PA-2 is a novel compound that we have designed to improve the therapeutic index of aspirin. The phospho-modification of the carboxylic acid moiety of aspirin results in a remarkable safety profile, as well as improved efficacy. In ER+ breast Nilotinib cancer cell lines, PA-2 exhibits 6- to 8-fold improvement in potency as compared to aspirin. In vivo, administration of PA-2 has a dramatic effect on the growth of ER+ MCF7 xenografts, achieving a complete inhibition of tumor growth, triggered by a cytokinetic effect involving apoptosis induction, inhibition of cell proliferation and induction of cell cycle arrest. Our work has identified an oxidative stress-initiated and p53- dependent signaling cascade that plays a fundamental role in the anticancer effect of PA-2. Modulation of oxidative stress is increasingly recognized to be a potential anticancer strategy. ER+ breast cancer cell lines such as MCF7 displayed an increased susceptibility to cell death in response to the induction of RONS. Phospho-NSAIDs, as a class of novel anticancer drugs, elevate oxidative stress in cancer cells as a common proximal event that causes the induction of cancer cell death. Indeed, PA-2 induces oxidative stress in ER+ breast cancer cells in vitro and in MCF7 xenografts in vivo. The major RONS species involved were mitochondrial superoxide anion and nitric oxide. Remarkably, induction of oxidative stress by PA-2 appears to be exclusive to tumor tissues, as there was no significant elevation of oxidative stress in tumor-free animals. Elevation of oxidative stress at the beginning of PA-2 treatment was inversely correlated with tumor volume at the end point, suggesting that oxidative stress appears to be a dominant mediator of the anticancer effect of PA-2. It should be noted that the detection of mitochondrial superoxide anion and nitric oxide as reported here does have limitations, being in many instances qualitative indicators due to possible additional oxidation products with overlapping fluorescence spectra. Nevertheless, given its strong predictive value, oxidative stress induction, as determined here, may serve as a potential biomarker in future studies for identifying patient populations sensitive or resistant to the anticancer effect of PA-2. Induction of oxidative stress by PA-2 in ER+ breast cancer is highly consequential, as it activates redox-sensitive downstream signaling that ultimately contributes to its growth inhibitory effect. A pivotal downstream target is the tumor suppressor, p53. Intracellular levels of p53 are tightly regulated through its ubiquitylation by MDM2 and subsequent degradation by the 26S proteasome, and p53 expression is frequently silenced in ER+ breast cancer. PA-2 strongly induced p53 acetylation, a post-translational modification that destabilizes the p53-MDM2 interaction, leading to the accumulation of p53 and its transcriptional activation. Furthermore, PA-2 prompted the mitochondrial translocation of p53, where it can regulate the opening of the PTP to directly induce mitochondrial-dependent cell death.