Metastasis Suppressor NME1 Modulates Choice of Double-Strand Break Repair Pathways in Melanoma Cells by Enhancing Alternative NHEJ while Inhibiting NHEJ and HR.

Gemma Puts,Devin Snyder,Michal Zalzman,Benjamin Portney,Lena Mclaughlin,Feyruz Rassool,Mary Leonard,Rachel Abbotts,Stuart Jarrett,Ying Wang,Nicolette Matsangos,Richard Vincent,David Kaetzel

doi:10.3390/ijms21165896

Abstract

Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.

Highlights

Metastasis suppressors are a unique class of genes (MSGs) that suppress metastatic potential of cancer cells without affecting their growth characteristics in vitro and in vivo [1]
While immunostaining of untreated WM793 cells with anti-NME1 antibody was diffuse and exclusively extra-nuclear, γ-IR treatment resulted in complete translocation of NME1 into nuclear foci. γ-H2AX-positive and NME1-positive foci were absent from nuclei in the absence of γ-IR treatment
Approximately γ-H2AX-positive foci and NME1-positive foci were largely colocalized (40%; Figure 1F). These concordant observations obtained in two cell lines of distinct cancer origins indicate induction of double-stranded breaks (DSBs) stimulates profound translocation of NME1 into the nucleus and recruitment of NME1 to focal structures within the nucleus that are associated with double-strand break repair (DSBR)

Summary

Introduction

Metastasis suppressors are a unique class of genes (MSGs) that suppress metastatic potential of cancer cells without affecting their growth characteristics in vitro and in vivo [1]. Repair of double-stranded breaks in DNA (DSBs) via homologous recombination (HR) pathways requires trimming of overhanging 3 -termini by 3 -5 exonuclease such as MRE11 and this activity can sometimes occur during non-homologous end joining (NHEJ) [16]. The reduced expression of NME1 would appear likely to confer genomic instability and acquisition of progression-driving alterations in DNA structure (e.g., point mutations, insertions/deletions, translocations). In this regard, we showed that point mutations disabling the 3 -5 exonuclease activity of NME1 are associated with loss of metastasis suppressor function in melanoma cells [19]

Methods

Results

Conclusion