a new target for cancer treatment, the authors wrote in The Journal of Clinical Investigation.
As evidence of the association between miRNAs and cancer mounts, it has become clear that the small, non-coding RNAs can act either as oncogenes or tumor suppressors by inhibiting “key protein-coding genes involved in cancer development and progression,” according to research team. At the same time, it has been found that viral infections frequently alter miRNA expression profiles.
Notably, a recent study found that the changes in miRNA expression in chronic HBV patients was more closely aligned to the changes seen in patients with hepatocellular carcinoma than those with acute HBV infection, “suggesting the contribution of altered miRNAs to [liver cancer] genesis from chronic HBV infection,” the investigators noted.
Yet, just how the perturbation of cellular miRNAs influences cancer development and progression is unclear.
In 2008, the scientists from the Beijing Institute of Biotechnology reported that hematopoietic pre-B cell leukemia transcription factor-interacting protein, or HPIP, regulates cancer cell growth through the activation of AKT and ERK, which can activate the cancer-related mTOR signaling pathway.
HPIP is also a co-repressor for the transcription factor PBX, which influences organogenesis and tumorigenesis, and interacts with estrogen receptor in breast cancer cells to promote proliferation, in vitro migration, and in vivo tumor growth, according to the JCI paper.
To further investigate this, the research team applied two target-prediction programs to screen for miRNAs that target HPIP, pinpointing three: miR-148a, miR-148b, and miR-152. However, miR-148a was found to decrease HPIP in a variety of cells when over-expressed, while its inhibition boosted HPIP expression. Further experimentation indicated that the miRNA suppresses HPIP expression by directly targeting its 3' UTR.
Given HPIP's association with AKT and ERK, the investigators examined whether miR-148a can repress their activation and, if so, if it does so by targeting HPIP.
“Western blot analysis showed that miR-148a over-expression in HepG2 cells decreased the phosphorylation levels of AKT and ERK1/2, whereas knockdown of miR-148a with miR-148a inhibitor enhanced AKT and ERK1/2 phosphorylation, though their total levels remained unchanged,” they wrote in JCI.
Meantime, transfection of miR-148a-expressing HepG2 cells with HPIP resulted in a reversal of AKT and ERK inhibition, while the introduction of HPIP-targeting siRNAs “abolished the ability of miR-148a to repress AKT and ERK,” confirming that the miRNA represses AKT and ERK by inhibiting HPIP.
Further investigation showed that miR-148a's inhibitory effect on AKT and ERK via HPIP extended to the mTOR pathway. However, the team's experiments indicated that mTOR is not itself a direct target of the miRNA. Instead, miR-148a and its effect on HPIP influences mTOR expression through the AKT/ERK/FOXO4/AFT5 pathway.
The researchers then turned their attention to HBx, an HBV-encoded protein implicated in the molecular pathogenesis of liver cancer, testing whether it has an effect on miR-148a expression by transfecting normal hepatocytes with HBx, its deletion mutant, or large hepatitis delta antigen.
“Expression of HBx, but not the C-terminal deletion mutant HBx and L-HDAg, inhibited miR-148a expression, suggesting that HBx inhibition of miR-148a is specific,” they wrote. Additional experimentation indicated that HBx inhibits the miRNA by suppressing recruitment of p53 as its promoter.
Having established that miR-148a regulates the mTOR pathway, the researchers looked at whether the miRNA would impact cancer cell growth, finding that its over-expression reduced the proliferation of three different liver cancer cell lines while its inhibition had the opposite effect. The miRNA also was found to inhibit epithelial-to-mesenchymal transition, invasion, and metastasis of HBx-expressing hepatocarcinoma cells by inhibiting HPIP-mediated mTOR signaling.
Further, miR-148a was found to have similar effects in a mouse model, and to be down-regulated in tissue samples from patients with HBV-related liver cancer and negatively correlated with HPIP, which is up-regulated in these patients.
Collectively, the data indicate that miR-148a “plays important roles in the development and progression of both virus- and non-virus-associated cancers,” and that HPIP is a “key mediator of virus-related carcinogenesis and progression,” the team wrote.
“Although HPIP up-regulation in patients with cancer may be due to miR-148a down-regulation, we cannot exclude other mechanisms,” they added, noting that the epithelial-to-mesenchymal transition “is an important step toward tumor invasion and metastasis” and can be induced by a variety of different molecules.
Still, since miR-148a and HPIP are upstream regulators of AKT, ERK, and mTOR signaling, “we believe that miR-148a and HPIP are critical regulators of EMT,” they concluded. “The important role of miR-148a and HPIP in cancer suggests that miR-148a activation or HPIP inhibition may be a useful strategy for cancer treatment.”