Identification of New Pathogenic Variants of Hereditary Diffuse Gastric Cancer.

Purpose: Hereditary diffuse gastric cancer (HDGC) presents a significant genetic predisposition, notably linked to mutations in the CDH1 and CTNNA1. However, the genetic basis for over half of HDGC cases remains unidentified. The aim of this study is to identify novel pathogenic variants in HDGC and evaluate their protein expression. Materials and Methods: Among 20 qualifying families, two were selected based on available pedigree and DNA. Whole genome sequencing (WGS) on DNA extracted from blood and whole exome sequencing (WES) on DNA from formalin-fixed paraffin-embedded tissues were performed to find potential pathogenic variants in HDGC. After selection of a candidate variant, functional validation and enrichment analysis were performed. Results: As a result of WGS, three candidate germline mutations (EPHA5, MCOA2, and RHOA) were identified in one family. After literature review and in silico analyses, the RHOA mutation (R129W) was selected as a candidate. This mutation was found in two gastric cancer patients within the family. In functional validation, it showed RhoA overexpression and a higher GTP-bound state in the RhoaR129W mutant. Decreased phosphorylation at Ser127/397 suggested altered YAP1 regulation in the Rho-ROCK pathway. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses linked RhoAR129W overexpression to changed migration/adhesion in MKN1 cell line. However, this RHOA mutation (R129W) was not found in index patients in other families. Conclusion: The RHOA mutation (R129W) emerges as a potential causative gene for HDGC, but only in one family, indicating a need for further studies to understand its role in HDGC pathogenesis fully.

The Effect of Clostridium butyricum on Gut Microbial Changes and Functional Profiles of Metabolism in High-fat Diet-fed Rats Depending on Age and Sex.

Background/Aims: A high-fat diet (HFD) causes dysbiosis and promotes inflammatory responses in the colon. This study aims to evaluate the effects of Clostridium butyricum on HFD-induced gut microbial changes in rats. Methods: Six-week-old Fischer-344 rats with both sexes were given a control or HFD during 8 weeks, and 1-to-100-fold diluted Clostridium butyricum were administered by gavage. Fecal microbiota analyses were conducted using 16S ribosomal RNA metagenomic sequencing and predictive functional profiling of microbial communities in metabolism. Results: A significant increase in Ruminococcaceae and Lachnospiraceae, which are butyric acid-producing bacterial families, was observed in the probiotics groups depending on sex. In contrast, Akkermansia muciniphila, which increased through a HFD regardless of sex, and decreased in the probiotics groups. A. muciniphila positively correlated with Claudin-1 expression in males (P < 0.001) and negatively correlated with the expression of Claudin-2 (P = 0.042), IL-1ß (P = 0.037), and IL-6 (P = 0.044) in females. In terms of functional analyses, a HFD decreased the relative abundances of M00131 (carbohydrate metabolism module), M00579, and M00608 (energy metabolism), and increased those of M00307 (carbohydrate metabolism), regardless of sex. However, these changes recovered especially in male C. butyricum groups. Furthermore, M00131, M00579, and M00608 showed a positive correlation and M00307 showed a negative correlation with the relative abundance of A. muciniphila (P < 0.001). Conclusion: The beneficial effects of C. butyricum on HFD-induced gut dysbiosis in young male rats originate from the functional profiles of carbohydrate and energy metabolism.

Targeting the m6A RNA methyltransferase METTL3 attenuates the development of kidney fibrosis.

