Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Intragenic DNA methylation of PITX1 and the adjacent long non-coding RNA C5orf66-AS1 are prognostic biomarkers in patients with head and neck squamous cell carcinomas

  • Verena Sailer ,

    Contributed equally to this work with: Verena Sailer, Arthur Charpentier

    Roles Formal analysis, Methodology, Writing – original draft

    Affiliations Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, United States of America, Caryl and Israel Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY, United States of America

  • Arthur Charpentier ,

    Contributed equally to this work with: Verena Sailer, Arthur Charpentier

    Roles Data curation, Formal analysis

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

  • Joern Dietrich,

    Roles Data curation, Formal analysis, Software

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

  • Timo J. Vogt,

    Roles Formal analysis, Writing – review & editing

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

  • Alina Franzen,

    Roles Formal analysis, Writing – review & editing

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

  • Friedrich Bootz,

    Roles Conceptualization, Funding acquisition, Writing – review & editing

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

  • Dimo Dietrich ,

    Roles Conceptualization, Data curation, Formal analysis, Methodology, Visualization, Writing – original draft, Writing – review & editing

    dimo.dietrich@gmail.com

    ‡ These authors contributed equally as joint senior authors of this work.

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

  • Andreas Schroeck

    Roles Writing – review & editing

    ‡ These authors contributed equally as joint senior authors of this work.

    Affiliation University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery, Bonn, Germany

Intragenic DNA methylation of PITX1 and the adjacent long non-coding RNA C5orf66-AS1 are prognostic biomarkers in patients with head and neck squamous cell carcinomas

  • Verena Sailer, 
  • Arthur Charpentier, 
  • Joern Dietrich, 
  • Timo J. Vogt, 
  • Alina Franzen, 
  • Friedrich Bootz, 
  • Dimo Dietrich, 
  • Andreas Schroeck
PLOS
x

Abstract

Background

Patients with squamous cell cancer of the head and neck region (HNSCC) are at risk for disease recurrence and metastases, even after initial successful therapy. A tissue-based biomarker could be beneficial to guide treatment as well as post-treatment surveillance. Gene methylation status has been recently identified as powerful prognostic biomarker in HNSCC. We therefore evaluated the methylation status of the homeobox gene PITX1 and the adjacent long intergenic non-coding RNA (lincRNA) C5orf66-AS1 in publicly available datasets.

Methods

Gene methylation and expression data from 528 patients with HNSCC included in The Cancer Genome Atlas (TCGA, there obtained by using the Infinium HumanMethylation450 BeadChip Kit) were evaluated and methylation and expression levels of PITX1 and lincRNA C5orf66-AS1 was correlated with overall survival and other parameters. Thus, ten beads targeting PITX1 exon 3 and three beads targeting lincRNA C5orf66-AS1 were identified as significant candidates. The mean methylation of these beads was used for further correlation and the median was employed for dichotomization.

Results

Both PITX1 exon 3 and lincRNA C5orf66-AS1 were significantly higher methylated in tumor tissue than in normal adjacent tissue (NAT) (PITX1 exon 3: tumor tissue 58.1%, NAT: 31.7%, p<0.001; lincRNA C5orf66-AS1: tumor tissue: 27.4%, NAT: 18.9%, p<0.001). In a univariate analysis, hypermethylation of both loci was significantly associated with the risk of death (univariate: exon 3: Hazard ratio (HR): 4.97 [1.78–16.71], p = 0.010, lincRNA C5orf66-AS1: Hazard ratio (HR): 12.23 [3.01–49.74], p<0.001). PITX1 exon 3 and lincRNA C5orf66-AS1 methylation was also significantly correlated with tumor localization, T category, human papilloma virus (HPV)-negative and p16-negative tumors and tumor grade. Kaplan-Meier analysis showed, that lincRNA C5orf66-AS1 hypomethylation was significantly associated with overall survival (p = 0.001) in the entire cohort as well in a subgroup of HPV-negative tumors (p = 0.003) and in patients with laryngeal tumors (p = 0.022).

Conclusion

Methylation status of PITX1 and even more so of lincRNA C5orf66-AS1 is a promising prognostic biomarker in HNSCC, in particular for HPV-negative patients. Further prospective evaluation is warranted.

Introduction

Squamous cell carcinoma of the head and neck region (HNSCC) is a common malignancy with 5-year survival rates ranging from 61% for laryngeal cancers to 63% for cancers of the oral cavity and pharynx [1]. For 2017, 63,030 new cases are estimated in the United States and 13,360 patients are estimated to die of cancer-related causes [2].

Besides cigarette smoking and alcohol, infection with high-risk human papilloma viruses (HPV) like HPV16 has now been well established as an important risk factor in HNSCC development. HPV-positive tumors are predominantly located in the oropharynx and the incident of these tumors is increasing [3]. Moreover, since a positive HPV-status is associated with better overall survival, HPV-positive tumors have now been recognized as a distinct entity in the new 8th edition of the TNM-classification [4, 5]. Molecular features of an additional “atypical” HNSCC subtype, i.e. female patients with oral cavity tumors, who are HPV-negative and have no smoking history, have recently been described. These tumors exhibit a CpG island methylator phenotype (CIMP) with striking genomic stability, but are characterized by gene silencing through methylation events [6].

