Zhao Hui Wu

Sequential Modification of NEMO/IKKγ by SUMO-1 and Ubiquitin Mediates NF-κB Activation by Genotoxic Stress

Abstract The transcription factor NF-κB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-κB signaling pathways is NEMO/IKKγ, the regulatory subunit of the cytoplasmic IκB kinase (IKK) complex. While NF-κB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Here, we show that genotoxic stress causes nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-κB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets.

Molecular Linkage Between the Kinase ATM and NF-κB Signaling in Response to Genotoxic Stimuli

The transcription factor NF-κB modulates apoptotic responses induced by genotoxic stress. We show that NF-κB essential modulator (NEMO), the regulatory subunit of IκB kinase (IKK) (which phosphorylates the NF-κB inhibitor IκB), associates with activated ataxia telangiectasia mutated (ATM) after the induction of DNA double-strand breaks. ATM phosphorylates serine-85 of NEMO to promote its ubiquitin-dependent nuclear export. ATM is also exported in a NEMO-dependent manner to the cytoplasm, where it associates with and causes the activation of IKK in a manner dependent on another IKK regulator, a protein rich in glutamate, leucine, lysine, and serine (ELKS). Thus, regulated nuclear shuttling of NEMO links two signaling kinases, ATM and IKK, to activate NF-κB by genotoxic signals.

ATM and NEMO-dependent ELKS ubiquitination coordinates TAK1-mediated IKK activation in response to genotoxic stress

Activation of the transcription factor NF-κB by multiple genotoxic stimuli modulates cancer cell survival. This response is mediated by a conserved pathway involving the nuclear ATM kinase and cytoplasmic IκB kinase (IKK); however, the molecular link between them remains incompletely understood. Here we show that ATM activates the IKK kinase TAK1 in a manner dependent on IKKγ/NEMO and ELKS (a protein rich in glutamate, leucine, lysine, and serine). K63-linked polyubiquitination of ELKS, dependent on the ubiquitin ligase XIAP and the conjugating enzyme UBC13, allows ELKS association with TAK1 via its ubiquitin-binding subunits TAB2/3. Although NEMO mutants defective in ubiquitin binding permit ATM-dependent TAK1 activation, they block NEMO association with ELKS and IKK activation. Thus, ATM- and NEMO-dependent ubiquitination of ELKS leads to the ubiquitin-dependent assembly of TAK1/TAB2/3 and NEMO/IKK complexes, resulting in IKK and NF-κB activation following genotoxic stimuli.

Many faces of NF-κB signaling induced by genotoxic stress

The nuclear factor-κB (NF-κB) family of dimeric transcription factors plays pivotal roles in physiologic and pathologic processes, including immune and inflammatory responses and development and progression of various human cancers. Inactive NF-κB dimers normally exist in the cytoplasm in association with inhibitor proteins belonging to the inhibitor of NF-κB (IκB) family of related proteins. Activation of NF-κB involves its release from IκB and subsequent nuclear translocation to induce expression of target genes. Intense research effort has revealed many distinct signaling pathways and mechanisms of NF-κB activation induced by immune and inflammatory stimuli. These aspects of NF-κB biology have been amply reviewed in the literature. However, those that involve DNA-damaging agents are less well understood, and multiple conflicting pathways and mechanisms have been described in the literature. In this review, we summarize the proposed mechanisms of NF-κB activation by various DNA-damaging agents, discuss the significance of such activation in the context of cancer treatment, and highlight some of the critical questions that remain to be addressed in future studies.

