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1.
J Med Virol ; 96(5): e29521, 2024 May.
Article in English | MEDLINE | ID: mdl-38727013

ABSTRACT

Methylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex-determining region Y-box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under-researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV-DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV-DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid-based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43-0.62), 0.88 (95% CI: 0.80-0.97), and 0.88 (95% CI: 0.75-1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40-0.58), 0.80 (95% CI: 0.68-0.93), and 0.92 (95% CI: 0.811-1.00), respectively. In HPV16/18-negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.


Subject(s)
DNA Methylation , Paired Box Transcription Factors , Papillomavirus Infections , SOXB1 Transcription Factors , Triage , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Humans , Female , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Neoplasms/genetics , SOXB1 Transcription Factors/genetics , Adult , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/virology , Middle Aged , Triage/methods , Paired Box Transcription Factors/genetics , Papillomavirus Infections/diagnosis , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , Sensitivity and Specificity , Biomarkers, Tumor/genetics , China , Precancerous Conditions/diagnosis , Precancerous Conditions/genetics , Young Adult , Early Detection of Cancer/methods , Colposcopy
2.
PeerJ ; 12: e17376, 2024.
Article in English | MEDLINE | ID: mdl-38784389

ABSTRACT

Background: Condyloma acuminatum (CA) is caused by low-risk human papillomavirus, and is characterized by high recurrence after treatment. The RNA modification N6-methyladenosine (m6A) plays an important role during diverse viral infections, including high-risk HPV infection in cervical cancer. However, it is unclear whether low-risk HPV infection changes the RNA m6A methylation in CA. Methods: High-throughputm6A-sequencing was performed to profile the transcriptome-wide mRNA modifications of CA tissues infected by LR-HPVs and the paired normal tissues from CA patients. We further investigated the regulation of alternative splicing by RNA binding proteins (RBPs) with altered m6A modification and constructed a regulatory network among these RBPs, regulated alternative splicing events (RASEs) and regulated alternative splicing genes (RASGs) in CA. Results: The results show that the m6A level in CA tissues differed from that in the paired controls. Furthermore, cell cycle- and cell adhesion- associated genes with m6A modification were differentially expressed in CA tissues compared to the paired controls. In particular, seven RNA binding protein genes with specific m6A methylated sites, showed a higher or lower expression at the mRNA level in CA tissues than in the paired normal tissues. In addition, these differentially expressed RNA binding protein genes would regulate the alternative splicing pattern of apoptotic process genes in CA tissue. Conclusions: Our study reveals a sophisticated m6A modification profile in CA tissue that affects the response of host cells to HPV infection, and provides cues for the further exploration of the roles of m6A and the development of a novel treatment strategy for CA.


Subject(s)
Alternative Splicing , Condylomata Acuminata , RNA-Binding Proteins , Humans , Alternative Splicing/genetics , Condylomata Acuminata/genetics , Condylomata Acuminata/virology , Condylomata Acuminata/metabolism , Condylomata Acuminata/pathology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Female , Adenosine/analogs & derivatives , Adenosine/metabolism , Methylation , Adult , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptome , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Papillomavirus Infections/pathology , RNA Methylation
3.
Syst Biol Reprod Med ; 70(1): 113-123, 2024 Dec.
Article in English | MEDLINE | ID: mdl-38743820

ABSTRACT

As demonstrated in previous research, hsa_circ_0052602 (circODC1) is dynamically expressed in HPV-positive cervical cancer (CC). CircODC1 expression was quantified using qRT-PCR, and its role in CC cell growth was assessed via loss-of-function assays. Interactions between miR-607 and circODC1 or ODC1 were confirmed using bioinformatics and mechanistic assays. The association of FOXA1 with the circODC1 promoter was validated through ChIP and luciferase reporter assays. CircODC1 was highly expressed in HPV-positive CC cell lines, and its depletion significantly impeded malignant processes such as proliferation, migration, and invasion. We found that ODC1 also played an oncogenic role in HPV-positive CC cells. CircODC1 was shown to positively regulate ODC1 as a ceRNA, competitively binding to miR-607 to counteract its suppression of ODC1. HPV-associated FOXA1 was identified as a potential transcription factor of circODC1. Restoration experiments showed that overexpression of circODC1 could counterbalance the inhibitory effect of FOXA1 knockdown. These findings offer new insights into therapeutic strategies for HPV-positive CC patients.


Subject(s)
Cell Proliferation , Hepatocyte Nuclear Factor 3-alpha , Ornithine Decarboxylase , Uterine Cervical Neoplasms , Female , Humans , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 3-alpha/metabolism , Hepatocyte Nuclear Factor 3-alpha/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , RNA, Circular/genetics , RNA, Circular/metabolism , Uterine Cervical Neoplasms/virology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/metabolism , Ornithine Decarboxylase/genetics , Ornithine Decarboxylase/metabolism
4.
Genes (Basel) ; 15(5)2024 Apr 27.
Article in English | MEDLINE | ID: mdl-38790189

ABSTRACT

BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.


Subject(s)
Chromatin Assembly and Disassembly , DNA-Binding Proteins , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Oncogene Proteins, Viral , Papillomavirus E7 Proteins , Repressor Proteins , Uterine Cervical Neoplasms , Humans , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Uterine Cervical Neoplasms/virology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Papillomavirus E7 Proteins/genetics , Papillomavirus E7 Proteins/metabolism , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Chromatin Assembly and Disassembly/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Cell Line, Tumor , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Human papillomavirus 16/pathogenicity , Carcinogenesis/genetics , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , Papillomavirus Infections/metabolism , Gene Expression Regulation, Neoplastic
5.
J Med Virol ; 96(4): e29571, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38563330

ABSTRACT

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17ß-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.


Subject(s)
Laurencia , MicroRNAs , Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Female , Humans , Mice , Animals , Laurencia/genetics , Papillomavirus Infections/complications , Papillomavirus Infections/drug therapy , Papillomavirus Infections/genetics , Uterine Cervical Neoplasms/pathology , MicroRNAs/genetics , Mice, Transgenic , Carcinogenesis , Papillomaviridae/genetics
6.
Clin Epigenetics ; 16(1): 56, 2024 Apr 20.
Article in English | MEDLINE | ID: mdl-38643219

ABSTRACT

BACKGROUND: Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. RESULTS: Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12-100.00], and specificity of 91% [95% CI 62.26-99.53] to 96% [95% CI 79.01-99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00-1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77-70.84] to 89% [95% CI 67.20-98.03], specificity of 93% [95% CI 84.07-97.38] to 96% [95% CI 79.01-99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68-0.89] to 0.99 [95% CI 0.97-1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45-92.69], specificity 95% [95% CI 88.64-98.18], and ROC AUC = 0.925 [95% CI 0.878-0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11-80.44), specificity of 94% (95% CI 88.30-97.40), and ROC AUC = 0.884 (95% CI 0.822-0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers. CONCLUSIONS: This 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.


Subject(s)
Carcinoma, Squamous Cell , Papillomavirus Infections , Uterine Cervical Neoplasms , Humans , Female , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Developing Countries , Papillomavirus Infections/diagnosis , Papillomavirus Infections/genetics , Genetic Markers , DNA Methylation , Carcinoma, Squamous Cell/genetics , Papillomaviridae/genetics , Vaginal Smears/methods , Tumor Suppressor Proteins/genetics
7.
Sci Rep ; 14(1): 9062, 2024 04 20.
Article in English | MEDLINE | ID: mdl-38643268

ABSTRACT

Head and neck cancer is the sixth most common malignancy worldwide, with the relatively low 5-year survival rate, mainly because it is diagnosed at a late stage. Infection with HPV is a well known aetiology, which affects the nature of these cancers and patients' survival. Besides, it is considered that the main driving force for this type of cancer could be epigenetics. In this study we aimed to find potential epigenetic biomarkers, by integrating miRNome, methylome, and transcriptome analyses. From the fresh head and neck cancer tissue samples, we chose a group for miRNome, methylome and transcriptome profiling, in comparison to adequate control samples. Bioinformatics analyses are performed in R v4.2.2. Count normalisation and group differential expression for mRNA and the previously obtained miRNA count data was performed with DESeq2 v1.36. Gene set enrichment analysis was performed and visualised using gProfiler2 v0.2.1 Identification of miRNA targets was performed by querying in miRTarBase using multiMiR v1.18.0. Annotation of CpG sites merging into islands was obtained from RnBeads.hg19 v1.28.0. package. For the integrative analysis we performed kmeans clustering using stats v4.2.2 package, using 8-12 clusters and nstart 100. We found that transcriptome analysis divides samples into cancers and controls clusters, with no relation to HPV status or cancer anatomical location. Differentially expressed genes (n = 2781) were predominantly associated with signalling pathways of tumour progression. We identified a cluster of genes under the control of the transcription factor E2F that are significantly underexpressed in cancer tissue, as well as T cell immunity genes and genes related to regulation of transcription. Among overexpressed genes in tumours we found those that belong to cell cycle regulation and vasculature. A small number of genes were found significantly differentially expressed in HPV-positive versus HPV-negative tumours (for example NEFH, ZFR2, TAF7L, ZNF541, and TYMS). In this comprehensive study on an overlapping set of samples where the integration of miRNome, methylome and transcriptome analysis were performed for head and neck cancer, we demonstrated that the majority of genes were associated exclusively with miRNome or methylome and, to a lesser extent, under the control of both epigenetic mechanisms.


Subject(s)
Head and Neck Neoplasms , MicroRNAs , Papillomavirus Infections , Humans , Epigenome , Papillomavirus Infections/genetics , Head and Neck Neoplasms/genetics , MicroRNAs/genetics , Gene Expression Profiling , Epigenesis, Genetic
8.
Invest Ophthalmol Vis Sci ; 65(4): 24, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38597722

ABSTRACT

Purpose: Conjunctival squamous cell carcinoma (conjSCC) is more prevalent and aggressive in sub-Saharan African countries compared with the rest of the world. This study aims to compare the genomic, immunophenotypic, and histologic features between patients from the United States and Ethiopia, to identify etiopathogenic mechanisms and unveil potential treatment strategies. Methods: We compared histologic features and mutational profiles using whole exome sequencing, high-risk human papillomavirus (HPV) status, PD-L1 expression, and tumor-infiltrating lymphocytes in conjSCC tumors of patients from Ethiopia (ETH; n = 25) and the United States (from MD Anderson [the MDA cohort]; n = 29). Genomic alterations were compared with SCCs from other anatomic sites using data from The Cancer Genome Atlas. Results: Solar elastosis was seen in 78% of ETH and 10% of MDA samples. Thicker tumors had higher density of CD8+ and CD3+ cells. HPV status was similar between the cohorts (ETH = 21% and MDA = 28%). The mean tumor mutation burden (TMB) was significantly higher in conjSCC (3.01/Mb, log10) and cutaneous SCC compared other SCC subtypes. ETH samples had higher TMB compared to the MDA cohort (3.34 vs. 2.73). Mutations in genes associated with ultraviolet light (UV) signature were most frequently encountered (SBS7b = 74% and SBS7a = 72%), with higher prevalence in the ETH cohort, whereas SBS2 and SBS13 signatures were more common among MDA HPV+ conjSCCs. Conclusions: Our findings suggest that UV exposure may play a major role in conjSCC, with a higher prevalence in the ETH cohort compared with the MDA cohort, where HPV also contributes.


Subject(s)
Papillomavirus Infections , Ultraviolet Rays , Humans , Papillomavirus Infections/genetics , Genomics , Conjunctiva , Black People
9.
BMC Cancer ; 24(1): 442, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38600473

ABSTRACT

Head and neck cancers, particularly oropharyngeal cancers (OPC), have been increasingly associated with human papillomavirus (HPV) infections, specifically HPV16. The current methods for HPV16 detection primarily rely on p16 staining or PCR techniques. However, it is important to note the limitations of conventional PCR, as the presence of viral DNA does not always indicate an ongoing viral infection. Moreover, these tests heavily rely on the availability of tissue samples, which can present challenges in certain situations. In this study, we developed a RT-qPCR biplex approach to detect HPV16 oncogenes E6 and E7 RNA in saliva samples from OPC patients. Salivary supernatant was used as the liquid biopsy source. We successfully obtained RNA from salivary supernatant, preserving its integrity as indicated by the detection of several housekeeping genes. Our biplex approach accurately detected E6 and E7 RNA in HPV16-positive cell lines, tissues, and finally in OPC salivary samples. Importantly, the assay specifically targeted HPV16 and not HPV18. This biplexing technique allowed for reduced sample input without compromising specificity. In summary, our approach demonstrates the potential to detect viable HPV16 in saliva from OPC patients. Since the assay measures HPV16 RNA, it provides insights into the transcriptional activity of the virus. This could guide clinical decision-making and treatment planning for individuals with HPV-related OPC.


Subject(s)
Oncogene Proteins, Viral , Oropharyngeal Neoplasms , Papillomavirus Infections , Humans , Human papillomavirus 16/genetics , Saliva/metabolism , Papillomavirus Infections/diagnosis , Papillomavirus Infections/genetics , Papillomavirus Infections/complications , Oncogene Proteins, Viral/genetics , Oropharyngeal Neoplasms/pathology , RNA , Polymerase Chain Reaction , Papillomavirus E7 Proteins/genetics
10.
J Med Virol ; 96(4): e29580, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38566572

ABSTRACT

A persistent infection with human papillomavirus (HPV) can induce precancerous lesions of the cervix that may ultimately develop into cancer. Cervical cancer development has been linked to altered microRNA (miRNA) expression, with miRNAs regulating anchorage-independent growth being particularly important for the progression of precancerous lesions to cancer. In this study, we set out to identify and validate targets of miR-129-5p, a previously identified tumor suppressive miRNA involved in anchorage-independent growth and HPV-induced carcinogenesis. We predicted 26 potential miR-129-5p targets using online databases, followed by KEGG pathway enrichment analysis. RT-qPCR and luciferase assays confirmed that 3'UTR regions of six genes (ACTN1, BMPR2, CAMK4, ELK4, EP300, and GNAQ) were targeted by miR-129-5p. Expressions of ACTN1, CAMK4, and ELK4 were inversely correlated to miR-129-5p expression in HPV-transformed keratinocytes, and their silencing reduced anchorage-independent growth. Concordantly, miR-129-5p overexpression decreased protein levels of ACTN1, BMPR2, CAMK4 and ELK4 in anchorage-independent conditions. Additionally, c-FOS, a downstream target of ELK4, was downregulated upon miR-129-5p overexpression, suggesting regulation through the ELK4/c-FOS axis. ACTN1 and ELK4 expression was also upregulated in high-grade precancerous lesions and cervical cancers, supporting their clinical relevance. In conclusion, we identified six targets of miR-129-5p involved in the regulation of anchorage-independent growth, with ACTN1, BMPR2, ELK4, EP300, and GNAQ representing novel targets for miR-129-5p. For both ACTN1 and ELK4 functional and clinical relevance was confirmed, indicating that miR-129-5p-regulated ACTN1 and ELK4 expression contributes to HPV-induced carcinogenesis.


Subject(s)
MicroRNAs , Papillomavirus Infections , Precancerous Conditions , Uterine Cervical Neoplasms , Female , Humans , Human Papillomavirus Viruses , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Cell Line, Tumor , MicroRNAs/genetics , MicroRNAs/metabolism , Keratinocytes/metabolism , Keratinocytes/pathology , Carcinogenesis/genetics , Carcinogenesis/pathology , Precancerous Conditions/pathology , Cell Proliferation/genetics , ets-Domain Protein Elk-4 , Actinin/genetics
11.
J Acquir Immune Defic Syndr ; 96(2): 190-195, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38630441

ABSTRACT

BACKGROUND: People living with HIV (PLWH) have substantially increased incidence of anal precancer and cancer. There are very little data regarding genomic disturbances in anal precancers among PLWH. In this study, specific chromosomal variants were identified in anal squamous intraepithelial lesions. METHODS: Overall, 63 anal biopsy specimens (27 low-grade intraepithelial lesions [LSIL] and 36 high-grade intraepithelial lesions [HSIL]) were collected from PLWH obtained as part of anal cancer screening in our NYC-based health system. Data on patient demographics, anal cytological, and high-risk human papillomavirus (HR-HPV) diagnoses were collected. Specimens were tested for a panel of chromosomal alterations associated with HPV-induced oncogenesis using fluorescence in situ hybridization, and analyses compared the associations of these alterations with clinical characteristics. RESULTS: Gains of 3q26, 5p15, 20q13, and cen7 were detected in 42%, 31%, 31%, and 19% of HSIL compared with 7%, 0%, 4%, and 0% of LSIL, respectively. If at least 1 abnormality was observed, 89% had a 3q26 gain. In lesions with 5p15 gains, 20q13 gains co-occurred in 91% of cases, while cen7 gain only co-occurred with the other 3 alterations. The sensitivity and specificity of any alteration to predict HSIL were 47% (95% CI: 30%-65%) and 93% (95% CI: 76%-99%), respectively. CONCLUSIONS: Genomic alterations seen in HPV-associated cancers may help distinguish anal LSIL from HSIL. 3q26 amplification may be an early component of anal carcinogenesis, preceding 5p16, 20q13, and/or chr7. IMPACT: Insights into potential genomic biomarkers for discriminating high-risk anal precancers are shared.


Subject(s)
Anus Neoplasms , DNA Copy Number Variations , HIV Infections , Precancerous Conditions , Humans , Anus Neoplasms/genetics , Anus Neoplasms/virology , Male , HIV Infections/complications , Female , Middle Aged , Adult , DNA Copy Number Variations/genetics , Precancerous Conditions/genetics , Precancerous Conditions/virology , Precancerous Conditions/pathology , Papillomavirus Infections/complications , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , Squamous Intraepithelial Lesions/genetics , Squamous Intraepithelial Lesions/virology
12.
mBio ; 15(5): e0072924, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38624210

ABSTRACT

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.


Subject(s)
DNA, Viral , Oncogene Proteins, Viral , Papillomavirus Infections , Uterine Cervical Neoplasms , Virus Integration , Humans , Female , Uterine Cervical Neoplasms/virology , Uterine Cervical Neoplasms/genetics , Virus Integration/genetics , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus Infections/virology , Papillomavirus Infections/genetics , DNA, Viral/genetics , Human papillomavirus 16/genetics , Human papillomavirus 18/genetics , Cell Line, Tumor , Oncogenes/genetics , Polyadenylation
13.
Clin Exp Pharmacol Physiol ; 51(6): e13864, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38679464

ABSTRACT

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.


Subject(s)
Carcinoma, Squamous Cell , Human papillomavirus 16 , Methyltransferases , Microfilament Proteins , Papillomavirus Infections , RNA-Binding Proteins , Uterine Cervical Neoplasms , Female , Humans , Adenosine/analogs & derivatives , Adenosine/metabolism , Carcinoma, Squamous Cell/virology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Methylation , Methyltransferases/metabolism , Methyltransferases/genetics , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/genetics , Papillomavirus E7 Proteins/metabolism , Papillomavirus Infections/genetics , Papillomavirus Infections/virology , Papillomavirus Infections/metabolism , Papillomavirus Infections/pathology , Repressor Proteins , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Uterine Cervical Neoplasms/virology , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/metabolism
14.
Viruses ; 16(4)2024 03 25.
Article in English | MEDLINE | ID: mdl-38675844

ABSTRACT

Chromosomal instability (CIN) and aneuploidy are hallmarks of cancer. CIN is defined as a continuous rate of chromosome missegregation events over the course of multiple cell divisions. CIN causes aneuploidy, a state of abnormal chromosome content differing from a multiple of the haploid. Human papillomavirus (HPV) is a well-known cause of squamous cancers of the oropharynx, cervix, and anus. The HPV E6 and E7 oncogenes have well-known roles in carcinogenesis, but additional genomic events, such as CIN and aneuploidy, are often required for tumor formation. HPV+ squamous cancers have an increased frequency of specific types of CIN, including polar chromosomes. CIN leads to chromosome gains and losses (aneuploidies) specific to HPV+ cancers, which are distinct from HPV- cancers. HPV-specific CIN and aneuploidy may have implications for prognosis and therapeutic response and may provide insight into novel therapeutic vulnerabilities. Here, we review HPV-specific types of CIN and patterns of aneuploidy in squamous cancers, as well as how this impacts patient prognosis and treatment.


