The promising role of PAX1 (aliases: HUP48, OFC2) gene methylation in cancer screening

1 INTRODUCTION

PAX1 (OMIM: 167,411) gene, a member of the PAX family located on chromosome 20p11.2, is essential to regulate fetal development. (Schnittger et al., 1992). Members of the PAX family typically contain a paired-box domain and a paired-type homeodomain, which are essential during fetal development and play critical roles during development of the vertebral column (Wallin et al., 1994). PAX1 plays a role in sclerotome differentiation and interacts with homeobox (HOX) genes, which play a prominent role in normal fetal development and controlling cell proliferation (Cillo, Cantile, Faiella, & Boncinelli, 2001). Substitution or deletion of this gene in mice produces variants associated with vertebral malformations and Klippel–Feil syndrome (Hol et al., 1996; McGaughran, Oates, Donnai, Read, & Tassabehji, 2003). Methylation of PAX1 promoter is an important epigenetic regulation associating to the development and the metastasis of the tumor. PAX1 gene in cervical and ovarian cancer is silenced by methylation and is considered as a tumor suppressor gene (Chang et al., 2014; Hassan, Hafez, Kamel, & Zekri, 2017; Kan et al., 2014; Kong, Du, Wang, Yang, & Zhang, 2015; Su et al., 2009).

2 PAX1 METHYLATION AND CERVICAL CANCER

2.2 Relationships between PAX1 methylation and cervical cancer screening

Epigenetic studies demonstrate that DNA methylation could be a symbolic event of carcinogenesis. Several kinds of DNA methylation are reported as strongly associated with CINs and cervical cancer, including those in sex-determining region Y-box 1 (SOX1; OMIM: 602148), PAX1, LIM homeobox transcription factor 1A (OMIM: 600298), NK6 transcription factor-related locus 1 (OMIM: 602563), and Wilms tumor 1 (OMIM: 607102) (Chang et al., 2014; Lai et al., 2008; Lim et al., 2010; Lorincz, 2016; Vasiljevic, Scibior-Bentkowska, Brentnall, Cuzick, & Lorincz, 2014). Among these genes, multiple studies confirmed PAX1 methylation as the most highly correlated with CIN progression and cervical carcinogenesis (Chang et al., 2014; Chao et al., 2013; Chen et al., 2016; Huang et al., 2010; Kan et al., 2014; Lai et al., 2010, 2014; Luan et al., 2017; Tian et al., 2017; Xu et al., 2015).

In 2008, Lai et al. (2008) first reported that PAX1 was abnormally methylated in association with cervical cancer, and the PAX1 gene was silenced by hyper methylation and low expressed in these biopsies of cervical cancer (Lai et al., 2008). Several studies found that PAX1 methylation increased along with increased disease grade in the following order: PAX1 methylation in squamous cell carcinoma (SCC) > high-grade squamous intraepithelial lesion (HSIL) > low-grade squamous intraepithelial lesion (LSIL) > normal tissue (Lai et al., 2008; Lim et al., 2010; Xu et al., 2015). Detection of high-grade cervical lesions in patients with atypical squamous cells of undetermined significance (ASCUS) remains a challenge in the screening and diagnosis of cervical cancer. PAX1 methylation demonstrated better performance as a marker than results of a high-risk HPV-DNA test for the detection of high-grade lesions (CIN2+) in ASCUS cases; however, PAX1 methylation allows for the screening out of a majority of low-grade ASCUS cases (Li et al., 2015; Wang, 2014). A result from 443 cervical scraping samples showed that PAX1 detection alone had a sensitivity and specificity of 86% and 85%, respectively, for the detection of CIN3+ lesions, whereas when used as a co-test with the Pap test, the sensitivity and specificity were 89% and 83%, respectively (Kan et al., 2014). Additionally, our previous studies found a significant association between methylated PAX1 and CIN3+ or worse in combination with HPV16/18, with sensitivities and specificities of methylated PAX1 with HPV16/18 for CIN3+ detection at 89.2% and 76.0%, respectively (Liou et al., 2016), whereas dual methylation testing for PAX1/zinc protein finger 582 (OMIM: 615600) combined with HPV-16/18 genotyping resulted in 100% identification of carcinoma in situ or SCC (Tian et al., 2017). Meta-analyses also supported the utility of PAX1 methylation as an auxiliary biomarker in cervical cancer screening. One meta-analysis reviewed 1,385 subjects with various stages of CIN and normal cervical pathology, finding that the sensitivity and specificity of PAX1 methylation in CIN3+ vs. normal samples were 0.77 and 0.92, respectively (Nikolaidis et al., 2015). Additionally, 15 individual studies showed that single PAX1 methylation allowed the accurate differential diagnosis of cervical cancer/HSIL patients from normal individuals with a sensitivity of 0.80 and a specificity of 0.89 (Kong et al., 2015).

