ITCH nuclear translocation and H1.2 polyubiquitination negatively regulate the DNA damage response.

The downregulation of the DNA damage response (DDR) enables aggressive tumors to achieve uncontrolled proliferation against replication stress, but the mechanisms underlying this process in tumors are relatively complex. Here, we demonstrate a mechanism through which a distinct E3 ubiquitin ligase, ITCH, modulates DDR machinery in triple-negative breast cancer (TNBC). We found that expression of a nuclear form of ITCH was significantly increased in human TNBC cell lines and tumor specimens. Phosphorylation of ITCH at Ser257 by AKT led to the nuclear localization of ITCH and ubiquitination of H1.2. The ITCH-mediated polyubiquitination of H1.2 suppressed RNF8/RNF168-dependent formation of 53BP1 foci, which plays important roles in DDR. Consistent with these findings, impaired ITCH nuclear translocation and H1.2 polyubiquitination sensitized cells to replication stress and limited cell growth and migration. AKT activation of ITCH-H1.2 axis may confer TNBC cells with a DDR repression to counteract the replication stress and increase cancer cell survivorship and growth potential.

Triptolide-Assisted Phosphorylation of p53 Suppresses Inflammation-Induced NF-κB Survival Pathways in Cancer Cells.

Chronic inflammation plays important roles in cancer initiation and progression. Resolving chronic inflammation or blocking inflammatory signal transduction may prevent cancer development. Here, we report that the combined low-dose use of two anti-inflammatory drugs, aspirin and triptolide, reduces spontaneous lung cancer incidence from 70% to 10% in a mouse model. Subsequent studies reveal that such treatment has little effect on resolving chronic inflammatory conditions in the lung, but it significantly blocks the NF-κB-mediated expression of proliferation and survival genes in cancer cells. Furthermore, triptolide and aspirin induce distinct mechanisms to potentiate each other to block NF-κB nuclear localization stimulated by inflammatory cytokines. While aspirin directly inhibits IκB kinases (IKKs) to phosphorylate IκBα for NF-κB activation, triptolide does not directly target IKKs or other factors that mediate IKK activation. Instead, it requires p53 to inhibit IκBα phosphorylation and degradation. Triptolide binds to and activates p38α and extracellular signal-regulated kinase 1/2 (ERK1/2), which phosphorylate and stabilize p53. Subsequently, p53 competes with IκBα for substrate binding to IKKß and thereby blocks IκBα phosphorylation and NF-κB nuclear translocation. Inhibition of p38α and ERK1/2 or p53 mutations could abolish the inhibitory effects of triptolide on NF-κB. Our study defines a new p53-dependent mechanism for blocking NF-κB survival pathways in cancer cells.

Chromosomal instability triggered by Rrm2b loss leads to IL-6 secretion and plasmacytic neoplasms.

Chronic inflammation has a tight cause-and-effect relationship with DNA damage by inflicting tissue damage and increasing cancer risk. Rrm2b, a key enzyme in de novo deoxyribonucleotide synthesis, is involved in DNA damage repair, but its role in cancer development has yet to be demonstrated. In this work, Rrm2b gene loss led to severe numerical and structural chromosome abnormalities that caused ATM activation, inducing p-Ser85 IKKγ/NEMO and IκB kinase (IKK). NF-κB consequently induced by IKK triggered sustained IL-6 expression that constitutively activated STAT3 in Rrm2b-deficient cells. High plasma interleukin-6 (IL-6) and associated hematologic disorders were observed in Rrm2b-/- mice, and 30%-40% of aged Rrm2b heterozygous knockout mice developed plasma cell neoplasms and suffered from progressive splenomegaly and ascites. The genetic ablation of IL-6 suppressed STAT3 induction and delayed disease onset in Rrm2b-/- mice, extending their lifespan. Thus, Rrm2b plays a crucial role in maintaining chromosomal stability and preventing chronic-inflammation-associated tumorigenesis.

MicroRNA-657 promotes tumorigenesis in hepatocellular carcinoma by targeting transducin-like enhancer protein 1 through nuclear factor kappa B pathways.

UNLABELLED: Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Dysregulation of miR-657 has been observed in several types of cancers, but its biological function is still largely unknown. Our results showed that miR-657 expression can be induced by hepatitis viral proteins and is significantly increased in hepatocellular carcinoma (HCC) tissues. Moreover, introduction of miR-657 dramatically increases proliferation and colony formation of HCC cells in vitro and induces tumor development in immunodeficient mice. Further studies showed that miR-657 directly targets the transducin-like enhancer protein 1 (TLE1) 3' untranslated region (UTR) and activates nuclear factor kappa B (NF-κB) pathways that contribute to hepatocarcinogenesis. CONCLUSION: This study identified a mechanism whereby miRNA-657 contributed to HCC through novel cancer pathways and provides new insights into the potential molecular mechanisms of hepatic carcinogenesis.

