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The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

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The search for genetic variants that act as causative factors in human diseases by disrupting the normal splicing process has primarily focused on single nucleotide variants (SNVs). It is worth noting that insertions or deletions (indels) have also been sporadically reported as causative disease variants through their potential impact on the splicing process. In this study, to perform identification of indels inducing exon extension/shrinkage events, we used individual-specific genomes and RNA sequencing (RNA-seq) data pertaining to the corresponding individuals and identified 12 exon extension/shrinkage events that were potentially induced by indels that disrupted authentic splice sites or created novel splice sites in 235 normal individuals. By evaluating the impact of these abnormal splicing events on the resulting transcripts, we found that five events led to the generation of premature termination codons (PTCs), including those occurring within genes associated with genetic disorders. Our analysis revealed that the potential functions of indels have been underexamined, and it is worth considering the possibility that indels may affect splice site usage, using RNA-seq data to discover novel potentially disease-associated mutations.

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Genome analysis in cancer has focused mainly on elucidating the function and regulatory mechanisms of genes that exhibit differential expression or mutation in cancer samples compared to normal samples. Recently, transcriptome analysis revealed that abnormal splicing events in cancer samples could contribute to cancer pathogenesis. Moreover, splicing variants in cancer reportedly generate diverse cancer antigens. Although abnormal splicing events are expected to be potential targets in cancer immunotherapy, the exploration of such targets and their biological significance in cancer have not been fully understood. In this study, to explore subtype-specific alternative splicing events, we conducted a comprehensive analysis of splicing events for each breast cancer subtype using large-scale splicing data derived from The Cancer Genome Atlas and found subtype-specific alternative splicing patterns. Analyses indicated that genes that produce subtype-specific alternative splicing events are potential novel targets for immunotherapy against breast cancer. The subtype-specific alternative splicing events identified in this study, which were not identified by mutation or differential expression analysis, bring new significance to previously overlooked splicing events.

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The initiation of human pregnancy is marked by the implantation of an embryo into the uterine environment; however, the underlying mechanisms remain largely elusive. To address this knowledge gap, we developed hormone-responsive endometrial organoids (EMO), termed apical-out (AO)-EMO, which emulate the in vivo architecture of endometrial tissue. The AO-EMO comprise an exposed apical epithelium surface, dense stromal cells, and a self-formed endothelial network. When cocultured with human embryonic stem cell-derived blastoids, the three-dimensional feto-maternal assembloid system recapitulates critical implantation stages, including apposition, adhesion, and invasion. Endometrial epithelial cells were subsequently disrupted by syncytial cells, which invade and fuse with endometrial stromal cells. We validated this fusion of syncytiotrophoblasts and stromal cells using human blastocysts. Our model provides a foundation for investigating embryo implantation and feto-maternal interactions, offering valuable insights for advancing reproductive medicine.

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INTRODUCTION: Osimertinib is an irreversible EGFR tyrosine kinase inhibitor approved for the first-line treatment of patients with metastatic NSCLC harboring EGFR exon 19 deletions or L858R mutations. Patients treated with osimertinib invariably develop acquired resistance by mechanisms involving additional EGFR mutations, MET amplification, and other pathways. There is no known involvement of the oncogenic MUC1-C protein in acquired osimertinib resistance. METHODS: H1975/EGFR (L858R/T790M) and patient-derived NSCLC cells with acquired osimertinib resistance were investigated for MUC1-C dependence in studies of EGFR pathway activation, clonogenicity, and self-renewal capacity. RESULTS: We reveal that MUC1-C is up-regulated in H1975 osimertinib drug-tolerant persister cells and is necessary for activation of the EGFR pathway. H1975 cells selected for stable osimertinib resistance (H1975-OR) and MGH700-2D cells isolated from a patient with acquired osimertinib resistance are found to be dependent on MUC1-C for induction of (1) phospho (p)-EGFR, p-ERK, and p-AKT, (2) EMT, and (3) the resistant phenotype. We report that MUC1-C is also required for p-EGFR, p-ERK, and p-AKT activation and self-renewal capacity in acquired osimertinib-resistant (1) MET-amplified MGH170-1D #2 cells and (2) MGH121 Res#2/EGFR (T790M/C797S) cells. Importantly, targeting MUC1-C in these diverse models reverses osimertinib resistance. In support of these results, high MUC1 mRNA and MUC1-C protein expression is associated with a poor prognosis for patients with EGFR-mutant NSCLCs. CONCLUSIONS: Our findings reveal that MUC1-C is a common effector of osimertinib resistance and is a potential target for the treatment of osimertinib-resistant NSCLCs.

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BACKGROUND: Whole-genome doubling (WGD) is a common mutation in cancer. Various studies have suggested that WGD is associated with a poor prognosis in cancer. However, the detailed association between WGD occurrence and prognosis remains unclear. In this study, we aimed to elucidate the mechanism by which WGD affects prognosis using sequencing data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) and The Cancer Genome Atlas. METHODS: Whole-genome sequencing data of 23 cancer types were downloaded from PCAWG project. We defined the WGD event in each sample using the WGD status annotated using PCAWG. We used MutationTimeR to predict the relative timings of mutations and loss of heterozygosity (LOH) in WGD, thus evaluating their association with WGD. We also analyzed the association between WGD-associated factors and patient prognosis. RESULTS: WGD was associated with several factors, e.g., length of LOH regions. Survival analysis using WGD-associated factors revealed that longer LOH regions and LOH in chr17 were associated with poor prognosis in samples with WGD (WGD samples) and samples without WGD (nWGD samples). In addition to these two factors, nWGD samples showed that the number of mutations in tumor suppressor genes was associated with prognosis. Moreover, we explored the genes associated with prognosis in both samples separately. CONCLUSION: The prognosis-related factors in WGD samples differed significantly compared with those in nWGD samples. This study emphasizes the need for different treatment strategies for WGD and nWGD samples.

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Recent studies have shown that some silent mutations can be harmful to various processes. In this study, we performed a comprehensive in silico analysis to elucidate the effects of silent mutations on cancer pathogenesis using exome sequencing data derived from the Cancer Genome Atlas. We focused on the codon optimality scores of silent mutations, which were defined as the difference between the optimality of synonymous codons, calculated using the codon usage table. The relationship between cancer evolution and silent mutations showed that the codon optimality score of the mutations that occurred later in carcinogenesis was significantly higher than of those that occurred earlier. In addition, mutations with higher scores were enriched in genes involved in the cell cycle and cell division, while those with lower scores were enriched in genes involved in apoptosis and cellular senescence. Our results demonstrate that some silent mutations can be involved in cancer pathogenesis.

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BACKGROUND: Alzheimer's disease (AD) is one of the neurodegenerative diseases and characterized by the appearance and accumulation of amyloid-ß (Aß) aggregates and phosphorylated tau with aging. The aggregation of Aß, which is the main component of senile plaques, is closely associated with disease progression. AppNL-G-F mice, a mouse model of AD, have three familial AD mutations in the amyloid-ß precursor gene and exhibit age-dependent AD-like symptoms and pathology. Gut-brain interactions have attracted considerable attention and inflammatory bowel disease (IBD) has been associated with a higher risk of dementia, especially AD, in humans. However, the underlying mechanisms and the effects of intestinal inflammation on the brain in AD remain largely unknown. Therefore, we aimed to investigate the effects of intestinal inflammation on AD pathogenesis. METHODS: Wild-type and AppNL-G-F mice at three months of age were fed with water containing 2% dextran sulfate sodium (DSS) to induce colitis. Immune cells in the brain were analyzed using single-cell RNA sequencing (scRNA-seq) analysis, and the aggregation of Aß protein in the brain was analyzed via immunohistochemistry. RESULTS: An increase in aggregated Aß was observed in the brains of AppNL-G-F mice with acute intestinal inflammation. Detailed scRNA-seq analysis of immune cells in the brain showed that neutrophils in the brain increased after acute enteritis. Eliminating neutrophils by antibodies suppressed the accumulation of Aß, which increased because of intestinal inflammation. CONCLUSION: These results suggest that neutrophils infiltrate the AD brain parenchyma when acute colitis occurs, and this infiltration is significantly related to disease progression. Therefore, we propose that neutrophil-targeted therapies could reduce Aß accumulation observed in early AD and prevent the increased risk of AD due to colitis.

