Decrease of Nibrin expression in chronic hypoxia is associated with hypoxia-induced chemoresistance in some brain tumour cells

Sophie Cowman,Yuen Ngan Fan,Barry Pizer,Violaine Sée

doi:10.1186/s12885-019-5476-9

Sophie Cowman, Yuen Ngan Fan + Show 2 more

Open Access

https://doi.org/10.1186/s12885-019-5476-9

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Abstract

BackgroundSolid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. We aimed to elucidate the impact of hypoxia on the sensitivity of MB cells to chemo- and radiotherapy.MethodsWe used two MB cell line (D283-MED and MEB-Med8A) and a widely used glioblastoma cell line (U87MG) for comparison. We applied a range of molecular and cellular techniques to measure cell survival, cell cycle progression, protein expression and DNA damage combined with a transcriptomic micro-array approach in D283-MED cells, for global gene expression analysis in acute and chronic hypoxic conditions.ResultsIn D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to chemotherapy and X-ray irradiation. This acquired resistance upon chronic hypoxia was present but less pronounced in MEB-Med8A cells. Using transcriptomic analysis in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated (ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway.ConclusionTogether our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB in D283-MED cells, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols.

Highlights

Solid tumours are less oxygenated than normal tissues
This study demonstrates that the dampening of the DNA damage response to double strand break (DSB) triggered by chronic hypoxia strongly contributes to treatment resistance in hypoxic conditions in MB cells
We used two MB cell lines: D283-MED [37, 38], and MEB-Med8A [39, 40], to assess the effects of hypoxia pre-conditioning on cell sensitivity to chemotherapeutic treatment and X-ray irradiation

Summary

Introduction

Solid tumours are less oxygenated than normal tissues This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. Medulloblastoma (MB) is a malignant embryonal brain tumour originating from neural stem cells or granule-cell progenitors of the cerebellum, due to a deregulation of signalling pathways involved in neuronal development such as Wnt or Sonic Hedgehog (SHH) [1]. It is one of the most common brain tumours in children accounting for 15–20% of all paediatric CNS tumours. Recent studies have explored the use of proton beam therapy for MB, which triggers fewer detrimental side effects, as a result of reduction in irradiation of healthy brain tissue [7]

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