RRM2B suppresses activation of the oxidative stress pathway and is up-regulated by p53 during senescence.

RRM2B is the DNA damage-inducible small subunit of ribonucleotide reductase, the rate-limiting enzyme in de novo deoxyribonucleoside triphosphate synthesis. Although RRM2B is implicated in DNA repair and the maintenance of mitochondrial DNA content, the regulation and function of RRM2B in senescence have not been previously established. Here, we show that RRM2B is highly induced in a p53-dependent manner during senescence in primary human fibroblast IMR90 cells and is expressed at higher levels in senescent precancerous human prostatic intraepithelial neoplasm lesions compared to adjacent normal prostate glands. Paradoxically, silencing RRM2B expression leads to an increase in the level of reactive oxygen species, mitochondrial membrane depolarization, and premature senescence in a p38MAPK- and p53-dependent manner in young fibroblasts. Consistently, induction of senescence is accelerated in Rrm2b deficient mouse embryo fibroblasts. Our data demonstrate that RRM2B is induced by stress signals prior to the onset of senescence and prevents premature oxidative stress-induced senescence.

p53R2 inhibits the proliferation of human cancer cells in association with cell-cycle arrest.

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells.

ATM-mediated serine 72 phosphorylation stabilizes ribonucleotide reductase small subunit p53R2 protein against MDM2 to DNA damage.

Ribonucleotide reductase small subunit p53R2 was identified as a p53 target gene that provides dNTP for DNA damage repair. However, the slow transcriptional induction of p53R2 in RNA may not be rapid enough for prompt DNA damage repair, which has to occur within a few hours of damage. Here, we demonstrate that p53R2 becomes rapidly phosphorylated at Ser(72) by ataxia telangiectasia mutated (ATM) within 30 min after genotoxic stress. p53R2, as well as its heterodimeric partner RRM1, are associated with ATM in vivo. Mutational studies further indicate that ATM-mediated Ser(72) phosphorylation is essential for maintaining p53R2 protein stability and conferring resistance to DNA damage. The mutation of Ser(72) on p53R2 to alanine results in the hyperubiquitination of p53R2 and reduces p53R2 stability. MDM2, a ubiquitin ligase for p53, interacts and facilitates ubiquitination of the S72A-p53R2 mutant more efficiently than WT-p53R2 after DNA damage in vivo. Our results strongly suggest a novel mechanism for the regulation of p53R2 activity via ATM-mediated phosphorylation at Ser(72) and MDM2-dependent turnover of p53R2 dephosphorylated at the same residue.

Functional in vivo interactions between JNK1 and JNK2 isoforms in obesity and insulin resistance.

The c-Jun N-terminal kinases (JNKs) are key regulators of inflammation and interfere with insulin action in cultured cells and whole animals. Obesity increases total JNK activity, and JNK1, but not JNK2, deficiency results in reduced adiposity and improved insulin sensitivity. Interestingly, a higher-than-normal level of JNK activation is observed in Jnk2(-/-) mice, particularly in the liver, indicating an interaction between the isoforms that might have masked the metabolic activity of JNK2 in isolated mutant mice. To address the role of the JNK2 isoform in metabolic homeostasis, we intercrossed Jnk1(-/-) and Jnk2(-/-) mice and examined body weight and glucose metabolism in the resulting mutant allele combinations. Among all of the viable genotypes examined, we observed only reduced body weight and increased insulin sensitivity in Jnk1(-/-) and Jnk1(+/-)Jnk2(-/-) mice. These two groups of mice also exhibited reduced total JNK activity and cytokine expression in liver tissue compared with all other genotypes examined. These data indicate that the JNK2 isoform is also involved in metabolic regulation, but its function is not obvious when JNK1 is fully expressed because of regulatory crosstalk between the two isoforms.

Loss of hepatic NF-kappa B activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation.

A major link between inflammation and cancer is provided by NF-kappaB transcription factors. Ikkbeta(Deltahep) mice, which specifically lack IkappaB kinase beta (IKKbeta), an activator of NF-kappaB, in hepatocytes, are unable to activate NF-kappaB in response to proinflammatory stimuli, such as TNF-alpha. Surprisingly, Ikkbeta(Deltahep) mice are hypersusceptible to diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Because defective NF-kappaB activation promotes sustained c-Jun N-terminal kinase (JNK) activation in cells exposed to TNF-alpha, whose expression is induced by DEN, and JNK activity is required for normal hepatocyte proliferation, we examined whether increased susceptibility to DEN-induced hepatocarcinogenesis in Ikkbeta(Deltahep) mice requires JNK activation. Hepatocytes express both JNK1 and JNK2, but previous studies indicate that JNK1 is more important for hepatocyte proliferation. We therefore investigated this hypothesis using mice homozygous for a JNK1 deficiency either in wild-type or Ikkbeta(Deltahep) backgrounds. In both cases, mice lacking JNK1 were much less susceptible to DEN-induced hepatocarcinogenesis. This impaired tumorigenesis correlated with decreased expression of cyclin D and vascular endothelial growth factor, diminished cell proliferation, and reduced tumor neovascularization. Whereas hepatocyte-specific deletion of IKKbeta augmented DEN-induced hepatocyte death and cytokine-driven compensatory proliferation, disruption of JNK1 abrogated this response. In addition to underscoring the importance of JNK1-mediated hepatocyte death and compensatory proliferation, these results strongly suggest that the control of tissue renewal through the IKK and JNK pathways plays a key role in liver carcinogenesis.