These data suggested the efficacy of PAX1 methylation as a biomarker for cervical cancer screening, and that it plays a guiding role in triage management of LSIL, HSIL, and SCC patients, as well as displays higher accuracy than single HPV-DNA testing. These findings suggest that incorporating PAX1-methylation detection into current cervical cancer-screening protocols (Figure 1) will promote the accurate screening of women requiring treatment, reduce unnecessary referrals for colposcopy and biopsy, and ease the burden on patients and medical resources.

3 PAX1 METHYLATION IN OTHER TUMORS

Aside from cervical cancer, PAX1 also displays hypermethylation in other tumors and offers great promise as a marker for cancer detection (Table 1). Detection of PAX1-methylation levels in oral scrapings or oral swabs indicated that PAX1-methylation levels and positive rates increased along with disease severity (SCC > precancerous lesions > normal oral mucosa) (Cheng et al., 2017, 2016; Huang et al., 2014), but decreased following cancer excision. However, these levels increased again at subsequent sites of recurrence in some cases at ~3- to ~4-months prior to recurrence (Cheng et al., 2016). These studies suggest PAX1 methylation as an effective biomarker for oral cancer detection and the prediction of oral cancer recurrence.

In epithelial ovarian cancer (EOC), PAX1 was significantly hypermethylated in HPV16/18-infected EOC tissues (Hassan et al., 2017). Another study showed that levels of PAX1 methylation were significantly higher in esophageal squamous cell carcinoma (Huang, Wang, et al., 2017), colorectal cancer (Huang, Tan, et al., 2017), and head and neck squamous cell carcinoma (Guerrero-Preston et al., 2014), and the PAX1 protein levels were lower in endometrial carcinoma (Liu et al., 2016). Moreover, detection of PAX1 methylation displayed relatively good sensitivity and specificity for the diagnosis of these tumors and holds great promise for tumor screening or as a prognostic marker.

