Cervical cancer is primarily caused by the infection of high-risk human papillomavirus (hrHPV). Moreover, tumor immune microenvironment plays a significant role in the tumorigenesis of cervical cancer. Therefore, it is necessary to comprehensively identify predictive biomarkers from immunogenomics associated with cervical cancer prognosis. The Cancer Genome Atlas (TCGA) public database has stored abundant sequencing or microarray data, and clinical data, offering a feasible and reliable approach for this study. In the present study, gene profile and clinical data were downloaded from TCGA, and the Immunology Database and Analysis Portal (ImmPort) database. Wilcoxon-test was used to compare the difference in gene expression. Univariate analysis was adopted to identify immune-related genes (IRGs) and transcription factors (TFs) correlated with survival. A prognostic prediction model was established by multivariate cox analysis. The regulatory network was constructed and visualized by correlation analysis and Cytoscape, respectively. Gene functional enrichment analysis was performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 204 differentially expressed IRGs were identified, and 22 of them were significantly associated with the survival of cervical cancer. These 22 IRGs were actively involved in the JAK-STAT pathway. A prognostic model based on 10 IRGs (APOD, TFRC, GRN, CSK, HDAC1, NFATC4, BMP6, IL17RD, IL3RA, and LEPR) performed moderately and steadily in squamous cell carcinoma (SCC) patients with FIGO stage I, regardless of the age and grade. Taken together, a risk score model consisting of 10 novel genes capable of predicting survival in SCC patients was identified. Moreover, the regulatory network of IRGs associated with survival (SIRGs) and their TFs provided potential molecular targets.

Head and neck squamous cell carcinoma (HNSCC), especially oropharyngeal squamous cell carcinoma (OPSCC), has recently been found to be significantly associated with human papillomavirus (HPV) infection. The incidence of OPSCC has been increasing and surpassed the number of cervical cancer cases in the United States. Although HPV-associated OPSCC has a relatively better prognosis than HPV-negative cancer, approximately 20% of HPV-associated HNSCC patients show a poor prognosis or therapeutic response, and the molecular mechanism behind this outcome in the intermediate-risk group is yet to be elucidated. These biological differences between HPV-associated HNSCC and HPV-negative HNSCC are partly explained by the differences in mutation patterns. However, recent reports have revealed that epigenetic dysregulation, such as dysregulated DNA methylation, is a strikingly common pathological feature of human malignancy. Notably, viral infections can induce aberrant DNA methylation, leading to carcinogenesis, and HPV-associated HNSCC cases tend to harbor a higher amount of aberrantly methylated DNA than HPV-negative HNSCC cases. Furthermore, recent comprehensive genome-wide DNA-methylation analyses with large cohorts have revealed that a sub-group of HPV-associated HNSCC correlates with increased DNA methylation. Accordingly, in this review, we provide an overview of the relationship between DNA methylation and HPV-associated HNSCC.

Screening plays a key role in secondary prevention of cervical cancer. High-risk human papillomavirus (hrHPV) testing, a highly sensitive test but with limited specificity, has become the gold standard frontline for screening programs. Thus, the importance of effective triage strategies, including DNA methylation markers, has been emphasized. Despite the potential reported in individual studies, methylation markers still require validation before being recommended for clinical practice. This systematic review and meta-analysis aimed to evaluate the performance of DNA methylation-based biomarkers for detecting high-grade intraepithelial lesions (HSIL) in hrHPV-positive women.Hence, PubMed, Scopus, and Cochrane databases were searched for studies that assessed methylation in hrHPV-positive women in cervical scrapes. Histologically confirmed HSIL was used as endpoint and QUADAS-2 tool enabled assessment of study quality. A bivariate random-effect model was employed to pool the estimated sensitivity and specificity as well as positive (PPV) and negative (NPV) predictive values.Twenty-three studies were included in this meta-analysis, from which cohort and referral population-based studies corresponded to nearly 65%. Most of the women analyzed were Dutch, and CADM1, FAM19A4, MAL, and miR124-2 were the most studied genes. Pooled sensitivity and specificity were 0.68 (CI 95% 0.63-0.72) and 0.75 (CI 95% 0.71-0.80) for cervical intraepithelial neoplasia (CIN) 2+ detection, respectively. For CIN3+ detection, pooled sensitivity and specificity were 0.78 (CI 95% 0.74-0.82) and 0.74 (CI 95% 0.69-0.78), respectively. For pooled prevalence, PPV for CIN2+ and CIN3+ detection were 0.514 and 0.392, respectively. Furthermore, NPV for CIN2+ and CIN3+ detection were 0.857 and 0.938, respectively.This meta-analysis confirmed the great potential of DNA methylation-based biomarkers as triage tool for hrHPV-positive women in cervical cancer screening. Standardization and improved validation are, however, required. Nevertheless, these markers might represent an excellent alternative to cytology and genotyping for colposcopy referral of hrHPV-positive women, allowing for more cost-effective screening programs.© 2023. The Author(s).

The number of deaths from cervical cancer in the United States has decreased substantially since the implementation of widespread cervical cancer screening and has declined from 2.8 to 2.3 deaths per 100 000 women from 2000 to 2015.To update the US Preventive Services Task Force (USPSTF) 2012 recommendation on screening for cervical cancer.The USPSTF reviewed the evidence on screening for cervical cancer, with a focus on clinical trials and cohort studies that evaluated screening with high-risk human papillomavirus (hrHPV) testing alone or hrHPV and cytology together (cotesting) compared with cervical cytology alone. The USPSTF also commissioned a decision analysis model to evaluate the age at which to begin and end screening, the optimal interval for screening, the effectiveness of different screening strategies, and related benefits and harms of different screening strategies.Screening with cervical cytology alone, primary hrHPV testing alone, or cotesting can detect high-grade precancerous cervical lesions and cervical cancer. Screening women aged 21 to 65 years substantially reduces cervical cancer incidence and mortality. The harms of screening for cervical cancer in women aged 30 to 65 years are moderate. The USPSTF concludes with high certainty that the benefits of screening every 3 years with cytology alone in women aged 21 to 29 years substantially outweigh the harms. The USPSTF concludes with high certainty that the benefits of screening every 3 years with cytology alone, every 5 years with hrHPV testing alone, or every 5 years with both tests (cotesting) in women aged 30 to 65 years outweigh the harms. Screening women older than 65 years who have had adequate prior screening and women younger than 21 years does not provide significant benefit. Screening women who have had a hysterectomy with removal of the cervix for indications other than a high-grade precancerous lesion or cervical cancer provides no benefit. The USPSTF concludes with moderate to high certainty that screening women older than 65 years who have had adequate prior screening and are not otherwise at high risk for cervical cancer, screening women younger than 21 years, and screening women who have had a hysterectomy with removal of the cervix for indications other than a high-grade precancerous lesion or cervical cancer does not result in a positive net benefit.The USPSTF recommends screening for cervical cancer every 3 years with cervical cytology alone in women aged 21 to 29 years. (A recommendation) The USPSTF recommends screening every 3 years with cervical cytology alone, every 5 years with hrHPV testing alone, or every 5 years with hrHPV testing in combination with cytology (cotesting) in women aged 30 to 65 years. (A recommendation) The USPSTF recommends against screening for cervical cancer in women younger than 21 years. (D recommendation) The USPSTF recommends against screening for cervical cancer in women older than 65 years who have had adequate prior screening and are not otherwise at high risk for cervical cancer. (D recommendation) The USPSTF recommends against screening for cervical cancer in women who have had a hysterectomy with removal of the cervix and do not have a history of a high-grade precancerous lesion or cervical cancer. (D recommendation).

DNA methylation is the main epigenetic event for gene silencing and is associated with carcinogenesis. In this meta-analysis, we evaluated the association between the methylation of the promoter regions of APC, CADM1, CCNA1, CDH1, DAPK, FHIT, HIC1, MAL, MGMT, hMLH1, P16, PAX1, RAR-β, and RASSF1 genes and the risk of cervical cancer development and progression. Overall, 194 eligible studies were identified assessing the associations of promoter methylation status of aforementioned genes with low- and high-grade squamous intraepithelial lesions (LSIL and HSIL) and cervical cancer development. The majority of studies were conducted on Caucasian and Asian populations, whereas rare studies were available on the African population. Promoter methylation frequencies were shown to be significantly higher in LSIL and HSIL cervical cancer cases as compared to control specimens for CADM1, CCNA1, CDH1, DAPK1, FHIT, MAL, P16, PAX1, RAR-β, and RASSF1 genes. A moderate association was found between HIC promoter methylation, whereas APC, MGMT, and hMLH1 promoter methylation was not correlated with cervical cancer development. Promoter methylation could be considered as a noninvasive biomarker for early cervical lesions, making them highly promising targets for a personalized therapeutic approach.

Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.© 2024. The Author(s).

DNA methylation alteration, such as global hypomethylation and localized hypermethylation, within the promoters of tumor suppressor genes, is an important risk factor in cervical cancer. The potential use of DNA methylation detection, in cervical cancer screening or triage of mildly abnormal cytology, has recently been demonstrated. In particular, PAX1 DNA methylation testing was approved as an adjunct to cytology, in Taiwan, and is now undergoing registration trials in China. However, the function of PAX1 in cancer biology remains largely unknown. Here, we show that PAX1 inhibits malignant phenotypes upon oncogenic stress. Specifically, PAX1 expression inhibited the phosphorylation of multiple kinases, after challenges with oncogenic growth factors such as EGF and IL-6. Analogously, PAX1 activated a panel of phosphatases, including DUSP1, 5, and 6, and inhibited EGF/MAPK signaling. PAX1 also interacted with SET1B, increasing histone H3K4 methylation and DNA demethylation of numerous phosphatase-encoding genes. Furthermore, hypermethylated PAX1 associated with poor prognosis in cervical cancer. Taken together, this study reveals, for the first time, the functional relevance of PAX1 in cancer biology, and further supports the prospect of targeting multifold oncogenic kinase cascades, which jointly contribute to multiresistance, via epigenetic reactivation of PAX1.

To compare the performance of cytology, HPV16/18 genotyping and PAX1/SOX1 methylation for the triage of high‐risk HPV‐positive cervical samples.

In China, the national cervical cancer screening protocol involves initial testing for high-risk human papillomavirus (hrHPV), followed by cytology for hrHPV-positive cases. This study evaluates the effectiveness of PAX1 methylation (PAX1) analysis in identifying precancerous or cancerous lesions in cervical samples from Chinese women positive for non-16/18 hrHPV strains.Between February 2022 and March 2023, 281 cervical samples from non-16/18 hrHPV-positive women underwent cytological examination and PAX1 analysis. The study assessed the statistical relationship between PAX1 levels and the presence of cervical lesions, comparing the diagnostic performance of PAX1 to conventional cytology.A significant association was found between PAX1 methylation levels and the risk of CIN2 + and CIN3 + lesions, with 47 instances of CIN2 + detected. Odds ratios (ORs) for moderate and high PAX1 levels were 8.86 (95% CI: 2.24-42.17) and 166.32 (95% CI: 47.09-784.97), respectively. The area under the ROC curve for PAX1 in identifying CIN2 + lesions was 0.948 (95% CI: 0.895-0.99). PAX1 demonstrated similar sensitivity and negative predictive value (NPV) to cytology but reduced the colposcopy referral rate from 47.7% with cytology alone to 25.6% with PAX1, showing superior specificity and positive predictive value across age groups.PAX1 methylation is a strong indicator of CIN2 + and CIN3 + risk, offering diagnostic performance comparable to cytology with the added benefit of reduced unnecessary colposcopy referrals. These findings support the use of PAX1 analysis as a reliable tool for triaging non-16/18 hrHPV-positive women in outpatient settings.© 2024. The Author(s).

Although aberrant methylation of PAX1 is closely associated with cervical cancer (CC), PAX1 methylation (PAX1m) and its role in CC remain to be elucidated. Here, we clarified the biological function of PAX1 in CC. First, PAX1m in ThinPrep cytologic test samples was measured via quantitative methylation‐specific PCR. The results showed that PAX1 promoter methylation levels were significantly increased in CC patients (p < 0.001). We also found that PAX1 promoter methylation levels were positively correlated with tumor purity but negatively correlated with immune‐infiltration via public databases. Then, CRISPR‐based methylation perturbation tools (dCas9‐Tet1) were constructed to further demonstrate that DNA methylation participates in the regulation of PAX1 expression directly. Gain‐ and loss‐of‐function experiments were used to show that PAX1 overexpression restrained proliferation, migration and improved cisplatin sensitivity by interfering with the WNT/TIMELESS axis in CC cells. Additionally, Co‐immunoprecipitation assays further confirmed the interaction between PAX1 and TCF7L2. Taken together, our results suggested that a tumor suppressor role of PAX1 in CC and that CRISPR‐based PAX1 demethylation editing might be a promising therapeutic strategy for CC.

PAX1 gene methylation plays an important role in the development of cervical cancer. However, its prognostic value after radiotherapy for locally advanced cervical cancer is unknown, so this study aimed to investigate the value of PAX1 gene methylation for predicting the sensitivity of radiotherapy for cervical cancer.We selected 125 patients with primary cervical cancer who underwent concurrent chemo-radiotherapy as the study population, quantitative methylation-specific polymerase chain reaction (QMSP) was used for detecting PAX1 methylation status of cervical exfoliated cells. Logistic regression model was used to analyze the risk factors associated with the short-term efficacy and to establish a prediction model of radiotherapy sensitivity based on PAX1 gene methylation. Cell viability after radiation of Hela and SiHa cells transfected with PAX1 or control vector was evaluated by CCK8. Furthermore, RNA-Seq analyses identified different expressed genes (DEGs) in PAX1 overexpressed SiHa cells. Gene Ontology (GO) and pathway enrichment analysis was carried out to determine the biological function of DEGs.PAX1 methylation level was associated with HPV16/18-positive rate. PAX1 hypomethylation was found to be a risk factor for tumor residual after chemo-radiotherapy. A nomogram containing the risk factors for PAX1 methylation status, lymph node metastasis, pathological type and tumor size was further constructed to predict the probability of tumor residual after chemo-radiotherapy (AUC = 0.823, 95% CI 0.736-0.910). High PAX1 protein level was more likely to cause radioresistance in both Hela and SiHa cells. Transcriptomic sequencing of PAX1 overexpressed and control cells identified 615 differentially expressed genes, and GO enrichment analysis suggested that PAX1 may be involved in the regulation of signaling receptor activity and response to viruses.PAX1 hypomethylation status could be used as a promising biomarker to predict radioresistance in cervical cancer. This further provides a new idea for the individualized treatment strategy of simultaneous radiotherapy for cervical cancer.© 2023. The Author(s).

Despite rapid improvements in DNA methylation tools for cervical cancer screening, few robust, exploratory studies have been performed using the combination of two host genes, EPB41L3 and JAM3, newly developed assays.A review of abnormal liquid-based cytology and/or high-risk human papillomavirus (hrHPV) data from outpatient clinics in the study center from March 2018 to March 2019 was performed. Eligible patients with definitive histological pathology results were included, and their residual cytology samples were assessed for EPB41L3 and JAM3 methylation. The diagnostic accuracies of various screening strategies for definitive pathology and for cervical intraepithelial neoplasia (CIN) 2 or more severe lesions (CIN2+) were compared.In total, 306 patients were successfully tested; 301 cases with cervical histological pathology were included in the final analysis, including 118 (39.2%) and 183 (60.8%) cases of inflammation/CIN1 and CIN2+, respectively. Regarding CIN2+ detection, methylation status and hrHPV plus methylation had similar positive predictive values (0.930 and 0.954, respectively, p = 0.395). Additionally, hrHPV, methylation, and hrHPV plus methylation had similar negative predictive values (0.612, 0.679, and 0.655, p = 0.677) that were significantly higher than that of cytology alone (0.250, p values 0.012, 0.001, and 0.001, respectively). For 49 cases with negative hrHPV results, positive methylation alone was able to differentiate CIN2+ from inflammation/CIN1.Methylation of both EPB41L3 and JAM3 is an accurate and feasible screening method for CIN2+.

To study the predictive value of the DNA methylation levels of JAM3, SOX1, SLIT2, C13ORF18, and TERT in the cervical intraepithelial neoplasia prognosis.In the present study, 139 cases were collected and followed up for 24 months. The DNA methylation levels of JAM3, SOX1, SLIT2, C13ORF18, and TERT were tested from their exfoliated cells. One-way ANOVA, receiver operating characteristic (ROC) curve analyses were conducted to analyze the data.The DNA methylation of the five genes was associated with prognosis of CIN. The levels of methylation increased as the progression of lesion for the prognosis. For CIN1, difference between DNA methylation of JAM3, SOX1, SLIT2, and C13ORF18 had significance statistically (P < 0.001). Sensitivity (95.2%) and specificity (93.1%) of JAM3 were the highest compared with other genes for the prognosis of CIN1. In addition, for CIN2/3, DNA methylation of JAM3, SOX1, SLIT2, TERT, and C13ORF18 had difference statistically (P < 0.001). JAM3 were also the highest in sensitivity (95.2%) and specificity (93.1%) compared with other genes for the prognosis of CIN2/3.Our data suggest for the first time that DNA methylation levels are associated with prognosis of CIN significantly. DNA methylation levels of some genes, especially JAM3, may serve as markers for the prediction of the CIN prognosis, including CIN1 nature prognosis and CIN2/3 after treatment.

