A patient-derived cell model for malignant transformation in IDH-mutant glioma.

Malignant transformation (MT) is commonly seen in IDH-mutant gliomas. There has been a growing research interest in revealing its underlying mechanisms and intervening prior to MT at the early stages of the transforming process. Here we established a unique pair of matched 3D cell models: 403L, derived from a low-grade glioma (LGG), and 403H, derived from a high-grade glioma (HGG), by utilizing IDH-mutant astrocytoma samples from the same patient when the tumor was diagnosed as WHO grade 2 (tumor mutational burden (TMB) of 3.96/Mb) and later as grade 4 (TMB of 70.07/Mb), respectively. Both cell models were authenticated to a patient's sample retaining endogenous expression of IDH1 R132H. DNA methylation profiles of the parental tumors referred to LGG and HGG IDH-mutant glioma clusters. The immunopositivity of SOX2, NESTIN, GFAP, OLIG2, and beta 3-Tubulin suggested the multilineage potential of both models. 403H was more prompt to cell invasion and developed infiltrative HGG in vivo. The differentially expressed genes (DEGs) from the RNA sequencing analysis revealed the tumor invasion and aggressiveness related genes exclusively upregulated in the 403H model. Pathway analysis showcased an enrichment of genes associated with epithelial-mesenchymal transition (EMT) and Notch signaling pathways in 403H and 403L, respectively. Mass spectrometry-based targeted metabolomics and hyperpolarized (HP) 1-13C pyruvate in-cell NMR analyses demonstrated significant alterations in the TCA cycle and fatty acid metabolism. Citrate, glutamine, and 2-HG levels were significantly higher in 403H. To our knowledge, this is the first report describing the development of a matched pair of 3D patient-derived cell models representative of MT and temozolomide (TMZ)-induced hypermutator phenotype (HMP) in IDH-mutant glioma, providing insights into genetic and metabolic changes during MT/HMP. This novel in vitro model allows further investigation of the mechanisms of MT at the cellular level.

Molecular Detection of Anaplasma phagocytophilum in Cats in Europe and Associated Risk Factors.

Infections with Anaplasma (A.) phagocytophilum in cats seem to be rare. The study aimed to determine whether infections in cats are underestimated and to identify the risk factors for infection. Blood samples of 1015 cats across Europe (2017-2022), sent to IDEXX Laboratories, Germany, were tested for A. phagocytophilum DNA. The influence of the cats' origin on A. phagocytophilum infection was assessed by univariable analysis, while multivariable logistic regression evaluated associations with the cats' sex and age, and the years, and seasonality of the samples' submission. Furthermore, univariable linear regression was used to determine patterns in PCR orders. The number of submitted samples increased significantly during the 6 years (p = 0.042). Anaplasma phagocytophilum DNA was detected in 76/1015 of cats (7.5%, 95% CI 6.0-9.3%). Infections were significantly more common in Northern compared to Central (p < 0.001, OR: 8.70) and Southern Europe (p < 0.001, OR: 39.94). A significantly higher likelihood for infections during the summer compared with winter (p = 0.047, OR: 3.13) was found. Bacteremia with A. phagocytophilum in European cats is not uncommon. Anaplasma phagocytophilum infection should be considered an important risk, particularly in Northern Europe. Effective tick prevention is crucial for managing feline health across Europe, not just in the Mediterranean region.

Exploiting the therapeutic vulnerability of IDH-mutant gliomas with zotiraciclib.

Isocitrate dehydrogenase (IDH)-mutant gliomas have distinctive metabolic and biological traits that may render them susceptible to targeted treatments. Here, by conducting a high-throughput drug screen, we pinpointed a specific susceptibility of IDH-mutant gliomas to zotiraciclib (ZTR). ZTR exhibited selective growth inhibition across multiple IDH-mutant glioma in vitro and in vivo models. Mechanistically, ZTR at low doses suppressed CDK9 and RNA Pol II phosphorylation in IDH-mutant cells, disrupting mitochondrial function and NAD+ production, causing oxidative stress. Integrated biochemical profiling of ZTR kinase targets and transcriptomics unveiled that ZTR-induced bioenergetic failure was linked to the suppression of PIM kinase activity. We posit that the combination of mitochondrial dysfunction and an inability to adapt to oxidative stress resulted in significant cell death upon ZTR treatment, ultimately increasing the therapeutic vulnerability of IDH-mutant gliomas. These findings prompted a clinical trial evaluating ZTR in IDH-mutant gliomas towards precision medicine ( NCT05588141 ). Highlights: Zotiraciclib (ZTR), a CDK9 inhibitor, hinders IDH-mutant glioma growth in vitro and in vivo . ZTR halts cell cycle, disrupts respiration, and induces oxidative stress in IDH-mutant cells.ZTR unexpectedly inhibits PIM kinases, impacting mitochondria and causing bioenergetic failure.These findings led to the clinical trial NCT05588141, evaluating ZTR for IDH-mutant gliomas.

Ultrahigh Resolution Lipid Mass Spectrometry Imaging of High-Grade Serous Ovarian Cancer Mouse Models.

No effective screening tools for ovarian cancer (OC) exist, making it one of the deadliest cancers among women. Considering little is known about the detailed progression and metastasis mechanism of OC at a molecular level, it is crucial to gain more insights on how metabolic and signaling alterations accompany its development. Herein, we present a comprehensive study using ultra-high-resolution Fourier transform ion cyclotron resonance matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to investigate the spatial distribution and alterations of lipids in ovarian tissues collected from double knockout (n = 4) and a triple mutant mouse models (n = 4) of high-grade serous ovarian cancer (HGSC). Lipids belonging to a total of 15 different classes were annotated and their abundance changes compared to those in healthy mouse reproductive tissue (n = 4), mapping onto major lipid pathways involved in OC progression. From intermediate-stage OC to advanced HGSC, we provide a direct visualization of lipid distributions and their biological links to inflammatory response, cellular stress, cell proliferation, and other processes. We also show the ability to distinguish tumors at different stages from healthy tissues via a number of highly specific lipid biomarkers, providing targets for future panels that could be useful in diagnosis.

Combined inhibition of topoisomerase I and poly(ADP-ribose) polymerase: A synergistic therapeutic strategy for glioblastoma with phosphatase and tensin homolog deficiency.

Background: Deletions or loss-of-function mutations in phosphatase and tensin homolog (PTEN) are common in glioblastoma (GBM) and have been associated with defective DNA damage repair. Here we investigated whether PTEN deficiency presents a vulnerability to a simultaneous induction of DNA damage and suppression of repair mechanisms by combining topoisomerase I (TOP1) and PARP inhibitors. Methods: Patient-derived GBM cells and isogenic PTEN-null and PTEN-WT glioma cells were treated with LMP400 (Indotecan), a novel non-camptothecin TOP1 inhibitor alone and in combination with a PARP inhibitor, Olaparib or Niraparib. RNAseq analysis was performed to identify treatment-induced dysregulated pathways. Results: We found that GBM cells lacking PTEN expression are highly sensitive to LMP400; however, rescue of the PTEN expression reduces sensitivity to the treatment. Combining LMP400 with Niraparib leads to synergistic cytotoxicity by inducing G2/M arrest, DNA damage, suppression of homologous recombination-related proteins, and activation of caspase 3/7 activity significantly more in PTEN-null cells compared to PTEN-WT cells. LMP400 and Niraparib are not affected by ABCB1 and ABCG2, the major ATP-Binding Cassette (ABC) drug efflux transporters expressed at the blood-brain barrier (BBB), thus suggesting BBB penetration which is a prerequisite for potential brain tumor treatment. Animal studies confirmed both an anti-glioma effect and sufficient BBB penetration to prolong survival of mice treated with the drug combination. Conclusions: Our findings provide a proof of concept for the combined treatment with LMP400 and Niraparib in a subset of GBM patients with PTEN deficiency.

