Profiling and characterization of Epstein-Barr virus encoded BamHI-A rightward transcript microRNAs in nasopharyngeal carcinoma

Abstract

Persistent latent infection of Epstein-Barr Virus (EBV) is associated with various human malignancies such as Burkitt’s lymphoma, gastric (GC) and nasopharyngeal carcinoma (NPC). Several studies have reported the abundance of EBV-encoded alternatively spliced BamHI A rightward transcripts (BARTs) in these malignancies, of which NPC confers the highest tissue expression. However, extensive investigations of their potential use as tissue- and/or blood-based NPC biomarkers, particularly for Hong Kong cases, remain elusive.

Comparison of microRNA (miRNA) profiles between tumor and non-tumor specimens from 5 paired NPC cases reveal 29/39 of EBV miRNAs were significantly upregulated in tumor tissues versus their non-tumor counterparts (P<0.05). Microarray-based results also indicate that EBV miRNAs, particularly from the BART clusters, are generally more upregulated than miRNAs of human origin. Of the upregulated EBV miRNAs, 12 were chosen for further validation in 15 additional paired NPC cases by qRT-PCR and yielded consistent results with the array. BART6-5p, 6-3p, 14, 18-5p, 19-3p and 2-5p were also distinctly detected in the serum of NPC patients, and positively correlated with corresponding tumor copy numbers.

Given that successfully validated EBV BARTs were also present at functional levels in tumors. Their functional role(s) were further investigated to identify potential oncogenic ‘driver’ BART miRNA(s). Like miRNAs of human origin, BARTs can also downregulate ‘target’ genes conferring 3’UTR sites with partial homology to their miRNA seed region. Bioinformatic tools were used to generate putative target genes of upregulated BARTs, whereby functional clustering reveals Wnt signaling as the most enriched pathway. Experimental target validation shows partial redundancy between several Wnt inhibitors (APC, GSK3B, BTRC, NLK) by a subset of BARTs (6-3p, 14, 17-5p, 19-3p). Gain- and loss-of-function studies pinpoint BART19-3p as a potential Wnt ‘driver’. Given its ability to enhance .-catenin stability and attenuate ubiquitin-mediated proteolysis to promote intranuclear s-catenin accumulation, BART19-3p-mediated TCF/LEF activation promoted tumor growth (via CCND1), self-renewal (via CMYC, CD44), EMT (via SNAIL) and invasion (via MMP7) in EBV negative cell lines. BART19-3p silencing in C666 sufficiently rescued Wnt regulatory systems and modulated Wnt/Hedgehog crosstalk via GSK3B/BTRC-mediated GLI3 processing.

In addition to BART miRNA profiling, the mechanism-based approach of this study provides novel and interesting insights into EBV-mediated oncogenesis. Partially redundant and likely additive effects of BARTs may be important in finetuning the dose-dependent effects of aberrant signal transduction pathways in NPC. A better understanding of BART-mediated regulation of Wnt signaling and their ability to recapitulate frequently observed nuclear accumulation of t-catenin in primary NPC specimens also helps pinpoint potential druggable targets (e.g. BART19-3p). Considering the difficulties in targeting mutation-independent aberrant Wnt signaling in cancer and the advent of miRNA-based targeted therapy, ‘driver’ BART(s) may be promising therapeutic targets for improving the currently poor treatment outcomes of advanced stage NPC. Alternatively, the specificity of BARTs within NPC tumors and patient sera could also be harnessed to develop less invasive means for early detection, which like majority of cancers yields better prognosis.