In vitro transformation of primary human hepatocytes: Epigenetic changes and stemness properties.

Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.

Human Papillomavirus infection in senegalese female sex workers.

OBJECTIVES: Several studies have documented the HPV genotypes in the Senegalese general population. The objective was to explore the HPV genotype distribution in Senegalese FSWs in order to assess the potential relevance of currently-available vaccines. METHODS: Vaginal swabs samples collected as part of the National Integrated Biological and Behavioral Survey in 14 regions throughout the country were randomly selected for HPV testing using bead-based multiplex genotyping (TS-MPG). RESULTS: Among the 436 FSW samples analyzed, the overall HPV prevalence was 79.8% (N = 348), with 70.1% (N = 244) cases presenting as multiple infections. High Risk HPV genotypes affecting at least 10% of FSWs included in order of decreasing frequency: 52, 16, 35, 51, 33, 31, 18, and 45. Sixty-seven (15.4%) FSWs were HIV positive and they were significantly more affected by HPV (94% vs 77%; p < 0.01) than seronegative FSWs as well as infections with multiple genotype. CONCLUSION: The present study indicates that FSW in Senegal experience a high burden of HPV infection with a high frequency of coinfection with HIV and multiple HPV genotypes. Public health interventions for this key population should include an earlier cervical dysplasia/cancer detection and preventative measures such as vaccination programs that must consider the HPV genotype distribution.

Sensitive Multiplex Real-time RT-qPCR Assay for the Detection of Filoviruses.

Filoviruses are important etiological agents of emergent diseases with high mortality rates. Traditionally, filovirus fever diseases have primarily been a burden of African countries; however, global interconnectedness has increased the probability of the worldwide spread of filoviruses. Therefore, national healthcare organizations need tools for managing filovirus risk, including diagnostic kits based on real-time reverse transcription PCR (RT-qPCR), as this is the most suitable method for diagnosing filovirus fever diseases. Here we describe a real-time RT-qPCR assay for filovirus detection. This assay is a further development of our previously reported EBOV (Zaire)-Fl kit. Two sets (FiloA-Fl and FiloB-Fl) of real-time RT-qPCR assays for the detection of filoviruses were developed and evaluated using armored RNA phage particles (ARs) as positive controls. The limit of detection of the assay was 5x102 copies/ml of the AR-positive control for the FiloA-Fl set and 5x103 copies/ml of the AR-positive control for the FiloB-Fl set. Our assay provides a rapid and sensitive tool for detecting filoviruses. The high specificity and sensitivity of the assay make it useful for clinical and epidemiologic investigations in the field of filovirus fever diseases and their etiological agents.

Primary Breast Burkitt's Lymphoma in an HIV-Infected Woman.

A 30-year-old HIV positive woman presented with a multifocal mass tumour associated with axillary and lateral-cervical lymphadenopathy in the right breast. Laboratory examination of the biopsy confirmed a case of mammary Burkitt's lymphoma with a nodular infiltration of the breast. Antiretroviral treatment and chemotherapy were effective to control the tumour. Although Burkitt's lymphoma rarely involves the breasts, it should be considered during routine breast examination of African woman.

Epstein-Barr virus down-regulates tumor suppressor DOK1 expression.

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.

Human papillomavirus infection in women in four regions of Senegal.

Cervical cancer is the most frequent cancer among women in Senegal. However, there are few data concerning the human papillomavirus (HPV) types inducing neoplasia and cervical cancers and their prevalence in the general population of Senegal. The aim of this study is to determine the prevalence of HPV infection in Senegalese women aged 18 years and older in Dakar Region and three other regions. Cervical samples were collected from 498 women aged 18-80 years (mean, 42.1 years) in Dakar Region. Also, 438 samples were collected from three other regions: Thiès, Saint-Louis, and Louga. The samples were screened for 21 HPV genotypes using an HPV type-specific E7 PCR bead-based multiplex genotyping assay (TS-MPG). The prevalence of high risk (HR)-HPV in Dakar Region was 17.4%. HPV 52 (3.2%) was the most prevalent HPV type, followed by HPV 31 (3.0%) and HPV 16, 45, and 53 (all 2.8%). In the Thiès, Saint-Louis, and Louga Regions, the prevalence of HR-HPV was 23.2%, 13.1%, and 19.4%, respectively. The study revealed the specificity of HPV prevalence in Dakar Region and other regions of Senegal. The observed patterns show some differences compared with other regions of the world. These findings raise the possibility that, in addition to HPV 16 and HPV 18, other HPV types should be considered for a vaccination program in Senegal. However, additional studies to determine the HPV type distribution in cervical cancer specimens in Senegal are required to further corroborate this hypothesis.

