The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2′deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and hematopoietic malignancy (64% in Burkitt’s lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV-induced hepatocellular carcinoma (HCC). HBx interacts with several cell signaling molecules, leading to activation of various transcription factors including nuclear factor-kappaB (NF-κB). Activated NF-κB signaling is implicated in many human cancers including HCC. Here, we present evidence that the NF-κB signaling activator, tumor necrosis factor (TNF)-α, induces the accumulation of HBx in cells by increasing protein stability due to reduced proteasomal degradation. The effects of TNF-α on HBx protein stability are mediated via activated NF-κB effector kinases IKKα and IKKβ and p65. The non-IKK-phosphorylable p65-S534A mutant did not induce HBx protein stability; hence, phosphorylation of p65 by IKK is a key step in TNF-α-induced stabilization of HBx. Phospho-p65 showed higher affinity to HBx compared with the non-phosphorylable p65 mutant, suggesting that the interaction of phospho-p65 with HBx might be important for HBx stabilization. We also show that the increased level of HBx in cells cooperates with TNF-α toward activation of NF-κB and expression of NF-κB-regulated genes, indicating a positive feedback loop between HBx and NF-κB signaling. Overall, our study provides evidence for interplay between HBx and NF-κB signaling, which may account for HBV-mediated liver carcinogenesis.

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.Human papillomaviruses (HPVs) are double-stranded small DNA viruses, comprising more than 100 members, that are the causative agents of several human diseases, including cancers (25). According to their tropisms, HPVs are subdivided into mucosal and cutaneous types. High-risk mucosal HPV types, such as HPV type 16 (HPV16) and HPV18, are etiological agents of cervical cancer and other anogenital cancers (25). Their ability to induce cellular immortalization and transformation is attributed primarily to the viral oncoproteins E6 and E7 (20,25). While E6 prevents apoptosis by inducing the degradation of the tumor suppressor p53 through the proteasome system, E7 disrupts cell cycle regulation by inactivating pRb (20,25). In addition, HPV16 E6 and E7 alter several other cellular signaling pathways by interacting with a number of cellular proteins, enhancing their carcinogenic properties (20,38,39,41).A large subgroup of cutaneous HPV types belonging to the beta genus of the HPV phylogenetic tree has been proposed to be involved in the development of nonmelanoma skin cancer (NMSC), since they were isolated for the first time from patients suffering from a rare autosomal recessive genetic disorder called epidermodysplasia verruciformis (EV), characterized by susceptibility to beta HPV infection and NMSC development at sun-exposed anatomical regions (7). Subsequent epidemiological studies have also provided lines of evidence for a possible oncogenic role of beta HPV types in non-EV patients, including the healthy population and immunocompromised individuals, e.g., organ transplant recipients (7, 51).Our group has previously reported t...

The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2′-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis.