Retraction Note: Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

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Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

Signal transducer and activator of transcription 3 (STAT3) has a critical role in regulating cell fate, inflammation and immunity1,2. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization3,4. It remains unknown whether other factors promote STAT3 activation through different mechanisms. Here we show that STAT3 is post-translationally S-palmitoylated at the SRC homology 2 (SH2) domain, which promotes the dimerization and transcriptional activation of STAT3. Fatty acids can directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase that regulates STAT3. Cytokine stimulation increases STAT3 palmitoylation by promoting the association between ZDHHC19 and STAT3, which is mediated by the SH3 domain of GRB2. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, and impairs the cytokine- and fatty-acid-induced activation of STAT3. ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas. High levels of ZDHHC19 correlate with high levels of nuclear STAT3 in patient samples. In addition, knockout of ZDHHC19 in lung squamous cell carcinoma cells significantly blocks STAT3 activity, and inhibits the fatty-acid-induced formation of tumour spheres as well as tumorigenesis induced by high-fat diets in an in vivo mouse model. Our studies reveal that fatty-acid- and ZDHHC19-mediated palmitoylation are signals that regulate STAT3, which provides evidence linking the deregulation of palmitoylation to inflammation and cancer.

Auto-fatty acylation of transcription factor RFX3 regulates ciliogenesis.

Defects in cilia have been associated with an expanding human disease spectrum known as ciliopathies. Regulatory Factor X 3 (RFX3) is one of the major transcription factors required for ciliogenesis and cilia functions. In addition, RFX3 regulates pancreatic islet cell differentiation and mature ß-cell functions. However, how RFX3 protein is regulated at the posttranslational level remains poorly understood. Using chemical reporters of protein fatty acylation and mass spectrometry analysis, here we show that RFX3 transcriptional activity is regulated by S-fatty acylation at a highly conserved cysteine residue in the dimerization domain. Surprisingly, RFX3 undergoes enzyme-independent, "self-catalyzed" auto-fatty acylation and displays preferences for 18-carbon stearic acid and oleic acid. The fatty acylation-deficient mutant of RFX3 shows decreased homodimerization; fails to promote ciliary gene expression, ciliogenesis, and elongation; and impairs Hedgehog signaling. Our findings reveal a regulation of RFX3 transcription factor and link fatty acid metabolism and protein lipidation to the regulation of ciliogenesis.

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.

USP10 inhibits genotoxic NF-κB activation by MCPIP1-facilitated deubiquitination of NEMO.

DNA damage-induced activation of the transcription factor NF-κB plays an important role in the cellular response to genotoxic stress. However, uncontrolled NF-κB activation upon DNA damage may lead to deleterious consequences. Although the mechanisms mediating genotoxic NF-κB activation have been elucidated, how this signalling is terminated remains poorly understood. Here, we show that the CCCH-type zinc finger-containing protein MCPIP1 (monocyte chemotactic protein-1-induced protein-1; also known as ZC3H12A) is induced upon genotoxic treatment in an NF-κB-dependent manner. MCPIP1 upregulation reduces NEMO linear ubiquitylation, resulting in decreased activation of IKK and NF-κB. NEMO ubiquitylation is decreased through the deubiquitinase USP10, which interacts with NEMO via MCPIP1 upon genotoxic stress. USP10 association with NEMO leads to removal of NEMO-attached linear polyubiquitin chains and subsequent inhibition of the genotoxic NF-κB signalling cascade. Consistently, USP10 is required for MCPIP1-mediated inhibition of genotoxic NF-κB activation and promotion of apoptosis. Thus, by mediating USP10-dependent deubiquitination of NEMO, MCPIP1 induction serves as a negative feedback mechanism for attenuating genotoxic NF-κB activation.

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.

NF-κB-dependent microRNA-125b up-regulation promotes cell survival by targeting p38α upon ultraviolet radiation.

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 IKKß-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.

DNA damage induces NF-κB-dependent microRNA-21 up-regulation and promotes breast cancer cell invasion.

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.

15-Lipoxygenase-1-enhanced Src-Janus kinase 2-signal transducer and activator of transcription 3 stimulation and monocyte chemoattractant protein-1 expression require redox-sensitive activation of epidermal growth factor receptor in vascular wall remodeling.

To understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates signal transducer and activator of transcription 3 (STAT3), we studied the role of epidermal growth factor receptor (EGFR). 15(S)-HETE stimulated tyrosine phosphorylation of EGFR in a time-dependent manner in vascular smooth muscle cells (VSMCs). Interference with EGFR activation blocked 15(S)-HETE-induced Src and STAT3 tyrosine phosphorylation, monocyte chemoattractant protein-1 (MCP-1) expression and VSMC migration. 15(S)-HETE also induced tyrosine phosphorylation of Janus kinase 2 (Jak2) in VSMCs, and its inhibition substantially reduced STAT3 phosphorylation, MCP-1 expression, and VSMC migration. In addition, Src formed a complex with EGFR and Jak2, and its inhibition completely blocked Jak2 and STAT3 phosphorylation, MCP-1 expression, and VSMC migration. 15(S)-HETE induced the production of H(2)O(2) via an NADPH oxidase-dependent manner and its scavengers, N-acetyl cysteine (NAC) and catalase suppressed 15(S)-HETE-stimulated EGFR, Src, Jak2, and STAT3 phosphorylation and MCP-1 expression. Balloon injury (BI) induced EGFR, Src, Jak2, and STAT3 phosphorylation, and inhibition of these signaling molecules attenuated BI-induced MCP-1 expression and smooth muscle cell migration from the medial to the luminal surface resulting in reduced neointima formation. In addition, inhibition of EGFR blocked BI-induced Src, Jak2, and STAT3 phosphorylation. Similarly, interference with Src activation suppressed BI-induced Jak2 and STAT3 phosphorylation. Furthermore, adenovirus-mediated expression of dnJak2 also blocked BI-induced STAT3 phosphorylation. Consistent with the effects of 15(S)-HETE on the activation of EGFR-Src-Jak2-STAT3 signaling in VSMCs in vitro, adenovirus-mediated expression of 15-lipoxygenase 1 (15-Lox1) enhanced BI-induced EGFR, Src, Jak2, and STAT3 phosphorylation leading to enhanced MCP-1 expression in vivo. Blockade of Src or Jak2 suppressed BI-induced 15-Lox1-enhanced STAT3 phosphorylation, MCP-1 expression, and neointima formation. In addition, whereas dominant negative Src blocked BI-induced 15-Lox1-enhanced Jak2 phosphorylation, dnJak2 had no effect on Src phosphorylation. Together, these observations demonstrate for the first time that the 15-Lox1-15(S)-HETE axis activates EGFR via redox-sensitive manner, which in turn mediates Src-Jak2-STAT3-dependent MCP-1 expression leading to vascular wall remodeling.

15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires Src-mediated Egr-1-dependent rapid induction of FGF-2 expression.

To understand the mechanisms underlying 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE]-induced angiogenesis, we studied the role of Egr-1. 15(S)-HETE induced Egr-1 expression in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Blockade of Egr-1 via forced expression of its dominant-negative mutant attenuated 15(S)-HETE-induced HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. 15(S)-HETE-induced Egr-1 expression requires Src activation. In addition, adenovirus-mediated expression of dominant-negative mutant of Src blocked 15(S)-HETE's effects on migration and tube formation of HDMVECs and Matrigel plug angiogenesis. 15(S)-HETE induced fibroblast growth factor-2 (FGF-2) expression rapidly via Src-mediated production of Egr-1. Cloning and mutational analysis of FGF-2 promoter revealed that Egr-1 binding site proximal to transcription start site is required for 15(S)-HETE-induced FGF-2 expression. Neutralizing antibody-mediated suppression of FGF-2 function also attenuated the effects of 15(S)-HETE on HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. Furthermore, in contrast to wild-type mice, 12/15-LOX(-/-) mice exhibited decreased Matrigel plug angiogenesis in response to AA, which was rescued by 15(S)-HETE. On the basis of these observations, we conclude that 15(S)-HETE-induced angiogenesis requires Src-mediated Egr-1-dependent rapid induction of FGF-2. These findings may suggest that 15(S)-HETE could be a potential endogenous regulator of pathologic angiogenesis associated with atherosclerosis and restenosis.

[Tumor cell-tumor endothelial cell adhesion mediated by alphavbeta3 and alphavbeta5 molecules].

OBJECTIVE: To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro. METHODS: The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture. RESULTS: The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5. CONCLUSION: alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.

Protein Lipidation in Cell Signaling and Diseases: Function, Regulation, and Therapeutic Opportunities.

