RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination.

End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.

The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing.

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

Quantitative Proteomics of Tissue-Infiltrating T Cells From CRC Patients Identified Lipocalin-2 Induces T-Cell Apoptosis and Promotes Tumor Cell Proliferation by Iron Efflux.

T cells play the most pivotal roles in antitumor immunity; the T-cell proteome and the differentially expressed proteins in the tumor immune microenvironment have rarely been identified directly from the clinical samples, especially for tumors that lack effective immunotherapy targets, such as colorectal cancer (CRC). In this study, we analyzed the protein expression pattern of the infiltrating T cells isolated from CRC patients using quantitative proteomics. CD4+ and CD8+ T cells were isolated from clinical samples and labeled by tandem mass tag reagents, and the differentially expressed proteins were quantified by mass spectrometry. The T-cell proteome profiling revealed dysfunctions in these tumor-infiltrating T cells. Specifically, antitumor immunity was suppressed because of differentially expressed metal ion transporters and immunity regulators. For the first time, lipocalin-2 (LCN2) was shown to be significantly upregulated in CD4+ T cells. Quantitative proteomic analysis of LCN2-overexpressed Jurkat cells showed that LCN2 damaged T cells by changes in iron transport. LCN2 induced T-cell apoptosis by reducing cellular iron concentration; moreover, the iron that was transported to the tumor microenvironment aided tumor cell proliferation, promoting tumor development. Meanwhile, LCN2 also influenced tumor progression through immune cytokines and cholesterol metabolism. Our results demonstrated that LCN2 has immunosuppressive functions that can promote tumor development; therefore, it is a potential immunotherapy target for CRC.

Quantitative Proteomic Analysis Reveals Functional Alterations of the Peripheral Immune System in Colorectal Cancer.

Colorectal cancer (CRC) is characterized by high morbidity, high mortality, and limited response to immunotherapies. The peripheral immune system is an important component of tumor immunity, and enhancements of peripheral immunity help to suppress tumor progression. However, the functional alterations of the peripheral immune system in CRC are unclear. Here, we used mass spectrometry-based quantitative proteomics to establish a protein expression atlas for the peripheral immune system in CRC, including plasma and five types of immune cells (CD4+ T cells, CD8+ T cells, monocytes, natural killer cells, and B cells). Synthesizing the results of the multidimensional analysis, we observed an enhanced inflammatory phenotype in CRC, including elevated expression of plasma inflammatory proteins, activation of the inflammatory pathway in monocytes, and increased inflammation-related ligand-receptor interactions. Notably, we observed tumor effects on peripheral T cells, including altered cell subpopulation ratios and suppression of cell function. Suppression of CD4+ T cell function is mainly mediated by high expression levels of protein tyrosine phosphatases. Among them, the expression of protein tyrosine phosphatase receptor type J (PTPRJ) gradually increased with CRC progression; knockdown of PTPRJ in vitro could promote T cell activation, thereby enhancing peripheral immunity. We also found that the combination of leucine-rich α-2 glycoprotein 1 (LRG1) and apolipoprotein A4 (APOA4) had the best predictive ability for colorectal cancer and has the potential to be a biomarker. Overall, this study provides a comprehensive understanding of the peripheral immune system in CRC. It also offers insights regarding the potential clinical utilities of these peripheral immune characteristics as diagnostic indicators and therapeutic targets.

Proteogenomic analysis identifies neoantigens and bacterial peptides as immunotherapy targets in colorectal cancer.

Considerable progress has recently been made in cancer immunotherapy, including immune checkpoint blockade, cancer vaccine, and adoptive T cell methods. The lack of effective targets is a major cause of the low immunotherapy response rate in colorectal cancer (CRC). Here, we used a proteogenomic strategy comprising immunopeptidomics, whole exome sequencing, and 16 S ribosomal DNA sequencing analyses of 8 patients with CRC to identify neoantigens and bacterial peptides that can serve as antitumor targets. This study directly identified several personalized neoantigens and bacterial immunopeptides. Immunoassays showed that all neoantigens and 5 of 8 bacterial immunopeptides could be recognized by autologous T cells. Additionally, T cell receptor (TCR) αß sequencing revealed the TCR repertoire of epitope-reactive CD8+ T cells. Functional studies showed that T cell receptor-T (TCR-T) could be activated by epitope pulsed lymphoblastoid cells. Overall, this study comprehensively profiled the CRC immunopeptidome, revealing several neoantigens and bacterial peptides with potential to serve as immunotherapy targets in CRC.

