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P74. PAX6: An Epigenetically-Regulated Candidate Tumor Suppressor in HPV-Associated Head and Neck Cancers

Thursday, March 5, 2026
4:09 PM - 4:16 PM MST
Location: 105A-C
Introduction

By epigenome-wide methylomic analyses, we have previously identified differentially methylated genes in HPV-positive (+) vs. HPV-negative (-) head and neck squamous cell carcinomas (HNSCC). Paired box gene 6 (PAX6), a lineage-specific tumor suppressor, was identified as epigenetically downregulated in most HPV+ HNSCC. We examined the impact of PAX6 manipulation in HPV+ and HPV- HNSCC cell lines.

Methods

PAX6 expression was analyzed by reverse transcriptase-PCR and Western blot in CRL3239 (HPV16+), HTB43 (HPV-), and its HPV E6/E7-transfected counterpart, CRL3212. The effects of PAX6 modulation by siRNA knockdown or full-length plasmid-mediated overexpression on cell proliferation (MTT), anchorage-independent growth (soft agar assay), and the transcriptome (RNA-Seq) were assessed.

Results

PAX6 was confirmed to be expressed in CRL3212 and HTB43, but absent in CRL3239. siRNA knockdown and forced overexpression were confirmed in all cell lines. In PAX6-expressing cell lines, siRNA knockdown resulted in increased proliferation [CRL3212 (p < 0.001), HTB43 (p < 0.001)] and colony formation in soft agar [CRL3212 (p=0.05) and HTB43 (p=0.04)] compared to empty vector controls. In CRL3239, forced expression resulted in decreased proliferation (p=0.0086) and colony formation (p=0.04). Pooled RNA-seq analysis following PAX6 silencing revealed significant enrichment of cell cycle and oncogenic signaling pathways, including MYC (q <0.001), E2F targets (q <0.001), and G2/M checkpoint regulation (q <0.001).

Conclusions

We have demonstrated that PAX6 is an epigenetically mediated tumor suppressor gene in HNSCC cell lines, with its function mediated by specific oncogenic and cell cycle pathways. Further studies are warranted to confirm the role of PAX6 in HPV-associated HNSCC carcinogenesis and its potential in preventive and therapeutic strategies.

Learning Objectives:

  • To elucidate the tumor suppressor function of PAX6 in HPV-associated head and neck squamous cell carcinoma
  • To understand how HPV affects tumor biology in head and neck squamous cell carcinoma