RESUMEN
Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.
Asunto(s)
Proteínas de Unión al ADN/genética , Factor 4A Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/genética , Papillomavirus Humano 18/genética , Proteínas Oncogénicas Virales/genética , ARN Mensajero/genética , Regiones no Traducidas 5'/genética , Línea Celular Tumoral , Codón Iniciador/genética , Proteínas de Unión al ADN/biosíntesis , Femenino , Regulación Viral de la Expresión Génica/genética , Células HEK293 , Células HaCaT , Células HeLa , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Viral/genética , Serina-Treonina Quinasas TOR/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) is highly prevalent in HIV-positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross-sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross-sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki-67 (cellular proliferation marker) and HPV-E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki-67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki-67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited.
Asunto(s)
Canal Anal/metabolismo , Neoplasias del Ano/metabolismo , Biomarcadores de Tumor/biosíntesis , Carcinoma in Situ/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Infecciones por VIH/metabolismo , Homosexualidad Masculina/estadística & datos numéricos , Antígeno Ki-67/biosíntesis , Adulto , Alphapapillomavirus/metabolismo , Alphapapillomavirus/fisiología , Canal Anal/patología , Neoplasias del Ano/diagnóstico , Neoplasias del Ano/genética , Biomarcadores de Tumor/genética , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/genética , Estudios Transversales , Metilación de ADN , Infecciones por VIH/genética , Infecciones por VIH/virología , Humanos , Inmunohistoquímica/métodos , Masculino , Proteínas Oncogénicas Virales/biosíntesis , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Estudios RetrospectivosRESUMEN
Resveratrol is a representative polyphenol of dietderived putative cancer chemopreventive agents, which have attracted increasing interest in the cancer chemoprevention community. The inhibition of the action of human papillomavirus (HPV) E6 and E7 has been considered a key approach for cervical cancer therapy. Resveratrol has been shown to induce the apoptosis, and reduce both the viability and mitotic index of a number of cancer cell lines, including human cervical cancer cells. In the present study, it was confirmed that resveratrol inhibited the HPV E6 mRNA, HPV E6 protein and phosphorylated retinoblastoma protein (ppRb1) levels, and increased the p53 protein levels in HeLa and Ca Ski cells, as well as in subcutaneous tumor tissue grown from HeLa cells. Highrisk HPV uses a bicistronic RNA to control E6 and E7 genes simultaneously. On the whole, the present study demonstrates that resveratrol inhibits cervical cancer development by suppressing the transcription and translation of E6 and E7, and also by promoting the apoptosis and G1/S phase transition arrest. These findings may provide the basis for the development of resveratrol as a candidate for cervical cancer therapy.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Papillomavirus Humano 18/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Resveratrol/farmacología , Transcripción Genética/efectos de los fármacos , Neoplasias del Cuello Uterino/metabolismo , Femenino , Células HeLa , Humanos , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.
Asunto(s)
Proteínas de la Cápside , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales , Multimerización de Proteína , Proteínas Recombinantes de Fusión , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.
