Publications Repository - Helmholtz-Zentrum Dresden-Rossendorf

Introduction:

Focal adhesion proteins (FAPs) have been shown to essentially contribute to cancer cell therapy resistance. Based on our previous finding that integrins partially control DNA repair processes, we here aim at characterizing the function of FAPs in the DNA damage response. Among others, we identified Synemin, an intermediate filament protein, as novel DNA repair regulator and highly potential novel cancer target in head and neck squamous cell carcinoma (HNSCC).

Methods and materials:

A novel 3D High Throughput esiRNA Screen (3DHTesiRNAs) using (3D)-laminin-rich extracellular matrix (lr-ECM) was established. Screening for residual double strand breaks (DSBs) and clonogenic radiation survival was performed in UTSCC15-pEGFP-53BP1 HNSCC cells upon esiRNA-mediated FAPs knockdown and X-ray exposure (6 Gy). The top 2 targets were validated in a panel of 10 3D lr-ECM HNSCC cell cultures regarding γH2AX/53BP1 foci and clonogenic survival. Immunostaining and 3D chromatin fractionation (CF) of Synemin prior and post irradiation (IR) were performed. Upon Synemin knockdown, DNA repair assay for NHEJ and HR as well as Western Blotting for protein expression and phosphorylation were employed.

Results:

Among a number of interesting novel targets found in our 3DHTesiRNAs, Synemin turned out as novel determinant of HNSCC radiosensitivity. Synemin silencing led to radiosensitization of 3D HNSCC cell cultures. Intriguingly, we showed that Synemin knockdown resulted in a 40% reduction in NHEJ without affecting HR. Concomitantly, phosphorylation of ATM Ser1981, DNA-PKcs Ser2056 and c-Abl Tyr412 were diminished relative to controls. Associated with these observations, we found a dramatic Synemin accumulation in the perinuclear area, which is accompanied by an increased interaction of Synemin with chromatin.

Conclusion:

Our data indicate the interfilament protein Synemin as a new important determinant of DNA repair and radioresistance in HNSCC cells. Ongoing research is focusing on evaluating the molecular mechanism how Synemin participates in NHEJ and chromatin organization.