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.

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.

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.

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.

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.

Liver Immune Microenvironment and Metastasis from Colorectal Cancer-Pathogenesis and Therapeutic Perspectives.

A drastic difference exists between the 5-year survival rates of colorectal cancer patients with localized cancer and distal organ metastasis. The liver is the most favorable organ for cancer metastases from the colorectum. Beyond the liver-colon anatomic relationship, emerging evidence highlights the impact of liver immune microenvironment on colorectal liver metastasis. Prior to cancer cell dissemination, hepatocytes secrete multiple factors to recruit or activate immune cells and stromal cells in the liver to form a favorable premetastatic niche. The liver-resident cells including Kupffer cells, hepatic stellate cells, and liver-sinusoidal endothelial cells are co-opted by the recruited cells, such as myeloid-derived suppressor cells and tumor-associated macrophages, to establish an immunosuppressive liver microenvironment suitable for tumor cell colonization and outgrowth. Current treatments including radical surgery, systemic therapy, and localized therapy have only achieved good clinical outcomes in a minority of colorectal cancer patients with liver metastasis, which is further hampered by high recurrence rate. Better understanding of the mechanisms governing the metastasis-prone liver immune microenvironment should open new immuno-oncology avenues for liver metastasis intervention.

Multi-omic analysis suggests tumor suppressor genes evolved specific promoter features to optimize cancer resistance.

Tumor suppressor genes (TSGs) exhibit distinct evolutionary features. We speculated that TSG promoters could have evolved specific features that facilitate their tumor-suppressing functions. We found that the promoter CpG dinucleotide frequencies of TSGs are significantly higher than that of non-cancer genes across vertebrate genomes, and positively correlated with gene expression across tissue types. The promoter CpG dinucleotide frequencies of all genes gradually increase with gene age, for which young TSGs have been subject to a stronger evolutionary pressure. Transcription-related features, namely chromatin accessibility, methylation and ZNF263-, SP1-, E2F4- and SP2-binding elements, are associated with gene expression. Moreover, higher promoter CpG dinucleotide frequencies and chromatin accessibility are positively associated with the ability of TSGs to resist downregulation during tumorigenesis. These results were successfully validated with independent datasets. In conclusion, TSGs evolved specific promoter features that optimized cancer resistance through achieving high expression in normal tissues and resistance to downregulation during tumorigenesis.

NOTCH3, a crucial target of miR-491-5p/miR-875-5p, promotes gastric carcinogenesis by upregulating PHLDB2 expression and activating Akt pathway.

Aberrant Notch activation has been implicated in multiple malignancies and the identification of NOTCH receptors and related pathways is critical for targeted therapy. In this study, we aim to delineate the most prominent dysregulated NOTCH receptor and comprehensively reveal its deregulation in gastric cancer (GC). In the four Notch members, NOTCH3 was found uniformly upregulated and associated with poor clinical outcomes in multiple GC datasets. siRNA-mediated NOTCH3 knockdown demonstrated antitumor effects by suppressing cell proliferation, inhibiting monolayer formation, and impairing cell invasion abilities. Its depletion also induced early and late apoptosis. NOTCH3 was confirmed to be a direct target of two tumor suppressor microRNAs (miRNAs), namely miR-491-5p and miR-875-5p. The activation of NOTCH3 is partly due to the silence of these two miRNAs. Through RNA-seq profiling and functional validation, PHLDB2 was identified as a potent functional downstream modulator for NOTCH3 in gastric carcinogenesis. PHLDB2 expression demonstrated a positive correlation with NOTCH3, but was negatively correlated with miR-491-5p. Akt-mTOR was revealed as the downstream signaling of PHLDB2. The NOTCH3-PHLDB2-Akt co-activation was found in 33.7% GC patients and the activation of this axis predicted poor clinical outcome. GC cells treated with siNOTCH3, siPHLDB2, miR-491-5p, miR-875-5p, were more sensitive to Cisplatin and 5-FU. Taken together, the NOTCH3-PHLDB2-Akt cascade plays oncogenic role in gastric carcinogenesis and serves as a therapeutic target. Our study provided insights into Notch-mediated underlying molecular mechanisms and implied translational potential.

The Intersection between Oral Microbiota, Host Gene Methylation and Patient Outcomes in Head and Neck Squamous Cell Carcinoma.

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host-microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

FGF18-FGFR2 signaling triggers the activation of c-Jun-YAP1 axis to promote carcinogenesis in a subgroup of gastric cancer patients and indicates translational potential.

Fibroblast growth factor receptor type 2 (FGFR2) has emerged as a key oncogenic factor that regulates gastric cancer (GC) progression, but the underlying mechanism of FGF-FGFR2 signaling pathway remains largely unknown. To identify the potential molecular mechanisms of the oncogenic FGFR2 in gastric carcinogenesis and convey a novel therapeutic strategy, we profiled the FGFR alterations and analyzed their clinical associations in TCGA and Hong Kong GC cohorts. We found that FGFR2 overexpression in GC cell lines and primary tumors predicted poor survival and was associated with advanced stages of GC. Functionally, growth abilities and cell cycle progression of GC were inhibited by inactivation of ERK-MAPK signal transduction after FGFR2 knockdown, while apoptosis was promoted. Meanwhile, the first-line anti-cancer drug sensitivity was enhanced. RNA-seq analysis further revealed that YAP1 signaling serves as a significant downstream modulator and mediates the oncogenic signaling of FGFR2. When stimulating FGFR2 by rhFGF18, we observed intensified F-actin, nuclear accumulation of YAP1, and overexpression of YAP1 targets, but these effects were attenuated by either FGFR2 depletion or AZD4547 administration. Additionally, the FGF18-FGFR2 signaling upregulated YAP1 expression through activating c-Jun, an effector of MAPK signaling. In our cohort, 28.94% of GC cases were characterized as FGFR2, c-Jun, and YAP1 co-positive and demonstrated worse clinical outcomes. Remarkably, we also found that co-targeting FGFR2 and YAP1 by AZD4547 and Verteporfin synergistically enhanced the antitumor effects in vitro and in vivo. In conclusion, we have identified the oncogenic FGF-FGFR2 regulates YAP1 signaling in GC. The findings also highlight the translational potential of FGFR2-c-Jun-YAP1 axis, which may serve as a prognostic biomarker and therapeutic target for GC.

MCM family in gastrointestinal cancer and other malignancies: From functional characterization to clinical implication.

Despite the recent advances in cancer research and treatment, gastrointestinal (GI) cancers remain the most common deadly disease worldwide. The aberrant DNA replication serves as a major source of genomic instability and enhances cell proliferation that contributes to tumor initiation and progression. Minichromosome maintenance family (MCMs) is a well-recognized group of proteins responsible for DNA synthesis. Recent studies suggested that dysregulated MCMs lead to tumor initiation, progression, and chemoresistance via modulating cell cycle and DNA replication stress. Their underlying mechanisms in various cancer types have been gradually identified. Furthermore, multiple studies have investigated the association between MCMs expression and clinicopathological features of cancer patients, implying that MCMs might serve as prominent prognostic biomarkers for GI cancers. This review summarizes the current knowledge on the oncogenic role of MCM proteins and highlights their clinical implications in various malignancies, especially in GI cancers. Targeting MCMs might shed light on the potential for identifying novel therapeutic strategies.

AMOTL1 enhances YAP1 stability and promotes YAP1-driven gastric oncogenesis.

Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1-YAP1-CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1-CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.

Gastric cancer: genome damaged by bugs.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. The role of the microorganisms in gastric tumorigenesis attracts much attention in recent years. These microorganisms include bacteria, virus, and fungi. Among them, Helicobacter pylori (H. pylori) infection is by far the most important risk factor for GC development, with special reference to the early-onset cases. H. pylori targets multiple cellular components by utilizing various virulence factors to modulate the host proliferation, apoptosis, migration, and inflammatory response. Epstein-Barr virus (EBV) serves as another major risk factor in gastric carcinogenesis. The virus protein, EBER noncoding RNA, and EBV miRNAs contribute to the tumorigenesis by modulating host genome methylation and gene expression. In this review, we summarized the related reports about the colonized microorganism in the stomach and discussed their specific roles in gastric tumorigenesis. Meanwhile, we highlighted the therapeutic significance of eradicating the microorganisms in GC treatment.

ER-residential Nogo-B accelerates NAFLD-associated HCC mediated by metabolic reprogramming of oxLDL lipophagy.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome that elevates the risk of hepatocellular carcinoma (HCC). Although alteration of lipid metabolism has been increasingly recognized as a hallmark of cancer cells, the deregulated metabolic modulation of HCC cells in the NAFLD progression remains obscure. Here, we discovers an endoplasmic reticulum-residential protein, Nogo-B, as a highly expressed metabolic modulator in both murine and human NAFLD-associated HCCs, which accelerates high-fat, high-carbohydrate diet-induced metabolic dysfunction and tumorigenicity. Mechanistically, CD36-mediated oxLDL uptake triggers CEBPß expression to directly upregulate Nogo-B, which interacts with ATG5 to promote lipophagy leading to lysophosphatidic acid-enhanced YAP oncogenic activity. This CD36-Nogo-B-YAP pathway consequently reprograms oxLDL metabolism and induces carcinogenetic signaling for NAFLD-associated HCCs. Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome.

Targeting the Oncogenic FGF-FGFR Axis in Gastric Carcinogenesis.

Gastric cancer (GC) is one of the most wide-spread malignancies in the world. The oncogenic role of signaling of fibroblast growing factors (FGFs) and their receptors (FGFRs) in gastric tumorigenesis has been gradually elucidated by recent studies. The expression pattern and clinical correlations of FGF and FGFR family members have been comprehensively delineated. Among them, FGF18 and FGFR2 demonstrate the most prominent driving role in gastric tumorigenesis with gene amplification or somatic mutations and serve as prognostic biomarkers. FGF-FGFR promotes tumor progression by crosstalking with multiple oncogenic pathways and this provides a rational therapeutic strategy by co-targeting the crosstalks to achieve synergistic effects. In this review, we comprehensively summarize the pathogenic mechanisms of FGF-FGFR signaling in gastric adenocarcinoma together with the current targeted strategies in aberrant FGF-FGFR activated GC cases.

Mechanotransduction and Cytoskeleton Remodeling Shaping YAP1 in Gastric Tumorigenesis.

The essential role of Hippo signaling pathway in cancer development has been elucidated by recent studies. In the gastrointestinal tissues, deregulation of the Hippo pathway is one of the most important driving events for tumorigenesis. It is widely known that Yes-associated protein 1 (YAP1) and WW domain that contain transcription regulator 1 (TAZ), two transcriptional co-activators with a PDZ-binding motif, function as critical effectors negatively regulated by the Hippo pathway. Previous studies indicate the involvement of YAP1/TAZ in mechanotransduction by crosstalking with the extracellular matrix (ECM) and the F-actin cytoskeleton associated signaling network. In gastric cancer (GC), YAP1/TAZ functions as an oncogene and transcriptionally promotes tumor formation by cooperating with TEAD transcription factors. Apart from the classic role of Hippo-YAP1 cascade, in this review, we summarize the current investigations to highlight the prominent role of YAP1/TAZ as a mechanical sensor and responder under mechanical stress and address its potential prognostic and therapeutic value in GC.

New guidelines for DNA methylome studies regarding 5-hydroxymethylcytosine for understanding transcriptional regulation.

Many DNA methylome profiling methods cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Because 5mC typically acts as a repressive mark whereas 5hmC is an intermediate form during active demethylation, the inability to separate their signals could lead to incorrect interpretation of the data. Is the extra information contained in 5hmC signals worth the additional experimental and computational costs? Here we combine whole-genome bisulfite sequencing (WGBS) and oxidative WGBS (oxWGBS) data in various human tissues to investigate the quantitative relationships between gene expression and the two forms of DNA methylation at promoters, transcript bodies, and immediate downstream regions. We find that 5mC and 5hmC signals correlate with gene expression in the same direction in most samples. Considering both types of signals increases the accuracy of expression levels inferred from methylation data by a median of 18.2% as compared to having only WGBS data, showing that the two forms of methylation provide complementary information about gene expression. Differential analysis between matched tumor and normal pairs is particularly affected by the superposition of 5mC and 5hmC signals in WGBS data, with at least 25%-40% of the differentially methylated regions (DMRs) identified from 5mC signals not detected from WGBS data. Our results also confirm a previous finding that methylation signals at transcript bodies are more indicative of gene expression levels than promoter methylation signals. Overall, our study provides data for evaluating the cost-effectiveness of some experimental and analysis options in the study of DNA methylation in normal and cancer samples.

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.

E2F6 functions as a competing endogenous RNA, and transcriptional repressor, to promote ovarian cancer stemness.

Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long-term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen-mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c-Kit, by epigenetic silencing the tumor suppressor miR-193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c-KIT, but downregulated miR-193a. Luciferase assays further confirmed that microRNA-193a targets both E2F6 and c-Kit. Interestingly, ChIP-PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR-193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR-193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR-193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen-mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA-193a activity, promoting ovarian cancer stemness and tumorigenesis.