Role of HOPX in Head and Neck Squamous Cell Carcinoma

Objectives: Homeodomain Only Protein X (HOPX) is an atypical homeobox gene which plays a key role in cardiac development.  It has recently been shown to be downregulated in a variety of malignancies, including head and neck squamous cell carcinoma (HNSCC), perhaps as a result of epigenetic silencing of the HOPX promoter. This project aimed to assess the expression and function of HOPX in various stages of HNSCC development. Methods: Expression of the 5 splice variants of HOPX, NKX2-5 and SRF was assessed by qPCR in a panel of normal oral keratinocytes, oral dysplasia cell lines and HNSCC cell lines. Expression of HOPX was investigated ex vivo in normal, dysplastic and HNSCC tissues by immunohistochemistry.  Low expressing cell lines were treated with 5-aza-2 ’deoxycytidine, trichostatin A or a combination of both. HOPX transcript variant 5 was transiently transfected into low expressing cells, followed by assessment of NKX2-5 expression and markers of terminal differentiation. Results: HOPX expression was reduced in immortalized oral keratinocytes, oral dysplasia cell lines and HNSCC cell lines at transcript level.  HOPX was also down-regulated in tissues progressively through dysplasias to HNSCCs. HOPXv4 and v5 were re-expressed after treatment with 5-aza-2’deoxycytidine and trichostatin A, which resulted in a reduction in NKX2-5 expression in H357 cells. Transient transfection with HOPXv5 resulted in no change in NKX2-5 expression and variable expression of involucrin in dysplasia and HNSCC cell lines. Conclusion: HOPX expression is reduced in immortal cells from all stages of HNSCC development. The role of HOPX and its splice variants in keratinocyte differentiation and immortalisation is unclear and will form the basis of further investigation.