The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes.

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2 However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2 -mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Proteomic Landscape of Exosomes Reveals the Functional Contributions of CD151 in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Patients with TNBC have poor overall survival because of limited molecular therapeutic targets. Recently, exosomes have been recognized as key mediators in cancer progression, but the molecular components and function of TNBC-derived exosomes remain unknown. The main goal of this study was to reveal the proteomic landscape of serum exosomes derived from ten patients with TNBC and 17 healthy donors to identify potential therapeutic targets. Using a tandem mass tag-based quantitative proteomics approach, we characterized the proteomes of individual patient-derived serum exosomes, identified exosomal protein signatures specific to patients with TNBC, and filtered out differentially expressed proteins. Most importantly, we found that the tetraspanin CD151 expression levels in TNBC-derived serum exosomes were significantly higher than those exosomes from healthy subjects, and we validated our findings with samples from 16 additional donors. Furthermore, utilizing quantitative proteomics approach to reveal the proteomes of CD151-deleted exosomes and cells, we found that exosomal CD151 facilitated secretion of ribosomal proteins via exosomes while inhibiting exosome secretion of complement proteins. Moreover, we proved that CD151-deleted exosomes significantly decreased the migration and invasion of TNBC cells. This is the first comparative study of the proteomes of TNBC patient-derived and CD151-deleted exosomes. Our findings indicate that profiling of TNBC-derived exosomal proteins is a useful tool to extend our understanding of TNBC, and exosomal CD151 may be a potential therapeutic target for TNBC.

Targeting NADPH Oxidase and Integrin α5ß1 to Inhibit Neutrophil Extracellular Traps-Mediated Metastasis in Colorectal Cancer.

Metastasis leads to a high mortality rate in colorectal cancer (CRC). Increased neutrophil extracellular traps (NETs) formation is one of the main causes of metastasis. However, the mechanism of NETs-mediated metastasis remains unclear and effective treatments are lacking. In this study, we found neutrophils from CRC patients have enhanced NETs formation capacity and increased NETs positively correlate with CRC progression. By quantitative proteomic analysis of clinical samples and cell lines, we found that decreased secreted protein acidic and rich in cysteine (SPARC) results in massive NETs formation and integrin α5ß1 is the hub protein of NETs-tumor cell interaction. Mechanistically, SPARC regulates the activation of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) pathway by interacting with the receptor for activated C kinase 1 (RACK1). Over-activated NADPH oxidase generates more reactive oxygen species (ROS), leading to the release of NETs. Then, NETs upregulate the expression of integrin α5ß1 in tumor cells, which enhances adhesion and activates the downstream signaling pathways to promote proliferation and migration. The combination of NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and integrin α5ß1 inhibitor ATN-161 (Ac-PHSCN-NH2) effectively suppresses tumor progression in vivo. Our work reveals the mechanistic link between NETs and tumor progression and suggests a combination therapy against NETs-mediated metastasis for CRC.

Scalable Asymmetric Syntheses of Foslevodopa and Foscarbidopa Drug Substances for the Treatment of Parkinson's Disease.

Foslevodopa (FLD, levodopa 4'-monophosphate, 3) and foscarbidopa (FCD, carbidopa 4'-monophosphate, 4) were identified as water-soluble prodrugs of levodopa (LD, 1) and carbidopa (CD, 2), respectively, which are useful for the treatment of Parkinson's disease. Herein, we describe asymmetric syntheses of FLD (3) and FCD (4) drug substances and their manufacture at pilot scale. The synthesis of FLD (3) employs a Horner-Wadsworth-Emmons olefination reaction followed by enantioselective hydrogenation of the double bond as key steps to introduce the α-amino acid moiety with the desired stereochemistry. The synthesis of FCD (4) features a Mizoroki-Heck reaction followed by enantioselective hydrazination to install the quaternary chiral center bearing a hydrazine moiety.

Replication fork stalling elicits chromatin compaction for the stability of stalling replication forks.

DNA replication forks in eukaryotic cells stall at a variety of replication barriers. Stalling forks require strict cellular regulations to prevent fork collapse. However, the mechanism underlying these cellular regulations is poorly understood. In this study, a cellular mechanism was uncovered that regulates chromatin structures to stabilize stalling forks. When replication forks stall, H2BK33, a newly identified acetylation site, is deacetylated and H3K9 trimethylated in the nucleosomes surrounding stalling forks, which results in chromatin compaction around forks. Acetylation-mimic H2BK33Q and its deacetylase clr6-1 mutations compromise this fork stalling-induced chromatin compaction, cause physical separation of replicative helicase and DNA polymerases, and significantly increase the frequency of stalling fork collapse. Furthermore, this fork stalling-induced H2BK33 deacetylation is independent of checkpoint. In summary, these results suggest that eukaryotic cells have developed a cellular mechanism that stabilizes stalling forks by targeting nucleosomes and inducing chromatin compaction around stalling forks. This mechanism is named the "Chromsfork" control: Chromatin Compaction Stabilizes Stalling Replication Forks.

Mass spectrometry-based screening identifies circulating immunoglobulinA-α1-microglobulin complex as potential biomarker in immunoglobulin A nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is characterized by predominant IgA deposition in the glomerular mesangium. Previous studies have proved that renal-deposited IgA in IgAN came from circulating IgA1-containing complexes (CICs). METHODS: To explore the composition of CICs in IgAN, we isolated CICs from IgAN patients and healthy controls and then quantitatively analyzed them by mass spectrometry. Meanwhile, the isolated CICs were used to treat human mesangial cells to monitor mesangial cell injury. Using the protein content and injury effects, the key constituent in CICs was identified. Then the circulating levels of identified key constituent-IgA complex were detected in an independent population by an in-house-developed enzyme-linked immunosorbent assay. RESULTS: By comparing the proteins of CICs between IgAN patients and controls, we found that 14 proteins showed significantly different levels. Among them, α1-microglobulin content in CICs was associated with not only in vitro mesangial cell proliferation and monocyte chemoattractant protein 1 secretion, but also in vivo estimated glomerular filtration rate (eGFR) levels and tubulointerstitial lesions in IgAN patients. Moreover, we found α1-microglobulin was prone to bind aberrant glycosylated IgA1. Additionally, elevated circulating IgA-α1-microglobulin complex levels were detected in an independent IgAN population and IgA-α1-microglobulin complex levels were correlated with hypertension, eGFR levels and Oxford T- scores in these IgAN patients. CONCLUSIONS: Our results suggest that the IgA-α1-microglobulin complex is an important constituent in CICs and that circulating IgA-α1-microglobulin complex detection might serve as a potential noninvasive biomarker detection method for IgAN.

Novel Physics-Based Ensemble Modeling Approach That Utilizes 3D Molecular Conformation and Packing to Access Aqueous Thermodynamic Solubility: A Case Study of Orally Available Bromodomain and Extraterminal Domain Inhibitor Lead Optimization Series.

Drug design with patient centricity for ease of administration and pill burden requires robust understanding of the impact of chemical modifications on relevant physicochemical properties early in lead optimization. To this end, we have developed a physics-based ensemble approach to predict aqueous thermodynamic crystalline solubility, with a 2D chemical structure as the input. Predictions for the bromodomain and extraterminal domain (BET) inhibitor series show very close match (0.5 log unit) with measured thermodynamic solubility for cases with low crystal anisotropy and good match (1 log unit) for high anisotropy structures. The importance of thermodynamic solubility is clearly demonstrated by up to a 4 log unit drop in solubility compared to kinetic (amorphous) solubility in some cases and implications thereof, for instance on human dose. We have also demonstrated that incorporating predicted crystal structures in thermodynamic solubility prediction is necessary to differentiate (up to 4 log unit) between solubility of molecules within the series. Finally, our physics-based ensemble approach provides valuable structural insights into the origins of 3-D conformational landscapes, crystal polymorphism, and anisotropy that can be leveraged for both drug design and development.

Mitosis-specific MRN complex promotes a mitotic signaling cascade to regulate spindle dynamics and chromosome segregation.

The MRE11-RAD50-NBS1 (MRN) complex is well known for participating in DNA damage response pathways in all phases of cell cycle. Here, we show that MRN constitutes a mitosis-specific complex, named mMRN, with a protein, MMAP. MMAP directly interacts with MRE11 and is required for optimal stability of the MRN complex during mitosis. MMAP colocalizes with MRN in mitotic spindles, and MMAP-deficient cells display abnormal spindle dynamics and chromosome segregation similar to MRN-deficient cells. Mechanistically, both MMAP and MRE11 are hyperphosphorylated by the mitotic kinase, PLK1; and the phosphorylation is required for assembly of the mMRN complex. The assembled mMRN complex enables PLK1 to interact with and activate the microtubule depolymerase, KIF2A, leading to spindle turnover and chromosome segregation. Our study identifies a mitosis-specific version of the MRN complex that acts in the PLK1-KIF2A signaling cascade to regulate spindle dynamics and chromosome distribution.