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Mutations that affect phenotypes have been identified primarily as those that directly alter amino acid sequences or disrupt splice sites. However, some mutations not located in functionally important sites can also affect phenotypes, such as splice-site-creating mutations (SCMs). To investigate how frequent exon extension/shrinkage events induced by SCMs occur in normal individuals, we used personal genome sequencing data and transcriptome data of the corresponding individuals and identified 371 exon extension/shrinkage events in normal individuals. This number was about three times higher than the number of pseudo-exon activation events identified in the previous study. The average numbers of exon extension and exon shrinkage events in each sample were 3.3 and 11.2, respectively. We also evaluated the impact of exon extension/shrinkage events on the resulting transcripts and their protein products and found that 40.2% of the identified events may have possible functional impacts by either generating premature termination codons in transcripts or affecting protein domains. Our results indicated that a certain fraction of SCMs identified in this study can be pathogenic mutations by creating novel splice sites.

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Regulatory T cells (Tregs) are normally born in the thymus and activated in secondary lymphoid tissues to suppress immune responses in the lymph node and at sites of inflammation. Tregs are also resident in various tissues or accumulate in damaged tissues, which are now called tissue Tregs, and contribute to homeostasis and tissue repair by interacting with non-immune cells. We have shown that Tregs accumulate in the brain during the chronic phase in a mouse cerebral infarction model, and these Tregs acquire the characteristic properties of brain Tregs and contribute to the recovery of neurological damage by interacting with astrocytes. However, the mechanism of tissue Treg development is not fully understood. We developed a culture method that confers brain Treg characteristics in vitro. Naive Tregs from the spleen were activated and efficiently amplified by T-cell receptor (TCR) stimulation in the presence of primary astrocytes. Furthermore, adding IL-33 and serotonin could confer part of the properties of brain Tregs, such as ST2, peroxisome proliferator-activated receptor γ (PPARγ), and serotonin receptor 7 (Htr7) expression. Transcriptome analysis revealed that in vitro generated brain Treg-like Tregs (induced brain Tregs; iB-Tregs) showed similar gene expression patterns as those in in vivo brain Tregs, although they were not identical. Furthermore, in Parkinson's disease models, in which T cells have been shown to be involved in disease progression, iB-Tregs infiltrated into the brain more readily and ameliorated pathological symptoms more effectively than splenic Tregs. These data indicate that iB-Tregs contribute to our understanding of brain Treg development and could also be therapeutic for inflammatory brain diseases.

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Despite recent therapeutic advances for multiple myeloma (MM), relapse is very common. Here, we conducted longitudinal single-cell transcriptome sequencing (scRNA-seq) of MM cells from a patient with relapsed MM, treated with multiple anti-myeloma drugs. We observed five subclusters of MM cells, which appeared and/or disappeared in response to the therapeutic pressure, and identified cluster 3 which emerged during lenalidomide treatment and disappeared after proteasome inhibitor (PI) treatment. Among the differentially expressed genes in cluster 3, we found a candidate drug-response gene; pellino E3 ubiquitin-protein ligase family member 2 (PELI2), which is responsible for PI-induced cell death in in vitro assay. Kaplan-Meier survival analysis of database revealed that higher expression of PELI2 is associated with a better prognosis. Our integrated strategy combining longitudinal scRNA-seq analysis, in vitro functional assay, and database analysis would facilitate the understanding of clonal dynamics of MM in response to anti-myeloma drugs and identification of drug-response genes.