Since recurrence and metastasis is common after initial treatment, biomarkers need to be identified that can aid in stratification of treatment and surveillance after first-line curative therapy, in particular in HPV-negative patients [7]. More systemic therapeutic regimens than ever are now available for HNSCC patients, including immunotherapy [8]. Therefore, a prognostic biomarker can have a real impact in terms of influencing therapeutic strategy. A comprehensive review on molecular biomarkers in head and neck cancer is provided by Juodzbalys et al. [9].

Aberrant DNA methylation is found frequently in cancer and the methylation status of various functional genomic loci, e.g. promoters and bodies (exons and introns) of translated genes or genes encoding long non-coding RNAs (lncRNAs), can be used as biomarker. LncRNAs are longer than 200 base pairs and do not code for proteins but can influence cancer development and progression [10]. They can be classified as, amongst others, sense/antisense or long intergenic. Long intergenic non-coding RNA are localized between two genes and are involved in chromatin remodeling [11]. Sense/antisense non-coding RNA overlap one or more exons of other transcripts on the same (sense) or opposite (antisense) strand [12]. In addition, DNA methylation of long non-coding RNA promoter regions occurs during cancer development [13]. Promoter methylation of paired like homeodomain 2 (PITX2) transcription factor is a well-established and validated prognostic biomarker in various malignancies, including carcinomas of the prostate, breast, biliary tract, and lung [1420]. Recent publications suggest a role of methylation status of PITX3 and PITX2 and adjacent long non-coding RNA (PANCR) as promising new biomarkers in HNSCC [21, 22]. Another gene in this family, the transcription factor PITX1, might exhibit tumor-suppressing properties by regulating p53 transcription [23]. Moreover, PITX1-mediated p120RasGAP activation negatively influences Ras signaling [24, 25]. These data point to a role of PITX1 as potential tumor suppressor and dysregulation could influence cancer development. A germline single nucleotide polymorphism in the PITX1 gene has been identified as a susceptibility locus for colorectal cancer [26]. Like other genes in this family, PITX1 is an essential transcription factor in embryogenesis and is involved in mouth and hindlimb formation and pituitary development [2729]. Chromosomal rearrangements involving the PITX1 gene result in Liebenberg syndrome with partial arm-to-leg transformation [30]. PITX1 is located on chromosome 5q31, in very close proximity to the long intergenic non-coding RNA C5orf66-AS1, which is also called Epist [31, 32]. So far, only few studies have investigated lincRNA C5orf66-AS1. Feng et al. found a significant difference of expression when comparing tissue of oral squamous cell cancer with normal mucosa [32]. Yu et al. also observed decreasing expression of lincRNA C5orf66-AS1 from normal pituitary tissue to non-invasive to invasive pituitary null cell adenomas. They performed an additional in silico analysis and predicted PITX1 as target gene of lincRNA C5orf66-AS1. Interestingly, PITX1 expression is also decreased in several malignant tumors like gastric, colon and bladder cancer as well as HNSCC [25, 33, 34]. Reduced PITX1 expression is associated with treatment response in the latter [34]. The molecular mechanisms of the reduced expression of lincRNA C5orf66-AS1 and PITX1 in tumor tissue are incompletely understood and might be the result of epigenetic regulation. Aberrant PITX1 methylation has been found in salivary gland adenoid cystic carcinoma and is associated with survival in clear cell renal cell carcinomas [35, 36].

In the present study, methylation status of both PITX1 and lincRNA C5orf66-AS1 as well as corresponding mRNA levels were investigated and correlated with overall survival and clinicopathological parameters to evaluate its potential as a prognostic biomarker in HNSCC.

Methods

Ethical approval

The present study is based entirely upon data generated by the TCGA research network (www.cancergenome.nih.gov). All patients included in TCGA have been enrolled following strict human subjects protection guidelines, informed consent and IRB (Institutional Review Board) review of protocols. All patients provided informed consent (written).

Patients

527 head and neck squamous cell carcinoma patients included in the TCGA HNSC cohort with follow-up data were included and analyzed retrospectively. Data from normal adjacent tissues (NATs) were available for 50 of these patients. Data for PITX1 mRNA was available for 540 patients. Methylation data for both gene loci was available for 578 samples each. Clinicopathologic parameters of the whole cohort are listed in S1 Table. Survival was defined as time to death by any cause (overall survival, OS) and censored after five years (60 months), in order to exclude deaths that were not HNSCC-related [6].

