SUMMARY The NF-κB activating kinase IKKβ suppresses early chemically-induced liver tumorigenesis by inhibiting hepatocyte death and compensatory proliferation. To study IKKβ’s role in late tumor promotion and progression, we developed a transplant system that allows initiated mouse hepatocytes to form hepatocellular carcinomas (HCC) in host liver after a long latency. Deletion of IKKβ long after initiation accelerated HCC development and enhanced proliferation of tumor initiating cells. These effects of IKKβ/NF-κB were cell autonomous and correlated with increased accumulation of reactive oxygen species (ROS) that led to JNK and STAT3 activation. Hepatocyte-specific STAT3 ablation prevented HCC development. The negative crosstalk between NF-κB and STAT3, which is also evident in human HCC, is a critical regulator of liver cancer development and progression.

NF-jB/Rel transcription factors have recently emerged as crucial regulators of cell survival. Activation of NF-jB antagonizes programmed cell death (PCD) induced by tumor necrosis factor-receptors (TNF-Rs) and several other triggers. This prosurvival activity of NF-jB participates in a wide range of biological processes, including immunity, lymphopoiesis and development. It is also crucial for pathogenesis of various cancers, chronic inflammation and certain hereditary disorders. This participation of NF-jB in survival signaling often involves an antagonism of PCD triggered by TNF-Rfamily receptors, and is mediated through a suppression of the formation of reactive oxygen species (ROS) and a control of sustained activation of the Jun-N-terminal kinase (JNK) cascade. Effectors of this antagonistic activity of NF-jB on this ROS/JNK pathway have been recently identified. Indeed, further delineating the mechanisms by which NF-jB promotes cell survival might hold the key to developing new highly effective therapies for treatment of widespread human diseases.

JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics. JNKs are serine threonine protein kinases and members of the MAPK family (1-3). JNKs can be expressed as 10 different isoforms by mRNA alternative splicing of three highly related genes, JNK1, JNK2, and JNK3 (4). Although JNK1 and JNK2 are ubiquitous, JNK3 is principally present in the brain, cardiac muscle, and testis (4, 5). JNK activation by extracellular stimuli, such as stress or cytokines, leads to the phosphorylation of several transcription factors, and cellular substrates implicated in cell survival and proliferation, insulin receptor signaling, and mRNA stabilization (6-9). Because these pathways are related to the pathogenesis of several diseases, including diabetes, cancer, atherosclerosis, stroke, and Alzheimer's and Parkinson's diseases, JNKs represent valuable targets in the development of new therapies (10).JNKs bind to scaffold proteins and substrates containing a D-domain, consensus sequence of which is R/KXXXXLXL (11, 12). JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases (13). The JNK-JIP1 interaction is mediated by a specific, high affinity D-domain on JIP1, the critical features of which were elucidated by Barr and colleagues (14). Overexpression of either the D-domain of JIP1 or the full-length protein potently inhibits JNK signaling in the cell (15). The minimal region of JIP1, consisting of a single D-domain, has been identified as a JNK inhibitor (14, 16). A peptide corresponding to the D-domain of JIP1 (amino acids 153-163; pepJIP1), inhibits JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 and displays remarkable selectivity with little inhibition of the closely related Erk and p38 MAPKs (17).The mechanism of JNK1 inhibition by pepJIP1 is mainly because of the competition of pepJIP1 with the D-domains of substrat...

NF-B/Rel transcription factors are central to controlling programmed cell death (PCD).ARF signaling, suggesting that it mediates a novel protective mechanism activated by NF-B. Indeed, our data indicate that this activity involves a control of inhibitory Bcl-2 phosphorylation. The data also suggest that Twist-1 and -2 play an important role in NF-B-dependent chemoresistance.