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The first cell fate commitment during mammalian development is the specification of the inner cell mass and trophectoderm. This irreversible cell fate commitment should be epigenetically regulated, but the precise mechanism is largely unknown in humans. Here, we show that naïve human embryonic stem (hES) cells can transdifferentiate into trophoblast stem (hTS) cells, but primed hES cells cannot. Our transcriptome and methylome analyses reveal that a primate-specific miRNA cluster on chromosome 19 (C19MC) is active in naïve hES cells but epigenetically silenced in primed ones. Moreover, genome and epigenome editing using CRISPR/Cas systems demonstrate that C19MC is essential for hTS cell maintenance and C19MC-reactivated primed hES cells can give rise to hTS cells. Thus, we reveal that C19MC activation confers differentiation potential into trophoblast lineages on hES cells. Our findings are fundamental to understanding the epigenetic regulation of human early development and pluripotency.

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BACKGROUND: Open chromatin is associated with gene transcription. Previous studies have shown that the density of mutations in open chromatin regions is lower than that in flanking regions because of the higher accessibility of DNA repair machinery. However, in several cancer types, open chromatin regions show an increased local density of mutations in activated regulatory regions. Although the mutation distribution within open chromatin regions in cancer cells has been investigated, only few studies have focused on their functional implications in cancer. To reveal the impact of highly mutated open chromatin regions on cancer, we investigated the association between mutations in open chromatin regions and their possible functions. METHODS: Whole-genome sequencing data of 18 cancer types were downloaded from the PanCancer Analysis of Whole Genomes and Catalog of Somatic Mutations in Cancer. We quantified the mutations located in open chromatin regions defined by The Cancer Genome Atlas and classified open chromatin regions into three categories based on the number of mutations. Then, we investigated the chromatin state, amplification, and possible target genes of the open chromatin regions with a high number of mutations. We also analyzed the association between the number of mutations in open chromatin regions and patient prognosis. RESULTS: In some cancer types, the proportion of promoter or enhancer chromatin state in open chromatin regions with a high number of mutations was significantly higher than that in the regions with a low number of mutations. The possible target genes of open chromatin regions with a high number of mutations were more strongly associated with cancer than those of other open chromatin regions. Moreover, a high number of mutations in open chromatin regions was significantly associated with a poor prognosis in some cancer types. CONCLUSIONS: These results suggest that highly mutated open chromatin regions play an important role in cancer pathogenesis and can be effectively used to predict patient prognosis.

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Cancer-related mutations have been mainly identified in protein-coding regions. Recent studies have demonstrated that mutations in non-coding regions of the genome could also be a risk factor for cancer. However, the non-coding regions comprise 98% of the total length of the human genome and contain a huge number of mutations, making it difficult to interpret their impacts on pathogenesis of cancer. To comprehensively identify cancer-related non-coding mutations, we focused on recurrent mutations in non-coding regions using somatic mutation data from COSMIC and whole-genome sequencing data from The Cancer Genome Atlas (TCGA). We identified 21 574 recurrent mutations in non-coding regions that were shared by at least two different samples from both COSMIC and TCGA databases. Among them, 580 candidate cancer-related non-coding recurrent mutations were identified based on epigenomic and chromatin structure datasets. One of such mutation was located in RREB1 binding site that is thought to interact with TEAD1 promoter. Our results suggest that mutations may disrupt the binding of RREB1 to the candidate enhancer region and increase TEAD1 expression levels. Our findings demonstrate that non-coding recurrent mutations and coding mutations may contribute to the pathogenesis of cancer.

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BACKGROUND: No effective molecular targeted therapy has been established for SCC. We conducted a comprehensive study of SCC patients using RNA-sequencing and TCGA dataset to clarify the driver oncogene of SCC. METHOD: Forty-six samples of 23 patients were totally analyzed with RNA-sequencing. We then searched for candidate-oncogenes of SCC using the TCGA database. To identify candidate oncogenes, we used the following 2 criteria: (1) the genes of interest were overexpressed in tumor tissues of SCC patients in comparison to normal tissues; and (2) using an integrated mRNA expression and DNA copy number profiling analysis using the TCGA dataset, the DNA copy number of the genes was positively correlated with the mRNA expression. RESULT: We identified 188 candidate-oncogenes. Among those, the high expression of SLC38A7 was a strong prognostic marker that was significantly associated with a poor prognosis in terms of both overall survival (OS) and recurrence-free survival in the TCGA dataset (P < 0.05). