Replication Stress-Activated DNA Damage Response Enhances the Radioresistance of Glioblastoma Stem Cells

Elizabeth McDonald

Introduction: Glioblastomas (GBM) are a highly aggressive primary brain cancer predominantly made of abnormal astrocytes1,2,3. Of all malignant solid tumors, GBM is one of the deadliest and least responsive to treatment3 with a median survival time of less than 12 months. Current treatment is neurosurgical resection, followed with temozolomide and radiotherapy4.The high mortality rate is linked to development of radiation resistance and inevitable recurrence following treatment, 6. Inappropriate activation of glioblastoma cancer stem cells (GSC) can contribute to GBM radio resistance, recurrence, and, ultimately, poor patient prognosis. GBM resistance to treatment is linked to GSC subpopulations that have an increased DNA damage repair response (DDR)1. Replicative stress (RS) may induce heightened DDR, allowing the CSC to overcome damage from chemoradiation leading to growth and radioresistance1,3. This paper seeks to examine the role of RS activation of DDR in GSCs and radioresistance. Methods: Primary GBM cell lines from resected tumors were derived and maintained2. GSC lines were paired with differentiated GBM cell lines and used to examine the impact of high levels of RS on DDR activation and radio resistance. Western blot analysis confirmed increased GSC markers Sox2 and Nestin, as well as constitutive activation and upregulation of DDR proteins ATR and Chk1 in GSC cultures2. Low concentration of aphidicolin is known to lead to RS and DDR activation. To test if exogenous RS could enhance radioresistance by stimulating the DDR was test by incubating bulk cultures (E2, G7, R10) with 0.05 mmol/L aphidicolin for 72 hours then irradiating them2. Results: Phosphorylation of RPA subunit 32 (RPA32) at Ser4 and Ser8 is specific marker of RS. It was not detected in asynchronous undamaged cells, but exacerbation of RS by low doses of UV radiation elicited greater phosphorylation of RPA 32 in GSC than paired tumor bulk populations2. Diffuse nuclear staining also confirmed RS2. Non-GSC exposed to exogenous RS at low levels developed radiation resistance in vitro, confirming the role of RS as a source of novel radiation resistance. GBM CSCs repaired DNA double strand breaks more efficiently and had upregulated repair proteins when compared to paired differentiated tumor cells. Conclusions: Constitutive DDR activation or RS could provide a cancer stem cell-specific targets with significant clinical potential for patients with GBM. Discovering more about CSC biology and the role of RS-activated DDR has the potential to help overcome radioresistance in GBM and lead to improved clinical outcomes.

  1. Morgan MA, Canman CE. Replication stress: An achilles’ heel of glioma cancer stem-like cells. Cancer Res. 2018; 78:6713-6716.
  2. Carruthers RD, Ahmed SU, Ramachandran S, et al. Replication stress drives constitutive activation of the DNA damage response and radioresistance in glioblastoma stem-like cells. Cancer Res. 2018; 78:5060-5071.
  3. Alexander BM, Cloughesy TF. Adult glioblastoma. J Clin Oncol. 2017; 35:2402-2409.
  4. Auffinger B, Tobias AL, Han Y, et al. Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy. Cell Death Differ. 2014; 21:1119-1131.
  5. Ahmed SU, Carruthers R, Gilmour L, Yildirim S, Watts C, Chalmers AJ. Selective inhibition of parallel DNA damage response pathways optimizes radiosensitization of glioblastoma stem-like cells. Cancer Res. 2015; 75:4416-4428.
  6. Carruthers R, Ahmed SU, Strathdee K, et al. Abrogation of radioresistance in glioblastoma stem-like cells by inhibition of ATM kinase. Mol Oncol. 2015; 9:192-203.