DNA Methylation, mRNA and lncRNA expression, and HPV/p16 analyses

Data was processed as described previously [21]. In brief, gene methylation data were downloaded from the UCSC Xena browser (www.xena.ucsc.edu). TCGA methylation analysis has been performed using the Infinium HumanMethylation450 BeadChip (Illumina, Inc., San Diego, CA, USA). Methylation levels (Beta-values) were calculated as previously described [3739]. In brief, Beta-values were defined as: Beta = (Intensity_Methylated) / (Intensity_Methylated—Intensity_Unmethylated + α) [40]. The constant offset α was set to 0. Beta-values (values between 0 and 1) were multiplied with the factor 100% in order to shown percent methylation (0 to 100%). The following Illumina beads (numbered according to Fig 1) were investigated: cg00874891 (1), cg15922699 (2), cg04223420 (3), cg12673103 (4), cg18606375 (5), cg21614303 (6), cg08373003 (7), cg01966335 (8), cg24495017 (9), cg09713684(10), cg18443359 (11), cg14994060 (12), cg16258223 (13), cg11591267 (14), cg17034591 (15), cg04847174 (16), cg15528437 (17), cg03827835 (18), cg02037307 (19), cg05171952 (20), cg02213684 (21), cg02948884 (22), cg25648267 (23), cg15105206 (24), cg09839170 (25), cg01213519 (26), cg22488797 (27), cg07274716 (28), cg03347590 (29), cg23064601 (30), cg00089224 (31), cg19802165 (32), cg01830023 (33), cg24462476 (34), cg13441766 (35), cg15421305 (36), cg26509691 (37), cg00396667 (38), cg13715631 (39), cg11512280 (40), cg23341163 (41), cg11788465 (42), cg03654472 (43), cg06566775 (44), cg12622597 (45), cg22827250 (46), cg04101060 (47), cg02495310 (48), cg26972058 (49), cg08206318 (50), cg02100373 (51), cg08255782 (52), cg25330797 (53), cg12129103 (54), cg06933574 (55).

thumbnail
Fig 1. Organization of the PITX1 and C5orf66-AS1 genes.

Genomic organization of the PITX1 gene and C5orf66-AS1 genes and target regions of Illumina HumanMethylation450 BeadChip beads. Significant beads were grouped within the homeobox containing exon 3 and lincRNA C5orf66-AS1. Grouped beads are labeled with ‘*’ and ‘**’, respectively. The information was taken from Ensembl Homo sapiens version GRCh38.p10.

https://doi.org/10.1371/journal.pone.0192742.g001

In addition, RNAseq data was obtained. RNAseq data generation has been described before for TCGA [41]. C5orf66-AS1 (ENSG00000249082.1) lincRNA expression levels were obtained from TANRIC (http://ibl.mdanderson.org/tanric/_design/basic/download.html) [42]. Prevalence of HPV-infection was reported by using two different methods: p16 status by immunohistochemistry (termed p16-positive/negative) or viral integration assessed by RNAseq data (termed HPV-positive/negative).

Statistics

SPSS version 24 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Kaplan-Meier and Cox proportional hazard regression analyses were performed for survival analyses. Correlation between different parameters was tested using the Spearman’s rank correlation coefficient (ϱ). Mann-Whitney-U test, one-way analysis of variance (one-way ANOVA), and Fisher’s exact test were used for comparison between groups. P-values lower than 0.05 were considered significant.

Results

Identifying significantly methylated genomic regions

Fifty-five bead types for both gene loci were investigated (Fig 1) in this cohort of n = 527 patients. For PITX1, ten bead types were identified, which provided clinically significant information and matched to CpG islands on exon 3. Four beads were identified for lincRNA C5orf66-AS1. Two beads (cg18443359, cg15105206) are located within the second exon and one bead (cg03654472) targets the intron of C5orf66-AS1-201. One bead (cg01830023) is located 32 base pairs downstream from the antisense RNA.

Fifty out of 55 beads were differentially methylated between normal and tumor tissue. Inverse correlation between mRNA expression and methylation was found in 32/55 beads. Twenty-five out of 55 beads were significantly differential expression between HPV-positive and HV-negative cases. When evaluating methylation as continuous variable, 17/55 beads showed significant association with overall survival.

Of the ten beads selected for analysis in PITX1 exon 3, all but two beads were significantly associated with overall survival. All three beads selected for analysis in lincRNA C5orf66-AS1 were significantly associated with overall survival. The mean methylation of the respective beads was used for subsequent analyses.

PITX1 exon 3 and lincRNA C5orf66-AS1 methylation in tumor and normal adjacent tissue

Both PITX1 exon 3 and lincRNA C5orf66-AS1 were significantly higher methylated in tumor tissue than in normal adjacent tissue (PITX1 exon 3: tumor tissue 58.1%, NAT: 31.7%, p<0.001; lincRNA C5orf66-AS1: tumor tissue: 27.4%, NAT: 18.9%, p<0.001; Fig 2). Fig 1 shows, that the methylation was investigated not at a promoter region, but at an intragenic region within PITX1 exon 3. While methylation in promoter regions result in general in gene silencing, the functional consequence of intragenic methylation is less clearly defined. Both gene silencing and gene expression might ensue [43]. However, in the present dataset PITX1 mRNA was also evaluated and was negatively correlated with PITX1 exon 3 (Spearman’s ϱ = -0.347, p<0.001) as well as lincRNA C5orf66-AS1 methylation (Spearman’s ϱ = -0.350, p<0.001). This indicates gene silencing as result of DNA methylation.

thumbnail
Fig 2. PITX2 and C5orf66-AS1 methylation distribution in tissues.

