NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance.

The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11-RAD50-NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the 'writer' of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.

Exosome circATP8A1 induces macrophage M2 polarization by regulating the miR-1-3p/STAT6 axis to promote gastric cancer progression.

Circular RNAs (circRNAs) play important roles in gastric cancer progression but the regulatory role of circRNAs in controlling macrophage function remains elusive. Exosomes serve as cargo for circRNAs and play a crucial role as mediators in facilitating communication between cancer cells and the tumor microenvironment. In this study, we found that circATP8A1, a previously unreported circular RNA, is highly expressed in both gastric cancer tissues and exosomes derived from plasma. Increased circATP8A1 was associated with advanced TNM stage and worse prognosis in patients with gastric cancer. We showed that  the circATP8A1 knockdown significantly inhibited gastric cancer proliferation and invasion in vitro and in vivo. Functionally, exosome circATP8A1 induced the M2 polarization of macrophages through the STAT6 pathway instead of the STAT3 pathway. Mechanistically, circATP8A1 was shown to activate the STAT6 pathway through competitive binding to miR-1-3p, as confirmed by Fluorescence In Situ Hybridization (FISH), RNA immunoprecipitation, RNA pulldown, and Luciferase reporter assays. The reversal of circATP8A1-induced STAT6 pathway activation and macrophage polarization was observed upon blocking miR-1-3p. Macrophages treated with exosomes from gastric cancer cells overexpressing circATP8A1 were able to promote gastric cancer migration, while knockdown of circATP8A1 reversed these effects in vivo. In summary, exosome-derived circATP8A1 from gastric cancer cells induce macrophages M2 polarization via the circATP8A1/miR-1-3p/STAT6 axis, and tumor progression. Our results highlight circATP8A1 as a potential prognostic biomarker and therapeutic target in gastric cancer.

Metal-enriched HSP90 nanoinhibitor overcomes heat resistance in hyperthermic intraperitoneal chemotherapy used for peritoneal metastases.

Clinical hyperthermic intraperitoneal chemotherapy (HIPEC) is regarded as a potential treatment that can prolong survival of patients with peritoneal metastases after cytoreductive surgery. However, treated tumor cells are prone to becoming heat resistant to HIPEC therapy through high expression of heat shock proteins (HSPs). Here, a carrier-free bifunctional nanoinhibitor was developed for HIPEC therapy in the management of peritoneal metastases. Self-assembly of the nanoinhibitor was formed by mixing Mn ion and epigallocatechin gallate (EGCG) in a controllable manner. Such nanoinhibitor directly inhibited HSP90 and impaired the HSP90 chaperone cycle by reduced intracellular ATP level. Additionally, heat and Mn ion synergistically induced oxidative stress and expression of caspase 1, which activated GSDMD by proteolysis and caused pyroptosis in tumor cells, triggering immunogenic inflammatory cell death and induced maturation of dendritic cells through the release of tumor antigens. This strategy to inhibit heat resistance in HIPEC presented an unprecedented paradigm for converting "cold" tumors into "hot" ones, thus significantly eradicating disseminated tumors located deep in the abdominal cavity and stimulating immune response in peritoneal metastases of a mouse model. Collectively, the nanoinhibitor effectively induced pyroptosis of colon tumor cells under heat conditions by inhibiting heat stress resistance and increasing oxidative stress, which may provide a new strategy for treatment of colorectal peritoneal metastases.

Pyruvate Kinase M2 Tetramerization Protects against Hepatic Stellate Cell Activation and Liver Fibrosis.

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The in vitro and in vivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs in vitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis in vivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.

Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA-damaging chemotherapeutic agents.

The bromodomain-containing protein 7 (BRD7) is a tumour suppressor protein with critical roles in cell cycle transition and transcriptional regulation. Whether BRD7 is regulated by post-translational modifications remains poorly understood. Here, we find that chemotherapy-induced DNA damage leads to the rapid degradation of BRD7 in various cancer cell lines. PARP-1 binds and poly(ADP)ribosylates BRD7, which enhances its ubiquitination and degradation through the PAR-binding E3 ubiquitin ligase RNF146. Moreover, the PARP1 inhibitor Olaparib significantly enhances the sensitivity of BRD7-positive cancer cells to chemotherapeutic drugs, while it has little effect on cells with low BRD7 expression. Taken together, our findings show that PARP1 induces the degradation of BRD7 resulting in cancer cell resistance to DNA-damaging agents. BRD7 might thus serve as potential biomarker in clinical trial for the prediction of synergistic effects between chemotherapeutic drugs and PARP inhibitors.

