VSTM2A reverses immunosuppression in colorectal cancer by antagonizing the PD-L1/PD-1 interaction.

Immunoglobulin (Ig) VSTM2A (V-set and transmembrane domain containing 2A) is a top-ranked secretory protein frequently silenced during colorectal carcinogenesis; however, its role in immune modulation remains largely unknown. Bioinformatic and immunohistochemistry analysis of human colorectal specimens and Vstm2a+/- knockout mice indicated that VSTM2A positively correlated with CD8a and immune infiltration in both physiological and pathological conditions. We then utilized liquid chromatography-mass spectrometry to pinpoint programmed death ligand 1 (PD-L1) as a membrane receptor of VSTM2A. A series of in vitro biochemistry assays further revealed the binding pattern and kinetics between VSTM2A and PD-L1 proteins through their IgV domains at a dissociation constant of 0.7-2.5 nM. Recombinant VSTM2A protein inhibited the PD-1/PD-L1 interaction and induced NFAT response element (RE) luciferase activity dose dependently. Furthermore, interleukin (IL)-2 production from DO11.10 T cells upon co-culture with mouse non-T splenocytes was upregulated in the presence of VSTM2A conditioned medium. Finally, tumor killing assay and ex vivo data from human peripheral blood mononuclear cells and autologous dendritic cell-T cell co-culture demonstrated that VSTM2A significantly enhanced immune activation via the release of granzyme B and interferon (IFN)-γ cytokines. In conclusion, our study demonstrates the tumor-extrinsic role of VSTM2A in sterically blocking the PD-L1/PD-1 interaction at a picomole to nanomole affinity, which leads to the enhanced anti-tumor effect of cytotoxic T cells.

RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis.

The N6-methyladenosine (m6A) RNA-binding protein YTHDF1 is frequently overexpressed in colorectal cancer and drives chemotherapeutic resistance. To systematically identify druggable targets in colorectal cancer with high expression of YTHDF1, this study used a CRISPR/Cas9 screening strategy that revealed RUVBL1 and RUVBL2 as putative targets. RUVBL1/2 were overexpressed in primary colorectal cancer samples and represented independent predictors of poor patient prognosis. Functionally, loss of RUVBL1/2 preferentially impaired the growth of YTHDF1-high colorectal cancer cells, patient-derived primary colorectal cancer organoids, and subcutaneous xenografts. Mechanistically, YTHFD1 and RUVBL1/2 formed a positive feedforward circuit to accelerate oncogenic translation. YTHDF1 bound to m6A-modified RUVBL1/2 mRNA to promote translation initiation and protein expression. Coimmunoprecipitation and mass spectrometry identified that RUVBL1/2 reciprocally interacted with YTHDF1 at 40S translation initiation complexes. Consequently, RUVBL1/2 depletion stalled YTHDF1-driven oncogenic translation and nascent protein biosynthesis, leading to proliferative arrest and apoptosis. Ribosome sequencing revealed that RUVBL1/2 loss impaired the activation of MAPK, RAS, and PI3K-AKT signaling induced by YTHDF1. Finally, the blockade of RUVBL1/2 by the pharmacological inhibitor CB6644 or vesicle-like nanoparticle-encapsulated siRNAs preferentially arrested the growth of YTHDF1-expressing colorectal cancer in vitro and in vivo. Our findings show that RUVBL1/2 are potential prognostic markers and druggable targets that regulate protein translation in YTHDF1-high colorectal cancer. Significance: RUVBL1/2 inhibition is a therapeutic strategy to abrogate YTHDF1-driven oncogenic translation and overcome m6A dysregulation in colorectal cancer.

High Soluble Fiber Promotes Colorectal Tumorigenesis Through Modulating Gut Microbiota and Metabolites in Mice.

BACKGROUND & AIMS: Dietary fibers are mainly fermented by the gut microbiota, but their roles in colorectal cancer (CRC) are largely unclear. Here, we investigated the associations of different fibers with colorectal tumorigenesis in mice. METHODS: Apcmin/+ mice and C57BL/6 mice with azoxymethane (AOM) injection were used as CRC mouse models. Mice were fed with mixed high-fiber diet (20% soluble fiber and 20% insoluble fiber), high-inulin diet, high-guar gum diet, high-cellulose diet, or diets with different inulin dose. Germ-free mice were used for validation. Fecal microbiota and metabolites were profiled by shotgun metagenomic sequencing and liquid chromatography-mass spectrometry, respectively. RESULTS: Mixed high-fiber diet promoted colorectal tumorigenesis with increased tumor number and tumor load in AOM-treated and Apcmin/+ mice. Antibiotics use abolished the pro-tumorigenic effect of mixed high-fiber diet, while transplanting stools from mice fed with mixed high-fiber diet accelerated tumor growth in AOM-treated germ-free mice. We therefore characterized the contribution of soluble and insoluble fiber in CRC separately. Our results revealed that soluble fiber inulin or guar gum, but not insoluble fiber cellulose, promoted colorectal tumorigenesis in AOM-treated and Apcmin/+ mice. Soluble fiber induced gut dysbiosis with Bacteroides uniformis enrichment and Bifidobacterium pseudolongum depletion, accompanied by increased fecal butyrate and serum bile acids and decreased inosine. We also identified a positive correlation between inulin dosage and colorectal tumorigenesis. Moreover, transplanting stools from mice fed with high-inulin diet increased colonic cell proliferation and oncogene expressions in germ-free mice. CONCLUSION: High-dose soluble but not insoluble fiber potentiates colorectal tumorigenesis in a dose-dependent manner by dysregulating gut microbiota and metabolites in mice.