Kidney fibrosis is a major mechanism underlying chronic kidney disease (CKD). N6-methyladenosine (m6A) RNA methylation is associated with organ fibrosis. We investigated m6A profile alterations and the inhibitory effect of RNA methylation in kidney fibrosis in vitro (TGF-ß-treated HK-2 cells) and in vivo (unilateral ureteral obstruction [UUO] mouse model). METTL3-mediated signaling was inhibited using siRNA in vitro or the METTL3-specific inhibitor STM2457 in vivo and in vitro. In HK-2 cells, METTL3 protein levels increased in a dose- and time-dependent manner along with an increase in the cellular m6A levels. In the UUO model, METTL3 expression and m6A levels were significantly increased. Transcriptomic and m6A profiling demonstrated that epithelial-to-mesenchymal transition- and inflammation-related pathways were significantly associated with RNA m6A methylation. Genetic and pharmacologic inhibition of METTL3 in HK-2 cells decreased TGF-ß-induced fibrotic marker expression. STM2457-induced inhibition of METTL3 attenuated the degree of kidney fibrosis in vivo. Furthermore, METTL3 protein expression was significantly increased in the tissues of CKD patients with diabetic or IgA nephropathy. Therefore, targeting alterations in RNA methylation could be a potential therapeutic strategy for treating kidney fibrosis.

Graphislactone A, a Fungal Antioxidant Metabolite, Reduces Lipogenesis and Protects against Diet-Induced Hepatic Steatosis in Mice.

Graphislactone A (GPA), a secondary metabolite derived from a mycobiont found in the lichens of the genus Graphis, exhibits antioxidant properties. However, the potential biological functions and therapeutic applications of GPA at the cellular and animal levels have not yet been investigated. In the present study, we explored the therapeutic potential of GPA in mitigating non-alcoholic fatty liver disease (NAFLD) and its underlying mechanisms through a series of experiments using various cell lines and animal models. GPA demonstrated antioxidant capacity on a par with that of vitamin C in cultured hepatocytes and reduced the inflammatory response induced by lipopolysaccharide in primary macrophages. However, in animal studies using an NAFLD mouse model, GPA had a milder impact on liver inflammation while markedly attenuating hepatic steatosis. This effect was confirmed in an animal model of early fatty liver disease without inflammation. Mechanistically, GPA inhibited lipogenesis rather than fat oxidation in cultured hepatocytes. Similarly, RNA sequencing data revealed intriguing associations between GPA and the adipogenic pathways during adipocyte differentiation. GPA effectively reduced lipid accumulation and suppressed lipogenic gene expression in AML12 hepatocytes and 3T3-L1 adipocytes. In summary, our study demonstrates the potential application of GPA to protect against hepatic steatosis in vivo and suggests a novel role for GPA as an underlying mechanism in lipogenesis, paving the way for future exploration of its therapeutic potential.

Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability.

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.

Transglutaminase 2 Prevents Premature Senescence and Promotes Osteoblastic Differentiation of Mesenchymal Stem Cells through NRF2 Activation.

Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, kinase, and protein disulfide isomerase (PDI) activities. Of these, transamidase-mediated modification of proteins regulates apoptosis, differentiation, inflammation, and fibrosis. TG2 is highly expressed in mesenchymal stem cells (MSCs) compared with differentiated cells, suggesting a role of TG2 specific for MSC characteristics. In this study, we report a new function of TG2 in the regulation of MSC redox homeostasis. During in vitro MSC expansion, TG2 is required for cell proliferation and self-renewal by preventing premature senescence but has no effect on the expression of surface antigens and oxidative stress-induced cell death. Moreover, induction of differentiation upregulates TG2 that promotes osteoblastic differentiation. Molecular analyses revealed that TG2 mediates tert-butylhydroquinone, but not sulforaphane, -induced nuclear factor erythroid 2-related factor 2 (NRF2) activation in a transamidase activity-independent manner. Differences in the mechanism of action between two NRF2 activators suggest that PDI activity of TG2 may be implicated in the stabilization of NRF2. The role of TG2 in the regulation of antioxidant response was further supported by transcriptomic analysis of MSC. These results indicate that TG2 is a critical enzyme in eliciting antioxidant response in MSC through NRF2 activation, providing a target for optimizing MSC manufacturing processes to prevent premature senescence.

METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m6A modifications and splicing factors.