Asunto(s)
Anticuerpos/inmunología , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/inmunología , Proteínas Represoras/biosíntesis , Proteínas Represoras/inmunología , Animales , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Plásmidos , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunologíaRESUMEN
Human papillomaviruses (HPVs) produce a large number of alternatively spliced mRNAs, including a number of differently spliced mRNAs with the potential to produce E2 protein. To identify the alternatively spliced HPV16 mRNA with the highest ability to produce E2 protein, we have generated E2 cDNA expression plasmids representing the most common, alternatively spliced E2 mRNAs, and assessed their translational potential. Our results revealed that an mRNA initiated at the HPV16 late promoter p670 and spliced from the HPV16 5'-splice site SD880 to the HPV16 3'-splice site SA2709, located immediately upstream of the E2 ATG, produced higher levels of E2 than any of the other alternatively spliced, E2-encoding mRNAs. Utilization of a known, alternative 3'-splice site located upstream of the E2 ATG named SA2582, generated mRNAs with lower ability to produce E2 than mRNAs spliced to SA2709. Finally, analysis of HPV16 mRNA splicing demonstrated that SA2709 was more efficiently spliced to the upstream 5'-splice site SD880 than to the upstream 5'-splice site SD226. In conclusion, the HPV16 mRNA with the greatest ability to produce E2 protein is generated from the HPV16 late promoter and is spliced between HPV16 5'-splice site SD880 and HPV16 3'-splice site SA2709.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Empalme Alternativo , ARN Mensajero , ARN ViralRESUMEN
Colchicine, a plant-derived alkaloid with relatively low toxicity on normal human epidermal keratinocytes (HEKn), has selective inhibitory effect on the growth of CaSki (HPV16-positive) and HeLa (HPV18-positive) human cervical cancer cell lines via the induction of apoptosis. Colchicine (2.5, 5.0 and 10.0 ng/ml) significantly reduced the expression of human papilloma virus (HPV) 16 E6/E7 mRNA and protein in CaSki and HeLa cells. Moreover, reduced expression of E6 and E7 induced by Colchicine resulted in the up-regulation of tumor suppressor proteins, p53 and Rb, as well as down-regulation of phospho Rb (pRb) protein. In addition, Bax, cytosolic cytochrome c and cleaved caspase-3 protein were increased while Bcl-2 protein was decreased significantly by 48 h of Colchicine treatment. These results implied that Colchicine could be explored as a potent candidate agent for the treatment and prevention of HPV-associated cervical cancer without deleterious effects.
Asunto(s)
Colchicina/farmacología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/virología , Apoptosis/efectos de los fármacos , Femenino , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificación , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/efectos de los fármacos , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma/biosíntesis , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Previous microRNA (miR) microarray analysis revealed that miR218 is downregulated in cervical cancer tissues. The present study aimed to further evaluate the expression of miR218 in cervical cancer specimens, determine the association between its expression with disease progression, and investigate the roles of miR218 in cervical cancer cells. Tissue specimens were obtained from 80 patients with cervical squamous cell carcinoma, 30 patients with highgrade cervical intraepithelial neoplasia [(CIN) II/III] and 15 patients with lowgrade CIN (CINI); in addition, 60 plasma samples were obtained from patients with cervical cancer, and 15 normal cervical tissue specimens and 30 plasma samples were obtained from healthy women. These samples were used for analysis of miR218 expression via reverse transcription-quantitative PCR. In addition, tumor cells were transfected with miR218 mimics, human papillomavirus (HPV)16 E6/E7 small interfering RNA, or their respective negative controls to determine the viability, colony formation, migration and invasion of cells using MTT, colony formation, wound healing and Transwell assays, respectively. Target genes of miR218 were bioinformatically predicted and analyzed using Gene Ontology (GO) terms. The results revealed that miR218 was downregulated in the tumor tissues and plasma of patients with cervical cancer, with expression associated with the advanced clinicopathological characteristics of patients, including HPV positivity, tumor size, blood vessel invasion and lymph node metastasis. Furthermore, miR218 overexpression reduced tumor cell viability and xenograft growth, and suppressed tumor cell migration and invasion. HPV was detected in 75% of the 80 patients with cervical cancer, and HPV positivity was inversely associated with miR218 expression. In addition, bioinformatics analysis predicted that roundabout guidance receptor 1 (ROBO1) was a target gene of miR218; miR218 overexpression significantly reduced ROBO1 levels. Furthermore, GO analysis revealed that ROBO1 was involved in regulating cell proliferation, adhesion and migration, and the cell cycle. In conclusion, the findings of the present study suggested that miR218 may possess antitumor activities in cervical cancer.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Genes Supresores de Tumor , ARN Neoplásico/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Femenino , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , ARN Neoplásico/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3'UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3'UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif "AUUUA". Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3'UTR, indicating that the AU-rich elements of the 3'UTR are not contributing to RNA stability. Instead, the three "AUUUA" motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.
Asunto(s)
Transformación Celular Viral , Queratinocitos/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Estabilidad del ARN , ARN Viral/metabolismo , Proteínas Represoras/biosíntesis , Neoplasias del Cuello Uterino/metabolismo , Adenoviridae , Fusión Celular , Femenino , Vectores Genéticos , Humanos , Queratinocitos/patología , Proteínas Oncogénicas Virales/genética , ARN Viral/genética , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND: Increasing evidence indicates an etiological role of human papillomavirus (HPV) in head and neck cancers, particularly oropharyngeal squamous cell carcinoma (OPSCC). However, the association between HPV and other cancers, including esophageal and tongue remains unclear. This study delineated the molecular characteristics of HPV18 E6 and E7 in esophageal (EC109 and EC9706) and tongue (Tca83) cancer cell lines with reference to cervical cancer (HeLa). METHODS: We analysed the HPV transcription profiles of esophageal and tongue cancer cells through Next-generation RNA sequencing, and the role of HPV18 E6 and E7 in these cells was assessed via siRNA approach, Western blotting and immunofluorescence assays. RESULTS: Overall, the HPV transcription profiles of esophageal and tongue cancer cells mimicked that of cervical cancer cells, with notable disruption of E2, and expression of E6, spliced E6 (E6*), E7, E1 and L1 transcripts. As with cervical cancer cells, p53 and its downstream transactivation target, p21, were found to be the major targets of E6 in esophageal and tongue cancer cell lines. Intriguingly, E7 preferentially targeted p130 in the two esophageal cancer cell lines, instead of pRb as in cervical cancer. Tca83 exhibited an E7 to E6 transcript ratio comparable to HeLa (cervix), targeted the ERK1/2 and MMP2 pathways, and was dependent on E6 and E7 to survive and proliferate. In contrast, both the esophageal cancer cell lines were distinct from HeLa in these aspects. CONCLUSIONS: This is the first study that delineates transcript expression and protein interaction of HPV18 E6 and E7 in esophageal and tongue cancer cell lines, suggesting that HPV plays a role in inducing these cancers, albeit via distinct pathways than those observed in cervical cancer.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Neoplasias Esofágicas/virología , Carcinoma de Células Escamosas de Esófago/virología , Papillomavirus Humano 18/fisiología , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Neoplasias de la Lengua/virología , Línea Celular Tumoral , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Células HeLa , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas E7 de Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Neoplasias de la Lengua/metabolismo , Neoplasias de la Lengua/patologíaRESUMEN
The homeodomain transcription factor SIX1 plays a critical role in embryogenesis, is not expressed in normal adult tissue, but is expressed in many malignancies, including cervical cancer. SIX1 drives the progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward malignancy: HKc/HPV16 express high levels of SIX1 mRNA and protein; overexpression of SIX1 in HKc/HPV16 produces pre-malignant, differentiation-resistant lines (HKc/DR); SIX1 overexpression in HKc/DR induces tumorigenicity. In this paper, we explore the consequences of inhibition of SIX1 expression in premalignant HKc/DR. Only partial inhibition of SIX1 expression could be obtained in HKc/DR by RNA interference. Decreased SIX1 expression (up to 80%) in HKc/DR resulted in slower proliferation, decreased HPV16-E6/E7 mRNA levels, and increased p53 protein levels. Gene expression changes induced in HKc/DR by anti-SIX1 shRNA were indicative of mesenchymal-epithelial transition (MET) and changes in TGF-beta signaling. We conclude that HPV16-transformed cells depend on SIX1 for survival, HPV16 E6/E7 gene expression and epithelial-mesenchymal transition.