Ultrahigh resolution lipid mass spectrometry imaging of high-grade serous ovarian cancer mouse models.

No effective screening tools for ovarian cancer (OC) exist, making it one of the deadliest cancers among women. Considering that little is known about the detailed progression and metastasis mechanism of OC at a molecular level, it is crucial to gain more insights into how metabolic and signaling alterations accompany its development. Herein, we present a comprehensive study using ultra-high-resolution Fourier transform ion cyclotron resonance matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to investigate the spatial distribution and alterations of lipids in ovarian tissues collected from double knockout (n = 4) and triple mutant mouse models (n = 4) of high-grade serous ovarian cancer (HGSOC). Lipids belonging to a total of 15 different classes were annotated and their abundance changes were compared to those in healthy mouse reproductive tissue (n = 4), mapping onto major lipid pathways involved in OC progression. From intermediate-stage OC to advanced HGSC, we provide direct visualization of lipid distributions and their biological links to inflammatory response, cellular stress, cell proliferation, and other processes. We also show the ability to distinguish tumors at different stages from healthy tissues via a number of highly specific lipid biomarkers, providing targets for future panels that could be useful in diagnosis.

Case report: Oligodendroglioma, IDH-mutant and 1p/19q-codeleted, associated with a germline mutation in PMS2.

Most tumors, including brain tumors, are sporadic. However, a small subset of CNS tumors are associated with hereditary cancer conditions like Lynch Syndrome (LS). Here, we present a case of an oligodendroglioma, IDH-mutant and 1p/19q-codeleted, and LS with a germline pathogenic PMS2 mutation. To our knowledge, this has only been reported in a few cases in the literature. While the family history is less typical of LS, previous studies have indicated the absence of a significant family history in patient cohorts with PMS2 mutations due to its low penetrance. Notably, only a handful of studies have worked on characterizing PMS2 mutations in LS, and even fewer have looked at these mutations in the context of brain tumor development. This report aims to add to the limited literature on germline PMS2 mutations and oligodendrogliomas. It highlights the importance of genetic testing in neuro-oncology.

Targeted Microchip Capillary Electrophoresis-Orbitrap Mass Spectrometry Metabolomics to Monitor Ovarian Cancer Progression.

The lack of effective screening strategies for high-grade serous carcinoma (HGSC), a subtype of ovarian cancer (OC) responsible for 70-80% of OC related deaths, emphasizes the need for new diagnostic markers and a better understanding of disease pathogenesis. Capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) offers high selectivity and sensitivity for ionic compounds, thereby enhancing biomarker discovery. Recent advances in CE-MS include small, chip-based CE systems coupled with nanoelectrospray ionization (nanoESI) to provide rapid, high-resolution analysis of biological specimens. Here, we describe the development of a targeted microchip (µ) CE-HRMS method, with an acquisition time of only 3 min and sample injection volume of 4nL, to analyze 40 target metabolites in serum samples from a triple-mutant (TKO) mouse model of HGSC. Extracted ion electropherograms showed sharp, baseline resolved peak shapes, even for structural isomers such as leucine and isoleucine. All calibration curves of the analytes maintained good linearity with an average R2 of 0.994, while detection limits were in the nM range. Thirty metabolites were detected in mouse serum with recoveries ranging from 78 to 120%, indicating minimal ionization suppression and good accuracy. We applied the µCE-HRMS method to biweekly-collected serum samples from TKO and TKO control mice. A time-resolved analysis revealed characteristic temporal trends for amino acids, nucleosides, and amino acid derivatives. These metabolic alterations are indicative of altered nucleotide biosynthesis and amino acid metabolism in HGSC development and progression. A comparison of the µCE-HRMS dataset with non-targeted ultra-high performance liquid chromatography (UHPLC)-MS results showed identical temporal trends for the five metabolites detected with both platforms, indicating the µCE-HRMS method performed satisfactorily in terms of capturing metabolic reprogramming due to HGSC progression while reducing the total data collection time three-fold.

Space- and Time-Resolved Metabolomics of a High-Grade Serous Ovarian Cancer Mouse Model.

The dismally low survival rate of ovarian cancer patients diagnosed with high-grade serous carcinoma (HGSC) emphasizes the lack of effective screening strategies. One major obstacle is the limited knowledge of the underlying mechanisms of HGSC pathogenesis at very early stages. Here, we present the first 10-month time-resolved serum metabolic profile of a triple mutant (TKO) HGSC mouse model, along with the spatial lipidome profile of its entire reproductive system. A high-coverage liquid chromatography mass spectrometry-based metabolomics approach was applied to longitudinally collected serum samples from both TKO (n = 15) and TKO control mice (n = 15), tracking metabolome and lipidome changes from premalignant stages to tumor initiation, early stages, and advanced stages until mouse death. Time-resolved analysis showed specific temporal trends for 17 lipid classes, amino acids, and TCA cycle metabolites, associated with HGSC progression. Spatial lipid distributions within the reproductive system were also mapped via ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and compared with serum lipid profiles for various lipid classes. Altogether, our results show that the remodeling of lipid and fatty acid metabolism, amino acid biosynthesis, TCA cycle and ovarian steroidogenesis are critical components of HGSC onset and development. These metabolic alterations are accompanied by changes in energy metabolism, mitochondrial and peroxisomal function, redox homeostasis, and inflammatory response, collectively supporting tumorigenesis.

Tumor Mutation Burden, Expressed Neoantigens and the Immune Microenvironment in Diffuse Gliomas.

BACKGROUND: A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. METHODS: Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). RESULTS: Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5% of IDH-wildtype gliomas (p = 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R = -0.46, p = 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. The Antigen Processing and Presentation (APP) score correlated with immune score and was surprisingly higher in NHMP versus HMP samples in IDH-wildtype gliomas, but higher in HMP versus NHMP in IDH-mutant gliomas. CONCLUSION: TMB was inversely correlated with immune score in IDH-wildtype gliomas and showed no correlation in IDH-mutant tumors. APP was correlated with immune score and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a larger glioma cohort are warranted.

Tumor mutational burden and immunotherapy in gliomas.

Tumor mutational burden (TMB) is an emerging biomarker for the prediction of immunotherapy success in solid tumors. Gliomas, however, do not demonstrate a correlation between TMB and immunotherapy efficacy. Here, we discuss the potential factors influencing this discordance, focusing on the impact of neoantigen immunogenicity, clonality, expression, and presentation.

Targeting CDK9 for the Treatment of Glioblastoma.

Glioblastoma is the most common and aggressive primary malignant brain tumor, and more than two-thirds of patients with glioblastoma die within two years of diagnosis. The challenges of treating this disease mainly include genetic and microenvironmental features that often render the tumor resistant to treatments. Despite extensive research efforts, only a small number of drugs tested in clinical trials have become therapies for patients. Targeting cyclin-dependent kinase 9 (CDK9) is an emerging therapeutic approach that has the potential to overcome the challenges in glioblastoma management. Here, we discuss how CDK9 inhibition can impact transcription, metabolism, DNA damage repair, epigenetics, and the immune response to facilitate an anti-tumor response. Moreover, we discuss small-molecule inhibitors of CDK9 in clinical trials and future perspectives on the use of CDK9 inhibitors in treating patients with glioblastoma.

Targeting progesterone signaling prevents metastatic ovarian cancer.

Effective cancer prevention requires the discovery and intervention of a factor critical to cancer development. Here we show that ovarian progesterone is a crucial endogenous factor inducing the development of primary tumors progressing to metastatic ovarian cancer in a mouse model of high-grade serous carcinoma (HGSC), the most common and deadliest ovarian cancer type. Blocking progesterone signaling by the pharmacologic inhibitor mifepristone or by genetic deletion of the progesterone receptor (PR) effectively suppressed HGSC development and its peritoneal metastases. Strikingly, mifepristone treatment profoundly improved mouse survival (∼18 human years). Hence, targeting progesterone/PR signaling could offer an effective chemopreventive strategy, particularly in high-risk populations of women carrying a deleterious mutation in the BRCA gene.

