Fractionation-Dependent Radiosensitization by Molecular Targeting of Nek1.

Isabel Freund,Markus Löbrich,Emmanouil Fokas,Daniel Martin,Stephanie Hehlgans,Claus Rödel,Franz Rödel,Michael Ensminger

doi:10.3390/cells9051235

Abstract

NIMA (never-in-mitosis gene A)-related kinase 1 (Nek1) is shown to impact on different cellular pathways such as DNA repair, checkpoint activation, and apoptosis. Its role as a molecular target for radiation sensitization of malignant cells, however, remains elusive. Stably transduced doxycycline (Dox)-inducible Nek1 shRNA HeLa cervix and siRNA-transfected HCT-15 colorectal carcinoma cells were irradiated in vitro and 3D clonogenic radiation survival, residual DNA damage, cell cycle distribution, and apoptosis were analyzed. Nek1 knockdown (KD) sensitized both cell lines to ionizing radiation following a single dose irradiation and more pronounced in combination with a 6 h fractionation (3 × 2 Gy) regime. For preclinical analyses we focused on cervical cancer. Nek1 shRNA HeLa cells were grafted into NOD/SCID/IL-2Rγc−/− (NSG) mice and Nek1 KD was induced by Dox-infused drinking water resulting in a significant cytostatic effect if combined with a 6 h fractionation (3 × 2 Gy) regime. In addition, we correlated Nek1 expression in biopsies of patients with cervical cancer with histopathological parameters and clinical follow-up. Our results indicate that elevated levels of Nek1 were associated with an increased rate of local or distant failure, as well as with impaired cancer-specific and overall survival in univariate analyses and for most endpoints in multivariable analyses. Finally, findings from The Cancer Genome Atlas (TCGA) validation cohort confirmed a significant association of high Nek1 expression with a reduced disease-free survival. In conclusion, we consider Nek1 to represent a novel biomarker and potential therapeutic target for drug development in the context of optimized fractionation intervals.

Highlights

Genetic screens for cell cycle mutants in the fungus Aspergillus nidulans resulted in the discovery of never-in-mitosis gene A (NIMA), a dual serine/threonine kinase required for mitotic entry [1].In mammals, 11 homologs named never-in-mitosis kinase-related kinases (Nek1-Nek11) have been reported with a 40% amino-acid similarity in their conserved kinase domains [2,3]
Cells 2020, 9, 1235 evidence indicate a prominent role of Nek1, the prototypic member of the family, in the DNA damage response (DDR), shown by its interaction with proteins involved in DNA repair pathways, cell cycle regulation and apoptosis [4,5]
We report that Nek1 knockdown (KD) in combination with a 6 h fractionation regime resulted in radiation sensitization and growth delay in a xenograft model of cervical cancer suggesting that Nek1 might represent a valuable molecular target

Summary

Introduction

Genetic screens for cell cycle mutants in the fungus Aspergillus nidulans resulted in the discovery of never-in-mitosis gene A (NIMA), a dual serine/threonine kinase required for mitotic entry [1].In mammals, 11 homologs named never-in-mitosis kinase-related kinases (Nek1-Nek11) have been reported with a 40% amino-acid similarity in their conserved kinase domains [2,3]. Cells 2020, 9, 1235 evidence indicate a prominent role of Nek, the prototypic member of the family, in the DNA damage response (DDR), shown by its interaction with proteins involved in DNA repair pathways, cell cycle regulation and apoptosis [4,5]. Nek is significantly up-regulated in cells exposed to ionizing radiation, alkylating agents, UV, cross linking agents and oxidative stress [6,7] and colocalizes with markers of the early DDR, such as phosphorylated H2AX [6,8]. A mechanistic role for Nek during homologous recombination (HR) was recently reported where it phosphorylates Rad at residue. Ser572 in a G2 phase specific manner [9]. This promotes Rad removal from chromatin and fosters proper completion of HR. A tousled-like kinase 1 (TLK1) > Nek1 > Ataxia Telangiectasia

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