Histogram showing the DNA methylation level (A: lincRNA C5orf66-AS1, B: PITX1 exon 3) in normal adjacent tissue (n = 50) and HNSCC samples (n = 528). Tumor tissue DNA was methylated significantly higher compared to corresponding normal adjacent tissue (PITX1 exon 3: p<0.001, lincRNA C5orf66-AS1: p<0.001; Mann-Whitney U test). In addition, two distinct peaks can be recognized in the DNA methylation level of lincRNA C5orf66-AS1, which are separated by the median cut-off.

https://doi.org/10.1371/journal.pone.0192742.g002

Association of PITX1 and lincRNA C5orf66-AS1 methylation status with clinicopathological parameters

Methylation and expression of both PITX1 mRNA and lincRNA C5orf66-AS1 were significantly associated with tumor location, p16 expression, HPV-status and complete resection (R0) (S1 Table). HPV-status as assessed by RNAseq was available for 279 patients, 243 of whom were HPV-negative and 36 patients were HPV-positive. Expression of p16, which is regarded as surrogate marker for HPV infection [44], was available for 115 patients: 74 patients were p16-negative and 41 patients were p16-positive. In general, a higher methylation and a lower mRNA expression of genes was seen in tumors located in the larynx and oral cavity as well as in HPV-negative tumors.

Association with overall survival

Cox proportional hazard analyses was performed using logarithmic value (base 2) of the methylation as continuous variable. The analysis as continuous variable without the introduction of a cut-off for result dichotomization allows for an accurate assessment of the biomarker performance without the risk of statistical artifacts resulting from overfitted cut-offs. In univariate analysis, both PITX1 exon 3 and lincRNA C5orf66-AS1 methylation was associated with an increased risk of death (hazard ratio (HR)PITX1exon3 = 1.63, 95%CI [1.12–2.38], p = 0.011; HRlncRNAC5orf66-AS1 = 1.47, 95%CI [1.16–1.86], p = 0.002). T category, nodal status and age were also associated with a risk of death in univariate analyses (p = 0.001, p = 0.016, p = 0.018, respectively; Table 1).

thumbnail
Table 1. Univariate and multivariate Cox proportional hazards analyses.

Univariate and multivariate Cox proportional hazards analyses on overall survival in 528 HNSCC patients stratified according to DNA methylation levels and clinico-pathologic variables. Methylation of lincRNA C5orf66-AS1 and PITX1 exon 3 as well as expression of PITX1 mRNA and lincRNA C5orf66-AS1 were analyzed as continuous variates logarithmized to base 2.

https://doi.org/10.1371/journal.pone.0192742.t001

Parameters that tested significant in univariate analyses were further analyzed in multivariate analyses. Here, T category remained as the only significant independent prognostic marker in multivariate Cox proportional hazard analysis.

Methylation status was dichotomized in hypo- and hypermethylation using the median as cut-off for both gene loci in the entire cohort (median PITX1 exon 3 methylation: 58.0%, median C5orf66-AS1 methylation: 27.4%). The median was also used to dichotomize RNA levels (median PITX1 mRNA: 2585 [normalized counts], median C5orf66-AS1 lincRNA: 0.61). lincRNA C5orf66-AS1 hypomethylation was significantly associated with an improved overall survival (p<0.001, Fig 3). Kaplan-Meier curves showed a trend towards better survival in patients with high PITX1 mRNA levels and hypomethylated PITX1 exon 3 gene locus, but this result failed to be statistically significant (p = 0.21 and p = 0.12, respectively). In addition, lincRNA C5orf66-AS1 RNA levels were not associated with survival (p = 0.22). An optimized cut-off might have resulted in a significant result for all parameters, but was not applied in order to avoid overfitting.

thumbnail
Fig 3. Kaplan-Meier survival analysis.

Kaplan-Meier survival analysis of overall survival of 526 HNSCC patients stratified according to DNA methylation status of lincRNA C5orf66-AS1. Patient samples were classified as hypo- and hypermethylated applying the median methylation level (27.4%) for dichotomization of the methylation levels.

https://doi.org/10.1371/journal.pone.0192742.g003

A subgroup analysis in different tumor locations (oral cavity, larynx and oropharynx) revealed a survival benefit for patients, whose laryngeal tumors were hypomethylated at the lincRNA C5orf66-AS1 locus (p = 0.022, Fig 4). A trend towards significance in survival was seen in oral cavity and oropharynx tumors. In contrast to oropharynx tumors, both oral cavity and larynx tumors are in general not associated with HPV-infection [45]. Further sub-stratification of tumor location by HPV-status might have elucidated upon the survival benefit for these patients, but was prevented by the lack of HPV data.

thumbnail
Fig 4. Kaplan-Meier survival analyses with regard to tumor localization.