4 POTENTIAL MECHANISMS OF PAX1 METHYLATION

In cancer development, hypermethylation of promoter regions containing CpG islands can inactivate tumor suppressor genes, thereby affecting genes associated with the cell cycle, DNA repair, cell–cell interactions, apoptosis, and angiogenesis (Herman & Baylin, 2003). In most vertebrates, PAX1 and PAX9 exhibit similar expression patterns and functions that belong to a highly conserved family of PAX genes, encode highly conserved transcription factors, and play roles in pattern formation during vertebrate embryogenesis (Paixao-Cortes, Salzano, & Bortolini, 2015). Studies show that PAX genes promote cell proliferation, cell-lineage specification, migration, and survival, and roles in tissue development and cellular differentiation in embryos (Dahl, Koseki, & Balling, 1997). In most cases, as fetal development progresses, PAX expression attenuates; however, in some tissues, PAX expression either persists into adult life or increases to exert functions, such as protection against stress-induced cell death (Cai et al., 2005). Studies PAX1 and PAX9 mutants show that PAX1 can compensate for the loss of PAX9, although not vice versa (Wilm, Dahl, Peters, Balling, & Imai, 1998). Little is known about the functional role of PAX1 in cancer biology. Liu et al. (2016) identified PAX1 protein levels as potential histopathology biomarkers for the differential diagnosis of malignant and premalignant endometrial lesions (Liu et al., 2016). Another study reported PAX1 expression-inducing tumor formation following subcutaneous injection of cultured cells expressing PAX1 into nude mice (Maulbecker & Gruss, 1993). Additionally, other studies showed that methylation of CpG islands in the PAX1 promoter region regulates cervical neoplasia (Chen et al., 2016; Kan et al., 2014), resulting in their characterization of PAX1 as a tumor suppressor gene. Although PAX1 functions have been hypothesized based on phenotypic outcomes associated with knockout models, PAX1 molecular functions and target genes remain largely unknown. Using mutation mouse models, studies have identified that PAX1 regulates epithelial cell death and proliferation during thymus and parathyroid organogenesis (Su, Ellis, Napier, Lee, & Manley, 2001). Studies investigating PAX1 reactivation in cervical cancer cell lines suggest that this can occur through curcumin and resveratrol administration through their effect on histone deacetylase accompanied by the downregulation of ubiquitin-like with PHD and RING finger domains 1 (OMIM: 607990), which regulates both DNA methylation and histone acetylation (Parashar & Capalash, 2016). Moreover, PAX1 methylation levels decrease along with increases in its mRNA expression after silencing of DNA methyltransferase 1 (DNMT1; OMIM: 126375), which plays a significant role in maintaining DNA methylation status and regulating the expression of tumor suppressor genes (Zhang et al., 2011). Similar results showed that curcumin, resveratrol, and DNMT1 influence PAX1 activity and might represent effective targets for treatment of cervical cancer (Parashar, Parashar, & Capalash, 2017). PAX1 methylation is also associated with NOTCH1 mutation and the Hedgehog pathway, which is regulated by HOX transcription factors and enhancer of split 1 (OMIM: 139605) (Bolos, Grego-Bessa, & de la Pompa, 2007; Forastiere, Koch, Trotti, & Sidransky, 2001; Guerrero-Preston et al., 2014; Koop et al., 2010; Landsman, Parent, & Hebrok, 2011; Mammucari et al., 2005; Manley & Capecchi, 1995; Mill et al., 2003; Sang, Roberts, & Coller, 2010; Schubert et al., 2005; Wall et al., 2009). Loss of ΝOTCH1 function due to mutation or the methylation-dependent silencing of downstream genes, such as PAX1, likely abrogates normal cell differentiation (Guerrero-Preston et al., 2014).

5 DISCUSSION AND PROSPECTS

PAX genes encode a family of nine transcription factors that act as cell-lineage-specific regulators of the tissues where they are normally expressed and are now recognized as important factors in cancer progression. Additionally, these factors might play previously unrecognized fundamental roles in balancing proliferation and differentiation signals. Numerous studies have demonstrated that PAX1 methylation plays an important role in the progression of cancers and contributes significantly to the sensitivity and specificity of cancer screening, especially for cervical cancer. In scrapings for cervical cancer, analyses indicated that PAX1 is silenced by hypermethylation. Moreover, PAX1 methylation plays a guiding role in the triage management of normal tissues, as well as CIN2, CIN3, LSIL, HSIL, and SCC patients. Although HPV testing is appealing for cervical cancer diagnosis, it cannot distinguish whether or not an HPV-positive result is associated with a clinically relevant lesion. Furthermore, these test results can be subject to overinterpretation and causing unnecessary panic. PAX1 methylation represents a novel biomarker that exhibits increased specific and accuracy for cervical cancer screening and diagnosis. There is increasing evidence that testing for methylated genes can replace cytology as a reflex test for HPV-positive women, and interim clinical guidance approves the use of such tests as an appropriate triage tool for HPV (Huh et al., 2015; Luttmer et al., 2016).

Here, we propose a screening strategy for cervical cancer that combines using the HPV testing and PAX1-methylation analysis as triage tests according to current evidence. In HPV-positive patients, the detection of PAX1 methylation is necessary for diagnosis of CIN3+ lesions (Figure 1) and will greatly benefit accurate cervical cancer screening, identify women that require treatment, and reduce unnecessary referrals for colposcopy and biopsy. However, additional standardization and large-scale clinical studies are needed to evaluate the efficacy of PAX1 methylation for cervical cancer screening and early detection. Additionally, further studies targeting the specific mechanisms associated with methylation-induced alterations in cellular activity are required to provide additional evidence supporting the clinical use of PAX1 methylation as a screening tool.

CONFLICTS OF INTEREST

The authors declare no competing financial interests.