Synemin, a novel regulator of DNA repair machinery and tyrosine kinases in head and neck cancers

Introduction: Focal adhesion proteins (FAPs) have been shown to be essential determinants of cancer therapy outcome. Our previous findings revealed the role of FAPs in DNA repair processes. Here, we characterized the unknown and novel functions of Synemin, an intermediate filament protein, which functions as FAP, as novel double strand break (DSB) repair and as tyrosine kinase regulator in head and neck squamous cell carcinoma (HNSCC).
Materials and methods: Using a novel 3D High Throughput RNAi Screen (3D HTP-RNAi-S), clonogenic survival and double strand breaks (DSB) repair in non- and -irradiated HNSCC were analyzed upon knockdown of 117 FAP. Confirmatory data were generated in a panel of 10 HNSCC cell lines. Reporter gene assays were applied to determine the efficiency in DNA DSB repair by non-homologous end joining (NHEJ) and homologous recombination (HR). Western blot was used to determine protein expression and phosphorylation. Immunoprecipitation (IP) and kinase activity profiling (PamGene) were carried out to determine Synemin interactome.
Results and Discussion: Among the potential FAP targets from our 3D HTP-RNAi-S, Synemin turned out as a novel and most promising candidate in controlling HNSCC radiosensitivity. Intriguingly, Synemin depletion induced a 40% reduced NHEJ activity while leaving HR unchanged. We demonstrated significant dephosphorylation of DNA-PKcs kinase, a key component of the NHEJ pathway, as well as reduced levels of Ku70 in Synemin depleted, irradiated HNSCC cells as compared to controls. We further demonstrated an almost comprehensive deactivation of 86 tyrosine kinases after Synemin silencing. Among these, bioinformatic analysis revealed c-Abl highly downregulated at 24 hours post irradiation in Synemin-depleted HNSCC cells. Co-IP revealed an interaction between Synemin and c-Abl suggesting those proteins to form a protein complex. Single, double and triple depletion of Synemin, DNA-PKcs, and c-Abl resulted in similar radiosensitization and DSB levels, suggesting Synemin to be located upstream of these DNA repair kinases.
Conclusion: Our data suggest the intermediate filament Synemin as a novel determinant of DNA repair, tyrosine kinase regulation and radiosensitivity of HNSCC cells. These observations further support the notion that DNA repair is controlled by cooperative interactions between nuclear, membrane and cytoplasmic proteins.