Kaplan-Meier survival analysis of overall survival in the subgroups of oropharyngeal (n = 33), oral (n = 171) and laryngeal (n = 72) carcinomas, respectively, included in the 528 HNSCC patients from the TCGA. Patients were stratified according to DNA methylation status of lincRNA C5orf66-AS1. Patient samples were classified as hypo- and hypermethylated applying the median methylation level for dichotomization of the methylation levels.

https://doi.org/10.1371/journal.pone.0192742.g004

When correlating lincRNA C5orf66-AS1 hypo- and hypermethylation with p16 expression, a trend towards significance was seen in survival (p16-positive: n = 41, p = 0.086; p16-negative: n = 74, p = 0.12, Fig 5), which was supported by Kaplan-Meier curves, but ultimately failed to reach significance. This may be explained by the fact, that p16 expression was only available for 115 patients. However, additional stratification by HPV-status revealed a significantly better overall survival for patients with HPV-negative tumors and lincRNA C5orf66-AS1 hypomethylation (n = 243, p = 0.032, Fig 5).

thumbnail
Fig 5. Kaplan-Meier survival analyses with regard to HPV-status.

Kaplan-Meier survival analysis of overall survival in p16-positive (A), p16-negative (B), and HPV-negative (C) patients included in the 528 HNSCC patients from the TCGA. Patients were stratified according to DNA methylation status of lincRNA C5orf66-AS1. Patient samples were classified as hypo- and hypermethylated applying the median methylation level for dichotomization of the methylation levels.

https://doi.org/10.1371/journal.pone.0192742.g005

Discussion

The present study evaluates for the first time the clinical utility of PITX1 and lincRNA C5orf66-AS1 methylation status as prognostic biomarker in HNSCC. Both parameters added significant information about risk of death in univariate analysis and hyomethylation of lincRNA C5orf66-AS1 is associated with better survival, in particular in patients with negative HPV-status. While overall survival rates have improved in HNSCC, no improvement has been achieved for patients with laryngeal carcinoma, which is not thought to be associated with HPV infection [4547]. As yet, only few prognostic biomarkers have been investigated in this patient cohort. For example, in silico analysis of expression data deposited in the Gene Expression Omnibus (GEO) identified spectrin, a cytoskleton protein, as a promising new biomarker [48]. Expression of the tyrosine kinase c-MET has been shown to be prognostic in oral squamous cell cancer, which is in general not associated with HPV-infection [49]. Expression of sonic hedgehog pathway related genes Gli-1 and Gli-2 was found to be associated with overall survival in a prospective study in patients with HPV-negative HNSCC [50]. Methylation status of lincRNA C5orf66-AS1 could emerge as a useful prognostic biomarker to identify HPV-negative patients at risk for disease recurrence and metastases, who might benefit from additional therapy like immune checkpoint inhibitors.

The rationale for investigating PITX1 and lincRNA C5orf66-AS1 is strong, since both are lost in some tumor tissues. Moreover, genome-wide association studies have identified single nucleotide polymorphism associated with susceptibility of colorectal cancer in East Asian patients, notably rs647161 (A/C) on 5q31.1, which matches the position of PITX1 [26]. This confirms a more prominent role of PITX1 dysregulation in cancer development than previously thought. The present study indicates, that lincRNA C5orf66-AS1 might also be involved in tumorigenesis, likely by acting as (post)-transcriptional regulator of PITX1. LincRNA C5orf66-AS1 is down-regulated in esophageal squamous cell cancer and re-expression inhibits migration and invasion in vitro [51]. Further functional validation studies are needed to address this hypothesis. Loss of PITX1 mRNA in the TCGA HNSCC dataset cannot be explained by genomic events like chromosomal perturbances or loss-of-function mutation. Only two out of 279 patients in the TCGA HNSCC dataset with HPV-status determined by RNAseq had a genomic alteration, one deletion and one -likely passenger- missense mutation, (cBioPortal, [52]). However, while this data is intriguing, a major limitation of our study is that the available RNAseq did not allow us to discriminate between different transcript variants. This would require a more detailed computational analysis and needs to be done in further studies [53]. Since our study is solely based on data generated by the TCGA network and we don’t have access to the tissues, we were not able to perform analyses of the specific transcripts. A detailed isoform-specific analysis should be performed in future studies.