Infrastructure and facilities for human biobanking in low- and middle-income countries: a situation analysis.

OBJECTIVE: To collect information on biobanking facilities in low- and middle-income countries (LMICs) as a first step towards establishing an LMIC biobank and cohort building network (BCNet) to support research, with a focus on cancer control. METHOD: Sixty centres were identified from sources including cancer centres, universities, hospitals, and public health facilities and invited to participate in a survey between December 2012 and March 2013. RESULTS: Of the 27 centres (45%) that responded, most have existed for <10 years. They store between 1,000 and 1,000,000 research samples as well as samples remaining after clinical diagnosis. Sample storage is mostly in freezers, although 45% (9/20) of the centres do not have regular access to electricity. Biobank managers, sample management systems, and mechanisms for follow-up using linkages are uncommon. Many (80%; 21/26) of the centres have regulations to govern research, but regulations for the use of biobank resources (samples and data) are not well developed. CONCLUSIONS: Biobanking facilities are being developed in LMICs. Shortcomings in international visibility, sample sharing regulations, standardization, quality assurance, and sample management systems could be alleviated by international networking. Stakeholders need to work together to increase access to high-quality biological resources for scientific research.

Oncoprotein E7 from beta human papillomavirus 38 induces formation of an inhibitory complex for a subset of p53-regulated promoters.

Our previous studies on cutaneous beta human papillomavirus 38 (HPV38) E6 and E7 oncoproteins highlighted a novel activity of IκB kinase beta (IKKß) in the nucleus of human keratinocytes, where it phosphorylates and stabilizes ΔNp73α, an antagonist of p53/p73 functions. Here, we further characterize the role of the IKKß nuclear form. We show that IKKß nuclear translocation and ΔNp73α accumulation are mediated mainly by HPV38 E7 oncoprotein. Chromatin immunoprecipitation (ChIP)/Re-ChIP experiments showed that ΔNp73α and IKKß are part, together with two epigenetic enzymes DNA methyltransferase 1 (DNMT1) and the enhancer of zeste homolog 2 (EZH2), of a transcriptional regulatory complex that inhibits the expression of some p53-regulated genes, such as PIG3. Recruitment to the PIG3 promoter of EZH2 and DNMT1 resulted in trimethylation of histone 3 on lysine 27 and in DNA methylation, respectively, both events associated with gene expression silencing. Decreases in the intracellular levels of HPV38 E7 or ΔNp73α strongly affected the recruitment of the inhibitory transcriptional complex to the PIG3 promoter, with consequent restoration of p53-regulated gene expression. Finally, the ΔNp73α/IKKß/DNMT1/EZH2 complex appears to bind a subset of p53-regulated promoters. In fact, the complex is efficiently recruited to several promoters of genes encoding proteins involved in DNA repair and apoptosis, whereas it does not influence the expression of the prosurvival factor Survivin. In summary, our data show that HPV38 via E7 protein promotes the formation of a multiprotein complex that negatively regulates the expression of several p53-regulated genes.

The human papillomavirus type 16 E7 oncoprotein induces a transcriptional repressor complex on the Toll-like receptor 9 promoter.

Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50-p65 and ERα induced by the E7 oncoprotein. The E7-mediated transcriptional complex also recruited the histone demethylase JARID1B and histone deacetylase HDAC1. The entire complex bound to a specific region on the TLR9 promoter, which resulted in decreased methylation and acetylation of histones upstream of the TLR9 transcriptional start site. The involvement of NF-κB and ERα in the TLR9 down-regulation by HPV16 E7 was fully confirmed in cervical tissues from human patients. Importantly, we present evidence that the HPV16-induced TLR9 down-regulation affects the interferon response which negatively regulates viral infection. Our studies highlight a novel HPV16-mediated mechanism that combines epigenetic and transcriptional events to suppress a key innate immune sensor.

Epstein - Barr virus transforming protein LMP-1 alters B cells gene expression by promoting accumulation of the oncoprotein ΔNp73α.

Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.

Transcriptional regulation of the human tumor suppressor DOK1 by E2F1.

The expression of the tumor suppressor DOK1 is repressed in a variety of human tumors as a result of hypermethylation of its promoter region. However, the molecular mechanisms by which DOK1 expression is regulated have been poorly investigated. Here, we show that the expression of DOK1 is regulated mainly by the transcription factor E2F1. We identified three putative E2F1 response elements (EREs) in the DOK1 promoter region. E2F1 had a relatively higher binding affinity for the ERE located between bp -498 and -486 compared with the other two EREs. E2F1 gene silencing strongly inhibited DOK1 expression. E2F1-driven DOK1 transcription occurred in the presence of cellular stresses, such as accumulation of DNA damage induced by etoposide. DOK1 silencing promoted cell proliferation and protected against etoposide-induced apoptosis, indicating that DOK1 acts as a key mediator of cellular stress-induced cell death. Most importantly, we observed that DNA methylation of the DOK1 core promoter region found in head and neck cancer cell lines hampered the recruitment of E2F1 to the DOK1 promoter and compromised DOK1 expression. In summary, our data show that E2F1 is a key factor in DOK1 expression and provide novel insights into the regulation of these events in cancer cells.

Prevalence of human papillomavirus types in cervical lesions from women in rural Western India.

Cervical cancer is the most common cancer among women in many areas of India which contributes for a fifth of the global burden of disease. Persistent infection with one of the high-risk human papillomaviruses (HPV) has been established as the cause for cervical cancer and the documentation of the prevalence of HPV types in cervical cancer in different regions of India is useful for a prevention program combining both screening and vaccination. In this study, the HPV type distribution and the frequency of p16(INK4a) immunoexpression have been determined in 125 cases of inflammatory lesions or grade 1 cervical intraepithelial neoplasia, 74 cases of grade 2, 72 cases of grade 3, and 113 cervical cancer cases diagnosed among women from rural Solapur and Osmanabad districts, Maharashtra. The overall prevalence of high-risk HPV was 37.6% in inflammatory lesions or grade 1 cervical intraepithelial neoplasia, 63.5% in grade 2, 97.2% in grade 3 and 92% in cervical cancer cases. HPV 16 and HPV 18 were detected in 80.6% of grade 3 cervical intraepithelial neoplasia and 86.5% of cervical cancer cases. 94.7% of the cervical cancer and 84.4% of the high grade lesions with a strong and full thickness staining for p16(INK4a) were positive for HPV infection; p16(INK4a) immunoexpression increased with worsening grade of cervical intraepithelial neoplasia. The HPV genotyping data showing a high HPV 16 and 18 prevalence in cancer specimens indicate that prophylactic HPV 16/18 vaccination would have a significant impact on the prevention of cervical cancer in India.

Human papillomavirus type 16 genetic variants: phylogeny and classification based on E6 and LCR.

