Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/ß-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

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.

Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis.

Massive expansion of immature and suppressive myeloid cells is a common feature of malignant solid tumors. Over-expression of cyclin-dependent kinase 20, also known as cell cycle-related kinase (CCRK), in hepatocellular carcinoma (HCC) correlates with reduced patient survival and low immunotherapy responsiveness. Beyond tumor-intrinsic oncogenicity, here we demonstrated that CCRK is upregulated in myeloid cells in tumor-bearing mice and in patients with HCC. Intratumoral injection of Ccrk-knockdown myeloid-derived suppressor cells (MDSCs) increased tumor-infiltrating CD8+T cells and suppressed HCC tumorigenicity. Using an indel mutant transgenic model, we showed that Ccrk inactivation in myeloid cells conferred a mature phenotype with elevated IL-12 production, driving Th1 responses and CD8+T cell cytotoxicity to reduce orthotopic tumor growth and prolong survival. Mechanistically, CCRK activates STAT3/E4BP4 signaling in MDSCs to acquire immunosuppressive activity through transcriptional IL-10 induction and IL-12 suppression. Taken together, our findings unravel mechanistic insights into MDSC-mediated immunosuppression and offer a therapeutic kinase-target for cancer immunotherapy.

Cold-Induced Reprogramming of Subcutaneous White Adipose Tissue Assessed by Single-Cell and Single-Nucleus RNA Sequencing.

Adipose browning has demonstrated therapeutic potentials in several diseases. Here, by conducting transcriptomic profiling at the single-cell and single-nucleus resolution, we reconstituted the cellular atlas in mouse inguinal subcutaneous white adipose tissue (iWAT) at thermoneutrality or chronic cold condition. All major nonimmune cells within the iWAT, including adipose stem and progenitor cells (ASPCs), mature adipocytes, endothelial cells, Schwann cells, and smooth muscle cells, were recovered, allowing us to uncover an overall and detailed blueprint for transcriptomes and intercellular cross-talks and the dynamics during white adipose tissue brown remodeling. Our findings also unravel the existence of subpopulations in mature adipocytes, ASPCs, and endothelial cells, as well as new insights on their interconversion and reprogramming in response to cold. The adipocyte subpopulation competent of major histocompatibility complex class II (MHCII) antigen presentation is potentiated. Furthermore, a subcluster of ASPC with CD74 expression was identified as the precursor of this MHCII+ adipocyte. Beige adipocytes are transdifferented from preexisting lipid generating adipocytes, which exhibit developmental trajectory from de novo differentiation of amphiregulin cells (Aregs). Two distinct immune-like endothelial subpopulations are present in iWAT and are responsive to cold. Our data reveal fundamental changes during cold-evoked adipose browning.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

Neuropathological signatures revealed by transcriptomic and proteomic analysis in Pten-deficient mouse models.

PTEN hamartoma tumour syndrome is characterised by mutations in the human PTEN gene. We performed transcriptomic and proteomic analyses of neural tissues and primary cultures from heterozygous and homozygous Pten-knockout mice. The somatosensory cortex of heterozygous Pten-knockout mice was enriched in immune response and oligodendrocyte development Gene Ontology (GO) terms. Parallel proteomic analysis revealed differentially expressed proteins (DEPs) related to dendritic spine development, keratinisation and hamartoma signatures. However, primary astrocytes (ASTs) from heterozygous Pten-knockout mice were enriched in the extracellular matrix GO term, while primary cortical neurons (PCNs) were enriched in immediate-early genes. In ASTs from homozygous Pten-knockout mice, cilium-related activity was enriched, while PCNs exhibited downregulation of forebrain neuron generation and differentiation, implying an altered excitatory/inhibitory balance. By integrating DEPs with pre-filtered differentially expressed genes, we identified the enrichment of traits of intelligence, cognitive function and schizophrenia, while DEPs in ASTs were significantly associated with intelligence and depression.

Targeting PPAR-gamma counteracts tumour adaptation to immune-checkpoint blockade in hepatocellular carcinoma.

OBJECTIVE: Therapy-induced tumour microenvironment (TME) remodelling poses a major hurdle for cancer cure. As the majority of patients with hepatocellular carcinoma (HCC) exhibits primary or acquired resistance to antiprogrammed cell death (ligand)-1 (anti-PD-[L]1) therapies, we aimed to investigate the mechanisms underlying tumour adaptation to immune-checkpoint targeting. DESIGN: Two immunotherapy-resistant HCC models were generated by serial orthotopic implantation of HCC cells through anti-PD-L1-treated syngeneic, immunocompetent mice and interrogated by single-cell RNA sequencing (scRNA-seq), genomic and immune profiling. Key signalling pathway was investigated by lentiviral-mediated knockdown and pharmacological inhibition, and further verified by scRNA-seq analysis of HCC tumour biopsies from a phase II trial of pembrolizumab (NCT03419481). RESULTS: Anti-PD-L1-resistant tumours grew >10-fold larger than parental tumours in immunocompetent but not immunocompromised mice without overt genetic changes, which were accompanied by intratumoral accumulation of myeloid-derived suppressor cells (MDSC), cytotoxic to exhausted CD8+ T cell conversion and exclusion. Mechanistically, tumour cell-intrinsic upregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) transcriptionally activated vascular endothelial growth factor-A (VEGF-A) production to drive MDSC expansion and CD8+ T cell dysfunction. A selective PPARγ antagonist triggered an immune suppressive-to-stimulatory TME conversion and resensitised tumours to anti-PD-L1 therapy in orthotopic and spontaneous HCC models. Importantly, 40% (6/15) of patients with HCC resistant to pembrolizumab exhibited tumorous PPARγ induction. Moreover, higher baseline PPARγ expression was associated with poorer survival of anti-PD-(L)1-treated patients in multiple cancer types. CONCLUSION: We uncover an adaptive transcriptional programme by which tumour cells evade immune-checkpoint targeting via PPARγ/VEGF-A-mediated TME immunosuppression, thus providing a strategy for counteracting immunotherapeutic resistance in HCC.

Compromised browning in white adipose tissue of ageing people.

BACKGROUND: Adipose tissue plays a pivotal role in the pathology of metabolic disorders. In the past decade, brown and brown-like adipose tissues were detected in adult humans and show therapeutic potential in ageing-related metabolic diseases. OBJECTIVE: This study investigated expressions of major brown adipose markers in white adipose tissue (WAT) of different ages. Their associations with metabolic parameters and key adipokines were interrogated. DESIGN: Cross-sectional study, 2019-2021. METHODS: We recruited 21 young, 67 middle-aged, and 34 older patients. Omental adipose tissues were collected, and expressions of key brown markers and adipokines and the adipocyte size were evaluated. The fat depot distribution was evaluated by computed tomography. RESULTS: UCP1 and PRDM16 mRNA expressions declined with ageing in WAT and were more associated with age, than with the body mass index (BMI). The increased visceral adipose tissue (VAT) amount, as well as the VAT to subcutaneous adipose tissue (SAT) ratio, was decreased in the highest tertile of UCP1 expression, while individuals in different PRDM16 mRNA tertiles exhibited similar fat distribution. UCP1 mRNA was positively correlated with ADIPOQ and the strength of the correlation declined with ageing. In contrast, the association between UCP1 and LEP was insignificant in young and middle-aged groups but became significantly correlated in the older-people group. We also found a positive correlation between UCP1 and PRDM16. CONCLUSIONS: PRDM16 and UCP1, despite their key functions in adipose browning, exhibit differential clinical correlations with metabolic features in human WAT in an age-dependent manner. These two genes may participate in the pathogenesis of ageing-related metabolic diseases, but with distinct mechanisms.

Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

Isoformic PD-1-mediated immunosuppression underlies resistance to PD-1 blockade in hepatocellular carcinoma patients.

OBJECTIVE: Immune checkpoint blockade (ICB) has improved cancer treatment, yet why most hepatocellular carcinoma (HCC) patients are resistant to PD-1 ICB remains elusive. Here, we elucidated the role of a programmed cell death protein 1 (PD-1) isoform, Δ42PD-1, in HCC progression and resistance to nivolumab ICB. DESIGN: We investigated 74 HCC patients in three cohorts, including 41 untreated, 28 treated with nivolumab and 5 treated with pembrolizumab. Peripheral blood mononuclear cells from blood samples and tumour infiltrating lymphocytes from tumour tissues were isolated for immunophenotyping. The functional significance of Δ42PD-1 was explored by single-cell RNA sequencing analysis and validated by functional and mechanistic studies. The immunotherapeutic efficacy of Δ42PD-1 monoclonal antibody was determined in HCC humanised mouse models. RESULTS: We found distinct T cell subsets, which did not express PD-1 but expressed its isoform Δ42PD-1, accounting for up to 71% of cytotoxic T lymphocytes in untreated HCC patients. Δ42PD-1+ T cells were tumour-infiltrating and correlated positively with HCC severity. Moreover, they were more exhausted than PD-1+ T cells by single T cell and functional analysis. HCC patients treated with anti-PD-1 ICB showed effective PD-1 blockade but increased frequencies of Δ42PD-1+ T cells over time especially in patients with progressive disease. Tumour-infiltrated Δ42PD-1+ T cells likely sustained HCC through toll-like receptors-4-signalling for tumourigenesis. Anti-Δ42PD-1 antibody, but not nivolumab, inhibited tumour growth in three murine HCC models. CONCLUSION: Our findings not only revealed a mechanism underlying resistance to PD-1 ICB but also identified anti-Δ42PD-1 antibody for HCC immunotherapy.

Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation.

BACKGROUND AND AIMS: Radiofrequency ablation (RFA) is an important curative therapy in hepatocellular carcinoma (HCC), but recurrence rate remains as high as all the other HCC therapeutic modalities. Methyltransferase 1 (METTL1), an enzyme for m 7 G tRNA modification, was reported to promote HCC development. Here, we assessed the role of METTL1 in shaping the immunosuppressive tumor microenvironment after insufficient RFA (iRFA). APPROACH AND RESULTS: By immunohistochemistry and multiplex immunofluorescence (mIF) staining, we showed that METTL1 expression was enhanced in post-RFA recurrent HCC, accompanied by increased CD11b + CD15 + polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and decreased CD8 + T cells. Mechanistically, heat-mediated METTL1 upregulation enhanced TGF-ß2 translation to form the immunosuppressive environment by induction of myeloid-derived suppressor cell. Liver-specific overexpression or knockdown of Mettl1 significantly affected the accumulation of PMN-MDSCs and subsequently affected CD8 + T cell infiltration. Complete RFA successfully eliminated the tumor, whereas iRFA-treated mice exhibited enhanced tumor growth and metastasis with increased PMN-MDSC accumulation and decreased CD8 + T cells compared to sham surgery. Interrupting METTL1-TGF-ß2-PMN-MDSC axis by anti-Ly6G antibody, or knockdown of hepatoma-intrinsic Mettl1 or Tgfb2 , or TGF-ß signaling blockade significantly mitigated tumor progression induced by iRFA and restored CD8 + T cell population. CONCLUSIONS: Our study sheds light on the pivotal role of METTL1 in modulating an immunosuppressive microenvironment and demonstrated that interrupting METTL1-TGF-ß2-PMN-MDSC axis could be a therapeutic strategy to restore antitumor immunity and prevent HCC recurrence after RFA treatment, meriting further clinical studies.

Fibrotic immune microenvironment remodeling mediates superior anti-tumor efficacy of a nano-PD-L1 trap in hepatocellular carcinoma.

The local microenvironment where tumors develop can shape cancer progression and therapeutic outcome. Emerging evidence demonstrate that the efficacy of immune-checkpoint blockade (ICB) is undermined by fibrotic tumor microenvironment (TME). The majority of hepatocellular carcinoma (HCC) develops in liver fibrosis, in which the stromal and immune components may form a barricade against immunotherapy. Here, we report that nanodelivery of a programmed death-ligand 1 (PD-L1) trap gene exerts superior efficacy in treating fibrosis-associated HCC when compared with the conventional monoclonal antibody (mAb). In two fibrosis-associated HCC models induced by carbon tetrachloride and a high-fat, high-carbohydrate diet, the PD-L1 trap induced significantly larger tumor regression than mAb with no evidence of toxicity. Mechanistic studies revealed that PD-L1 trap, but not mAb, consistently reduced the M2 macrophage proportion in the fibrotic liver microenvironment and promoted cytotoxic interferon gamma (IFNγ)+tumor necrosis factor α (TNF-α)+CD8+T cell infiltration to the tumor. Moreover, PD-L1 trap treatment was associated with decreased tumor-infiltrating polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) accumulation, resulting in an inflamed TME with a high cytotoxic CD8+T cell/PMN-MDSC ratio conductive to anti-tumor immune response. Single-cell RNA sequencing analysis of two clinical cohorts demonstrated preferential PD-L1 expression in M2 macrophages in the fibrotic liver, thus supporting the translational potential of nano-PD-L1 trap for fibrotic HCC treatment.

Accurate identification of structural variations from cancer samples.

Structural variations (SVs) are commonly found in cancer genomes. They can cause gene amplification, deletion and fusion, among other functional consequences. With an average read length of hundreds of kilobases, nano-channel-based optical DNA mapping is powerful in detecting large SVs. However, existing SV calling methods are not tailored for cancer samples, which have special properties such as mixed cell types and sub-clones. Here we propose the Cancer Optical Mapping for detecting Structural Variations (COMSV) method that is specifically designed for cancer samples. It shows high sensitivity and specificity in benchmark comparisons. Applying to cancer cell lines and patient samples, COMSV identifies hundreds of novel SVs per sample.

Development of Alkylated Hydrazides as Highly Potent and Selective Class I Histone Deacetylase Inhibitors with T cell Modulatory Properties.

Histone deacetylases (HDACs) are epigenetic regulators and additionally control the activity of non-histone substrates. We recently demonstrated that inhibition of HDAC8 overexpressed in various of cancers reduces hepatocellular carcinoma tumorigenicity in a T cell-dependent manner. Here, we present alkylated hydrazide-based class I HDAC inhibitors in which the n-hexyl side chain attached to the hydrazide moiety shows HDAC8 selectivity in vitro. Analysis of the mode of inhibition of the most promising compound 7d against HDAC8 revealed a substrate-competitive binding mode. 7d marked induced acetylation of the HDAC8 substrates H3K27 and SMC3 but not tubulin in CD4+ T lymphocytes, and significantly upregulated gene expressions for memory and effector functions. Furthermore, intraperitoneal injection of 7d (10 mg/kg) in C57BL/6 mice increased interleukin-2 expression in CD4+ T cells and CD8+ T cell proportion with no apparent toxicity. This study expands a novel chemotype of HDAC8 inhibitors with T cell modulatory properties for future therapeutic applications.

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.

The mitotic regulator RCC2 promotes glucose metabolism through BACH1-dependent transcriptional upregulation of hexokinase II in glioma.

Weighted gene co-expression network analysis (WGCNA) identified a cell-cycle module that is associated with poor prognosis and aggressiveness of glioma. One of the core members, Regulator of chromatin condensation 2 (RCC2) is a component of the chromosome passenger complex. Accumulating evidence suggests that RCC2 plays a vital role in the mitotic process and that abnormal RCC2 expression is involved in cancer development. Gene silencing experiments show that RCC2 is required for glioma cell proliferation and migration. RNA-Sequencing analysis reveals a dual role of RCC2 in both the cell cycle and metabolism. Specifically, RCC2 regulates G2/M progression via CDC2 phosphorylation at Tyrosine 15. Metabolomic analysis identifies a role for RCC2 in promoting the glycolysis and pentose phosphate pathway. RCC2 exerts effects on metabolism by stabilizing the transcription factor BACH1 at its C-terminus leading to the transcriptional upregulation of hexokinase 2 (HK2). These findings elucidate a novel PTEN/RCC2/BACH1/HK2 signaling axis that drives glioma progression through the dual regulation of mitotic cell cycle and glycolytic events.

Aberrant cholesterol metabolic signaling impairs antitumor immunosurveillance through natural killer T cell dysfunction in obese liver.

Obesity is a major risk factor for cancers including hepatocellular carcinoma (HCC) that develops from a background of non-alcoholic fatty liver disease (NAFLD). Hypercholesterolemia is a common comorbidity of obesity. Although cholesterol biosynthesis mainly occurs in the liver, its role in HCC development of obese people remains obscure. Using high-fat high-carbohydrate diet-associated orthotopic and spontaneous NAFLD-HCC mouse models, we found that hepatic cholesterol accumulation in obesity selectively suppressed natural killer T (NKT) cell-mediated antitumor immunosurveillance. Transcriptome analysis of human liver revealed aberrant cholesterol metabolism and NKT cell dysfunction in NAFLD patients. Notably, cholesterol-lowering rosuvastatin restored NKT expansion and cytotoxicity to prevent obesogenic diet-promoted HCC development. Moreover, suppression of hepatic cholesterol biosynthesis by a mammalian target of rapamycin (mTOR) inhibitor vistusertib preceded tumor regression, which was abolished by NKT inactivation but not CD8+ T cell depletion. Mechanistically, sterol regulatory element-binding protein 2 (SREBP2)-driven excessive cholesterol production from hepatocytes induced lipid peroxide accumulation and deficient cytotoxicity in NKT cells, which were supported by findings in people with obesity, NAFLD and NAFLD-HCC. This study highlights mTORC1/SREBP2/cholesterol-mediated NKT dysfunction in the tumor-promoting NAFLD liver microenvironment, providing intervention strategies that invigorating NKT cells to control HCC in the obesity epidemic.

Single-Hit Inactivation Drove Tumor Suppressor Genes Out of the X Chromosome during Evolution.

Cancer-related genes are under intense evolutionary pressure. In this study, we conjecture that X-linked tumor suppressor genes (TSG) are not protected by the Knudson's two-hit mechanism and are therefore subject to negative selection. Accordingly, nearly all mammalian species exhibited lower TSG-to-noncancer gene ratios on their X chromosomes compared with nonmammalian species. Synteny analysis revealed that mammalian X-linked TSGs were depleted shortly after the emergence of the XY sex-determination system. A phylogeny-based model unveiled a higher X chromosome-to-autosome relocation flux for human TSGs. This was verified in other mammals by assessing the concordance/discordance of chromosomal locations of mammalian TSGs and their orthologs in Xenopus tropicalis. In humans, X-linked TSGs are younger or larger in size. Consistently, pan-cancer analysis revealed more frequent nonsynonymous somatic mutations of X-linked TSGs. These findings suggest that relocation of TSGs out of the X chromosome could confer a survival advantage by facilitating evasion of single-hit inactivation. SIGNIFICANCE: This work unveils extensive trafficking of TSGs from the X chromosome to autosomes during evolution, thus identifying X-linked TSGs as a genetic Achilles' heel in tumor suppression.

Vitamin D3 and carbamazepine protect against Clostridioides difficile infection in mice by restoring macrophage lysosome acidification.

Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D3 and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/ß-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1ß (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D3 or carbamazepine suppressed pro-inflammatory cytokine expression in vitro. In mice, gavage with TcdB-hyperproducing C. difficile or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D3 and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D3 and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice.Abbreviations: ATP6V0B: ATPase H+ transporting V0 subunit b; ATP6V0C: ATPase H+ transporting V0 subunit c; ATP6V0E1: ATPase H+ transporting V0 subunit e1; ATP6V1H: ATPase H+ transporting V1 subunit H; CBZ: carbamazepine; CDI: C. difficile infection; CXCL: chemokine C-X-X motif ligand; IL: interleukin; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; LEF: lymphoid enhancer binding factor 1; MITF: melanocyte inducing transcription factor; NFKB: nuclear factor kappa B; PMA: phorbol 12-myristate 13-acetate; TcdA: Clostridial toxin A; TcdB: Clostridial toxin B; TFE3: transcription factor E3; TFEB: transcription factor EB.