The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover.

The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).

Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.

TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.

Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death.

Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP(-/-) or TRAF2(-/-) mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H(2)O(2)-induced cell death. We also found that RIP and TRAF2 form a complex upon H(2)O(2) exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H(2)O(2). Finally, our results demonstrate that activation of c-Jun NH(2)-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.

IKKbeta is required for prevention of apoptosis mediated by cell-bound but not by circulating TNFalpha.

IkappaB kinase beta (IKKbeta) is required for NF-kappaB activation and suppression of TNFalpha-mediated liver apoptosis. To investigate how IKKbeta suppresses apoptosis, we generated hepatocyte-specific Ikkbeta knockout mice, Ikkbeta(Deltahep), which exhibit little residual NF- kappaB activity but are healthy with normal liver function. Unexpectedly, Ikkbeta(Deltahep) mice are slightly more sensitive than controls to LPS-induced liver apoptosis but are highly susceptible to liver destruction following concanavalin A (ConA)-induced T cell activation. Unlike LPS, a potent inducer of circulating TNFalpha, ConA exerts cytotoxic effects through cell-bound TNFalpha, which activates type 1 and 2 TNF receptors (TNFR). While TNFR2 does not contribute to NF-kappaB activation, it is important for ConA-induced JNK activation, which is augmented by the absence of IKKbeta. Using JNK-deficient mice we show that JNK is required for ConA-induced liver damage. Thus, the antiapoptotic function of IKKbeta, which is most critical in situations that involve cell-bound TNFalpha, is mediated partially through attenuation of JNK activity.

JNK1 is required for maintenance of neuronal microtubules and controls phosphorylation of microtubule-associated proteins.

Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.

A central role for JNK in obesity and insulin resistance.

Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 2 diabetes mellitus, yet the molecular mechanisms of this association are poorly understood. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin action in cultured cells and are activated by inflammatory cytokines and free fatty acids, molecules that have been implicated in the development of type 2 diabetes. Here we show that JNK activity is abnormally elevated in obesity. Furthermore, an absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and enhanced insulin receptor signalling capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and a potential target for therapeutics.

Joint damage and inflammation in c-Jun N-terminal kinase 2 knockout mice with passive murine collagen-induced arthritis.

OBJECTIVE: Previous studies have demonstrated that inhibition of c-Jun N-terminal kinase (JNK) decreases joint destruction in the rat adjuvant arthritis model. The present study was undertaken to investigate whether selective loss of JNK-2 function decreases joint destruction in JNK-2 knockout mice, in order to determine the role of this isoform in inflammatory arthritis. METHODS: Passive collagen-induced arthritis (CIA) was induced in Jnk2(-/-) and wild-type mice by administering anti-type II collagen antibodies. Arthritis was assessed daily using a semiquantitative clinical scoring system. Fibroblast-like synoviocytes (FLS) were prepared from Jnk2(-/-) and wild-type mice, and JNK protein expression was determined by Western blot analysis. Matrix metalloproteinase 13 (MMP-13) expression was determined by Northern blot analysis, and activator protein 1 (AP-1) binding activity by electromobility shift assay (EMSA). RESULTS: The JNK protein level in Jnk2(-/-) mice with CIA was 22% of that in wild-type mice with CIA (P < 0.001), and mainly the 46-kd isoform was expressed in the former group. Surprisingly, clinical arthritis was slightly more severe in the Jnk2(-/-) mice. Histologic scores for synovial inflammation were not significantly different. However, Safranin O-stained sections from the Jnk2(-/-) mice exhibited significantly less joint damage. Although joint destruction was decreased in Jnk2(-/-) mice with CIA, EMSA and Northern blot analysis of total joint extracts revealed similar levels of AP-1 binding and MMP-13 expression in Jnk2(-/-) and wild-type mice. The lack of correlation with AP-1 activity and MMP expression was probably because non-FLS cells in the joint may express more JNK-1 than do FLS. CONCLUSION: JNK-2 is a determinant of matrix degradation, but it has little effect on inflammation in arthritis. Complete inhibition of MMP expression and joint destruction will likely require combined JNK-1 and JNK-2 inhibition.