PITX1 is an upstream inducer of RASAL1 and thus an important mediator of the Ras signaling pathway[54]. Loss of PITX1 and lincRNA C5orf66-AS1 by aberrant epigenetic regulation might point to an overactive Ras signaling pathway, thus identifying patients who could benefit from therapy targeting this pathway, e.g. sorafenib. In a recent analysis, a strong Ras signaling pathway was associated with short response to platinum-based chemotherapy plus cetuximab in HNSCC patients. The authors further investigated this finding in vitro and found indeed an overactive Ras signaling in a cetuximab-sensitive cell line [55]. The predictive potential of PITX1 and lincRNA C5orf66-AS1 methylation status should be investigated ideally in a prospective randomized clinical study.

This is the first study that investigates methylation status of PITX1 and lincRNA C5orf66-AS1 in patients with HNSCC. lincRNA C5orf66-AS1 methylation is emerging as promising new prognostic biomarker to guide clinical treatment.

Supporting information

S1 Table. Associations of C5orf66-AS1 and PITX1 mRNA expression and methylation with clinicopathologic parameters.

https://doi.org/10.1371/journal.pone.0192742.s001

(XLSX)

References

  1. 1. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, et al. Cancer treatment and survivorship statistics, 2016. CA: a cancer journal for clinicians. 2016;66(4):271–89. pmid:27253694
  2. 2. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA: a cancer journal for clinicians. 2017;67(1):7–30. pmid:28055103
  3. 3. Simard EP, Ward EM, Siegel R, Jemal A. Cancers with increasing incidence trends in the United States: 1999 through 2008. CA: a cancer journal for clinicians. 2012;62(2):118–28. pmid:22281605
  4. 4. Huang SH, O'Sullivan B. Overview of the 8th Edition TNM Classification for Head and Neck Cancer. Curr Treat Options Oncol. 2017;18(7):40. Epub 2017/05/31. pmid:28555375
  5. 5. Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-Tan PF, et al. Human papillomavirus and survival of patients with oropharyngeal cancer. The New England journal of medicine. 2010;363(1):24–35. pmid:20530316
  6. 6. Brennan K, Koenig JL, Gentles AJ, Sunwoo JB, Gevaert O. Identification of an atypical etiological head and neck squamous carcinoma subtype featuring the CpG island methylator phenotype. EBioMedicine. 2017;17:223–36. Epub 2017/03/21. pmid:28314692
  7. 7. Cooper JS, Pajak TF, Forastiere AA, Jacobs J, Campbell BH, Saxman SB, et al. Postoperative concurrent radiotherapy and chemotherapy for high-risk squamous-cell carcinoma of the head and neck. N Engl J Med. 2004;350(19):1937–44. pmid:15128893
  8. 8. Blasco MA, Svider PF, Raza SN, Jacobs JR, Folbe AJ, Saraf P, et al. Systemic therapy for head and neck squamous cell carcinoma: Historical perspectives and recent breakthroughs. Laryngoscope. 2017. pmid:28581126
  9. 9. Juodzbalys G, Kasradze D, Cicciu M, Sudeikis A, Banys L, Galindo-Moreno P, et al. Modern molecular biomarkers of head and neck cancer. Part I. Epigenetic diagnostics and prognostics: Systematic review. Cancer biomarkers: section A of Disease markers. 2016;17(4):487–502. Epub 2016/11/02. pmid:27802200
  10. 10. Kondo Y, Shinjo K, Katsushima K. Long non-coding RNAs as an Epigenetic Regulator in Human Cancers. Cancer Sci. 2017. Epub 2017/08/05. pmid:28776911
  11. 11. Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 2010;329(5992):689–93. Epub 2010/07/10. pmid:20616235
  12. 12. Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell. 2009;136(4):629–41. Epub 2009/02/26. pmid:19239885
  13. 13. Yan X, Hu Z, Feng Y, Hu X, Yuan J, Zhao SD, et al. Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers. Cancer Cell. 2015;28(4):529–40. Epub 2015/10/16. pmid:26461095
  14. 14. Dietrich D, Hasinger O, Banez LL, Sun L, van Leenders GJ, Wheeler TM, et al. Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy. The Journal of molecular diagnostics: JMD. 2013;15(2):270–9. Epub 2012/12/26. pmid:23266319
  15. 15. Dietrich D, Hasinger O, Liebenberg V, Field JK, Kristiansen G, Soltermann A. DNA methylation of the homeobox genes PITX2 and SHOX2 predicts outcome in non-small-cell lung cancer patients. Diagnostic molecular pathology: the American journal of surgical pathology, part B. 2012;21(2):93–104. Epub 2012/05/05. pmid:22555092