FBW7 suppresses cell proliferation and G2/M cell cycle transition via promoting γ-catenin K63-linked ubiquitylation.

FBW7 is an E3 ubiquitin ligase and frequently mutated in various types of cancer. As a component of SCF ubiquitin ligase complex, FBW7 usually targets the substrates via K11 or K48-linked ubiquitylation and subsequent degradation of target proteins. Nevertheless, the role of FBW7 in mediating non-degradable ubiquitin signaling remains unknown in human cancers. In this study, we identified γ-catenin as a new binding protein of FBW7 by TAP-MS (tandem affinity purification-mass spectrum). Knockdown of FBW7 did not affect the stability of γ-catenin, but significantly reduced the K63-linked ubiquitin of γ-catenin, resulting in decreased expression of γ-catenin downstream gene 14-3-3σ. Rescue experiment revealed that γ-catenin promoted the expression of 14-3-3σ in a K63-linked ubiquitin signaling dependent manner. Furthermore, we showed that FBW7 cooperated with γ-catenin to inhibit G2/M cell cycle transition and cell proliferation. Taken together, our study uncovered a novel mechanism that FBW7 associated with γ-catenin and promoted its K63-linked ubiquitylation, providing new insights in understanding the role of FBW7 in inhibiting G2/M cell cycle transition and tumor cell proliferation.

Transcription factor EHF interacting with coactivator AJUBA aggravates malignancy and acts as a therapeutic target for gastroesophageal adenocarcinoma.

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

Identification and validation of a PD-L1-related signature from mass spectrometry in gastric cancer.

BACKGROUND: According to the guidelines, PD-L1 expression is a critical indicator for guiding immunotherapy application. According to certain studies, regardless of PD-L1 expression, immunotherapy could be advantageous for individuals with gastric cancer. Therefore, new scoring systems or biomarkers are required to enhance treatment strategies. METHODS: Mass spectrometry and machine learning were used to search for strongly related PD-L1 genes, and the NMF approach was then used to separate gastric cancer patients into two categories. Differentially expressed genes (DEGs) between the two subtypes identified in this investigation were utilized to develop the UBscore predictive model, which was verified by the Gene Expression Omnibus (GEO) database. Coimmunoprecipitation, protein expression, and natural killing (NK) cell coculture experiments were conducted to validate the findings. RESULTS: A total of 123 proteins were identified as PD-L1 interactors that are substantially enriched in the proteasome complex at the mRNA level. Using random forest, 30 UPS genes were discovered in the GSE66229 cohort, and ANAPC7 was experimentally verified as one of 123 PD-L1 interactors. Depending on the expression of PD-L1 and ANAPC7, patients were separated into two subgroups with vastly distinct immune infiltration. Low UBscore was related to increased tumor mutation burden (TMB) and microsatellite instability-high (MSI-H). In addition, chemotherapy medications were more effective in individuals with a low UBscore. Finally, we discovered that ANAPC7 might lead to the incidence of immunological escape when cocultured with NK-92 cells. CONCLUSION: According to our analysis of the PD-L1-related signature in GC, the UBscore played a crucial role in prognosis and had a strong relationship with TMB, MSI, and chemotherapeutic drug sensitivity. This research lays the groundwork for improving GC patient prognosis and treatment response.

Mitophagy is a novel protective mechanism for drug-tolerant persister (DTP) cancer cells.

Drug-tolerant persister (DTP) cancer cells drive residual tumor and relapse. However, the mechanisms underlying DTP state development are largely unexplored. In a recent study, we determined that PINK1-mediated mitophagy favors DTP generation in the context of MAPK inhibition therapy. DTP cells that persist in the presence of a MAPK inhibitor exhibit mitochondriadependent metabolism. During DTP state development, MYC depletion alleviates the transcriptional repression of PINK1, resulting in PINK1 upregulation and mitophagy activation. PINK1-mediated mitophagy is essential for mitochondrial homeostasis in DTP cells. Either knockdown of PINK1 or inhibition of mitophagy eradicates DTP cells and achieves complete responses to MAPK inhibition therapy. This study reveals a novel role of mitophagy as a protective mechanism for DTP development.

Pleckstrin-2 promotes tumour immune escape from NK cells by activating the MT1-MMP-MICA signalling axis in gastric cancer.

Immune escape is a major challenge in tumour immunotherapy. Pleckstrin-2(PLEK2) plays a critical role in tumour progression, but its role in immune escape in gastric cancer (GC) remains uncharacterized. RNA sequencing was used to explore the differentially expressed genes in a GC cell line that was resistant to the antitumor effect of Natural killer (NK) cells. Apoptosis and the expression of IFN-γ and TNF-α were detected by flow cytometry (FCM). PLEK2 expression was examined by Western blotting and immunohistochemistry (IHC). PLEK2 was upregulated in MGC803R cells that were resistant to the antitumor effect of NK cells. PLEK2 knockout increased the sensitivity of GC cells to NK cell killing. PLEK2 expression was negatively correlated with MICA and positively correlated with MT1-MMP expression both in vitro and in vivo. PLEK2 promoted Sp1 phosphorylation through the PI3K-AKT pathway, thereby upregulating MT1-MMP expression, which ultimately led to MICA shedding. In mouse xenograft models, PLEK2 knockout inhibited intraperitoneal metastasis of GC cells and promoted NK cell infiltration. In summary, PLEK2 suppressed NK cell immune surveillance by promoting MICA shedding, which serves as a potential therapeutic target for GC.

PINK1-Mediated Mitophagy Promotes Oxidative Phosphorylation and Redox Homeostasis to Induce Drug-Tolerant Persister Cancer Cells.

The drug-tolerant persister (DTP) state enables cancer cells to evade cytotoxic stress from anticancer therapy. However, the mechanisms governing DTP generation remain poorly understood. Here, we observed that lung adenocarcinoma (LUAD) cells and organoids entered a quiescent DTP state to survive MAPK inhibitor treatment. DTP cells following MAPK inhibition underwent a metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS). PTEN-induced kinase 1 (PINK1), a serine/threonine kinase that initiates mitophagy, was upregulated to maintain mitochondrial homeostasis during DTP generation. PINK1-mediated mitophagy supported DTP cell survival and contributed to poor prognosis. Mechanistically, MAPK pathway inhibition resulted in MYC-dependent transcriptional upregulation of PINK1, leading to mitophagy activation. Mitophagy inhibition using either clinically applicable chloroquine or depletion of PINK1 eradicated drug tolerance and allowed complete response to MAPK inhibitors. This study uncovers PINK1-mediated mitophagy as a novel tumor protective mechanism for DTP generation, providing a therapeutic opportunity to eradicate DTP and achieve complete responses. SIGNIFICANCE: DTP cancer cells that cause relapse after anticancer therapy critically depend on PINK1-mediated mitophagy and metabolic reprogramming, providing a therapeutic opportunity to eradicate persister cells to prolong treatment efficacy.

Rad54L promotes bladder cancer progression by regulating cell cycle and cell senescence.

Bladder cancer (BCa) is the most prevalent cancer of the urinary system, but its pathogenesis is still poorly understood. Several reports have suggested that gene damage repair is highly correlated with tumor development and drug resistance, in which homologous recombination repair gene Rad54L seems to play an important role, through yet unclear mechanisms. Therefore, this study stratified cancer patients by Rad54L expression in BCa tissue, and high Rad54L expression was associated with a poor prognosis. Mechanistically, we demonstrate that high Rad54L expression promotes abnormal bladder tumor cell proliferation by changing the cell cycle and cell senescence. In addition, this study also suggests that Rad54L may be associated with p53, p21, and pRB in BCa tissue. In summary, this study exposes Rad54L as potential a prognostic biomarker and precision treatment target in BCa.

On-Demand Generation of Peroxynitrite from an Integrated Two-Dimensional System for Enhanced Tumor Therapy.

Nanosystem-mediated tumor radiosensitization strategy combining the features of X-ray with infinite penetration depth and high atomic number elements shows considerable application potential in clinical cancer therapy. However, it is difficult to achieve satisfactory anticancer efficacy using clinical radiotherapy for the majority of solid tumors due to the restrictions brought about by the tumor hypoxia, insufficient DNA damage, and rapid DNA repair during and after treatment. Inspired by the complementary advantages of nitric oxide (NO) and X-ray-induced photodynamic therapy, we herein report a two-dimensional nanoplatform by the integration of the NO donor-modified LiYF4:Ce scintillator and graphitic carbon nitride nanosheets for on-demand generation of highly cytotoxic peroxynitrite (ONOO-). By simply adjusting the Ce3+ doping content, the obtained nanoscintillator can realize high radioluminescence, activating photosensitive materials to simultaneously generate NO and superoxide radical for the formation of ONOO- in the tumor. Obtained ONOO- effectively amplifies therapeutic efficacy of radiotherapy by directly inducing mitochondrial and DNA damage, overcoming hypoxia-associated radiation resistance. The level of glutamine synthetase (GS) is downregulated by ONOO-, and the inhibition of GS delays DNA damage repair, further enhancing radiosensitivity. This work establishes a combinatorial strategy of ONOO- to overcome the major limitations of radiotherapy and provides insightful guidance to clinical radiotherapy.

Cycloruthenated Self-Assembly with Metabolic Inhibition to Efficiently Overcome Multidrug Resistance in Cancers.

The synthesis and the evaluation of the efficacy of a cycloruthenated complex, RuZ, is reported, to overcome multi-drug resistance (MDR) in cancer cells. RuZ can self-assemble into nanoaggregates in the cell culture medium, resulting in a high intracellular concentration of RuZ in MDR cancer cells. The self-assembly significantly decreases oxygen consumption and inhibits glycolysis, which decreases cellular adenosine triphosphate (ATP) levels. The decrease in ATP levels and its low affinity for the ABCB1 and ABCG2 transporters (which mediate MDR) significantly increase the retention of RuZ by MDR cancer cells. Furthermore, RuZ increases cellular oxidative stress, inducing DNA damage, and, in combination with the aforementioned effects of RuZ, increases the apoptosis of cancer cells. Proteomic profiling analysis suggests that the RuZ primarily decreases the expression of proteins that mediate glycolysis and aerobic mitochondrial respiration and increases the expression of proteins involved in apoptosis. RuZ inhibits the proliferation of 35 cancer cell lines, of which 7 cell lines are resistant to clinical drugs. It is also active in doxorubicin-resistant MDA-MB-231/Adr mouse tumor xenografts. To the best of our knowledge, the results are the first to show that self-assembled cycloruthenated complexes are efficacious in inhibiting the growth of MDR cancer cells.

Cross-Talk of Focal Adhesion-Related Gene Defines Prognosis and the Immune Microenvironment in Gastric Cancer.

Background: Focal adhesion, as the intermediary between tumor cells and extracellular matrix communication, plays a variety of roles in tumor invasion, migration, and drug resistance. However, the potential role of focal adhesion-related genes in the microenvironment, immune cell infiltration, and drug sensitivity of gastric cancer (GC) has not yet been revealed. Methods: The genetic and transcriptional perspectives of focal adhesion-related genes were systematically analyzed. From a genetic perspective, the focal adhesion index (FAI) was constructed based on 18 prognosis-related focus adhesion-related genes to evaluate the immune microenvironment and drug sensitivity. Then three prognosis-related genes were used for consistent clustering to identify GC subtypes. Finally, use FLT1, EGF, COL5A2, and M2 macrophages to develop risk signatures, and establish a nomogram together with clinicopathological characteristics. Results: Mutations in the focal adhesion-related gene affect the survival time and clinical characteristics of GC patients. FAI has been associated with a shorter survival time, immune signaling pathways, M2 macrophage infiltration, epithelial-mesenchymal transition (EMT) signaling, and diffuse type of GC. FAI recognizes ALK, cell cycle, and BMX signaling pathways inhibitors as sensitive agents for the treatment of GC. FLT1, EGF, and COL5A2 may distinguish GC subtypes. The established risk signature is of great significance to the prognostic evaluation of GC based on FLT1, EGF, and COL5A2 and M2 macrophage expression. Conclusion: The focal adhesion-related gene is a potential biomarker for the evaluation of the immune microenvironment and prognosis. This work emphasizes the potential impact of the focal adhesion pathway in GC therapy and highlights its guiding role in prognostic evaluation.

Transcription factor NFYA promotes G1/S cell cycle transition and cell proliferation by transactivating cyclin D1 and CDK4 in clear cell renal cell carcinoma.

NFYA (nuclear transcription factor Y, subunit A) is a CCAAT-binding transcription factor. Accumulating evidence suggests that NFYA plays an important role in breast, ovarian, lung and gastric cancer. However, the role of NFYA in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, it was discovered that the expression of NFYA is elevated in tissues of ccRCC patient and high NFYA expression is linked to poor overall survival in ccRCC patient. Inhibition of G1/S cell cycle transition and decreased cell proliferation were observed upon NFYA knockdown in ccRCC cells. Moreover, further investigation revealed that NFYA binds directly to the promoter region of both CDK4 and cyclin D1 (CCND1) thus transactivating their expression, resulting in RB phosphorylation and the activation of subsequent E2F pathway activation. Taken together, these findings imply the oncogenic role of NFYA in ccRCC progression and its potential as a target for ccRCC therapy.

PARK2 promotes mitochondrial pathway of apoptosis and antimicrotubule drugs chemosensitivity via degradation of phospho-BCL-2.