Subject(s)
Aneuploidy , Chromosomal Instability , Papillomavirus Infections , Humans , Papillomavirus Infections/virology , Papillomavirus Infections/complications , Papillomavirus Infections/genetics , Papillomaviridae/genetics , Papillomaviridae/pathogenicity , Carcinoma, Squamous Cell/virology , Carcinoma, Squamous Cell/genetics , Neoplasms, Squamous Cell/virology , Neoplasms, Squamous Cell/genetics , Neoplasms, Squamous Cell/pathology , Female , Alphapapillomavirus/genetics , Alphapapillomavirus/pathogenicity , Human Papillomavirus Viruses
16.
JCI Insight ; 9(6)2024 Feb 08.
Article in English | MEDLINE | ID: mdl-38516891

ABSTRACT

BACKGROUNDTransrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.MethodsUsing whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.ResultsTR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.ConclusionOur data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.FundingNIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.


Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Oropharyngeal Neoplasms , Papillomavirus Infections , United States , Humans , Papillomavirus Infections/genetics , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Oropharyngeal Neoplasms/diagnosis , Oropharyngeal Neoplasms/genetics , Squamous Cell Carcinoma of Head and Neck , DNA, Neoplasm , Liquid Biopsy
17.
Gene ; 910: 148321, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38428621

ABSTRACT

Infection with human papillomavirus (HPV) is a major risk factor for head and neck squamous cell carcinoma (HNSCC). The objective of this study is to investigate the gene expression profiles and signaling pathways that are specific to HPV-positive HNSCC (HPV+ HNSCC). Moreover, a competing endogenous RNA (ceRNA) network analysis was utilized to identify the core gene of HPV+ HNSCC and potential targeted therapeutic drugs. Transcriptome sequencing analysis identified 3,253 coding RNAs and 3,903 non-coding RNAs (ncRNAs) that exhibited preferentially expressed in HPV+ HNSCC. Four key signaling pathways were selected through pathway enrichment analysis. By combining ceRNA network and protein-protein interaction (PPI) network topology analysis, RNA Polymerase II Associated Protein 2 (RPAP2), which also exhibited high expression in HPV+ HNSCC based on the TCGA database, was identified as the hub gene. Gene set enrichment analysis (GSEA) results revealed RPAP2's involvement in various signaling pathways, encompassing basal transcription factors, ubiquitin-mediated proteolysis, adherens junction, other glycan degradation, ATP-binding cassette (ABC) transporters, and oglycan biosynthesis. Five potential small molecule targeted drugs (enzastaurin, brequinar, talinolol, phenylbutazone, and afuresertib) were identified using the cMAP database, with enzastaurin showing the highest affinity for RPAP2. Cellular functional experiments confirmed the inhibitory effect of enzastaurin on cell viability of HPV+ HNSCC and RPAP2 expression levels. Additionally, enzastaurin treatment suppressed the expression levels of the top-ranked long non-coding RNA (lncRNA), circular RNA (circRNA), and microRNA (miRNA) in the ceRNA network. This study based on the ceRNA network provides valuable insights into the molecular mechanisms and potential therapeutic strategies for HPV+ HNSCC, and provide theoretical basis for the exploration of HPV+ HNSCC biomarkers and the development of targeted drugs.


Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , MicroRNAs , Papillomavirus Infections , RNA, Long Noncoding , Humans , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Transcriptome/genetics , RNA, Competitive Endogenous , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Papillomavirus Infections/drug therapy , Papillomavirus Infections/genetics , Gene Expression Profiling , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular , Gene Expression Regulation, Neoplastic , RNA, Long Noncoding/genetics , Carrier Proteins/genetics
18.
Genes (Basel) ; 15(3)2024 Feb 23.
Article in English | MEDLINE | ID: mdl-38540340

ABSTRACT

There is still much to learn about the epigenetic mechanisms controlling gene expression during carcinogenesis. When researching aberrant DNA methylation, active proliferative tumor cells from head and neck squamous cell cancer (HNSCC) can be used as a model. The aim of the study was to investigate the methylation status of CDKN1, CDKN2A, MYC, Smad3, SP1, and UBC genes in tumor tissue (control-normal tissue) in 50 patients (37 men and 13 women) with HPV-negative HNSCC. Methods: Bisulfite conversion methods and methyl-sensitive analysis of high-resolution melting curves were used to quantify the methylation of genes. In all patients and across various subgroups (tongue carcinoma, laryngeal and other types of carcinomas T2, T3, T4 status; age before and after 50 years; smoking and non-smoking), there are consistent differences in the methylation levels in the SP1 gene in tumor DNA compared to normal. Results: The methylation of the SP1 gene in tumor DNA suppresses its expression, hinders HNSCC cell proliferation regulation, and could be a molecular indicator of malignant cell growth. The study of DNA methylation of various genes involved in carcinogenesis is promising because hypermethylated promoters can serve as potential biomarkers of disease.


Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Papillomavirus Infections , Female , Humans , Male , Middle Aged , Carcinogenesis/genetics , Carcinoma, Squamous Cell/pathology , DNA/metabolism , DNA Methylation/genetics , Epithelial Cells/metabolism , Head and Neck Neoplasms/genetics , Papillomavirus Infections/genetics , Squamous Cell Carcinoma of Head and Neck/genetics
19.
Biol Cell ; 116(4): e202300072, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38514439

ABSTRACT

BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.


Subject(s)
Breast Neoplasms , Oncogene Proteins, Viral , Papillomavirus Infections , Female , Humans , Papillomavirus E7 Proteins/genetics , Papillomavirus E7 Proteins/metabolism , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Cyclin B1/metabolism , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Breast Neoplasms/genetics , Papillomavirus Infections/genetics , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism
20.
Cancer Lett ; 588: 216809, 2024 Apr 28.
Article in English | MEDLINE | ID: mdl-38471646

ABSTRACT

Human papillomavirus (HPV) is predominantly associated with HPV-related cancers, however, the precise mechanisms underlying the HPV-host epigenetic architectures in HPV carcinogenesis remain elusive. Here, we employed high-throughput chromosome conformation capture (Hi-C) to comprehensively map HPV16/18-host chromatin interactions. Our study identified the transcription factor Sp1 as a pivotal mediator in programming HPV-host interactions. By targeting Sp1, the active histone modifications (H3K27ac, H3K4me1, and H3K4me3) and the HPV-host chromatin interactions are reprogrammed, which leads to the downregulation of oncogenes located near the integration sites in both HPV (E6/E7) and the host genome (KLF5/MYC). Additionally, Sp1 inhibition led to the upregulation of immune checkpoint genes by reprogramming histone modifications in host cells. Notably, humanized patient-derived xenograft (PDX-HuHSC-NSG) models demonstrated that Sp1 inhibition promoted anti-PD-1 immunotherapy via remodeling the tumor immune microenvironment in cervical cancer. Moreover, single-cell transcriptomic analysis validated the enrichment of transcription factor Sp1 in epithelial cells of cervical cancer. In summary, our findings elucidate Sp1 as a key mediator involved in the programming and reprogramming of HPV-host epigenetic architecture. Inhibiting Sp1 with plicamycin may represent a promising therapeutic option for HPV-related carcinoma.


Subject(s)
Oncogene Proteins, Viral , Papillomavirus Infections , Uterine Cervical Neoplasms , Female , Humans , Chromatin/genetics , Epigenesis, Genetic , Human papillomavirus 16/metabolism , Human papillomavirus 18/genetics , Human papillomavirus 18/metabolism , Human Papillomavirus Viruses , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/metabolism , Papillomavirus Infections/genetics , Papillomavirus Infections/therapy , Transcription Factors/genetics , Tumor Microenvironment , Uterine Cervical Neoplasms/pathology
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