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1.
Artigo em Inglês | MEDLINE | ID: mdl-31631454

RESUMO

A considerable proportion of high grade cervical intraepithelial lesions (CIN2/3) are known to resolve on their own especially among young women. However, since reliable prognostic markers are still lacking, the diagnosis "CIN3" is still an indication for surgery which may result in overtreatment. It is conceivable that a combination of different, ideally independent molecular markers may provide more reliable results. In the present cross-sectional study two established triage markers, 3q26 amplification and a methylation signature, were evaluated in an age-dependent manner. The patient cohort comprised 60 patients with histologically confirmed CIN2/3 in two equally sized age groups (<30 years, ≥30 years). Cervical scrapes were analyzed by interphase fluorescence in situ hybridization for 3q26 amplification and methylation specific PCR (GynTect®) for six different genome regions. Both assays showed a significantly different pattern of test outcome independent of age (P=0.001). Moreover, the combination of both assays differed significantly for double positive and double negative cases when comparing the two age groups: In patients <30 years there were clearly less cases with positive methylation signature and amplification of 3q26 as in women ≥30 years (23% versus 63%, Bonferroni adjusted P=0.016). Of particular interest is the finding that double negative results were exclusive for the young age group (0% versus 27%, Bonferroni adjusted P=0.020). Since regression of CIN2/3 characteristically occurs among young women it is tempting to speculate that a double negative test result could be prognostic for regression of CIN2/3. This will have to be investigated further in a prospective longitudinal intervention study. This article is protected by copyright. All rights reserved.

2.
Philos Trans R Soc Lond B Biol Sci ; 374(1773): 20180295, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-30955486

RESUMO

Antibodies to the E6 and E7 oncoproteins of high-risk human papillomavirus (HPV) types are strongly associated with HPV-driven cancer, while antibodies against the capsid protein L1 are considered cumulative exposure markers. To test the hypothesis that L1 antibody levels are stable over time, whereas E6 and E7 levels undergo decay after cervical cancer (CxCa) treatment, we performed multiplex serology for HPV16 and 18 antigens E6, E7 and L1 in a post-treatment study of 184 patients with invasive CxCa that were characterized with a median follow-up time of 725 days, and 2-12 sera per patient. Antibody titers significantly decreased within the first six months for HPV16 E6 and E7 but not L1, and stabilized for the following 12 months on a high level, with few patients showing seroreversion. Of 67 patients seropositive for HPV16 E6 at diagnosis, 28 (41.8%) showed a decrease in antibody titers of at least 50% within the first 18 months. Similarly, of 50 HPV16 E7 seropositives, 33 (66.0%) showed decreasing antibody levels, whereas antibody decay was less frequent for HPV16 L1 (12 of 47, 25.5%). Using a power-law mathematical model to characterize antibody decay kinetics, the mean (±s.e.) durations to a 50% reduction in antibody titers within individual patients were estimated to be 56.9 (±26.1) and 56.3 (±19.0) days for HPV16 E6 and E7, respectively. In summary, HPV16 E6 and E7 antibodies undergo a slow but significant decrease in antibody titers within the first 6-18 months following CxCa treatment. However, larger studies are needed to confirm the utility of serology for prediction of disease progression and time to relapse based on antibody decay kinetics. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

3.
MBio ; 10(1)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755508

RESUMO

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.


Assuntos
Hipóxia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Oncogênicas Virais/biossíntese , Papillomaviridae/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Regulação para Baixo , Interações Hospedeiro-Patógeno , Humanos
4.
Dalton Trans ; 48(3): 936-944, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30565617

RESUMO

After more than 50 years of platinum-based anticancer research only three compounds are in clinical use worldwide. The use of the well-known lead compound of this class of anticancer agents, cisplatin, is limited by its side effects and varying resistance mechanisms. Therefore, we report on platinum(ii) compounds with asparagusic acid derivatives as ligands which show interesting anticancer results on cisplatin resistant cell lines.