N6-methyladenosine (m6A) modification in RNA affects various aspects of RNA metabolism and regulates gene expression. This modification is modulated by many regulatory proteins, such as m6A methyltransferases (writers), m6A demethylases (erasers), and m6A-binding proteins (readers). Previous studies have suggested that alterations in m6A regulatory proteins induce genome-wide alternative splicing in many cancer cells. However, the functional effects and molecular mechanisms of m6A-mediated alternative splicing have not been fully elucidated. To understand the consequences of this modification on RNA splicing in cancer cells, we performed RNA sequencing and analyzed alternative splicing patterns in METTL3-knockdown osteosarcoma U2OS cells. We detected 1,803 alternatively spliced genes in METTL3-knockdown cells compared to the controls and found that cell cycle-related genes were enriched in differentially spliced genes. A comparison of the published MeRIP-seq data for METTL14 with our RNA sequencing data revealed that 70-87% of alternatively spliced genes had an m6A peak near 1 kb of alternative splicing sites. Among the 19 RNA-binding proteins enriched in alternative splicing sites, as revealed by motif analysis, expression of SFPQ highly correlated with METTL3 expression in 12,839 TCGA pan-cancer patients. We also found that cell cycle-related genes were enriched in alternatively spliced genes of other cell lines with METTL3 knockdown. Taken together, we suggest that METTL3 regulates m6A-dependent alternative splicing, especially in cell cycle-related genes, by regulating the functions of splicing factors such as SFPQ.

Gut bacteria-derived 3-phenylpropionylglycine mitigates adipocyte differentiation of 3T3-L1 cells by inhibiting adiponectin-PPAR pathway.

BACKGROUND: Gut microbiota provide numerous types of metabolites that humans cannot produce and have a huge influence on the host metabolism. Accordingly, gut bacteria-derived metabolites can be employed as a resource to develop anti-obesity and metabolism-modulating drugs. OBJECTIVE: This study aimed to examine the anti-adipogenic effect of 3-phenylpropionylglycine (PPG), which is a glycine conjugate of bacteria-derived 3-phenylpropionic acid (PPA). METHODS: The effect of PPG on preadipocyte-to-adipocyte differentiation was evaluated in 3T3-L1 differentiation models and the degree of the differentiation was estimated by Oil red O staining. The molecular mechanisms of the PPG effect were investigated with transcriptome analyses using RNA-sequencing and quantitative real-time PCR. RESULTS: PPG suppressed lipid droplet accumulation during the adipogenic differentiation of 3T3-L1 cells, which is attributed to down-regulation of lipogenic genes such as acetyl CoA carboxylase 1 (Acc1) and fatty acid synthase (Fasn). However, other chemicals with chemical structures similar to PPG, including cinnamoylglycine and hippuric acid, had little effect on the lipid accumulation of 3T3-L1 cells. In transcriptomic analysis, PPG suppressed the expression of adipogenesis and metabolism-related gene sets, which is highly associated with downregulation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Protein-protein association network analysis suggested adiponectin as a hub gene in the network of genes that were differentially expressed genes in response to PPG treatment. CONCLUSION: PPG inhibits preadipocyte-to-adipocyte differentiation by suppressing the adiponectin-PPAR pathway. These data provide a potential candidate from bacteria-derived metabolites with anti-adipogenic effects.

Direct cell-to-cell transfer in stressed tumor microenvironment aggravates tumorigenic or metastatic potential in pancreatic cancer.

Pancreatic cancer exhibits a characteristic tumor microenvironment (TME) due to enhanced fibrosis and hypoxia and is particularly resistant to conventional chemotherapy. However, the molecular mechanisms underlying TME-associated treatment resistance in pancreatic cancer are not fully understood. Here, we developed an in vitro TME mimic system comprising pancreatic cancer cells, fibroblasts and immune cells, and a stress condition, including hypoxia and gemcitabine. Cells with high viability under stress showed evidence of increased direct cell-to-cell transfer of biomolecules. The resulting derivative cells (CD44high/SLC16A1high) were similar to cancer stem cell-like-cells (CSCs) with enhanced anchorage-independent growth or invasiveness and acquired metabolic reprogramming. Furthermore, CD24 was a determinant for transition between the tumorsphere formation or invasive properties. Pancreatic cancer patients with CD44low/SLC16A1low expression exhibited better prognoses compared to other groups. Our results suggest that crosstalk via direct cell-to-cell transfer of cellular components foster chemotherapy-induced tumor evolution and that targeting of CD44 and MCT1(encoded by SLC16A1) may be useful strategy to prevent recurrence of gemcitabine-exposed pancreatic cancers.

