Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents.

Sarah R Sabir,George D D Jones,Andrew M Fry,Navdeep K Sahota

doi:10.1371/journal.pone.0140975

Sarah R Sabir, George D D Jones + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0140975

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Journal: PloS one	Publication Date: Oct 26, 2015
Citations: 43	License type: CC BY 4.0

Affiliation: University of Leicester

Abstract

The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage.

Highlights

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the Western world
To explore how Nek11 might contribute to the DNA damage response (DDR) of CRC cells, a protocol was established that allowed cell cycle progression to be monitored by flow cytometry following Nek11 depletion and ionizing radiation (IR) exposure (Fig 1A)
Following IR exposure, cells depleted of Nek11 exhibited a substantial reduction in the G2/M fraction as compared to cells depleted with control oligonucleotides, with siNek11-2 causing a return to the basal level of G2/M cells (Fig 1E)

Summary

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the Western world. Current standard care for CRC patients following surgery involves chemotherapy combinations that usually include DNA damaging agents. Many patients receive FOLFIRI as first line therapy, a combination of folinic acid, 5-fluorouracil (5-FU) and irinotecan [1]. 5-FU is a pyrimidine analogue that blocks DNA synthesis through inhibiting DNA polymerase, while folinic acid potentiates the effect of 5-FU by inhibiting thymidylate synthase. Irinotecan is an inhibitor of topoisomerase I that causes single-strand DNA breaks, which are usually converted into double-strand breaks (DSBs). These activate the DNA damage checkpoints and cause arrest of the cell cycle at G1/S or G2/M.

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Conclusion