  16. 16. Hartmann O, Spyratos F, Harbeck N, Dietrich D, Fassbender A, Schmitt M, et al. DNA methylation markers predict outcome in node-positive, estrogen receptor-positive breast cancer with adjuvant anthracycline-based chemotherapy. Clin Cancer Res. 2009;15(1):315–23. Epub 2009/01/02. pmid:19118060
  17. 17. Nimmrich I, Sieuwerts AM, Meijer-van Gelder ME, Schwope I, Bolt-de Vries J, Harbeck N, et al. DNA hypermethylation of PITX2 is a marker of poor prognosis in untreated lymph node-negative hormone receptor-positive breast cancer patients. Breast cancer research and treatment. 2008;111(3):429–37. Epub 2007/10/30. pmid:17965955
  18. 18. Schatz P, Dietrich D, Koenig T, Burger M, Lukas A, Fuhrmann I, et al. Development of a diagnostic microarray assay to assess the risk of recurrence of prostate cancer based on PITX2 DNA methylation. The Journal of molecular diagnostics: JMD. 2010;12(3):345–53. Epub 2010/03/23. pmid:20304943
  19. 19. Uhl B, Dietrich D, Branchi V, Semaan A, Schaefer P, Gevensleben H, et al. DNA Methylation of PITX2 and PANCR Is Prognostic for Overall Survival in Patients with Resected Adenocarcinomas of the Biliary Tract. PloS one. 2016;11(10):e0165769. Epub 2016/11/01. pmid:27798672
  20. 20. Uhl B, Gevensleben H, Tolkach Y, Sailer V, Majores M, Jung M, et al. PITX2 DNA Methylation as Biomarker for Individualized Risk Assessment of Prostate Cancer in Core Biopsies. The Journal of molecular diagnostics: JMD. 2017;19(1):107–14. Epub 2016/12/13. pmid:27939865
  21. 21. Sailer V, Gevensleben H, Dietrich J, Goltz D, Kristiansen G, Bootz F, et al. Clinical performance validation of PITX2 DNA methylation as prognostic biomarker in patients with head and neck squamous cell carcinoma. PloS one. 2017;12(6):e0179412. pmid:28617833
  22. 22. Sailer V, Holmes EE, Gevensleben H, Goltz D, Droge F, de Vos L, et al. PITX2 and PANCR DNA methylation predicts overall survival in patients with head and neck squamous cell carcinoma. Oncotarget. 2016. pmid:27716615
  23. 23. Liu DX, Lobie PE. Transcriptional activation of p53 by Pitx1. Cell death and differentiation. 2007;14(11):1893–907. pmid:17762884
  24. 24. Tai WT, Chen YL, Chu PY, Chen LJ, Hung MH, Shiau CW, et al. Protein tyrosine phosphatase 1B dephosphorylates PITX1 and regulates p120RasGAP in hepatocellular carcinoma. Hepatology. 2016;63(5):1528–43. pmid:26840794
  25. 25. Kolfschoten IG, van Leeuwen B, Berns K, Mullenders J, Beijersbergen RL, Bernards R, et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell. 2005;121(6):849–58. pmid:15960973
  26. 26. Jia WH, Zhang B, Matsuo K, Shin A, Xiang YB, Jee SH, et al. Genome-wide association analyses in East Asians identify new susceptibility loci for colorectal cancer. Nature genetics. 2013;45(2):191–6. pmid:23263487
  27. 27. Lanctot C, Lamolet B, Drouin J. The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm. Development. 1997;124(14):2807–17. pmid:9226452
  28. 28. Tremblay JJ, Lanctot C, Drouin J. The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Molecular endocrinology. 1998;12(3):428–41. pmid:9514159
  29. 29. Graham A, McGonnell I. Limb development: Farewell to arms. Curr Biol. 1999;9(10):R368–70. pmid:10339420
  30. 30. Mennen U, Mundlos S, Spielmann M. The Liebenberg syndrome: in depth analysis of the original family. J Hand Surg Eur Vol. 2014;39(9):919–25. pmid:23940102
  31. 31. Crawford MJ, Lanctot C, Tremblay JJ, Jenkins N, Gilbert D, Copeland N, et al. Human and murine PTX1/Ptx1 gene maps to the region for Treacher Collins syndrome. Mamm Genome. 1997;8(11):841–5. pmid:9337397
  32. 32. Feng L, Houck JR, Lohavanichbutr P, Chen C. Transcriptome analysis reveals differentially expressed lncRNAs between oral squamous cell carcinoma and healthy oral mucosa. Oncotarget. 2017;8(19):31521–31. pmid:28415559
  33. 33. Qi DL, Ohhira T, Fujisaki C, Inoue T, Ohta T, Osaki M, et al. Identification of PITX1 as a TERT suppressor gene located on human chromosome 5. Molecular and cellular biology. 2011;31(8):1624–36. pmid:21300782
  34. 34. Takenobu M, Osaki M, Fujiwara K, Fukuhara T, Kitano H, Kugoh H, et al. PITX1 is a novel predictor of the response to chemotherapy in head and neck squamous cell carcinoma. Mol Clin Oncol. 2016;5(1):89–94. pmid:27330773
  35. 35. Bell A, Bell D, Weber RS, El-Naggar AK. CpG island methylation profiling in human salivary gland adenoid cystic carcinoma. Cancer. 2011;117(13):2898–909. Epub 2011/06/22. pmid:21692051
