C-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair.

Sara Sofia Deville,Nils Cordes,Luis Fernando Delgadillo Silva,Anne Vehlow

doi:10.3390/ijms21197277

Abstract

The intermediate filament synemin has been previously identified as novel regulator of cancer cell therapy resistance and DNA double strand break (DSB) repair. c-Abl tyrosine kinase is involved in both of these processes. Using PamGene technology, we performed a broad-spectrum kinase activity profiling in three-dimensionally, extracellular matrix grown head and neck cancer cell cultures. Upon synemin silencing, we identified 86 deactivated tyrosine kinases, including c-Abl, in irradiated HNSCC cells. Upon irradiation and synemin inhibition, c-Abl hyperphosphorylation on tyrosine (Y) 412 and threonine (T) 735 was significantly reduced, prompting us to hypothesize that c-Abl tyrosine kinase is an important signaling component of the synemin-mediated radioresistance pathway. Simultaneous targeting of synemin and c-Abl resulted in similar radiosensitization and DSB repair compared with single synemin depletion, suggesting synemin as an upstream regulator of c-Abl. Immunoprecipitation assays revealed a protein complex formation between synemin and c-Abl pre- and post-irradiation. Upon pharmacological inhibition of ATM, synemin/c-Abl protein-protein interactions were disrupted implying synemin function to depend on ATM kinase activity. Moreover, deletion of the SH2 domain of c-Abl demonstrated a decrease in interaction, indicating the dependency of the protein-protein interaction on this domain. Mechanistically, radiosensitization upon synemin knockdown seems to be associated with an impairment of DNA repair via regulation of non-homologous end joining independent of c-Abl function. Our data generated in more physiological 3D cancer cell culture models suggest c-Abl as further key determinant of radioresistance downstream of synemin.

Highlights

DNA repair processes are fundamentally regulated by several extra-nuclear factors via not well-characterized mechanisms
We reported that the intermediate filament synemin is involved in the response to X-ray irradiation in dependence on DNA-PKcs [23]
We further addressed the function of synemin in DNA damage repair (Figure 1G–I) and upon cisplatin (CDDP) treatment alone or in combination with X-ray irradiation in synemin-depleted HNSCC cell cultures (Figure 1J–L)

Summary

Introduction

DNA repair processes are fundamentally regulated by several extra-nuclear factors via not well-characterized mechanisms Some examples of such regulators are growth factor receptors and integrin cell adhesion molecules. Accumulating evidence suggests that cytoplasmic FAP signaling is linked to nuclear repair dynamics through interactions between the pro-survival non-receptor tyrosine kinase c-Abl and components of the DNA repair machinery, including DNA-PKcs, ATM, BRCA1 and RAD51 [11,12]. These observations suggest a crosstalk between cytoskeletal factors and nuclear DNA repair-associated processes

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Conclusion