Naturally occurring genetic variants of human papillomavirus type 16 (HPV16) are common and have previously been classified into 4 major lineages; European-Asian (EAS), including the sublineages European (EUR) and Asian (As), African 1 (AFR1), African 2 (AFR2), and North-American/Asian-American (NA/AA). We aimed to improve the classification of HPV16 variant lineages by using a large resource of HPV16-positive cervical samples collected from geographically diverse populations in studies on HPV and/or cervical cancer undertaken by the International Agency for Research on Cancer. In total, we sequenced the entire E6 genes and long control regions (LCRs) of 953 HPV16 isolates from 27 different countries worldwide. Phylogenetic analyses confirmed previously described variant lineages and subclassifications. We characterized two new sublineages within each of the lineages AFR1 and AFR2 that are robustly classified using E6 and/or the LCR. We could differentiate previously identified AA1, AA2, and NA sublineages, although they could not be distinguished by E6 alone, requiring the LCR for correct phylogenetic classification. We thus provide a classification system for HPV16 genomes based on 13 and 32 phylogenetically distinguishing positions in E6 and the LCR, respectively, that distinguish nine HPV16 variant sublineages (EUR, As, AFR1a, AFR1b, AFR2a, AFR2b, NA, AA1, and AA2). Ninety-seven percent of all 953 samples fitted this classification perfectly. Other positions were frequently polymorphic within one or more lineages but did not define phylogenetic subgroups. Such a standardized classification of HPV16 variants is important for future epidemiological and biological studies of the carcinogenic potential of HPV16 variant lineages.

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract.

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.

Comparative analysis of transforming properties of E6 and E7 from different beta human papillomavirus types.

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

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.

A million africans a year dying from cancer by 2030: what can cancer research and control offer to the continent?

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.

Aberrant DNA methylation of cancer-associated genes in gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST).

Epigenetic events have emerged as key mechanisms in the regulation of critical biological processes and in the development of a wide variety of human malignancies, including gastric cancer (GC), however precise gene targets of aberrant DNA methylation in GC remain largely unknown. Here, we have combined pyrosequencing-based quantitative analysis of DNA methylation in 98 GC cases and 64 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and in cancer tissue and non-tumorigenic adjacent tissue of an independent series of GC samples. A panel of 10 cancer-associated genes (CHRNA3, DOK1, MGMT, RASSF1A, p14ARF, CDH1, MLH1, ALDH2, GNMT and MTHFR) and LINE-1 repetitive elements were included in the analysis and their association with clinicopathological characteristics (sex, age at diagnosis, anatomical sub-site, histological sub-type) was examined. Three out of the 10 genes analyzed exhibited a marked hypermethylation, whereas two genes (ALDH2 and MTHFR) showed significant hypomethylation, in gastric tumors. Among differentially methylated genes, we identified new genes (CHRNA3 and DOK1) as targets of aberrant hypermethylation in GC, suggesting that epigenetic deregulation of these genes and their corresponding cellular pathways may promote the development and progression of GC. We also found that global demethylation of tumor cell genomes occurs in GC, consistent with the notion that abnormal hypermethylation of specific genes occurs concomitantly with genome-wide hypomethylation. Age and gender had no significant influence on methylation states, but an association was observed between LINE-1 and MLH1 methylation levels with histological sub-type and anatomical sub-site. This study identifies aberrant methylation patters in specific genes in GC thus providing information that could be exploited as novel biomarkers in clinics and molecular epidemiology of GC.

NF-kappaB protects human papillomavirus type 38 E6/E7-immortalized human keratinocytes against tumor necrosis factor alpha and UV-mediated apoptosis.

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.

Human papillomavirus type 16 E6 inhibits p21(WAF1) transcription independently of p53 by inactivating p150(Sal2).

HPV16 E6 deregulates G1/S cell cycle progression through p53 degradation preventing transcription of the CDK inhibitor p21(WAF1). However, additional mechanisms independent of p53 inactivation appear to exist. Here, we report that HPV16 E6 targets the cellular factor p150(Sal2), which positively regulates p21(WAF1) transcription. HPV16 E6 associates with p150(Sal2), inducing its functional inhibition by preventing its binding to cis elements on the p21(WAF1) promoter. A HPV16 E6 mutant, L110Q, which was unable to bind p150(Sal2), did not affect the ability of the cellular protein to bind p21(WAF1) promoter, underlining the linkage between these events. These data describe a novel mechanism by which HPV16 E6 induces cell cycle deregulation with a p53-independent pathway. The viral oncoprotein targets p150(Sal2), a positive transcription regulator of p21(WAF1) gene, preventing G1/S arrest and allowing cellular proliferation and efficient viral DNA replication.