Protein lipidation is an important co- or posttranslational modification in which lipid moieties are covalently attached to proteins. Lipidation markedly increases the hydrophobicity of proteins, resulting in changes to their conformation, stability, membrane association, localization, trafficking, and binding affinity to their co-factors. Various lipids and lipid metabolites serve as protein lipidation moieties. The intracellular concentrations of these lipids and their derivatives are tightly regulated by cellular metabolism. Therefore, protein lipidation links the output of cellular metabolism to the regulation of protein function. Importantly, deregulation of protein lipidation has been linked to various diseases, including neurological disorders, metabolic diseases, and cancers. In this review, we highlight recent progress in our understanding of protein lipidation, in particular, S-palmitoylation and lysine fatty acylation, and we describe the importance of these modifications for protein regulation, cell signaling, and diseases. We further highlight opportunities and new strategies for targeting protein lipidation for therapeutic applications.

Chemical Probe to Identify the Cellular Targets of the Reactive Lipid Metabolite 2- trans-Hexadecenal.

Lipid-derived electrophiles (LDEs) are reactive metabolites, which can covalently modify proteins and DNA and regulate diverse cellular processes. 2- trans-Hexadecenal (2-HD) is a byproduct of sphingolipid metabolism, involved in cytoskeletal reorganization, DNA damage, and apoptosis. In addition, the loss of ALDH3A2, an enzyme removing 2-HD in cells, is responsible for Sjörgen-Larsson Syndrome (SJS), suggesting that accumulation of 2-HD could lead to pathogenesis. However, the targets and the precise mechanisms of 2-HD are not well characterized. Herein, we report an alkyne-2-HD derivative as a bioorthogonal probe to explore the functions of 2-HD. We identified more than 500 potential cellular targets. Among them, the pro-apoptotic protein Bax can be covalently modified by 2-HD directly at the conserved Cys62 residue. Our work provided new chemical tools to explore the cellular functions of LDEs and revealed new mechanistic insights of the deregulation of lipid metabolism in diseases.

[Characteristics of endothelial cells derived from human hepatic cavernous hemangioma in vitro].

OBJECTIVE: To investigate the characteristics of endothelial cells derived from human cavernous hemangioma in morphology, phenotypes and functions. METHODS: Endothelial cells were isolated from human hepatic cavernous hemangioma. The morphological, and phenotypical and functional features of these cells were analyzed by transmission electron microscopy, fluorescence-activated cell sorter, RT-PCR, zymography, and confocal microscopy. Human liver sinusoidal endothelial cells (LSEC) were used as control. RESULTS: As compared with the LSEC, abnormally expanded endoplasmic reticulums and similarly arranged cytoplasmic vacuoles were found in the endothelial cells derived from hepatic cavernous hemangioma (HCHEC) by transmission electron microscopy. Flow cytometry showed that expression of alphavbeta3 was significantly increased in the HCHEC. The mRNA of vascular endothelial cell growth factor and angiopoietin 1 were more abundant in HCHEC than that in LSEC. Functional analysis indicated that the HCHEC exhibited strong activated angiogenesis capacity and formed abnormal capillary-like structures. HCHEC produced more pro-matrix metalloproteinase 2 (MMP-2) and the activated MMP-2 form as compared with the LSEC. Confocal microscopy revealed that MMP-2 was concentrated in those cytoplasmic granules of the HCHEC and was consistent with the distribution of the expanded endoplasmic reticulums. CONCLUSION: The endothelial cells derived from human cavernous hemangioma differ from the normal endothelial cells in morphology, phenotypes and functions.

Induction of miRNA-181a by genotoxic treatments promotes chemotherapeutic resistance and metastasis in breast cancer.

Acquired therapeutic resistance is the major drawback to effective systemic therapies for cancers. Aggressive triple-negative breast cancers (TNBC) develop resistance to chemotherapies rapidly, whereas the underlying mechanisms are not completely understood. Here we show that genotoxic treatments significantly increased the expression of miR-181a in TNBC cells, which enhanced TNBC cell survival and metastasis upon Doxorubicin treatment. Consistently, high miR-181a level associated with poor disease free survival and overall survival after treatments in breast cancer patients. The upregulation of miR-181a was orchestrated by transcription factor STAT3 whose activation depended on NF-κB-mediated IL-6 induction in TNBC cells upon genotoxic treatment. Intriguingly, activated STAT3 not only directly bound to MIR181A1 promoter to drive transcription but also facilitated the recruitment of MSK1 to the same region where MSK1 promoted a local active chromatin state by phosphorylating histone H3. We further identified BAX as a direct functional target of miR-181a, whose suppression decreased apoptosis and increased invasion of TNBC cells upon Dox treatment. These results were further confirmed by evidence that suppression of miR-181a significantly enhanced therapeutic response and reduced lung metastasis in a TNBC orthotopic model. Collectively, our data suggested that miR-181a induction had a critical role in promoting therapeutic resistance and aggressive behavior of TNBC cells upon genotoxic treatment. Antagonizing miR-181a may serve as a promising strategy to sensitize TNBC cells to chemotherapy and mitigate metastasis.

