Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers. Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that contributes to approximately 2% of all cancers by modulating a myriad of host cell activities. M6A is the most abundant RNA modification which is important for infection with HIV-1, HCV, Zika virus, KSHV and SV40 virus. The role of m6A modification and the associated enzymes in EBV infection and related oncogenic activities has not been explored. We investigated the role of m6A modification on EBV-infection and examined its effects on the proliferative activities of the virus which contributes to cancer. We showed that the m6A methyltransferase METTL14 is an important factor in EBV-induced oncogenesis. METTL14 was dramatically increased in EBV latently infected cells. Knock-down of METTL14 led to decreased latent viral gene expression. The essential EBV latent antigen EBNA3C was up-regulated by METTL14-mediated m6A modification, and its expression led to a feedback loop in up-regulation of METTL14 transcription, and the stability of its protein. We now provide a more comprehensive understanding of EBV and the host cell RNA processing machinery critical for regulation of EBV activities to maintain latent infection and its oncogenic properties.

The cellular adaptive response to hypoxia, mediated by high HIF1α levels includes metabolic reprogramming, restricted DNA replication and cell division. In contrast to healthy cells, the genome of cancer cells, and Kaposi’s sarcoma associated herpesvirus (KSHV) infected cells maintains replication in hypoxia. We show that KSHV infection, despite promoting expression of HIF1α in normoxia, can also restrict transcriptional activity, and promoted its degradation in hypoxia. KSHV-encoded vCyclin, expressed in hypoxia, mediated HIF1α cytosolic translocation, and its degradation through a non-canonical lysosomal pathway. Attenuation of HIF1α levels by vCyclin allowed cells to bypass the block to DNA replication and cell proliferation in hypoxia. These results demonstrated that KSHV utilizes a unique strategy to balance HIF1α levels to overcome replication arrest and induction of the oncogenic phenotype, which are dependent on the levels of oxygen in the microenvironment.

EBV latent antigen 3C (EBNA3C) is essential for EBV-induced primary B-cell transformation. Infection by EBV induces hypermethylation of a number of tumor suppressor genes, which contributes to the development of human cancers. The Ras association domain family isoform 1A (RASSF1A) is a cellular tumor suppressor, which regulates a broad range of cellular functions, including apoptosis, cell-cycle arrest, mitotic arrest, and migration. However, the expression of RASSF1A is lost in many human cancers by epigenetic silencing. In the present study, we showed that EBNA3C promoted B-cell transformation by specifically suppressing the expression of RASSF1A. EBNA3C directly interacted with RASSF1A and induced RASSF1A degradation via the ubiquitin-proteasome-dependent pathway. SCF^Skp2, an E3-ubiquitin ligase, was recruited by EBNA3C to enhance RASSF1A degradation. Moreover, EBNA3C decreased the transcriptional activity of RASSF1A promoter by enhancing its methylation through EBNA3C-mediated modulation of DNMTs expression. EBNA3C also inhibited RASSF1A-mediated cell apoptosis, disrupted RASSF1A-mediated microtubule and chromosomal stability, and promoted cell proliferation by upregulating Cyclin D1 and Cyclin E expression. Our data provides new details, which sheds light on additional mechanisms by which EBNA3C can induce B-cell transformation. This will also facilitate the development of novel therapeutic approaches through targeting of the RASSF1A pathway. Epstein-Barr virus (EBV) which is associated with multiple lymphoid and epithelial malignancies was the first recognized oncogenic virus in humans. EBNA3C, an essential latent antigen encoded by EBV interacts with numerous host transcription factors and plays an important role in the transformation of primary B-cells. RASSF1A, a tumor suppressor plays a vital role in regulating apoptosis, cell-cycle arrest, and mitotic arrest and is implicated in the development of a number of different cancers. We now demonstrate that EBNA3C can physically interact with RASSF1A and induce RASSF1A degradation through the ubiquitin-proteasome-dependent pathway. Further, the E3 ubiquitin ligase SCF^Skp2 was recruited by EBNA3C to mediate RASSF1A degradation. Moreover, RASSF1A mRNA expression was also suppressed by EBNA3C. EBNA3C repressed the transcriptional activity of the RASSF1A promoter through induction of its methylation by enhancing DNMT3a expression. EBNA3C regulation of RASSF1A promoted cell proliferation, inhibited RASSF1A-mediated apoptosis and disrupted RASSF1A-mediated microtubule and chromosomal stability. Overall, our results add to our understanding of the many strategies employed by EBNA3C to induce B-cell transformation, which will contribute to new therapeutics for interventions targeting EBV association malignancies.

Background and Aims: Menin is a nuclear scaffold protein that regulates gene transcription in an oftentimes tissue-specific manner. Our previous work showed that menin is over-expressed in colorectal cancer (CRC); however, the full spectrum of menin function in colonic neoplasia remains unclear. Herein, we aimed to uncover novel menin-regulated pathways important for colorectal carcinogenesis. Methods: RNA-Seq analysis identified that menin regulates LXR-target gene expressions in CRC cell lines. Isolated colonic epithelium from Men1^f/f;Vil1-Cre and Men1^f/f mice was used to validate the results in vivo. Cholesterol content was quantified via an enzymatic assay. Results: RNA-Seq analysis in the HT-29 CRC cell line identified that menin inhibition upregulated LXR-target genes, specifically ABCG1 and ABCA1, with protein products that promote cellular cholesterol efflux. Similar results were noted across other CRC cell lines and with different methods of menin inhibition. Consistent with ABCG1 and ABCA1 upregulation, and similarly to LXR agonists, menin inhibition reduced the total cellular cholesterol in both HT-29 and HCT-15 cells. To confirm the effects of menin inhibition in vivo, we assessed Men1^f/f;Vil1-Cre mice lacking menin expression in the colonic epithelium. Men1^f/f;Vil1-Cre mice were found to have no distinct baseline phenotype compared to control Men1^f/f mice. However, similarly to CRC cell lines, Men1^f/f;Vil1-Cre mice showed an upregulation of Abcg1 and a reduction in total cellular cholesterol. Promoting cholesterol efflux, either via menin inhibition or LXR activation, was found to synergistically suppress CRC cell growth under cholesterol-depleted conditions and when administered concomitantly with small molecule EGFR inhibitors. Conclusions: Menin represses the transcription of LXR-target genes, including ABCA1 and ABCG1 in the colonic epithelium and CRC. Menin inhibition conversely upregulates LXR-target genes and reduces total cellular cholesterol, demonstrating that menin inhibition may be an important mechanism for targeting cholesterol-dependent pathways in colorectal carcinogenesis.

Objectives: pH-responsive zeolite imidazolate framework-8 (ZIF-8) enables selective release of 5-fluorouracil (5-FU) within the acidic tumor microenvironment. However, the direct effects of ZIF-8 itself on cancer cells or surrounding tissues remain unclear. Since oral cancer involves interactions between epithelial tumor cells and stromal cells, comparing the effects of ZIF-8 on epithelial cancer cells and orofacial mesenchymal stem/stromal cells (OMSCs) is critical to understanding its broader biological impact. Methods: The effects of ZIF-8 on SCC7 epithelial cancer cells and OMSCs, including periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs), were evaluated using RNA sequencing, nuclear staining, live/dead assays, and immunocytochemistry. Cells were treated with 0, 1, 10, or 100 μg/mL ZIF-8. Based on nuclear staining results, live/dead viability assays were conducted on SCC7 and DPSCs treated with 0 or 100 μg/mL ZIF-8. Apoptosis-related markers (Bax, caspase-3, caspase-6, and caspase-10) were assessed following exposure to 100 μg/mL ZIF-8. Results: Transcriptomic analysis revealed that ZIF-8 not only facilitates selective 5-FU release but also directly induces apoptosis in SCC7 cells compared with 5-FU alone. At 100 μg/mL ZIF-8, SCC7 viability was significantly reduced, whereas OMSC viability was preserved. Nonviable SCC7 cells increased markedly compared with controls, while DPSCs showed no significant change. Apoptosis-related signaling was also elevated in SCC7 cells compared with DPSCs. Conclusions: ZIF-8 at 100 μg/mL selectively inhibits SCC7 growth while sparing OMSC viability and apoptosis.

The cellular adaptive response to hypoxia, mediated by high HIF1α levels includes metabolic reprogramming, restricted DNA replication and cell division. In contrast to healthy cells, the genome of cancer cells, and Kaposi’s sarcoma associated herpesvirus (KSHV) infected cells maintains replication in hypoxia. We show that KSHV infection, despite promoting expression of HIF1α in normoxia, can also restricts transcriptional activity, and promoted its degradation in hypoxia. KSHV-encoded vCyclin, expressed in hypoxia, mediated HIF1a cytosolic translocation, and its degradation through a non-canonical lysosomal pathway. Attenuation of HIF1α levels by vCyclin allowed cells to bypass the block to DNA replication and cell proliferation in the hypoxia. These results demonstrated that KSHV utilizes a unique strategy to balance HIF1α levels to overcome replication arrest and induction of the oncogenic phenotype, which are dependent on the levels of oxygen in the microenvironment.