  36. 36. Wei JH, Haddad A, Wu KJ, Zhao HW, Kapur P, Zhang ZL, et al. A CpG-methylation-based assay to predict survival in clear cell renal cell carcinoma. Nat Commun. 2015;6:8699. Epub 2015/10/31. pmid:26515236
  37. 37. Goltz D, Gevensleben H, Dietrich J, Ellinger J, Landsberg J, Kristiansen G, et al. Promoter methylation of the immune checkpoint receptor PD-1 (PDCD1) is an independent prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients following radical prostatectomy. Oncoimmunology. 2016;5(10):e1221555. pmid:27853645
  38. 38. Meller S, Zipfel L, Gevensleben H, Dietrich J, Ellinger J, Majores M, et al. CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients. Epigenetics. 2016:1–10. pmid:27689475
  39. 39. Goltz D, Gevensleben H, Grunen S, Dietrich J, Kristiansen G, Landsberg J, et al. PD-L1 (CD274) promoter methylation predicts survival in patients with acute myeloid leukemia. Leukemia. 2016. pmid:27840427
  40. 40. Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, Hou L, et al. Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics. 2010;11:587. Epub 2010/12/02. pmid:21118553
  41. 41. Cancer Genome Atlas Research N. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489(7417):519–25. pmid:22960745
  42. 42. Li J, Han L, Roebuck P, Diao L, Liu L, Yuan Y, et al. TANRIC: An Interactive Open Platform to Explore the Function of lncRNAs in Cancer. Cancer Res. 2015;75(18):3728–37. Epub 2015/07/26. pmid:26208906
  43. 43. Kulis M, Queiros AC, Beekman R, Martin-Subero JI. Intragenic DNA methylation in transcriptional regulation, normal differentiation and cancer. Biochimica et biophysica acta. 2013;1829(11):1161–74. Epub 2013/08/14. pmid:23938249
  44. 44. Jouhi L, Hagstrom J, Atula T, Makitie A. Is p16 an adequate surrogate for human papillomavirus status determination? Curr Opin Otolaryngol Head Neck Surg. 2017;25(2):108–12. Epub 2017/02/01. pmid:28141601
  45. 45. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 2000;92(9):709–20. Epub 2000/05/04. pmid:10793107
  46. 46. Pulte D, Brenner H. Changes in survival in head and neck cancers in the late 20th and early 21st century: a period analysis. The oncologist. 2010;15(9):994–1001. pmid:20798198
  47. 47. Gatta G, Botta L, Sanchez MJ, Anderson LA, Pierannunzio D, Licitra L, et al. Prognoses and improvement for head and neck cancers diagnosed in Europe in early 2000s: The EUROCARE-5 population-based study. European journal of cancer. 2015;51(15):2130–43. pmid:26421817
  48. 48. Yang SF, Bier-Laning CM, Adams W, Zilliox MJ. Candidate Biomarkers for HPV-Negative Head and Neck Cancer Identified via Gene Expression Barcode Analysis. Otolaryngol Head Neck Surg. 2016;155(3):416–22. pmid:27095047
  49. 49. Zhao D, Wang SH, Feng Y, Hua CG, Zhao J, Tang XF. Intratumoral c-Met expression is associated with vascular endothelial growth factor C expression, lymphangiogenesis, and lymph node metastasis in oral squamous cell carcinoma: implications for use as a prognostic marker. Human pathology. 2011;42(10):1514–23. pmid:21531000
  50. 50. Enzenhofer E, Parzefall T, Haymerle G, Schneider S, Kadletz L, Heiduschka G, et al. Impact of Sonic Hedgehog Pathway Expression on Outcome in HPV Negative Head and Neck Carcinoma Patients after Surgery and Adjuvant Radiotherapy. PloS one. 2016;11(12):e0167665. pmid:27918595
  51. 51. Wei G, Luo H, Sun Y, Li J, Tian L, Liu W, et al. Transcriptome profiling of esophageal squamous cell carcinoma reveals a long noncoding RNA acting as a tumor suppressor. Oncotarget. 2015;6(19):17065–80. pmid:26158411
  52. 52. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Science signaling. 2013;6(269):pl1. pmid:23550210
  53. 53. Zhang C, Zhang B, Lin LL, Zhao S. Evaluation and comparison of computational tools for RNA-seq isoform quantification. BMC Genomics. 2017;18(1):583. Epub 2017/08/09. pmid:28784092
  54. 54. Calvisi DF, Ladu S, Conner EA, Seo D, Hsieh JT, Factor VM, et al. Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer. J Hepatol. 2011;54(2):311–9. pmid:21067840
  55. 55. Bossi P, Bergamini C, Siano M, Cossu Rocca M, Sponghini AP, Favales F, et al. Functional Genomics Uncover the Biology behind the Responsiveness of Head and Neck Squamous Cell Cancer Patients to Cetuximab. Clinical cancer research: an official journal of the American Association for Cancer Research. 2016;22(15):3961–70. pmid:26920888