Synemin is a novel co-regulator of non-homologous end joining in head and neck cancer cells

Introduction:

Cancer therapy resistance is one of the major obstacles for higher cure rates. Novel key players of the resistome are focal adhesion proteins (FAPs). As FAPs are critically involved in DNA repair, we here characterize the yet unknown function of the FAP and intermediate filament protein Synemin (SYN) as a novel DNA repair regulator and potential cancer drug target in head and neck squamous cell carcinoma (HNSCC).

Methods and materials:

Our novel 3D High Throughput esiRNA Screen (3DHTesiRNAs) using laminin-rich extracellular matrix (lrECM) was conducted to measure radiation-induced residual DNA double strand breaks (DSBs; foci assay) and clonogenic radiation survival in UTSCC15-pEGFP-53BP1 cells. Validations were performed in 10 additional HNSCC cell lines in 3D lrECM. Upon SYN depletion, DSB repair reporter assays for non-homologous end joining (NHEJ) and homologous recombination (HR) as well as Western Blotting for protein expression and phosphorylation were carried out. SYN depleted cells with and without irradiation were analyzed for kinase activity profiling (PamGene) and protein interactome determination using a sequential immunoprecipitation/mass spectrometry approach.

Results and Discussion:

Among the targets found in the 3DHTesiRNAs, SYN turned out as one of the top FAP candidate determinants of HNSCC cell survival. SYN silencing radiosensitized HNSCC cells, while its exogenous overexpression induced radioprotection. We found an increased SYN/chromatin interaction and a marked perinuclear SYN accumulation post irradiation. Intriguingly, SYN depletion elicited a 40% reduction in NHEJ activity without affecting HR or alt-EJ. In line, ATM, DNA-PKcs and c-Abl phosphorylation as well as Ku70 expression strongly declined in SYN depleted and irradiated cells relative to controls and, in contrast, to the rescue of these protein modifications by SYN overexpression. Single, double and triple depletion of SYN, DNA-PKcs and c-Abl resulted in similar radiosensitization and DSB levels as observed for SYN only, suggesting its upstream role. In the kinome analysis we observed variable changes in the serine/threonine kinases, in contrast to the tyrosine kinases with a pronounce reduced kinase activity after SYN silencing.

Conclusion:

Our data suggest the intermediate filament SYN as a new important determinant of DNA repair, tyrosine kinome and radioresistance of HNSCC cells. These observations further support the notion that DNA repair is controlled by cooperative interactions between nuclear and cytoplasmic proteins.