Augmentation of the RNA m6A reader signature is associated with poor survival by enhancing cell proliferation and EMT across cancer types.

N6-Methyladenosine (m6A) RNA modification plays a critical role in the posttranscriptional regulation of gene expression. Alterations in cellular m6A levels and m6A-related genes have been reported in many cancers, but whether they play oncogenic or tumor-suppressive roles is inconsistent across cancer types. We investigated common features of alterations in m6A modification and m6A-related genes during carcinogenesis by analyzing transcriptome data of 11 solid tumors from The Cancer Genome Atlas database and our in-house gastric cancer cohort. We calculated m6A writer (W), eraser (E), and reader (R) signatures based on corresponding gene expression. Alterations in the W and E signatures varied according to the cancer type, with a strong positive correlation between the W and E signatures in all types. When the patients were divided according to m6A levels estimated by the ratio of the W and E signatures, the prognostic effect of m6A was inconsistent according to the cancer type. The R and especially the R2 signatures (based on the expression of IGF2BPs) were upregulated in all cancers. Patients with a high R2 signature exhibited poor prognosis across types, which was attributed to enrichment of cell cycle- and epithelial-mesenchymal transition-related pathways. Our study demonstrates common features of m6A alterations across cancer types and suggests that targeting m6A R proteins is a promising strategy for cancer treatment.

Polo-Like Kinase 1 Regulates Chromosomal Instability and Paclitaxel Resistance in Breast Cancer Cells.

PURPOSE: Chromosomal instability (CIN) contributes to intercellular genetic heterogeneity and has been implicated in paclitaxel (PTX) resistance in breast cancer. In this study, we explored polo-like kinase 1 (PLK1) as an important regulator of mitotic integrity and as a useful predictive biomarker for PTX resistance in breast cancer. METHODS: We performed PTX resistance screening using the human kinome CRISPR/Cas9 library in breast cancer cells. In vitro cell proliferation and apoptosis assays and in vivo xenograft experiments were performed to determine the effects of PLK1 on breast cancer cells. Immunofluorescence microscopy was used to measure the degree of multipolar cell division. RESULTS: Kinome-wide CRISPR/Cas9 screening identified various kinases involved in PTX resistance in breast cancer cells; among these, PLK1 was chosen for further experiments. PLK1 knockdown inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro and in vivo. Moreover, PLK1 silencing sensitized breast cancer cells and mouse xenograft tumor models to PTX cytotoxicity. Silencing of PLK1 induced the formation of multipolar spindles and increased the percentage of multipolar cells. In addition, PLK1 silencing resulted in the downregulation of BubR1 and Mad2 in breast cancer cells. Furthermore, PLK1 upregulation in primary breast cancer was associated with decreased overall patient survival based on the analysis of The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium databases. CONCLUSION: PLK1 plays an important role in PTX resistance by regulating CIN in breast cancer cells. Targeting PLK1 may be an effective treatment strategy for PTX-resistant breast cancers.

Comparative analysis of cancer gene mutations using targeted sequencing in matched primary and recurrent gastric cancers after chemotherapy.

BACKGROUND: Investigation of responsiveness-associated genes using longitudinal mutation analyses after standard treatments in recurrent gastric cancer (GC) is limited. OBJECTIVE: To evaluate the somatic mutations associated with resistance to combined treatment involving fluorouracil (FU) or platinum (PL) in advanced GC. METHODS: Samples from patients with advanced GC treated with FU or PL alone, or surgery plus FU/PL, were studied. GC patients who relapsed after standard chemotherapy (FU/PL) and with presence of tumor samples from initial diagnosis and recurrence were included. Targeted sequencing analysis of 143 cancer-related genes was performed using an Oncomine Comprehensive Cancer Panel. RESULTS: Matched samples of primary and recurrent lesions were analyzed in sixteen patients with GC. When genes with recurrent mutations in two or more patients were used as specific findings, a total of 26 genes were found. TP53 was the most predominantly increased allele frequency (AF) in recurrent GCs after standard treatment. The mutational AF of ERBB2, PTEN, and BRCA2 also commonly increased, suggesting the role of these mutations in treatment resistance, whereas the mutational AF of VLH, NF1, and STK11 frequently decreased in recurrent tumors, suggesting the role of these mutations in increasing sensitivity to treatment. TCGA gastric cancer data (n = 436) were analyzed, and mutation sites detected in 16 GC patients in this study were in agreement with TCGA cohort with some exceptions. Overall survival according to gene expression associated with chemotherapy responsiveness exhibited compatible patterns with gain or loss-of-function mutations of each gene. CONCLUSIONS: Mutations in TP53, ERBB2, PTEN, BRCA2, VHL, NF1, and STK11 are candidate somatic alterations related to chemoresistance in GC.

Predictive biomarkers for 5-fluorouracil and oxaliplatin-based chemotherapy in gastric cancers via profiling of patient-derived xenografts.

Gastric cancer (GC) is commonly treated by chemotherapy using 5-fluorouracil (5-FU) derivatives and platinum combination, but predictive biomarker remains lacking. We develop patient-derived xenografts (PDXs) from 31 GC patients and treat with a combination of 5-FU and oxaliplatin, to determine biomarkers associated with responsiveness. When the PDXs are defined as either responders or non-responders according to tumor volume change after treatment, the responsiveness of PDXs is significantly consistent with the respective clinical outcomes of the patients. An integrative genomic and transcriptomic analysis of PDXs reveals that pathways associated with cell-to-cell and cell-to-extracellular matrix interactions enriched among the non-responders in both cancer cells and the tumor microenvironment (TME). We develop a 30-gene prediction model to determine the responsiveness to 5-FU and oxaliplatin-based chemotherapy and confirm the significant poor survival outcomes among cases classified as non-responder-like in three independent GC cohorts. Our study may inform clinical decision-making when designing treatment strategies.

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2.

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas-/-)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.

CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers.

Metastatic or recurrent colorectal cancer (CRC) patients require systemic chemotherapy, but the therapeutic options of targeted agents remain limited. CRC patients with KRAS or BRAF gene mutations exhibit a worse prognosis and are resistant to anti-EGFR treatment. Previous studies have shown that the expression of anti-apoptotic protein BCL-XL is increased in CRC patients with KRAS/BRAF mutations, suggesting BCL-XL as a therapeutic target for this subgroup. Here, we performed genome-wide CRISPR/Cas9 screens of cell lines with KRAS mutations to investigate the factors required for sensitivity to BCL-XL inhibitor ABT-263 using single-guide RNAs (sgRNAs) that induce loss-of-function mutations. In the presence of ABT-263, sgRNAs targeting negative regulators of WNT signaling (resulting in WNT activation) were enriched, whereas sgRNAs targeting positive regulators of WNT signaling (resulting in WNT inhibition) were depleted in ABT-263-resistant cells. The activation of WNT signaling was highly associated with an increased expression ratio of anti- to pro-apoptotic BCL-2 family genes in CRC samples. Genetic and pharmacologic inhibition of WNT signaling using ß-catenin short hairpin RNA or TNIK inhibitor NCB-0846, respectively, augmented ABT-263-induced cell death in KRAS/BRAF-mutated cells. Inhibition of WNT signaling resulted in transcriptional repression of the anti-apoptotic BCL-2 family member, MCL1, via the functional inhibition of the ß-catenin-containing complex at the MCL1 promoter. In addition, the combination of ABT-263 and NCB-0846 exhibited synergistic effects in in vivo patient-derived xenograft (PDX) models with KRAS mutations. Our data provide a novel targeted combination treatment strategy for the CRC patient subgroup with KRAS or BRAF mutations.

Transglutaminase 2 mediates transcriptional regulation through BAF250a polyamination.