The molecular classification of cancer-associated fibroblasts on a pan-cancer single-cell transcriptional atlas.

BACKGROUND: Cancer-associated fibroblasts (CAFs), integral to the tumour microenvironment, are pivotal in cancer progression, exhibiting either pro-tumourigenic or anti-tumourigenic functions. Their inherent phenotypic and functional diversity allows for the subdivision of CAFs into various subpopulations. While several classification systems have been suggested for different cancer types, a unified molecular classification of CAFs on a single-cell pan-cancer scale has yet to be established. METHODS: We employed a comprehensive single-cell transcriptomic atlas encompassing 12 solid tumour types. Our objective was to establish a novel molecular classification and to elucidate the evolutionary trajectories of CAFs. We investigated the functional profiles of each CAF subtype using Single-Cell Regulatory Network Inference and Clustering and single-cell gene set enrichment analysis. The clinical relevance of these subtypes was assessed through survival curve analysis. Concurrently, we employed multiplex immunofluorescence staining on tumour tissues to determine the dynamic changes of CAF subtypes across different tumour stages. Additionally, we identified the small molecule procyanidin C1 (PCC1) as a target for matrix-producing CAF (matCAF) using molecular docking techniques and further validated these findings through in vitro and in vivo experiments. RESULTS: In our investigation of solid tumours, we identified four molecular clusters of CAFs: progenitor CAF (proCAF), inflammatory CAF (iCAF), myofibroblastic CAF (myCAF) and matCAF, each characterised by distinct molecular traits. This classification was consistently applicable across all nine studied solid tumour types. These CAF subtypes displayed unique evolutionary pathways, functional roles and clinical relevance in various solid tumours. Notably, the matCAF subtype was associated with poorer prognoses in several cancer types. The targeting of matCAF using the identified small molecule, PCC1, demonstrated promising antitumour activity. CONCLUSIONS: Collectively, the various subtypes of CAFs, particularly matCAF, are crucial in the initiation and progression of cancer. Focusing therapeutic strategies on targeting matCAF in solid tumours holds significant potential for cancer treatment.

H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling.

BACKGROUND: Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated. METHODS: The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC. RESULTS: Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis. CONCLUSION: This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.

Targeting the Hippo Pathway in Gastric Cancer and Other Malignancies in the Digestive System: From Bench to Bedside.

The Hippo pathway is an evolutionally conserved signaling cascade that controls organ size and tissue regeneration under physiological conditions, and its aberrations have been well studied to promote tumor initiation and progression. Dysregulation of the Hippo tumor suppressor signaling frequently occurs in gastric cancer (GC) and other solid tumors and contributes to cancer development through modulating multiple aspects, including cell proliferation, survival, metastasis, and oncotherapy resistance. In the clinic, Hippo components also possess diagnostic and prognostic values for cancer patients. Considering its crucial role in driving tumorigenesis, targeting the Hippo pathway may greatly benefit developing novel cancer therapies. This review summarizes the current research progress regarding the core components and regulation of the Hippo pathway, as well as the mechanism and functional roles of their dysregulation in gastrointestinal malignancies, especially in GC, and discusses the therapeutic potential of targeting the Hippo pathway against cancers.

MCM6 is a critical transcriptional target of YAP to promote gastric tumorigenesis and serves as a therapeutic target.

Rationale: Hyperactivation of Hippo-Yes-associated protein (YAP) signaling pathway governs tumorigenesis of gastric cancer (GC). Here we reveal that minichromosome maintenance complex component 6 (MCM6) is a critical transcriptional target of YAP in GC. We aim to investigate the function, mechanism of action, and clinical implication of MCM6 in GC. Methods: The downstream targets of YAP were screened by RNA sequencing (RNA-seq) and microarray, and further validated by chromatin immunoprecipitation PCR and luciferase reporter assays. The clinical implication of MCM6 was assessed in multiple GC cohorts. Biological function of MCM6 was evaluated in vitro, in patient-derived organoids, and in vivo. RNA-seq was performed to unravel downstream signaling of MCM6. Potential MCM6 inhibitor was identified and the effect of MCM6 inhibition on GC growth was evaluated. Results: Integrative RNA sequencing and microarray analyses revealed MCM6 as a potential YAP downstream target in GC. The YAP-TEAD complex bound to the promoter of MCM6 to induce its transcription. Increased MCM6 expression was commonly observed in human GC tissues and predicted poor patients survival. MCM6 knockdown suppressed proliferation and migration of GC cells and patient-derived organoids, and attenuated xenograft growth and peritoneal metastasis in mice. Mechanistically, MCM6 activated PI3K/Akt/GSK3ß signaling to support YAP-potentiated gastric tumorigenicity and metastasis. Furthermore, MCM6 deficiency sensitized GC cells to chemo- or radiotherapy by causing DNA breaks and blocking ATR/Chk1-mediated DNA damage response (DDR), leading to exacerbated cell death and tumor regression. As there are no available MCM6 inhibitors, we performed high-throughput virtual screening and identified purpureaside C as a novel MCM6 inhibitor. Purpureaside C not only suppressed GC growth but also synergized with 5-fluorouracil to induce cell death. Conclusions: Hyperactivated YAP in GC induces MCM6 transcription via binding to its promoter. YAP-MCM6 axis facilitates GC progression by inducing PI3K/Akt signaling. Targeting MCM6 suppresses GC growth and sensitizes GC cells to genotoxic agents by modulating ATR/Chk1-dependent DDR, providing a promising strategy for GC treatment.

Fangchinoline inhibits non-small cell lung cancer metastasis by reversing epithelial-mesenchymal transition and suppressing the cytosolic ROS-related Akt-mTOR signaling pathway.

Few drugs alleviate non-small cell lung cancer (NSCLC) metastasis effectively. Small molecular screening demonstrated that fangchinoline (Fan) reversed epithelial-mesenchymal transition (EMT) in NSCLC cells, inhibiting cell invasion and migration. RNA sequencing (RNA-seq) of Fan-treated NSCLC cells revealed that Fan potently quenched the NADP+ metabolic process. Molecular docking analysis revealed that Fan directly and specifically targeted NOX4. NOX4 was associated with poor prognosis in NSCLC in both The Cancer Genome Atlas (TCGA) and Hong Kong cohorts. In mitochondrial DNA-depleted ρ0 NSCLC cells, Fan decreased cytosolic reactive oxygen species (ROS) to inhibit the Akt-mTOR signaling pathway by directly promoting NOX4 degradation. In TCGA and Hong Kong cohorts, NOX4 upregulation acted as a driver event as it positively correlated with metastasis and oxidative stress. Single-cell RNA-seq indicated that NOX4 was overexpressed, especially in cancer cells, cancer stem cells, and endothelial cells. In mice, Fan significantly impeded subcutaneous xenograft formation and reduced metastatic nodule numbers in mouse lung and liver. Drug sensitivity testing demonstrated that Fan suppressed patient-derived organoid growth dose-dependently. Fan is a potent small molecule for alleviating NSCLC metastasis by directly targeting NOX4 and is a potential novel therapeutic agent.

FGF18, a prominent player in FGF signaling, promotes gastric tumorigenesis through autocrine manner and is negatively regulated by miR-590-5p.

Fibroblast growth factors (FGFs) and their receptors are significant components during fundamental cellular processes. FGF18 plays a distinctive role in modulating the activity of both tumor cells and tumor microenvironment. This study aims to comprehensively investigate the expression and functional role of FGF18 in gastric cancer (GC) and elucidate its regulatory mechanisms. The upregulation of FGF18 was detected in seven out of eleven (63.6%) GC cell lines. In primary GC samples, FGF18 was overexpressed in genomically stable and chromosomal instability subtypes of GC and its overexpression was associated with poor survival. Knocking down FGF18 inhibited tumor formation abilities, induced G1 phase cell cycle arrest and enhanced anti-cancer drug sensitivity. Expression microarray profiling revealed that silencing of FGF18 activated ATM pathway but quenched TGF-ß pathway. The key factors that altered in the related signaling were validated by western blot and immunofluorescence. Meanwhile, treating GC cells with human recombinant FGF18 or FGF18-conditioned medium accelerated tumor growth through activation of ERK-MAPK signaling. FGF18 was further confirmed to be a direct target of tumor suppressor, miR-590-5p. Their expressions showed a negative correlation in primary GC samples and more importantly, re-overexpression of FGF18 partly abolished the tumor-suppressive effect of miR-590-5p. Our study not only identified that FGF18 serves as a novel prognostic marker and a therapeutic target in GC but also enriched the knowledge of FGF-FGFR signaling during gastric tumorigenesis.

miR-375 is involved in Hippo pathway by targeting YAP1/TEAD4-CTGF axis in gastric carcinogenesis.

miR-375 is a tumor-suppressive microRNA (miRNA) in gastric cancer (GC). However, its molecular mechanism remains unclear. The aim of this study is to comprehensively investigate how miR-375 is involved in Hippo pathway by targeting multiple oncogenes. miR-375 expression in gastric cancer cell lines and primary GC was investigated by qRT-PCR. The regulation of YAP1, TEAD4, and CTGF expression by miR-375 was evaluated by qRT-PCR, western blot, and luciferase reporter assays, respectively. The functional roles of the related genes were examined by siRNA-mediated knockdown or ectopic expression assays. The clinical significance and expression correlation analysis of miR-375, YAP1, and CTGF were performed in primary GCs. TCGA cohort was also used to analyze the expression correlation of YAP1, TEAD4, CTGF, and miR-375 in primary GCs. miR-375 was down-regulated in GC due to promoter methylation and histone deacetylation. miR-375 downregulation was associated with unfavorable outcome and lymph node metastasis. Ectopic expression of miR-375 inhibited tumor growth in vitro and in vivo. Three components of Hippo pathway, YAP1, TEAD4 and CTGF, were revealed to be direct targets of miR-375. The expression of three genes showed a negative correlation with miR-375 expression and YAP1 re-expression partly abolished the tumor-suppressive effect of miR-375. Furthermore, CTGF was confirmed to be the key downstream of Hippo-YAP1 cascade and its knockdown phenocopied siYAP1 or miR-375 overexpression. YAP1 nuclear accumulation was positively correlated with CTGF cytoplasmic expression in primary GC tissues. Verteporfin exerted an anti-oncogenic effect in GC cell lines by quenching CTGF expression through YAP1 degradation. In short, miR-375 was involved in the Hippo pathway by targeting YAP1-TEAD4-CTGF axis and enriched our knowledge on the miRNA dysregulation in gastric tumorigenesis.

SRGAP1, a crucial target of miR-340 and miR-124, functions as a potential oncogene in gastric tumorigenesis.

Slit-Robo GTPase-activating protein 1 (SRGAP1) functions as a GAP for Rho-family GTPases and downstream of Slit-Robo signaling. We aim to investigate the biological function of SRGAP1 and reveal its regulation by deregulated microRNAs (miRNAs) in gastric cancer (GC). mRNA and protein expression of SRGAP1 were examined by quantitative reverse transcription PCR (qRT-PCR) and western blot. The biological role of SRGAP1 was demonstrated through siRNA-mediated knockdown experiments. The regulation of SRGAP1 by miR-340 and miR-124 was confirmed by western blot, dual luciferase activity assays and rescue experiments. SRGAP1 is overexpressed in 9 out of 12 (75.0%) GC cell lines. In primary GC samples from TCGA cohort, SRGAP1 shows gene amplification in 5/258 (1.9%) of cases and its mRNA expression demonstrates a positive correlation with copy number gain. Knockdown of SRGAP1 in GC cells suppressed cell proliferation, reduced colony formation, and significantly inhibited cell invasion and migration. Luciferase reporter assays revealed that SRGAP1 knockdown significantly inhibited Wnt/ß-catenin pathway. In addition, SRGAP1 was found to be a direct target of two tumor-suppressive miRNAs, miR-340 and miR-124. Concordantly, these two miRNAs were downregulated in primary gastric tumors and these decreasing levels w5ere associated with poor outcomes. Expression of miR-340 and SRGAP1 displayed a reverse relationship in primary samples and re-expressed SRGAP1, rescued the anti-cancer effects of miR-340. Taken together, these data strongly suggest that, apart from gene amplification and mutation, the activation of SRGAP1 in GC is partly due to the downregulation of tumor-suppressive miRNAs, miR-340 and miR-124. Thus SRGAP1 is overexpressed in gastric carcinogenesis and plays an oncogenic role through activating Wnt/ß-catenin pathway.

Preclinical analysis of the anti-tumor and anti-metastatic effects of Raf265 on colon cancer cells and CD26(+) cancer stem cells in colorectal carcinoma.

BACKGROUND: In colorectal carcinoma (CRC), activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition, the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore, we hypothesized that an ATP-competitive pan-Raf inhibitor, Raf265, is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS: HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS: Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition, anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally, the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS: This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC, providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC.