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. 2016 Dec 9:6:38758.
doi: 10.1038/srep38758.

HOPX functions as a tumour suppressor in head and neck cancer

Lee Fah Yap  1 , Sook Ling Lai  1 , Sathya Narayanan Patmanathan  1 , Ravindran Gokulan  1 , C Max Robinson  2 , Joe B White  3 , San Jiun Chai  4 , Pathmanathan Rajadurai  5 , Narayanan Prepageran  6 , Yew Toong Liew  6 , Victor Lopes  7 , Wenbin Wei  8   9 , Robert J Hollows  8 , Paul G Murray  8 , Daniel W Lambert  3 , Keith D Hunter  3 , Ian C Paterson  1
Affiliations

HOPX functions as a tumour suppressor in head and neck cancer

Lee Fah Yap et al. Sci Rep. .

Abstract

Head and neck squamous cell carcinoma (HNSCC) is generalized term that encompasses a diverse group of cancers that includes tumours of the oral cavity (OSCC), oropharynx (OPSCC) and nasopharynx (NPC). Genetic alterations that are common to all HNSCC types are likely to be important for squamous carcinogenesis. In this study, we have investigated the role of the homeodomain-only homeobox gene, HOPX, in the pathogenesis of HNSCC. We show that HOPX mRNA levels are reduced in OSCC and NPC cell lines and tissues and there is a general reduction of HOPX protein expression in these tumours and OPSCCs. HOPX promoter methylation was observed in a subset of HNSCCs and was associated with a worse overall survival in HPV negative tumours. RNAseq analysis of OSCC cells transfected with HOPX revealed a widespread deregulation of the transcription of genes related to epithelial homeostasis and ectopic over-expression of HOPX in OSCC and NPC cells inhibited cell proliferation, plating efficiency and migration, and enhanced sensitivity to UVA-induced apoptosis. Our results demonstrate that HOPX functions as a tumour suppressor in HNSCC and suggest a central role for HOPX in suppressing epithelial carcinogenesis.

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Figures

Figure 1
Figure 1. HOPX mRNA expression in OSCC and NPC.
The expression of HOPX was examined by RT-qPCR in normal oral keratinocytes (NOK), immortalised oral keratinocytes (OKF6), together with cell lines derived from dysplasias (D4, D19, D20, D35) and OSCCs (A) and in tissue samples of normal oral mucosa and OSCCs (B). A similar down-regulation of HOPX expression was seen in NPC cell lines (C) and tissues (D). Experiments were performed in triplicate and error bars indicate standard deviations.
Figure 2
Figure 2. HOPX protein expression in HNSCCs by immunohistochemistry.
The glands of secretory phase endometrium are known to express HOPX and were used as a positive control (A, arrowhead = gland). HOPX staining was strong in the keratinising layer of normal oral squamous epithelium (B,D and G, arrowhead = keratinising layer). By contrast, HOPX expression was reduced in oral premalignant lesions (C, arrowhead = keratinising layer). HOPX was absent in the majority of HNSCCs including OSCC, (D, * = malignant cells), OPSCC (F, * = malignant cells) and NPC (G, * = malignant cells). Focal HOPX staining co-localised with areas of keratinisation within tumours (E and F, arrowhead = keratinisation). Quantitation of HOPX expression using a quickscore method confirmed lower protein expression in premalignant lesions and OSCCs compared to normal oral squamous epithelium (H). Note: photomicrographs A, D-G HOPX Abcam ab57832, B and C HOPX Santa Cruz sc-30216. Experiment H used HOPX Santa Cruz sc-30216.
Figure 3
Figure 3. HOPX promoter methylation in HNSCC.
Quantitative analysis of HOPX-β promoter methylation in OSCC and NPC cell lines with normal oral keratinocytes and immortalised nasopharyngeal epithelial cell lines (A). Treatment of H413 OSCC cells with the demethylating drug, zebularine, for 5–10 days induced re-expression of HOPX (B). HOPX-β promoter methylation was associated with a reduction of HOPX expression as determined by comparing expression in matched tumour and normal data within the TCGA HNSCC dataset (C). Hypermethylated cases are shown in red, those with higher expression than their matched normal are shown in green, with the remainder shown in blue. Kaplan Meier survival analysis showed there was a significant association between HOPX-β promoter methylation and worse overall survival (D).
Figure 4
Figure 4. HOPX regulates the expression of genes involved in epithelial homeostasis.
Gene ontology (GO) analysis of down-regulated (A) and up-regulated (B) genes identified by RNASeq following ectopic expression of HOPX in H376 OSCC cells. Genes with over 1.5 fold change in expression in H376/HOPX cells compared to H376-pIRES were subjected to GO analysis. A similar GO analysis was performed on those genes down-regulated by HOPX in H376 and up-regulated in primary OSCCs (C) and those up-regulated by HOPX in H376 and down-regulated in tumours (D). Genes altered in H376/HOPX were compared with genes whose expression was altered in micro-dissected OSCCs (GSE35261 dataset16).
Figure 5
Figure 5. Tumour suppressive effects of HOPX in OSCC and NPC cells.
The proliferation (A), plating efficiency (B) and migration (C) of HOPX expressing H376 OSCC and HONE1 NPC cells was significantly reduced compared to vector transfected controls. HOPX expressing H376 and HONE1 cells were more sensitive to DNA damage induced by UVA irradiation as determined by the percentage of floating cells in the culture dish (D) after 24 hours and enhanced in apoptosis as determined by flow cytometric analysis of Annexin V staining 24 hours after irradiation (3 J/cm2 UVA; E). Results are shown as mean ± SD values of triplicates. *ρ < 0.05, **ρ < 0.01 and ***ρ < 0.001.
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References

    1. Ang K. K. et al.. Human papillomavirus and survival of patients with oropharyngeal cancer. The New England J. Med. 363, 24–35 (2010). - PMC - PubMed
    1. Zygogianni A. G. et al.. Oral squamous cell cancer: early detection and the role of alcohol and smoking. Head Neck Oncol. 3, 2 (2011). - PMC - PubMed
    1. Pathmanathan R., Prasad U., Sadler R., Flynn K. & Raab-Traub N. Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. The New England J. Med. 333, 693–698 (1995). - PubMed
    1. Cancer Genome Atlas N. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 517, 576–582 (2015). - PMC - PubMed
    1. Chen F. et al.. Hop is an unusual homeobox gene that modulates cardiac development. Cell. 110, 713–723 (2002). - PubMed

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