Bakary S. Sylla

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. Many oncogenic viruses exhibit cellular transforming properties, often involving oncogenes activation and tumor suppressor genes inactivation. The DOK1 gene is a newly identified tumor suppressor gene with altered expression via hypermethylation of its promoter in a variety of human cancers, including head and neck, lung, gastric and others. In addition, a correlation has been reported between DOK1 aberrant hypermethylation and the presence of oncogenic viruses such as hepatitis B virus (HBV) in hepatocellular carcinoma (HCC) and Epstein-Barr virus (EBV) in Burkitt's lymphoma-derived cell lines. Here we demonstrate for the first time that EBV is directly involved in the inhibition of DOK1 expression in B-cells. We show that EBV leads to epigenetic repression of DOK1 through increased DNA methylation of its promoter and H3K27 tri-methylation. The LMP1 oncoprotein plays a key role in the repression of DOK1 expression. It promotes the formation and the recruitment to the DOK1 promoter of transcriptionally inhibitory complexes composed of E2F1/pRB/DNMT1 and of EZH2 which is part of the polycomb repressive complex 2. Interestingly, one or more additional EBV protein(s) cooperate(s) with LMP1 in inducing massive DNA methylation at the DOK1 promoter, leading to the loss of E2F1 complexes recruitment and even stronger repression of DOK1 expression.

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.

Constitutive activation of NF-κB signaling is a key event in virus- and non-virus-induced carcinogenesis. We have previously reported that cutaneous human papillomavirus type 38 (HPV38) displays transforming properties in in vitro and in vivo experimental models. However, the involvement of NF-κB signaling in HPV38-induced cell growth transformation remains to be determined. In this study, we showed that HPV38 E6 and E7 activate NF-κB and that inhibition of the pathway with the IκBα superrepressor sensitizes HPV38E6E7-immortalized human keratinocytes to tumor necrosis factor alpha (TNF-α)- and UVB radiation-mediated apoptosis. Accordingly, inhibition of NF-κB signaling resulted in the downregulation of NF-κB-regulated antiapoptotic genes, including cIAP1, cIAP2, and xIAP genes. These findings demonstrate a critical role of NF-κB activity in the survival of HPV38E6E7-immortalized human keratinocytes exposed to cytokine or UV radiation. Our data provide additional evidence for cooperation between beta HPV infection and UV irradiation in skin carcinogenesis.

In Africa, there were an estimated 681,000 new cancer cases and 512,000 deaths in 2008. Projections to 2030 show a startling rise, with corresponding figures of 1.27 million cases and 0.97 million deaths resulting from population growth and aging alone. The figures make no assumptions about incidence rates which may increase due to the further introduction of tobacco and a more westernized lifestyle. The current situation in many parts of Africa with respect to health care systems suggests that improved cancer treatment would be an insufficient response to this increasing burden. Much could be achieved through cancer prevention by applying current knowledge about major risk factors and the natural history of the disease. For example, vaccination against hepatitis B virus and human papilloma viruses would prevent the occurrence of two of the most common cancers in Africa, liver and cervix, respectively, in the long‐term. Strong measures to prevent the widespread introduction of tobacco must be a priority. Early detection and treatment of cervical and breast cancers using approaches applicable now in Africa would provide immediate value, as would the management of human immunodeficiency virus (HIV) infection in respect to HIV‐associated malignancies. In parallel, further research is needed into the causes of cancer and the barriers to implementation of promising prevention strategies. Underpinning all is the need for African governments to look forward and prioritize cancer through national cancer control plans, to invest in public health infrastructure and to ensure the adequate training and support for people in cancer prevention and control. Given this core commitment from within Africa, international partners can provide complementary support in a cooperation that permits action now to mitigate the impending tragedy of cancer in the continent of Africa.

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia 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 haematopoietic 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.

ΔNp73α, a dominant-negative inhibitor of p53 and p73, exhibits antiapoptotic and transforming activity in in vitro models and is often found to be upregulated in human cancers. The mechanisms involved in the regulation of ΔNp73α protein levels in normal and cancer cells are poorly characterized. Here, we show that that IκB kinase beta (IKKβ) increases ΔNp73α protein stability independently of its ability to activate NF-κB. IKKβ associates with and phosphorylates ΔNp73α at serine 422 (S422), leading to its accumulation in the nucleus, where it binds and represses several p53-regulated genes. S422A mutation in ΔNp73α abolished IKKβ-mediated stabilization and inhibition of p53-regulated gene expression. Inhibition of IKKβ activity by chemical inhibitors, overexpression of dominant-negative mutants, or gene silencing by siRNA also resulted in ΔNp73α destabilization, which under these conditions was rapidly translocated into the cytoplasm and degraded by a calpain-mediated mechanism. We also present evidence for the IKKβ and ΔNp73α cross talk in cancer-derived cell lines and primary cancers. Our data unveil a new mechanism involved in the regulation of the p73 and p53 network.

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.

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.

Dok1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that negatively regulates cell growth and promotes migration. We now find that IκB kinase β (IKKβ) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKKβ phosphorylation of Dok1 depended on Dok1 S₄₃₉, S₄₄₃, S₄₄₆, and S₄₅₀. Recombinant IKKβ also phosphorylated Dok1 or Dok1 amino acids 430–481in vitro. TNF-α, IL-1, γ radiation, or IKKβ overexpression phosphorylated Dok1 S₄₄₃, S₄₄₆, and S₄₅₀in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S₄₃₉, S₄₄₃, S₄₄₆, and S₄₅₀mutated to A was not phosphorylated by IKKβin vivo. Surprisingly, mutant Dok1 A₄₃₉, A₄₄₃, A₄₄₆, and A₄₅₀differed from wild-type Dok1 in not inhibiting platelet-derived growth factor-induced extracellular signal-regulated kinase 1/2 phosphorylation or cell growth. Mutant Dok1 A₄₃₉, A₄₄₃, A₄₄₆, and A₄₅₀also did not promote cell motility, whereas wild-type Dok1 promoted cell motility, and Dok1 E₄₃₉, E₄₄₃, E₄₄₆, and E₄₅₀further enhanced cell motility. These data indicate that IKKβ phosphorylates Dok1 S₄₃₉S₄₄₃and S₄₄₆S₄₅₀after TNF-α, IL-1, or γ-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.