Aberrant nuclear factor-KB (NF-KB) signaling plays a role in cancer initiation and progression; thus, it represents a potential therapeutic target. We previously identified a mechanism of repression of NF-KB transcriptional activity and induction of apoptosis in colon cancer cells involving nuclear/nucleolar translocation of the RelA (p65) component of NF-KB. This response was stimulated by cellular stress-inducing agents, including aspirin, but not by tumor necrosis factor. Here, we investigate the upstream molecular mechanisms responsible for nucleolar targeting of RelA and show that aspirin activates the p38 mitogen-activated protein kinase (MAPK) pathway in colorectal cancer cells. We also show that aspirin causes rapid, ubiquitin-dependent degradation of cyclin D1, a known p38 target. Aspirin-induced p38 activation preceded cyclin D1 degradation, which was then followed by activation of the NF-KB pathway, suggesting a causative link. Indeed, chemical p38 inhibition (PD169316) and small interfering RNA directed against p38 blocked aspirin-induced cyclin D1 degradation, nucleolar translocation of RelA, and apoptosis. Furthermore, chemical inhibition of the cyclin D1/cyclindependent kinase 4 (CDK4) kinase complex, used as a surrogate for cyclin D1 degradation, caused nucleolar translocation of RelA, repression of KB-driven transcription, and apoptosis, thereby reproducing the effects of aspirin. In addition, we found that aspirin and the CDK4 inhibitor induced nucleolar translocation of RelA and apoptosis through a common mechanism involving the NH 2 -terminal nucleolar localization signal. Collectively, these data suggest that aspirin causes inhibition of cyclin D1/CDK4 through the p38 MAPK pathway. This inhibition stimulates the NF-KB pathway to induce nucleolar translocation of RelA and apoptosis. These novel findings have considerable relevance to the rational design of novel chemotherapeutic and chemopreventative strategies. [Cancer Res 2007;67(4):1660-9]

Stimulation of the NF-κB pathway can have proapoptotic or antiapoptotic consequences, and one mechanism that determines the outcome is the nuclear distribution of RelA. Certain stress stimuli induce nucleolar accumulation of RelA thereby mediating apoptosis, whereas others induce nucleoplasmic accumulation and inhibition of apoptosis. Here we investigated the mechanisms that regulate the nuclear distribution of RelA, specifically, the role of the ubiquitin/proteasome system. We found that stress-induced nucleolar translocation of RelA is preceded by ubiquitination of the protein. We also found that chemical proteasome inhibitors induce the ubiquitination and nucleolar translocation of RelA and that this is required for the apoptotic response to these agents. We show that the RelA nucleolar localization signal (amino acids 27-30) is a critical domain for ubiquitination of the protein but that the lysine residue within this motif is not a direct target. We show that RelA binds COMMD1, the rate-limiting component of the RelA ubiquitin ligase complex, in response to stress. Furthermore, we show that overexpression of COMMD1 promotes stress-mediated nucleolar targeting of RelA, whereas knockdown of COMMD1 blocks this effect, causing RelA to remain in the nucleoplasm. These data identify a new role for COMMD1 in regulating the nuclear/nucleolar distribution of RelA and suggest that ubiquitination acts as a signal for transport of RelA to the nucleolus. These findings have relevance to the design of chemopreventative/ anticancer agents that act by targeting RelA to the nucleolar compartment.

Understanding the mechanisms that underlie the antitumour activity of non-steroidal anti-inflammatory drugs (NSAIDs) against colorectal cancer will allow the development of more effective and specific chemopreventative agents. Modulation of the NF-jB pathway has been implicated as a key effector of the antitumour effect of aspirin, but the effects of non-aspirin NSAIDs on this pathway have yet to be fully defined. Here, we demonstrate that sulindac, sulindac sulfone and indomethacin activate the NF-jB pathway in colorectal cancer cells, as determined by western blot analysis of cytoplasmic levels of IjBa and immunocytochemical analysis of nuclear NFjB/RelA. Furthermore, we show that all of these NSAIDs induce nucleolar translocation of the RelA subunit of NFjB. Using RelA deleted for the previously described nucleolar localization signal, we demonstrate that this response is causally involved in the apoptotic effects of these agents. Finally, we demonstrate that NSAIDmediated nucleolar translocation of RelA is associated with downregulation of NF-jB-driven transcription and of the NF-jB target gene, ICAM-1. These data identify nucleolar translocation of RelA and the associated repression of the NF-jB-driven transcription as a central molecular mechanism of NSAID-mediated growth inhibition and apoptosis. As well as providing new understanding of the molecular determinants of RelA function, these findings also have relevance to the development of novel chemotherapeutic and chemopreventative agents.