Junctional adhesion molecules (JAMs) are members of an immunoglobulin subfamily expressed by leukocytes and platelets as well as by epithelial and endothelial cells, in which they localize to cell-cell contacts and are specifically enriched at tight junctions. The recent identification of extracellular ligands and intracellular binding proteins for JAMs suggests two functions for JAMs. JAMs associate through their extracellular domains with the leukocyte beta2 integrins LFA-1 and Mac-1 as well as with the beta1 integrin alpha4beta1. All three integrins are involved in the regulation of leukocyte-endothelial cell interactions. Through their cytoplasmic domains, JAMs directly associate with various tight junction-associated proteins including ZO-1, AF-6, MUPP1 and the cell polarity protein PAR-3. PAR-3 is part of a ternary protein complex that contains PAR-3, atypical protein kinase C and PAR-6. This complex is highly conserved through evolution and is involved in the regulation of cell polarity in organisms from Caenorhabditis elegans and Drosophila to vertebrates. These findings point to dual functions for JAMs: they appear to regulate both leukocyte/platelet/endothelial cell interactions in the immune system and tight junction formation in epithelial and endothelial cells during the acquisition of cell polarity.

Cervical cancer is the fourth most common cancer and the leading cause of mortality among women worldwide. Tumor metastasis is an important cause of poor prognosis. Determining the exact mechanisms of metastasis and potential targeted therapies is urgently needed. Junctional adhesion molecule 3 (JAM3) is an important member of the TJ tight junction (TJ) family, and its biological function in cervical cancer needs to be further clarified. We found that JAM3 was highly expressed in cervical cancer patients with lymph node metastasis and that high expression of JAM3 promoted cervical cancer cell metastasis both in vitro and in vivo. In addition, overexpression of JAM3 induces epithelial-mesenchymal transition (EMT). Moreover, silencing JAM3 suppressed cervical cancer cell migration and invasion in vitro. Finally, JAM3 overexpression activated the HIF-1α/VEGFA pathway. In conclusion, our results suggested that JAM3 promotes cervical cancer cell migration and invasion by activating the HIF-1α/VEGFA pathway. JAM3 may be a promising biomarker and effective therapeutic target for cervical cancer.© 2024. The Author(s).

Referring all women who tested positive for human papillomavirus (HPV) 16/18 to colposcopy may lead to potential over-referral issues. Triage tests based on cytology results face challenges in achieving accurate diagnoses. Our study aims to assess the clinical effectiveness of PAX1/JAM3 methylation (CISCER) test as a triage method for HPV 16/18-positive women.From November 2021 to December 2022, a total of 334 women who tested positive for HPV 16/18 and were referred to colposcopy at The Second Affiliated Hospital of Zhejiang University School of Medicine were studied. The clinical utility of the CISCER test, cytology, and the combination of CISCER with cytology as potential triage tests was compared.We observed a significant increase in the methylation levels of PAX1 gene and JAM3 gene in women with cervical intraepithelial neoplasia (CIN) grade 2 or severe (CIN2+). The CISCER test demonstrated superior triage performance over cytology, even when used in combination with cytology, showing a high sensitivity of 89.0% (95% confidence interval [CI] 82.9-95.1%) and specificity of 95.3% (95% CI 92.6-98.0%). It achieved an area under the curve of 0.921 (95% CI 0.877-0.966) and an odds ratio of 164.02 (95% CI 68.64-391.95). The immediate CIN2+ risk based on positive CISCER results would be 89.0% (95% CI 80.8-94.1%), with an estimated average of 1.12 referrals needed to detect one CIN2+ case. Moreover, CISCER triaging successfully identified all cancer patients and did not miss any CIN3+ cases among women aged ≥ 30.The PAX1/JAM3 methylation detection exhibited excellent accuracy in identifying cervical precancerous lesions in HPV 16/18-positive women and could be considered as a triage tool to reduce excessive referrals for colposcopy and overtreatment.© 2024. The Author(s).