Abstract
Non-small cell lung cancer (NSCLC) represents ~85% of the lung cancer cases. Despite recent advances in NSCLC treatment, the five-year survival rate is still around 23%. Radiotherapy is indicated in the treatment of both early and advanced stage NSCLC; however, treatment response in patients is heterogeneous. Thus, identification of new and more effective treatment combinations is warranted. We have identified Ubiquitin-specific protease 14 (USP14) s a regulator of major double-strand break (DSB) repair pathways in response to ionizing radiation (IR) by its impact on both non-homologous end joining (NHEJ) and homologous recombination (HR) in NSCLC. USP14 is a proteasomal deubiquitinase. IR treatment increases levels and DSB recruitment of USP14 in NSCLC cell lines. Genetic knockdown, using shUSP14 expression or pharmacological inhibition of USP14, using IU1, increases radiosensitization in NSCLC cell lines, as determined by a clonogenic survival assay. Moreover, shUSP14-expressing NSCLC cells show increased NHEJ efficiency, as indicated by chromatin recruitment of key NHEJ proteins, NHEJ reporter assay, and increased IR-induced foci formation by 53BP1 and pS2056-DNA-PKcs. Conversely, shUSP14-expressing NSCLC cells show decreased RPA32 and BRCA1 foci formation, suggesting HR-deficiency. These findings identify USP14 as an important determinant of DSB repair in response to radiotherapy and a promising target for NSCLC radiosensitization.
Highlights
Lung cancer is one of the most common malignancies worldwide and has become the leading cause of cancer-related death in USA
We report for the first time that Ubiquitin-specific protease 14 (USP14): (i) has a role in regulating homologous recombination (HR) and (ii) is a target to increase radiosensitization in non-small cell lung cancer (NSCLC)
USP14 undergoes IR-induced foci (IRIF) formation in NSCLC cells and its levels increase in chromatin-bound fractions in response to ionizing radiation (IR) treatment
Summary
Ubiquitination signaling at DSB sites by the E3 ubiquitin ligases RNF8 and RNF168 is crucial in determining DNA repair pathway choice by recruiting p53-binding protein 1 (53BP1) to DSBs. 53BP1, together with its partner protein(s) e.g., Rap1-interacting factor 1 (RIF1), inhibits BRCA1 (Breast Cancer gene 1)-CtIP (CTBP interacting protein) complex-dependent DSB end resection. This promotes rapid NHEJ of the DSB ends and inhibits the HR pathway. We have identified a novel nuclear role of USP14 as a negative regulator of NHEJ and DDR-associated ubiquitination signaling in autophagy-deficient prostate cancer (PCa) cells [21,22]. Targeting USP14 is a potential mechanism to enhance radiosensitization in NSCLC
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