The role of iRhom2 in the pathogenesis of head and neck squamous cell carcinomas (HNSCC)

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It has been proposed that the Notch signalling pathway plays a key role in the pathogenesis of head and neck squamous cell carcinomas (HNSCC). It has also been shown that the sheddase, ADAM17, whose intracellular trafficking and subsequent maturation is dependent on iRhom2, is key in the activation of the Notch pathway at the cell membrane, leading to the release of an intracellular domain for downstream activities. This thesis investigates the levels and distribution of iRhom2 and ADAM17 in HNSCC, and functional changes resulting from up-regulation of iRhom2 in HNSCC cell lines. Immunoblotting, using protein samples extracted from fresh, snap-frozen tissues (68 HNSCC and 27 paired normal tissues), and probing for iRhom2 identified relatively high expression levels of this protein in 43/68 tumour samples compared to 5/27 normal samples (p<0.05). Similar observations were obtained for ADAM17 expression, with high expression in 27/68 tumour samples compared to 4/27 normal samples (P<0.05). A positive correlation was observed between levels of expressions of iRhom2 and ADAM17 in these tissues (though not statistically significant), but only iRhom2 expression correlated with patient survival (p<0.05). Non-correlation of iRhom2 expression with other clinicopathological features was perhaps due to the relatively small number of samples investigated. Using a different cohort of HNSCC tissues, we also assessed levels of iRhom2 and ADAM17 and their intracellular distribution using tissue microarray (TMA) approach. Western blot results could not be corroborated with this methodology, given that most of the tissues stained negative for iRhom2, and 76/88 stained “highly positive” for ADAM17, with no observable specificity with ADAM17 staining pattern. Up-regulation of iRhom2 was achieved in the HNSCC cell lines, PE/CA-PJ15 and LIV37K; and in NOK (normal oral keratinocyte) cells and was, followed by shRNA knock-down of RHBDF2 (which codes for iRhom2). No observable differences were observed in the rate of cell replication when comparing wild-type, over-expressing and knock-down clones across each of the cell lines. However, a higher rate of cell migration was demonstrated in association with iRhom2 up-regulation, more in the HNSCC cell lines than the NOK. This higher rate of migration was shown to be reversed by the shRNA knock-down of RHBDF2, demonstrating that it is increased iRhom2 that is causative. Furthermore, a significant increase in the level of expression of mature ADAM17 was observed, following upregulation of iRhom2. iRhom2 and ADAM17 are both up-regulated in HNSCC, with up-regulation of iRhom2 associated with increased cell migration and decreased patient survival. Further experiments should aim to address whether it is the increased ADAM17 expression / activity and / or Notch activity that is the downstream effector of the observed effects. If will be important to expand the cohort of samples and cell lines used for this study to further validate the present findings, while also optimising some of the aspects that have not achieved significant results.