In vivo modeling of metastatic human high-grade serous ovarian cancer in mice.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.

NKX6.3 protects against gastric mucosal atrophy by downregulating ß-amyloid production.

BACKGROUND: Atrophic gastritis is characterized by loss of appropriate glands and reduction in gastric secretory function due to chronic inflammatory processes in gastric mucosa. Moreover, atrophic gastritis is considered as a precancerous condition of gastric cancer. However, little is known about the molecular mechanism underlying gastric mucosal atrophy and its contribution to gastric carcinogenesis. Thus, we hypothesized that transcription factor NKX6.3 might be involved in maintaining gastric epithelial homeostasis by regulating amyloid ß (Aß) production. AIM: To determine whether NKX6.3 might protect against gastric mucosal atrophy by regulating Aß production. METHODS: We identified NKX6.3 depletion induced cell death by cell count and Western blot assay. Production and mechanism of Aß oligomer were analyzed by enzyme-linked immunosorbent assay, Western blot, immunoprecipitation, real-time quantitative polymerase chain reaction and immunofluorescence analysis. We further validated the correlation between expression of NKX6.3, Helicobacter pylori CagA, Aß oligomer, apolipoprotein E (ApoE), and ß-secretase 1 (Bace1) in 55 gastric mucosae. RESULTS: NKX6.3 depletion increased both adherent and floating cell populations in HFE-145 cells. Expression levels of cleaved caspase-3, -9, and poly ADP ribose polymerase were elevated in floating HFE-145shNKX6.3 cells. NKX6.3 depletion produced Aß peptide oligomers, and increased expression of ApoE, amyloid precursor protein, Aß, Bace1, low-density lipoprotein receptor, nicastrin, high mobility group box1, and receptor for advanced glycosylation end product proteins. In immunoprecipitation assay, γ-secretase complex was stably formed only in HFE-145shNKX6.3 cells. In gastric mucosae with atrophy, expression of Aß peptide oligomer, ApoE, and Bace1 was detected and inversely correlated with NKX6.3 expression. Treatment with recombinant Aß 1-42 produced Aß oligomeric forms and decreased cell viability in HFE-145shNKX6.3 cells. Additionally, NKX6.3 depletion increased expression of inflammatory cytokines and cyclooxygenase-2. CONCLUSION: NKX6.3 inhibits gastric mucosal atrophy by regulating Aß accumulation and inflammatory reaction in gastric epithelial cells.

Multiple genetic mutations caused by NKX6.3 depletion contribute to gastric tumorigenesis.

NKX family members are involved in a variety of developmental processes such as cell fate determination in the central nervous system, gastrointestinal tract, and pancreas. However, whether NKX6.3 contributes to gastric carcinogenesis remains unclear. The objective of this study was to examine roles of NKX6.3 depletion in mutagenesis and gastric carcinogenesis, focusing on its effects on genetic alterations and expression of genes. Our results revealed that NKX6.3 depletion induced multiple genetic mutations in coding regions, including high frequency of point mutations such as cytosine-to-thymine and guanine-to-adenine transitions caused by aberrant expression of AICDA/APOBEC family in human gastric epithelial cells. Interestingly, NKX6.3 downregulated AICDA/APOBEC family, NFκB, and CBFß genes by acting as a transcription factor while inhibiting deaminase activity in gastric epithelial cells. Functional relevance of NKX6.3 was validated in xenograft mice injected with NKX6.3 depleting cells. NKX6.3 depletion resulted in tumor formation and mutations of tumor-associated genes, including p53 and E-cadherin. Moreover, expression levels of NKX6.3 and its target genes were analyzed in tumors derived from mice implanted with NKX6.3 depleting cells and tissue samples of gastric cancer patients. Our results indicate that NKX6.3 depletion in gastric epithelial cells activates AICDA/APOBEC family, leading to accumulation of genetic mutations and eventually driving the development of gastric cancers.

