Preferential Activation of the Hedgehog Pathway by Epigenetic Modulations in HPV Negative HNSCC Identified with Meta-Pathway Analysis

Elana J Fertig,Joseph A Califano,Daria A Gaykalova,Ana Markovic,Michael F Ochs,Christine H Chung,Leslie Cope,Ludmila V Danilova

doi:10.1371/journal.pone.0078127

Abstract

Head and neck squamous cell carcinoma (HNSCC) is largely divided into two groups based on their etiology, human papillomavirus (HPV)-positive and –negative. Global DNA methylation changes are known to drive oncogene and tumor suppressor expression in primary HNSCC of both types. However, significant heterogeneity in DNA methylation within the groups results in different transcriptional profiles and clinical outcomes. We applied a meta-pathway analysis to link gene expression changes to DNA methylation in distinguishing HNSCC subtypes. This approach isolated specific epigenetic changes controlling expression in HPV− HNSCC that distinguish it from HPV+ HNSCC. Analysis of genes identified Hedgehog pathway activation specific to HPV− HNSCC. We confirmed that GLI1, the primary Hedgehog target, showed higher expression in tumors compared to normal samples with HPV− tumors having the highest GLI1 expression, suggesting that increased expression of GLI1 is a potential driver in HPV− HNSCC. Our algorithm for integration of DNA methylation and gene expression can infer biologically significant molecular pathways that may be exploited as therapeutics targets. Our results suggest that therapeutics targeting the Hedgehog pathway may be of benefit in HPV− HNSCC. Similar integrative analysis of high-throughput coupled DNA methylation and expression datasets may yield novel insights into deregulated pathways in other cancers.

Highlights

Head and neck squamous cell carcinoma (HNSCC) is heterogeneous, arising in multiple sites within the head and neck region with diverse risk factors, including tobacco and alcohol use, and human papillomavirus (HPV) infection [1]
Not encoded in the analysis, meta-pathways identified from integrated DNA methylation and gene expression distinguished normal samples, HNSCC tumor samples, gender, HPV
Status, and tumor site, which were not distinguished robustly with hierarchical clustering. This improvement is similar to that observed with other non-negative matrix factorization (NMF)

Summary

Introduction

Head and neck squamous cell carcinoma (HNSCC) is heterogeneous, arising in multiple sites within the head and neck region with diverse risk factors, including tobacco and alcohol use, and human papillomavirus (HPV) infection [1]. The resulting analysis identified global patterns in gene expression driven by methylation changes in specific samples from the highthroughput data, without encoding information about the clinical phenotypes of samples. These patterns were associated with changes linked to tumor samples, HPV status, gender, and tumor subsite. The algorithm inferred pathways that reflect the biology of HNSCC [1] and identified novel coordinated DNA methylation and expression changes in GLI1 expression in a subset of HPV-negative HNSCC tumors

Methods

Results

Conclusion