Asunto(s)
Transformación Celular Viral , Proteínas de Homeodominio/metabolismo , Interacciones Huésped-Patógeno , Papillomavirus Humano 16/crecimiento & desarrollo , Queratinocitos/virología , Proteínas Oncogénicas Virales/biosíntesis , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas Represoras/biosíntesis , Línea Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Silenciador del Gen , Humanos , Transducción de SeñalRESUMEN
BACKGROUND: Several studies have reported the oncogenic role of human papillomavirus (HPV) in non-melanoma skin cancer (NMSC) carcinogenesis. Considering that HPV could affect tumor protein 53 (TP53) degradation via E6 oncoprotein, we evaluated the expression of TP53 according to HPV infection and E6 expression. METHODS: Biopsy specimens from 79 NMSCs (28 squamous cell carcinomas, 21 keratoacanthomas and 30 basal cell carcinomas) were enrolled. Nested PCR was used to detect mucosal HPV (mHPV) DNA. Genotyping was performed by reverse line hybridization. Expression of TP53 and E6 was evaluated by immunohistochemistry. RESULTS: mHPVs were detected in 34.2% (27/79) of NMSC, with 92.6% (25/27) of high-risk HPV (HR-HPV) types. HPV16-E6-positive expression was observed in all HPV16-positive samples. TP53 high expression was found in 51.4% (37/72) of specimens. In this group, 78.4% were HPV-negative (P = 0.014). TP53 expression was negative in 8/10 of HPV E6-positive specimens. Multivariate analysis showed that TP53 was associated with HPV infection independently of histopathologic type (P = 0.005). CONCLUSION: This study showed a high prevalence of mHPV in NMSC. Active infections assessed by E6 expression are associated with loss of p53 function, highlighting the involvement of mHPV in NMSC carcinogenesis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Infecciones por Papillomavirus , Proteínas Represoras/biosíntesis , Neoplasias Cutáneas , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Túnez/epidemiologíaRESUMEN
Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of the downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was associated with the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.
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Alphapapillomavirus/genética , Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Alphapapillomavirus/metabolismo , Cisplatino/farmacología , Daño del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Inestabilidad Genómica , Células HEK293 , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Oncogenes , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas E7 de Papillomavirus/genética , Cultivo Primario de Células , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Transducción de Señal , UbiquitinaciónRESUMEN
Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1ÌE4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Proteínas de Unión al ADN/metabolismo , Queratinocitos/metabolismo , Queratinocitos/virología , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/metabolismo , Animales , Autofagia , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Humanos , Ratones , Células 3T3 NIH , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Técnicas de Cultivo de Órganos , Piel/citología , Piel/metabolismo , Piel/virología , Transcripción GenéticaRESUMEN
Hypoxia is linked to therapeutic resistance and poor clinical prognosis for many tumor entities, including human papillomavirus (HPV)-positive cancers. Notably, HPV-positive cancer cells can induce a dormant state under hypoxia, characterized by a reversible growth arrest and strong repression of viral E6/E7 oncogene expression, which could contribute to therapy resistance, immune evasion and tumor recurrence. The present work aimed to gain mechanistic insights into the pathway(s) underlying HPV oncogene repression under hypoxia. We show that E6/E7 downregulation is mediated by hypoxia-induced stimulation of AKT signaling. Ablating AKT function in hypoxic HPV-positive cancer cells by using chemical inhibitors efficiently counteracts E6/E7 repression. Isoform-specific activation or downregulation of AKT1 and AKT2 reveals that both AKT isoforms contribute to hypoxic E6/E7 repression and act in a functionally redundant manner. Hypoxic AKT activation and consecutive E6/E7 repression is dependent on the activities of the canonical upstream AKT regulators phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) complex 2 (mTORC2). Hypoxic downregulation of E6/E7 occurs, at least in part, at the transcriptional level. Modulation of E6/E7 expression by the PI3K/mTORC2/AKT cascade is hypoxia specific and not observed in normoxic HPV-positive cancer cells. Quantitative proteome analyses identify additional factors as candidates to be involved in hypoxia-induced activation of the PI3K/mTORC2/AKT signaling cascade and in the AKT-dependent repression of the E6/E7 oncogenes under hypoxia. Collectively, these data uncover a functional key role of the PI3K/mTORC2/AKT signaling cascade for viral oncogene repression in hypoxic HPV-positive cancer cells and provide new insights into the poorly understood cross talk between oncogenic HPVs and their host cells under hypoxia.IMPORTANCE Oncogenic HPV types are major human carcinogens. Under hypoxia, HPV-positive cancer cells can repress the viral E6/E7 oncogenes and induce a reversible growth arrest. This response could contribute to therapy resistance, immune evasion, and tumor recurrence upon reoxygenation. Here, we uncover evidence that HPV oncogene repression is mediated by hypoxia-induced activation of canonical PI3K/mTORC2/AKT signaling. AKT-dependent downregulation of E6/E7 is only observed under hypoxia and occurs, at least in part, at the transcriptional level. Quantitative proteome analyses identify additional factors as candidates to be involved in AKT-dependent E6/E7 repression and/or hypoxic PI3K/mTORC2/AKT activation. These results connect PI3K/mTORC2/AKT signaling with HPV oncogene regulation, providing new mechanistic insights into the cross talk between oncogenic HPVs and their host cells.
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Hipoxia , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Papillomaviridae/fisiología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Línea Celular Tumoral , Regulación hacia Abajo , Interacciones Huésped-Patógeno , HumanosRESUMEN
Recent studies have demonstrated low specificity (false positive) of human papillomavirus (HPV) DNA testing for the screening and diagnosis of cervical samples. Therefore, we evaluated the performance of the HPV OncoCheck assay, which is an HPV E6/E7 mRNA-based assay, for the detection of 16 high-risk (HR)-HPVs including HPV 16 and HPV 18 genotypes in cervical samples using multiplex reverse transcriptase-quantitative PCR. In the present study, the analytical performance of the assay was evaluated using 16 HPV single strand DNAs. Clinical evaluation was performed using 319 Thinprep® liquid-based cytology samples obtained from women with cervical diseases, and the HPV OncoCheck assay results were compared with those of cytological diagnosis and sequence analysis. All 16 types of HPVs were detected with a minimum detection sensitivity of 100 copies per reaction and high specificity was observed. The sensitivity and specificity of the HPV OncoCheck assay for detecting high-grade lesions were 94.1% (95% confidence interval (CI), 0.875-0.975; pâ¯<â¯.0001) and 95.4% (95% CI, 0.868-0.989; pâ¯<â¯.0001), and sequence analysis were 99.4% (95% CI, 0.965-0.999; pâ¯<â¯.0001), and 98% (95% CI, 0.939-0.996; pâ¯<â¯.0001), respectively. Moreover, the agreement between the HPV OncoCheck assay and sequence analysis for the detection of HR-HPV was 98.8% (κâ¯=â¯0.98, 95% CI 0.967-0.996; pâ¯<â¯.0001). The results of this study showed high agreement and specificity with cytological diagnoses and sequence analysis. Future studies with histological follow- up are needed to determine whether use of the HPV OncoCheck assay in cervical screening may aid detection of the most significant cervical disease while reducing false-positive results.
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Pruebas de ADN del Papillomavirus Humano/métodos , Prueba de Papanicolaou , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Frotis Vaginal , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , ADN Complementario/análisis , ADN Viral/análisis , Femenino , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , ARN Mensajero/análisis , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Riesgo , Sensibilidad y Especificidad , Análisis de Secuencia de ARN , Manejo de Especímenes , Adulto JovenRESUMEN
Human papillomavirus (HPV) is a necessary but insufficient cause of a subset of oral squamous cell carcinomas (OSCCs) that is increasing markedly in frequency. To identify contributory, secondary genetic alterations in these cancers, we used comprehensive genomics methods to compare 149 HPV-positive and 335 HPV-negative OSCC tumor/normal pairs. Different behavioral risk factors underlying the two OSCC types were reflected in distinctive genomic mutational signatures. In HPV-positive OSCCs, the signatures of APOBEC cytosine deaminase editing, associated with anti-viral immunity, were strongly linked to overall mutational burden. In contrast, in HPV-negative OSCCs, T>C substitutions in the sequence context 5'-ATN-3' correlated with tobacco exposure. Universal expression of HPV E6*1 and E7 oncogenes was a sine qua non of HPV-positive OSCCs. Significant enrichment of somatic mutations was confirmed or newly identified in PIK3CA, KMT2D, FGFR3, FBXW7, DDX3X, PTEN, TRAF3, RB1, CYLD, RIPK4, ZNF750, EP300, CASZ1, TAF5, RBL1, IFNGR1, and NFKBIA Of these, many affect host pathways already targeted by HPV oncoproteins, including the p53 and pRB pathways, or disrupt host defenses against viral infections, including interferon (IFN) and nuclear factor kappa B signaling. Frequent copy number changes were associated with concordant changes in gene expression. Chr 11q (including CCND1) and 14q (including DICER1 and AKT1) were recurrently lost in HPV-positive OSCCs, in contrast to their gains in HPV-negative OSCCs. High-ranking variant allele fractions implicated ZNF750, PIK3CA, and EP300 mutations as candidate driver events in HPV-positive cancers. We conclude that virus-host interactions cooperatively shape the unique genetic features of these cancers, distinguishing them from their HPV-negative counterparts.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Proteínas de Neoplasias , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Femenino , Humanos , Masculino , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Neoplasias de la Boca/virología , Mutación , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Papillomaviridae/metabolismoRESUMEN
Scarce data are available on the expression of papillomavirus genome and the frequency of alternatively spliced E6E7 mRNAs in invasive cervical cancer. We carried out a comprehensive characterization of HPV expression by RNA-Seq analysis in 22 invasive cervical cancer with HPV16 or HPV18, characterizing the presence of integrated/episomal viral DNA, the integration sites in human genome and the proportion of alternative splicing products of E6 and E7 genes. The expression patterns suggested the presence of episomal and/or integrated viral DNA, with integration detected in most tumors, frequently occurring within human genes in HPV18+ and in intergenic regions in HPV16+ tumors. Alternative splicing of E6E7 transcripts showed E6*I as the most frequent isoform for both viral types, followed by E6*II and E6/E7 (unspliced) transcripts in HPV16+, and by E6/E7 in HPV18+ tumors. Previously described E6*VI and E6*V transcript isoforms for HPV16, and E6*X for HPV18, were rare or not detected.
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Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , RNA-Seq , Proteínas Represoras , Neoplasias del Cuello Uterino , Integración Viral , Empalme Alternativo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/biosíntesis , Proteínas E7 de Papillomavirus/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Although a vast amount of research underlines the roles of the HR HPV E6 and E7 oncogenes in HPV-induced carcinogenesis of cervical cancer, it remains unclear whether these oncogenes are also involved in the resistance of the cancer against chemotherapy. We examined the role of the HPV16 E6 oncogene in cisplatin resistance by analyzing its expression in newly established cisplatin-sensitive versus -resistant cervical cancer cell lines (CC7, CC10). Resistant variants were obtained by interval exposure treatment with 1-2 µM cisplatin for 8-9 months. Our results demonstrate that the expression level of HPV16 E6 directly correlates with the extent of cisplatin resistance in novel as well as established (SiHa) drug resistant cervical cancer cell lines. Overexpression of HPV16 E6 in cisplatin-naïve cells rendered these cells more resistant to cisplatin. Reducing E6 expression by JQ1 treatment reversed the drug resistant phenotype and strongly enhanced chemoresponse only in HPV-positive cisplatin-resistant variants and not in HPV-negative C33A cervical cancer cells. The level of E6 directly correlated with the extent of cisplatin sensitivity and was shown to be increased in newly established drug-resistant cell line variants, while reducing E6 expression using Brd4-inhibitors enhanced chemoresponse when co-delivered with cisplatin. Inhibition of Brd4 could represent a new therapeutic option by increasing treatment response in cervical cancer cells and might allow lower cisplatin dosages, thus reducing negative side effects.
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Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Papillomavirus Humano 16/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Represoras/biosíntesis , Factores de Transcripción/antagonistas & inhibidores , Neoplasias del Cuello Uterino/terapia , Neoplasias del Cuello Uterino/virología , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos/fisiología , Femenino , Humanos , Triazoles/farmacologíaRESUMEN
Here we show that treatment of the HPV16-positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan-induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down-regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1-negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half-life of the HPV16 E6 and E7 mRNAs renders HPV16-driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.