Rationale: Neoadjuvant chemotherapy has become the standard treatment of locally advanced breast cancer. Antimicrotubule drugs and DNA-damaging drugs are the most popular medicines used for neoadjuvant chemotherapy. However, we are unable to predict which chemotherapeutic drug will benefit to an individual patient. PARK2 as a tumor suppressor in breast cancer has been reported. While the role of PARK2 in chemotherapy response remains unknown. In this study, we explore the impact of PARK2 on chemosensitivity in breast cancer. Methods: PARK2 expression in breast cancer patients with different neoadjuvant chemotherapeutic regimens was studied using immunohistochemistry. Data was correlated to disease-free survival (DFS), overall survival and pathologic complete response (pCR). The functional roles of PARK2 were demonstrated by a series of in vitro and in vivo experiments. Including mass spectrometry, Co-immunoprecipitation, isolation of subcellular fractionation, fluorescence microscopy, in vivo ubiquitination assay and luciferase analyses. Results: Highly expressed PARK2 predicted better response to antimicrotubule drugs-containing regimen associated with higher rate of pathologic complete response (pCR). In contrast, PARK2 expression did not predict response to the DNA-damaging drugs regimen. Following antimicrotubule drugs treatment, levels of PARK2 was upregulated due to the repression of STAT3-mediated transcriptional inhibition of PARK2. Moreover, overexpression of PARK2 specifically rendered cells more sensitive to antimicrotubule drugs, but not to DNA-damaging drugs. Depletion of PARK2 enhanced resistance to antimicrotubule drugs. Mechanistically, PARK2 markedly activated the mitochondrial pathway of apoptosis after exposure to antimicrotubule drugs. This occurred through downregulating the antiapoptotic protein, phospho-BCL-2. BCL-2 phosphorylation can be specifically induced by antimicrotubule drugs, whereas DNA-damaging drugs do not. Notably, PARK2 interacted with phospho-BCL-2 (Ser70) and promoted ubiquitination of BCL-2 in an E3 ligase-dependent manner. Hence, PARK2 significantly enhanced the chemosensitivity of antimicrotubule drugs both in vitro and in vivo, while loss-of-function PARK2 mutants did not. Conclusions: Our findings explained why PARK2 selectively confers chemosensitivity to antimicrotubule drugs, but not to DNA-damaging drugs. In addition, we identified PARK2 as a novel mediator of antimicrotubule drugs sensitivity, which can predict response of breast cancer patients to antimicrotubule drugs-containing regime.

RCC2 promotes breast cancer progression through regulation of Wnt signaling and inducing EMT.

Regulator of chromosome condensation 2 (RCC2), also known as TD-60, is an RCC1 family member and plays an essential role in mitosis. However, the roles of RCC2 in breast cancer are still unclear. In this study, RCC2 was found to exert oncogenic activities in breast cancer. Samples of breast cancer tissue revealed an increased level of RCC2 and a high level of RCC2 was associated with poor overall survival rate of breast cancer patients. Overexpression of RCC2 significantly enhanced cell proliferation and migration abilities of breast cancer cells in vitro and in vivo. Mechanistically, RCC2 induced epithelial-mesenchymal transition (EMT) through the activation of Wnt signaling pathway. Collectively, our study indicates that RCC2 contributes to breast cancer progression and functions as an important regulator of EMT through the activation of Wnt signaling.

Correction to: High-performance gene expression and knockout tools using sleeping beauty transposon system.

[This corrects the article DOI: 10.1186/s13100-018-0139-y.].

High-performance gene expression and knockout tools using sleeping beauty transposon system.

BACKGROUND: Similar to retro-/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells. Sleeping Beauty (SB) transposon has adopted for integrating genes into host genomes in recent studies. However, SB-derived vector system for proteins purifying/tracking and gene knockout are still not available. RESULTS: In this study, we generated a series of vectors (termed as pSB vectors) containing Sleeping Beauty IRDR-L/R that can be transposed by SB transposase. Gateway cassette was combined to the pSB vectors to facilitate the cloning. Vectors with various tags, Flag, Myc, HA, V5 and SFB, were generated for multiple options. Moreover, we incorporated the CRISPR-Cas9 cassette into the pSB plasmids for gene knockout. Indeed, using one of these vectors (pSB-SFB-GFP), we performed Tandem Affinity Purification and identified that NFATc1 is a novel binding partner of FBW7. We also knocked out RCC2 and BRD7 using pSB-CRISPR vector respectively, and revealed the novel roles of these two proteins in mitosis. CONCLUSION: Our study demonstrated that the pSB series vectors are convenient and powerful tools for gene overexpression and knockout in mammalian cells, providing a new alternative approach for molecular cell biology research.