Investigating a Role for the Duchenne Muscular Dystrophy Gene in the Tumourigenesis of Head and Neck Cancer

Epithelial-derived head and neck squamous cell carcinomas (HNSCC) constitute 90% of all head and neck cancers and represent the sixth most common cancer worldwide which has a 50% five-year survival rate. Mutation of the Duchenne muscular dystrophy (DMD) gene is well established as a cause for neuromuscular disorders but increasing evidence has implicated DMD in the development and progression of major cancer types including those of the head and neck. The DMD gene encodes for several dystrophin protein products that have a range of roles outside of muscle. However, the details surrounding their involvement in tumourigenesis are poorly understood. Using primary HNSCC tumour RNA-Seq data from The Cancer Genome Atlas (TCGA) database paired with clinical data, the association of DMD expression with survival outcomes was assessed. Kaplan-Meier (KM) analysis showed that HNSCC individuals with high DMD expression were associated with a better overall survival outcome compared to individuals with low DMD expression, with a median survival difference of 26 months (log rank test p= 0.0083, n=526). Human papillomavirus (HPV) is one of the main biomarkers used for prognosis, where people who are HPV positive have a better outcome than those who are HPV negative. Interestingly, combined analysis in HPV positive individuals further stratified patients with a poorer overall survival (log rank test p= 0.045) suggesting DMD expression combined with HPV status may define a new subgroup with poor prognosis. Gene variant expression analysis showed that dystrophin protein 71 (Dp71) and its four isoforms (Dp71, Dp71a, Dp71b and Dp71ab) were predominantly expressed in primary HNSCC, and further KM analysis showed that high Dp71ab expression was associated with a better overall survival compared to patients with low Dp71ab expression with a median survival difference of 42 months (log rank test p= 0.0007, n=526). Protein expression analysis of dystrophin in 50 cases of HNSCC tissue using immunohistochemistry showed dystrophin expression in both the nucleus and cytoplasm of tumour cells and this was congruent with the RNAseq data showing that individuals with high nuclear dystrophin expression were vi associated with a better overall survival compared to individuals with low dystrophin expression (log rank test p=0.049, n=50). Dp71 expression was investigated in two HNSCC cell lines: FaDu a hypopharyngeal line with good endogenous Dp71 expression; and H314 an oral cavity line with no Dp71 expression. Cell migration was investigated through wound scratch assays and spheroid analysis however, none of the overexpressed Dp71 isoforms affected cell migration. H314 cells transfected with Dp71ab had altered nuclear morphology compared to control and there was a significant reduction in the number of normal nuclei (43.6%) and a concomitant increase in small regular (41.3%), small irregular (7.9%), and irregular (6.6%) nuclei. This suggested the presence of nuclear / mitotic damage which could affect cell proliferation. This was confirmed using WST1 proliferation assays and showed metabolic activity was significantly reduced in Dp71ab overexpressed FaDu (p = 0.0107) and H314 (p = 0.0172) cells. Overall, this work has for the first time identified a role for DMD, and specifically Dp71ab, expression in the tumorigeneses of HNSCC. The use of DMD/dystrophin expression in combination with HPV status may have added prognostic value and define additional subgroups that may better tailor treatment decisions. This work adds to the accumulating evidence for an important role of dystrophin in cancer which warrants further investigation in more complex disease models.