Consequences of higher DNA damage and higher constitutive activity of DNA damage response (DDR) proteins, especially Checkpoint kinase 1 (CHK1), in advanced malignancies such as Glioblastomas (GBM) are unknown. In this study we aim to assess the role of DNA damage modulator, CHK1, and its effectors in the pathogenesis of GBM.
1323 patient samples were assessed to identify CHK1 effectors and establish the clinical relevance of the key proteins in GBM. Small molecule inhibitors and siRNA approaches were used to dissect the mechanism. Cell viability, proliferation, monolayer, 3D culture and Tetracycline-inducible knockdown system were used to assess the functional impact on perturbing the newly identified pathway in U251MG and primary GBM cells. Finally, heterotopic and intracranial xenograft mouse models for human GBM were used validate the in vitro findings.
In contrast to normal brain clinical samples, higher expression and a strong positive correlation for CHK1 and CIP2A expression was observed in samples from glioma patients. Further, high CHK1 and CIP2A mRNA expression predicted for more aggressive form of the disease and reduced overall survival. Accordingly, GBM patients with higher CIP2A copy number had a lower probability of survival. Chemical and genetic inhibition of CHK1 resulted in decreased CIP2A expression. Functionally, using small molecules, siRNAs and inducible-knockdown systems we demonstrate that CHK1 and CIP2A promote viability, clonogenicity and the tumoursphere-forming capacity of GBM cells. Further, depletion of CIP2A induced senescence. Mechanistically, we demonstrate that CHK1 promotes pSer727-STAT3 which in turn regulates CIP2A transcription. Interestingly, CIP2A binds STAT3 and promotes pSer727-STAT3 levels both in vitro and in vivo thereby establishing a positive feedback between the two proteins. Finally, CIP2A depletion in the heterotopic and orthotopic murine models of human GBM results in decreased tumour growth and increased survival of mice.
Altogether, the results highlight the prognostic and therapeutic significance of CHK1 and CIP2A in human gliomas and provide novel mechanistic insights into GBM pathogenesis.
Clinical trial identification
All authors have declared no conflicts of interest.
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