Cell Origins of High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), is the most common and deadliest type of ovarian cancer. HGSC appears to arise from the ovary, fallopian tube, or peritoneum. As most HGSC cases present with widespread peritoneal metastases, it is often not clear where HGSC truly originates. Traditionally, the ovarian surface epithelium (OSE) was long believed to be the origin of HGSC. Since the late 1990s, the fallopian tube epithelium has emerged as a potential primary origin of HGSC. Particularly, serous tubal intraepithelial carcinoma (STIC), a noninvasive tumor lesion formed preferentially in the distal fallopian tube epithelium, was proposed as a precursor for HGSC. It was hypothesized that STIC lesions would progress, over time, to malignant and metastatic HGSC, arising from the fallopian tube or after implanting on the ovary or peritoneum. Many clinical studies and several mouse models support the fallopian tube STIC origin of HGSC. Current evidence indicates that STIC may serve as a precursor for HGSC in high-risk women carrying germline BRCA1 or 2 mutations. Yet not all STIC lesions appear to progress to clinical HGSCs, nor would all HGSCs arise from STIC lesions, even in high-risk women. Moreover, the clinical importance of STIC remains less clear in women in the general population, in which 85⁻90% of all HGSCs arise. Recently, increasing attention has been brought to the possibility that many potential precursor or premalignant lesions, though composed of microscopically-and genetically-cancerous cells, do not advance to malignant tumors or lethal malignancies. Hence, rigorous causal evidence would be crucial to establish that STIC is a bona fide premalignant lesion for metastatic HGSC. While not all STICs may transform into malignant tumors, these lesions are clearly associated with increased risk for HGSC. Identification of the molecular characteristics of STICs that predict their malignant potential and clinical behavior would bolster the clinical importance of STIC. Also, as STIC lesions alone cannot account for all HGSCs, other potential cellular origins of HGSC need to be investigated. The fallopian tube stroma in mice, for instance, has been shown to be capable of giving rise to metastatic HGSC, which faithfully recapitulates the clinical behavior and molecular aspect of human HGSC. Elucidating the precise cell(s) of origin of HGSC will be critical for improving the early detection and prevention of ovarian cancer, ultimately reducing ovarian cancer mortality.

Gastrokine 1 protein is a potential theragnostic target for gastric cancer.

BACKGROUND: Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer. METHODS: We identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay. RESULTS: In protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas. CONCLUSIONS: The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.

NKX6.3 Regulates Reactive Oxygen Species Production by Suppressing NF-kB and DNMT1 Activities in Gastric Epithelial Cells.

NKX6.3 plays an important role in gastric epithelial differentiation and also acts as a gastric tumor suppressor. The specific aim of this study was to determine whether NKX6.3 contributes to gastric mucosal barrier function by regulating reactive oxygen species (ROS) production. NKX6.3 reduced ROS production and regulated expression of anti-oxidant genes, including Hace1. In addition, NKX6.3 reduced DNMT1 expression and activity by down-regulating NF-kB family gene transcription. Silencing of Hace1 recovered ROS production, whereas knock-down of DNMT1 and NF-kB reduced ROS production and induced Hace1 expression by hypomethylating its promoter region. In addition, NKX6.3 inhibited CagA effects on cell growth, ROS production, and NF-kB and DNMT1 activity. In gastric mucosae and cancers, NKX6.3 and Hace1 expression was significantly reduced. The NKX6.3 expression was positively correlated with Hace1 and Nrf2 genes, but negatively correlated with DNMT1. Hypermethylation of Hace1 gene was observed only in gastric mucosae with H. pylori, atrophy and intestinal metaplasia. Thus, these results suggest that NKX6.3 inhibits ROS production by inducing the expression of Hace1 via down-regulating NF-kB and DNMT1 activity in gastric epithelial cells.