MicroRNAs in cancer therapeutic response: Friend and foe.

Cancer initiation and development engage extremely complicated pathological processes which involve alterations of a large number of cell signaling cascades and functional networks in temporal and spatial orders. During last decades, microRNAs (miRNAs), a class of non-coding RNAs, have emerged as critical players in cancer pathogenesis and progression by modulating many pathological aspects related to tumor development, growth, metastasis, and drug resistance. The major function of miRNAs is to post-transcriptionally regulate gene expression depending on recognition of complementary sequence residing in target mRNAs. Commonly, a particular miRNA recognition sequence could be found in a number of genes, which allows a single miRNA to regulate multiple functionally connected genes simultaneously and/or chronologically. Furthermore, a single gene can be targeted and regulated by multiple miRNAs. However, previous studies have demonstrated that miRNA functions are highly context-dependent, which leads to distinct pathological outcomes in different types of cancer as well as at different stages by alteration of the same miRNA. Here we summarize recent progress in studies on miRNA function in cancer initiation, metastasis and therapeutic response, focusing on breast cancer. The varying functions of miRNAs and potential application of using miRNAs as biomarkers as well as therapeutic approaches are further discussed in the context of different cancers.

Morphologic, phenotypic and functional characteristics of endothelial cells derived from human hepatic cavernous hemangioma.

BACKGROUNDS/AIMS: The pathogenesis of cavernous hemangiomas is largely unknown, and it is speculated that abnormal vasculogenesis and angiogenesis may be involved. In this study, the characteristics of cavernous hemangioma endothelial cells (CHECs) derived from the human liver were analyzed in terms of morphology, phenotype and function and compared with human liver sinusoidal endothelial cells (LSECs). METHODS AND RESULTS: By transmission electron microscopy, abnormally expanded endoplasmic reticulum (ER) and similarly arranged cytoplasmic vacuoles were only found in CHECs. Phenotypic analysis showed that the expression of alphavbeta3 was significantly increased in CHECs. mRNA expression of vascular endothelial growth factor A, and angiopoietins 1 and 2 was significantly increased in CHECs compared to LSECs. The functional analysis indicated that CHECs released more vascular endothelial growth factor A, produced significantly more pro-matrix metalloproteinase 2 (pro-MMP2) and activated MMP2, and exhibited higher procoagulant and fibrinolytic activities compared with LSECs. Confocal microscopy revealed that MMP2 was concentrated in some cytoplasmic granules of CHECs and was consistent with the distribution of expanded ER. CHECs exhibited more activated angiogenesis capacity and formed abnormal capillary-like structures in vitro. CONCLUSION: These results suggested that endothelial cells (ECs) derived from human cavernous hemangiomas differ from normal ECs in morphology, phenotype and function.

[Induction of anti-tumor immunity by HSP gp96-peptide complexes].

AIM: To immunize the mice with gp96-peptide complexes extracted and purified from different kinds of malignant tumor cells and to observe anti-tumor immunity induced by the complexes. METHODS: HSP gp96-peptide complexes were extracted and purified from different kinds of malignant tumor cells. Mice were immunized subcutaneously with the complexes from different sources at different doses. Then the mice were challenged with 2 x 10(5) tumor cells on the seventh day after the last immunization. Tumor incidence, weight and histology were observed and compared with those of control group. At the meantime, cytotoxicity activity and nitric oxide production of mouse peritoneal macrophages were detected in vitro after being stimulated with gp96-peptide complexes. RESULTS: The SDS-PAGE and Western blot showed that gp96-peptide complexes were purified successfully. The anti-tumor immunity was correlated with the dose of the complex and the immunized mice could resist the challenge of homogeneous tumor cells. NO secreted from the peritoneal macrophages stimulated with the complex was significantly higher than that of control group and the tumoricidal activity of the macrophages in vitro was markedly promoted by the complex. CONCLUSION: Mice immunized with appropriate dose of gp96-peptide complexes from H22 tumor cells can resist the challenge of syngeneic tumor cells. NO secreted by macrophages stimulated with the complex might play a key role in the anti-tumor immunity.