DNA Damage Induces NF-κB-dependent MicroRNA-21 Up-regulation and Promotes Breast Cancer Cell Invasion

Background: DNA damage response and miRNAs have been linked to cancer progression. Results: Genotoxic drug induces up-regulation of miR-21 in a NF-κB- and STAT3-dependent manner, which correlates with enhanced breast cancer cell invasion. Conclusion: Genotoxic NF-κB activation promotes breast cancer invasion via miR-21 induction. Significance: Genotoxic NF-κB signaling pathway may serve as a drug target to reduce therapeutic resistance and metastasis in breast cancer. NF-κB activation induced by genotoxic treatment in cancer cells has been associated with therapeutic resistance in multiple human malignancies. Therapeutic resistance also correlates with high metastatic potential in human cancers, including breast cancer. Whether genotoxic treatment-activated NF-κB also contributes to cancer metastasis following radiation and chemotherapy is unclear. Here, we show that chemotherapeutic drug-induced NF-κB activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL-6 induction mediated by genotoxic NF-κB activation. Moreover, genotoxic treatment also up-regulates oncogenic microRNA-21 (miR-21) expression through eliciting NF-κB recruitment to the miR-21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR-21 transcription. DNA damage-induced histone H3 phosphorylation via activated MSK1 creates an open chromatin structure for NF-κB/STAT3-driven transactivation of miR-21. NF-κB-dependent IL-6 up-regulation is responsible for STAT3 activation and recruitment to the miR-21 promoter upon genotoxic stress. Induction of miR-21 may enable cancer cells to elude DNA damage-induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4. Our data support a critical role of DNA damage-induced NF-κB activation in promoting cancer metastasis following genotoxic treatment, and NF-κB-dependent miR-21 induction may contribute to both therapeutic resistance and metastasis in breast cancer.

LUBAC regulates NF‐κB activation upon genotoxic stress by promoting linear ubiquitination of NEMO

The transcription factor nuclear factor κB (NF‐κB) regulates various cellular processes such as inflammation and apoptosis. The NF‐κB essential modulator (NEMO/IKKγ) is indispensable for NF‐κB activation by diverse stimuli including genotoxic stress. Here, we show that NEMO linear ubiquitination on K285/309 is critical for genotoxic NF‐κB activation. The E3 ligase linear ubiquitin chain assembly complex (LUBAC) facilitates NEMO linear ubiquitination upon genotoxic stress. Inhibiting LUBAC function interrupts the genotoxic NF‐κB signalling cascade by attenuating the activation of IKK and TAK1 in response to DNA damage. We further show that the linear ubiquitination of NEMO is a cytoplasmic event, potentially downstream of NEMO nuclear exportation. Moreover, ELKS ubiquitination appears to facilitate linear ubiquitination of NEMO through stabilizing NEMO:LUBAC association upon DNA damage. Deubiquitinating enzyme CYLD inhibits NEMO linear ubiquitination, possibly by disassembling both K63‐linked and linear polyubiquitin. We also found that abrogating linear ubiquitination of NEMO significantly increased genotoxin‐induced apoptosis, resulting in enhanced sensitivity to chemodrug in cancer cells. Therefore, LUBAC‐dependent NEMO linear ubiquitination is critical for genotoxic NF‐κB activation and protects cells from DNA damage‐induced apoptosis.

Induction of a pro‐apoptotic ATM–NF‐κB pathway and its repression by ATR in response to replication stress

The transcription factor NF‐κB has critical functions in biologic responses to genotoxic stimuli. Activation of NF‐κB in response to DNA double strand break (DSB) inducers can be mediated by ATM (ataxia telangiectasia mutated)‐dependent phosphorylation of NEMO (NF‐κB essential modulator). Here, we show that the replication stress inducers hydroxyurea (HU) and aphidicolin also activate this ATM‐dependent signalling pathway. We further show that ATR (ATM‐ and Rad3‐related) interacts with NEMO but surprisingly does not cause NEMO phosphorylation. Consequently, ATR represses NF‐κB activation induced by replication stress. Reduction or increase of ATR expression by RNA interference or overexpression increased or reduced ATM–NEMO association and NF‐κB activation induced by HU. Apoptosis gene expression and chromatin immunoprecipitation analyses indicated that HU and the DSB inducer etoposide caused complex patterns of NF‐κB‐dependent pro‐ and antiapoptotic gene expression with the overall outcome for the former being pro‐apoptotic, whereas the latter antiapoptotic. Thus, replication stress and DSB inducers activate NF‐κB through a conserved pathway with opposite biologic outcomes, and ATR antagonizes ATM function at least in part by competing for NEMO association.

NF-κB-dependent MicroRNA-125b Up-regulation Promotes Cell Survival by Targeting p38α upon Ultraviolet Radiation

Background: UV radiation-induced miRNA expression modulates cellular stress response. Results: UV up-regulates miR-125b expression via ATM-dependent NF-κB activation, which represses p38α expression. Conclusion: UV-induced miR-125b prevents prolonged p38α activation and thereby promotes cell survival. Significance: Up-regulation of miR-125b serves as a negative feedback mechanism to control p38α activity upon UV radiation, which may contribute to tumorigenesis of skin cancer. UV-induced stress response involves expression change of a myriad of genes, which play critical roles in modulating cell cycle arrest, DNA repair, and cell survival. Alteration of microRNAs has been found in cells exposed to UV, yet their function in UV stress response remains elusive. Here, we show that UV radiation induces up-regulation of miR-125b, which negatively regulates p38α expression through targeting its 3′-UTR. Increase of miR-125b depends on UV-induced NF-κB activation, which enhances miR-125b gene transcription upon UV radiation. The DNA damage-responsive kinase ATM (ataxia telangiectasia mutated) is indispensable for UV-induced NF-κB activation, which may regulate p38α activation and IKKβ-dependent IκBα degradation in response to UV. Consequently, repression of p38α by miR-125b prohibits prolonged hyperactivation of p38α by UV radiation, which is required for protecting cells from UV-induced apoptosis. Altogether, our data support a critical role of NF-κB-dependent up-regulation of miR-125b, which forms a negative feedback loop to repress p38α activation and promote cell survival upon UV radiation.

Rat MYH, a glycosylase for repair of oxidatively damaged DNA, has brain‐specific isoforms that localize to neuronal mitochondria

Mitochondrial genomes are exposed to a heavy load of reactive oxygen species (ROS) that damage DNA. Since in neurons, mitochondrial DNA integrity must be maintained over the entire mammalian life span, neuronal mitochondria most likely repair oxidatively damaged DNA. We show that the Escherichia coli MutY DNA glycosylase homolog (MYH) in rat (rMYH) involved in repair of oxidative damage is abundantly expressed in the rat brain, with isoforms that are exclusive to brain tissue. Confocal microscopy and western analyses reveal localization of rMYH in neuronal mitochondria. To assess involvement of MYH in the neuronal response to oxidative DNA damage, we used a rat model of respiratory hypoxia, in which acutely reduced blood oxygenation leads to generation of superoxide, and formation and subsequent removal of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG). Removal of 8OHdG is accompanied by a spatial increase in rMYH immunoreactivity in the brain and an increase in levels of one of the three mitochondrial MYH isoforms, suggesting that inducible and non‐inducible MYH isoforms exist in the brain. The mitochondrial localization of oxidative DNA damage repair enzymes in neurons may represent a specialized neuronal mechanism that safeguards mitochondrial genomes in the face of routine and accidental exposures to heavy loads of injurious ROS.

MicroRNA-22 promotes cell survival upon UV radiation by repressing PTEN.

DNA damage response upon UV radiation involves a complex network of cellular events required for maintaining the homeostasis and restoring genomic stability of the cells. As a new class of players involved in DNA damage response, the regulation and function of microRNAs in response to UV remain poorly understood. Here we show that UV radiation induces a significant increase of miR-22 expression, which appears to be dependent on the activation of DNA damage responding kinase ATM (ataxia telangiectasia mutated). Increased miR-22 expression may result from enhanced miR-22 maturation in cells exposed to UV. We further found that tumor suppressor gene phosphatase and tensin homolog (PTEN) expression was inversely correlated with miR-22 induction and UV-induced PTEN repression was attenuated by overexpression of a miR-22 inhibitor. Moreover, increased miR-22 expression significantly inhibited the activation of caspase signaling cascade, leading to enhanced cell survival upon UV radiation. Collectively, these results indicate that miR-22 is an important player in the cellular stress response upon UV radiation, which may promote cell survival via the repression of PTEN expression.