Abstract
Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic ‘hits’, compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.
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Abbreviations
- AOM:
-
azoxymethane
- ARF:
-
alternative reading frame
- ChIP:
-
chromatin immunoprecipitation
- DABG:
-
detection above background
- DDR:
-
DNA damage response
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DSS:
-
dextran-sodium-sulfate
- Ha-Ras Q61L:
-
amino-acid substitution at position 61 in Ha-RAS from glutamine (Q) to leucine (L)
- HBEC:
-
human bronchial epithelial cell
- hTERT:
-
human telomerase reverse transcriptase
- Ncstn−/−:
-
homozygous conditional nicastrin deletion
- PanIN:
-
pancreatic intraepithelial neoplasia
- pT68-Chk2:
-
phosphorylated at Thr68 Chk2
- Sa-β-gal:
-
senescence associated β-galactosidase
- siRNA:
-
small interfering RNA
- γH2AX:
-
phosphorylated at Ser139 Histone H2AX
- ATM-p:
-
phosphorylated at Ser1981 ATM
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Acknowledgements
We thank Proffessor M Oren for kindly providing the CM5 antibody and the anti-p53 siRNA, Dr. PP Pandolfi for kindly donating plasmids pGL2-p19ARF-Luc and p14ARF-Luc, Dr. Robert Strauss for donating plasmids shp53 pLKO.1 hygro and Dr. A Damalas and Antonia Daleziou for their technical support. Financial support was from the Danish Cancer Society, the Danish National Research Foundation, the European Commission FP7 projects: GENICA, INFLA-CARE, BioMedReg, DDResponse, INsPiRE and the NKUA-SARG projects (codes: 11351 and 8916). VG and MP are recipients of a Hellenic Association for Molecular Cancer Research scholarship, whereas Dr. A Kotsinas received an Empeirikeion Foundation fellowship.
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Evangelou, K., Bartkova, J., Kotsinas, A. et al. The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis. Cell Death Differ 20, 1485–1497 (2013). https://doi.org/10.1038/cdd.2013.76
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DOI: https://doi.org/10.1038/cdd.2013.76
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