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 p16 , 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.