BACKGROUND: Transglutaminase 2 (TG2) mediates protein modifications by crosslinking or by incorporating polyamine in response to oxidative or DNA-damaging stress, thereby regulating apoptosis, extracellular matrix formation, and inflammation. The regulation of transcriptional activity by TG2-mediated histone serotonylation or by Sp1 crosslinking may also contribute to cellular stress responses. OBJECTIVE: In this study, we attempted to identify TG2-interacting proteins to better understand the role of TG2 in transcriptional regulation. METHODS: Using a yeast two-hybrid assay to screen a HeLa cell cDNA library, we found that TG2 bound BAF250a, a core subunit of the cBAF chromatin remodeling complex, through an interaction between the TG2 barrel 1 and BAF250a C-terminal domains. RESULTS: TG2 was pulled down with a GST-BAF250a C-term fusion protein. Moreover, TG2 and BAF250a were co-fractionated using P11 chromatography, and co-immunoprecipitated. A transamidation reaction showed that TG2 mediated incorporation of polyamine into BAF250a. In glucocorticoid response-element reporter-expressing cells, TG2 overexpression increased the luciferase reporter activity in a transamidation-dependent manner. In addition, a comparison of genome-wide gene expression between wild-type and TG2-deficient primary hepatocytes in response to dexamethasone treatment showed that TG2 further enhanced or suppressed the expression of dexamethasone-regulated genes that were identified by a gene ontology enrichment analysis. CONCLUSION: Thus, our results indicate that TG2 regulates transcriptional activity through BAF250a polyamination.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Transglutaminase 2 crosslinks the glutathione S-transferase tag, impeding protein-protein interactions of the fused protein.

Glutathione S-transferase (GST) from Schistosoma japonicum has been widely used as a tag for affinity purification and pulldown of fusion proteins to detect protein-protein interactions. However, the reliability of this technique is undermined by the formation of GST-fused protein aggregates after incubation with cell lysates. It remains unknown why this aggregation occurs. Here, we demonstrate that the GST tag is a substrate of transglutaminase 2 (TG2), which is a calcium-dependent enzyme that polyaminates or crosslinks substrate proteins. Mutation analysis identified four glutamine residues in the GST tag as polyamination sites. TG2-mediated modification of the GST tag caused aggregate formation but did not affect its glutathione binding affinity. When incubated with cell lysates, GST tag aggregation was dependent on cellular TG2 expression levels. A GST mutant in which four glutamine residues were replaced with asparagine (GST4QN) exhibited a glutathione binding affinity similar to that of wild-type GST and could be purified by glutathione affinity chromatography. Moreover, the use of GST4QN as a tag reduced fused p53 aggregation and enhanced the induction of p21 transcription and apoptosis in cells treated with 5-fluorouracil (5-FU). These results indicated that TG2 interferes with the protein-protein interactions of GST-fused proteins by crosslinking the GST tag; therefore, a GST4QN tag could improve the reproducibility and reliability of GST pulldown experiments.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers.

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

Genome-scale CRISPR screening identifies cell cycle and protein ubiquitination processes as druggable targets for erlotinib-resistant lung cancer.

Erlotinib is highly effective in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. However, despite initial favorable responses, most patients rapidly develop resistance to erlotinib soon after the initial treatment. This study aims to identify new genes and pathways associated with erlotinib resistance mechanisms in order to develop novel therapeutic strategies. Here, we induced knockout (KO) mutations in erlotinib-resistant human lung cancer cells (NCI-H820) using a genome-scale CRISPR-Cas9 sgRNA library to screen for genes involved in erlotinib susceptibility. The spectrum of sgRNAs incorporated among erlotinib-treated cells was substantially different to that of the untreated cells. Gene set analyses showed a significant depletion of 'cell cycle process' and 'protein ubiquitination pathway' genes among erlotinib-treated cells. Chemical inhibitors targeting genes in these two pathways, such as nutlin-3 and carfilzomib, increased cancer cell death when combined with erlotinib in both in vitro cell line and in vivo patient-derived xenograft experiments. Therefore, we propose that targeting cell cycle processes or protein ubiquitination pathways are promising treatment strategies for overcoming resistance to EGFR inhibitors in lung cancer.