Assuntos
Antineoplásicos/farmacologia , Compostos Organoplatínicos/farmacologia , Tiofenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química , Relação Estrutura-Atividade , Tiofenos/química
5.
Nat Commun ; 9(1): 3166, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30093612

RESUMO

Endometrial cancer is the most commonly diagnosed cancer of the female reproductive tract in developed countries. Through genome-wide association studies (GWAS), we have previously identified eight risk loci for endometrial cancer. Here, we present an expanded meta-analysis of 12,906 endometrial cancer cases and 108,979 controls (including new genotype data for 5624 cases) and identify nine novel genome-wide significant loci, including a locus on 12q24.12 previously identified by meta-GWAS of endometrial and colorectal cancer. At five loci, expression quantitative trait locus (eQTL) analyses identify candidate causal genes; risk alleles at two of these loci associate with decreased expression of genes, which encode negative regulators of oncogenic signal transduction proteins (SH2B3 (12q24.12) and NF1 (17q11.2)). In summary, this study has doubled the number of known endometrial cancer risk loci and revealed candidate causal genes for future study.

6.
PLoS One ; 13(7): e0197561, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29979793

RESUMO

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations.

7.
Cancer Med ; 7(5): 1978-1987, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29608257

RESUMO

Epidemiological, biological, and molecular data suggest links between endometriosis and endometrial cancer, with recent epidemiological studies providing evidence for an association between a previous diagnosis of endometriosis and risk of endometrial cancer. We used genetic data as an alternative approach to investigate shared biological etiology of these two diseases. Genetic correlation analysis of summary level statistics from genomewide association studies (GWAS) using LD Score regression revealed moderate but significant genetic correlation (rg  = 0.23, P = 9.3 × 10-3 ), and SNP effect concordance analysis provided evidence for significant SNP pleiotropy (P = 6.0 × 10-3 ) and concordance in effect direction (P = 2.0 × 10-3 ) between the two diseases. Cross-disease GWAS meta-analysis highlighted 13 distinct loci associated at P ≤ 10-5 with both endometriosis and endometrial cancer, with one locus (SNP rs2475335) located within PTPRD associated at a genomewide significant level (P = 4.9 × 10-8 , OR = 1.11, 95% CI = 1.07-1.15). PTPRD acts in the STAT3 pathway, which has been implicated in both endometriosis and endometrial cancer. This study demonstrates the value of cross-disease genetic analysis to support epidemiological observations and to identify biological pathways of relevance to multiple diseases.

8.
Int J Mol Sci ; 19(2)2018 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-29439548

RESUMO

This study was designed to explore the role of human papillomavirus (HPV) in esophageal squamous cell carcinoma (ESCC). Fifty-five patients receiving diagnostic upper gastrointestinal endoscopy at Zomba Central Hospital or Queen Elizabeth Hospital in Blantyre (Malawi) in 2010, were included in our study. Formalin-fixed paraffin-embedded biopsies were collected for histopathological diagnosis. HPV DNA was detected using multiplex Quantitative PCR (qPCR) and in situ hybridization (ISH). p16INK4a staining served as a surrogate marker for HPV oncogene activity. Cell proliferation was determined by Ki-67 staining. Human immunodeficiency virus (HIV) status was evaluated by serology. Data on the consumption of alcohol and tobacco, and history of tuberculosis (TBC), oral thrush, and Herpes zoster, were obtained by questionnaire. Forty patients displayed ESCC, three displayed dysplastic epithelium, and 12 displayed normal epithelium. HPV16 was detected in six ESCC specimens and in one dysplastic lesion. Among HPV-positive patients, viral load varied from 0.001 to 2.5 copies per tumor cell. HPV DNA presence could not be confirmed by ISH. p16INK4a positivity correlated with the presence of HPV DNA (p = 0.03). Of particular note is that the Ki-67 proliferation index, in areas with diffuse nuclear or cytoplasmatic p16INK4a staining ≥50%, was significantly higher in HPV-positive tumors compared to the corresponding p16INK4a stained areas of HPV-negative tumors (p = 0.004). HPV infection in ESCC was not associated with the consumption of tobacco or alcohol, but there were significantly more patients drinking locally brewed alcohol among HPV-positive tumor patients compared to non-tumor patients (p = 0.02) and compared to HPV-negative tumor patients (p = 0.047). There was no association between HIV infection, history of TBC, Herpes zoster, oral thrush, or HPV infection, in ESCC patients. Our indirect evidence for viral oncogene activity is restricted to single tumor cell areas, indicative of the role of HPV16 in the development of ESCC. The inhomogeneous presence of the virus within the tumor is reminiscent of the "hit and run" mechanism discussed for ß-HPV types, such as HPV38.


Assuntos
Carcinoma de Células Escamosas/virologia , Neoplasias Esofágicas/virologia , Infecções por Papillomavirus/epidemiologia , Adulto , Idoso , Carcinoma de Células Escamosas/complicações , Neoplasias Esofágicas/complicações , Feminino , Papillomavirus Humano 16 , Humanos , Malaui , Masculino , Pessoa de Meia-Idade
9.
Int J Mol Sci ; 19(1)2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-29342962

RESUMO

(1) Background: Epithelial ovarian cancer (EOC) is the most lethal cancer of the female reproductive system. In an earlier study, we identified multiple genes as hypermethylated in tumors of patients with poor prognosis. The most promising combination of markers to predict a patient's outcome was CaMKIINα and RUNX3. Aim of this study was to functionally validate the importance of both genes. (2) Methods: IC50 measurements, cell cycle distribution-, proliferation, and migration experiments were conducted after transgene overexpression in two EOC cell lines. (3) Results: We showed that CaMKIINα has tumor suppressive functions in vitro and reduces proliferation, migration, and colony formation. However, it had no effect on the reversion of the resistance to cisplatin. RUNX3 exhibited dualistic functions related to cisplatin sensitivity and migration capacity, depending on the respective transcript variant (TV). A2780 cells expressing RUNX3 TV2-the promoter of which harbors a CpG (5'-C-phosphate-G-3') island and is potentially inactivated by hypermethylation-exhibited increased cisplatin sensitivity and reduced migration properties. However, RUNX3 TV1, not affected by CpG island methylation could be characterized as mediating resistance and enhancing migration in A2780. The higher resistance of RUNX3 TV1 transfected cells correlates with a reduction of cell proliferation. Moreover, RUNX3 TV1 expressing cells exhibit a reduced cell cycle arrest at the gap-2 or mitosis phase (G2/M) under cisplatin treatment comparable to resistant A2780 subcultures. (4) Conclusion: It appears that CaMKIINα and RUNX3 TV2 can reduce the malignant potential of EOC cells.


Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoflavonas/farmacologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteínas/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Biologia Computacional/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Ectópica do Gene , Feminino , Humanos , Regiões Promotoras Genéticas , Proteínas/genética , Transcrição Genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
10.
PLoS Pathog ; 14(1): e1006783, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29324843

RESUMO

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.


Assuntos
Carcinogênese/efeitos da radiação , Neoplasias Induzidas por Radiação/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Neoplasias Cutâneas/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Proteínas Virais/metabolismo , Animais , Betapapillomavirus/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Epiderme/efeitos da radiação , Feminino , Deleção de Genes , Genes p53/efeitos da radiação , Camundongos , Camundongos Transgênicos , Mutagênese/efeitos da radiação , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Induzidas por Radiação/patologia , Proteínas Oncogênicas Virais/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos da radiação , Proteínas Virais/genética
11.
BMC Res Notes ; 10(1): 532, 2017 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084579

RESUMO

BACKGROUND: Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. METHODS: Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. RESULTS: For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. CONCLUSIONS: Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.


Assuntos
Papillomaviridae , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Linfonodo Sentinela/virologia , Neoplasias do Colo do Útero/virologia , Biomarcadores , Linhagem Celular Tumoral , Feminino , Humanos , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , RNA Mensageiro , RNA Viral/isolamento & purificação , Recidiva , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
Clin Epigenetics ; 9: 118, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29090037

RESUMO

BACKGROUND: HPV DNA testing as a primary screening marker is being implemented in several countries. Due to the high HPV prevalence in the screening population, effective triage strategies for HPV-positive cases are required. The aim of this study was to evaluate the performance of a methylation-specific real-time PCR  assay (GynTect®) comprising six marker regions as a triage test. RESULTS: An analytical sensitivity of 0.1 ng genomic DNA corresponding to 15 SiHa cells was achieved. Absolute specificity was observed in the presence of 20 ng unmethylated genomic DNA. In a clinical setting, cervical scrapes of 306 women showing abnormal colposcopy were tested for cytology, HPV positivity, and the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Of all women, histopathological data were available. The overall sensitivity for GynTect to detect CIN3+ was 67.7% (95% CI 57.3%-77.1%) whereas sensitivity was significantly higher for women of age ≥ 30 years (p = 0.04). All cancer cases (n = 5) were detected by GynTect. The overall false positive rate (= 1-specificity) for women with no CIN was 17.4% (95% CI 12.5-23.1%), with a higher proportion among HPV-positive women (24.0%, 95% CI 16.0-33.6%). In a triage screening setting, where all women underwent HPV testing and the HPV positives in addition GynTect testing, the overall sensitivity would slightly decline but specificity would reach the maximum value of 88.7% (95% CI 83.7-92.6%). CONCLUSION: The GynTect® assay is a robust easy to use assay with high analytical sensitivity and specificity. Moreover, the performance of the assay based on cervical scrapes provides further evidence for the usefulness of methylation markers to detect HPV-positive women with clinically relevant disease.


Assuntos
Metilação de DNA , Papillomavirus Humano 16/genética , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/virologia , Adulto , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Feminino , Humanos , Programas de Rastreamento , Pessoa de Meia-Idade , Infecções por Papillomavirus/genética , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Triagem , Neoplasias do Colo do Útero/genética
13.
Oncotarget ; 8(39): 64670-64684, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-29029385

RESUMO

We previously identified associations with ovarian cancer outcome at five genetic loci. To identify putatively causal genetic variants and target genes, we prioritized two ovarian outcome loci (1q22 and 19p12) for further study. Bioinformatic and functional genetic analyses indicated that MEF2D and ZNF100 are targets of candidate outcome variants at 1q22 and 19p12, respectively. At 19p12, the chromatin interaction of a putative regulatory element with the ZNF100 promoter region correlated with candidate outcome variants. At 1q22, putative regulatory elements enhanced MEF2D promoter activity and haplotypes containing candidate outcome variants modulated these effects. In a public dataset, MEF2D and ZNF100 expression were both associated with ovarian cancer progression-free or overall survival time. In an extended set of 6,162 epithelial ovarian cancer patients, we found that functional candidates at the 1q22 and 19p12 loci, as well as other regional variants, were nominally associated with patient outcome; however, no associations reached our threshold for statistical significance (p<1×10-5). Larger patient numbers will be needed to convincingly identify any true associations at these loci.

14.
Oncotarget ; 8(31): 50930-50940, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28881617

RESUMO

We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10-3). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.

15.
Int J Mol Sci ; 18(10)2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-28937589

RESUMO

The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.


Assuntos
Biomarcadores Tumorais/análise , DNA Tumoral Circulante/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Sistema Livre de Células , DNA Viral/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas
16.
Int J Cancer ; 141(8): 1600-1614, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28670762

RESUMO

Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cisplatino/farmacologia , Dano ao DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Adutos de DNA/biossíntese , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fase G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pontos de Checagem da Fase M do Ciclo Celular , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Proteoma/metabolismo , Células Tumorais Cultivadas
17.
Proc Natl Acad Sci U S A ; 114(6): E990-E998, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28115701

RESUMO

Oncogenic human papillomaviruses (HPVs) are closely linked to major human malignancies, including cervical and head and neck cancers. It is widely assumed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes, that their intracellular p53 levels are reconstituted on E6/E7 repression, and that E6/E7 inhibition phenotypically results in cellular senescence. Here we show that hypoxic conditions, as are often found in subregions of cervical and head and neck cancers, enable HPV-positive cancer cells to escape from these regulatory principles: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does not result in cellular senescence, owing to hypoxia-associated impaired mechanistic target of rapamycin (mTOR) signaling via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also interfered with the senescence response of hypoxic HPV-positive cancer cells toward prosenescent chemotherapy in vitro. Collectively, these findings indicate that hypoxic HPV-positive cancer cells can induce a reversible state of dormancy, with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoirs for tumor recurrence on reoxygenation.


Assuntos
Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Papillomaviridae/genética , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HCT116 , Células HeLa , Células Hep G2 , Interações Hospedeiro-Patógeno/genética , Humanos , Hipóxia , Células MCF-7 , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Papillomaviridae/fisiologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
18.
Br J Cancer ; 116(4): 524-535, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28103614

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors (TFs) critical to somatic tumorigenesis. METHODS: All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery), and combined (9627 cases/30 845 controls; including additional individuals). RESULTS: The PAX8-target gene set was ranked 1/615 in the discovery (PGSEA<0.001; FDR=0.21), 7/615 in the replication (PGSEA=0.004; FDR=0.37), and 1/615 in the combined (PGSEA<0.001; FDR=0.21) studies. Adding other genes reported to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P<10-5 (including six with P<5 × 10-8). The pathway was also associated with differential gene expression after shRNA-mediated silencing of PAX8 in HeyA8 (PGSEA=0.025) and IGROV1 (PGSEA=0.004) SOC cells and several PAX8 targets near SOC risk loci demonstrated in vitro transcriptomic perturbation. CONCLUSIONS: Putative PAX8 target genes are enriched for common SOC risk variants. This finding from our agnostic evaluation is of particular interest given that PAX8 is well-established as a specific marker for the cell of origin of SOC.


Assuntos
Transformação Celular Neoplásica/genética , Cistadenocarcinoma Seroso/genética , Amplificação de Genes , Loci Gênicos , Predisposição Genética para Doença , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Humanos , Metanálise como Assunto , Análise em Microsséries , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Polimorfismo de Nucleotídeo Único
19.
Mol Carcinog ; 56(6): 1578-1589, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28059468

RESUMO

Progression from human papillomavirus-induced premalignant cervical intraepithelial neoplasia (CIN) to cervical cancer (CC) is driven by genetic and epigenetic events. Our microarray-based expression study has previously shown that inter-α-trypsin-inhibitor heavy chain 5 (ITIH5) mRNA levels in CCs were significantly lower than in high-grade precursor lesions (CIN3s). Therefore, we aimed to analyze in depth ITIH5 expression during cervical carcinogenesis in biopsy material and cell culture. Moreover, functional analyses were performed by ectopic expression of ITIH5 in different cell lines. We were able to confirm the validity of our microarray differential expression data by qPCR, demonstrating a clear ITIH5 downregulation in CC as compared with CIN2/3 or normal cervix. ITIH5 protein loss, evaluated by immunohistochemistry, was evident in 81% of CCs, whereas ITIH5 showed weak to moderate cytoplasmic staining in 91% of CIN2/3 cases. In addition, ITIH5 was strongly reduced or absent in seven CC cell lines and in three immortalized keratinocyte cell lines. Moreover, ITIH5 mRNA loss was associated with ITIH5 promoter methylation. ITIH5 expression could be restored in CC cell lines by pharmacological induction of DNA demethylation and histone acetylation. Functionally, ITIH5 overexpression significantly suppressed proliferation of SW756 cells and further resulted in a significant reduction of colony formation and cell migration in both CaSki and SW756 tumor models, but had no effect on invasion. Remarkably, ITIH5 overexpression did not influence the phenotype of HeLa cells. Taken together, ITIH5 gene silencing is a frequent event during disease progression, thereby providing evidence for a tumor suppressive role in cervical carcinogenesis.


Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Ovário/patologia , Proteínas Secretadas Inibidoras de Proteinases/genética , Neoplasias do Colo do Útero/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Metilação de DNA , Regulação para Baixo , Feminino , Genômica , Humanos , Ovário/metabolismo , Regiões Promotoras Genéticas , Proteínas Secretadas Inibidoras de Proteinases/análise , Neoplasias do Colo do Útero/patologia
20.
Dalton Trans ; 45(47): 18876-18891, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27897281

RESUMO

We report on platinum(ii) complexes with different cinnamic acid derivatives as ligands with cytotoxic activity against Cisplatin resistant ovarian cancer cell line subcultures of SKOV3 and A2780. A typical mechanism of action for platinum(ii) complexes as Cisplatin itself is binding to the DNA and inducing double-strand breaks. We examined the biological behavior of these potential drugs with 9-methylguanine using NMR spectroscopic methods and their DNA damage potential including γH2AX-foci analyses. X-ray diffraction methods have been used to elucidate the molecular structures of the platinum(ii) complexes. Interactions with the model protein lysozyme have been evaluated by different techniques including UV-Vis absorption spectroscopy, fluorescence and X-ray crystallography.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos Organoplatínicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/síntese química , Cinamatos/química , Humanos , Ligantes , Estrutura Molecular , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/química
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