α/ß-Hydrolase domain-containing 6 (ABHD6) negatively regulates the surface delivery and synaptic function of AMPA receptors.

In the brain, AMPA-type glutamate receptors are major postsynaptic receptors at excitatory synapses that mediate fast neurotransmission and synaptic plasticity. α/ß-Hydrolase domain-containing 6 (ABHD6), a monoacylglycerol lipase, was previously found to be a component of AMPA receptor macromolecular complexes, but its physiological significance in the function of AMPA receptors (AMPARs) has remained unclear. The present study shows that overexpression of ABHD6 in neurons drastically reduced excitatory neurotransmission mediated by AMPA but not by NMDA receptors at excitatory synapses. Inactivation of ABHD6 expression in neurons by either CRISPR/Cas9 or shRNA knockdown methods significantly increased excitatory neurotransmission at excitatory synapses. Interestingly, overexpression of ABHD6 reduced glutamate-induced currents and the surface expression of GluA1 in HEK293T cells expressing GluA1 and stargazin, suggesting a direct functional interaction between these two proteins. The C-terminal tail of GluA1 was required for the binding between of ABHD6 and GluA1. Mutagenesis analysis revealed a GFCLIPQ sequence in the GluA1 C terminus that was essential for the inhibitory effect of ABHD6. The hydrolase activity of ABHD6 was not required for the effects of ABHD6 on AMPAR function in either neurons or transfected HEK293T cells. Thus, these findings reveal a novel and unexpected mechanism governing AMPAR trafficking at synapses through ABHD6.

A rapid and easy protein N-terminal profiling strategy using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip.

Protein N-terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N-terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N-terminal peptide losses. In this study, a fast, one-step approach utilizing (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N-terminal peptide enrichment and analysis. Based on the characteristics of TMPP-derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision-induced dissociation or HCD fragmentation modes, first the SDS-PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP-modified N-terminal spectra. When utilizing a low amount of starting material (∼20 µg protein), a total of 581 yeast N-terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N-terminomic studies.

Identification of Caspase-6 as a New Regulator of Alternatively Activated Macrophages.

Alternatively activated macrophages (AAMs) play essential roles in the promotion of tissue remodeling, vasculogenesis, and tumor progression; however, the detailed mechanisms underlying the activation of AAMs remain largely unknown. Here, by using quantitative proteomic analysis, we identified 62 proteins that were up-regulated in IL-4-induced macrophages. Among these, Caspase-6 was increased significantly. Caspase-6 is important in the apoptotic signaling pathway; however, its role in non-apoptosis is also reported. Here, we first examined the non-apoptotic role of Caspase-6 in the alternative activation of macrophages after administration of IL-4, 4T1 tumor conditional medium, or co-culture with 4T1 cells. Both treatments promoted alternative activation of RAW264.7 cells and primary macrophages, whereas disruption of caspase-6 expression and activity could markedly suppress the biomarker levels of AAMs. Overexpression of Caspase-6 could significantly promote the activation of AAMs. Importantly, we further present evidence that caspase-6 could regulate breast cancer cell invasion by modulating MMP-2 and MMP-9 expression in 4T1 tumor-associated macrophages, as ablation of protein levels or activity of caspase-6 suppressed tumor cell invasion in vitro In conclusion, the observed results markedly expanded our views of the dynamic changes in protein composition during alternative activation of macrophages, and they revealed a critical new role of caspase-6 in regulating this cellular biological process, which suggested that caspase-6 might be a key nod molecule to regulate immunological steady-state and be a therapeutic candidate for tumor immunotherapy.

Ewing Tumor-associated Antigen 1 Interacts with Replication Protein A to Promote Restart of Stalled Replication Forks.

The replication protein A (RPA) complex binds single-stranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.

Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3ß.

Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3ß (Gsk3ß). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3ß, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101.

Quantitative nuclear proteomics identifies that miR-137-mediated EZH2 reduction regulates resveratrol-induced apoptosis of neuroblastoma cells.

Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials, and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4'-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction, and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment.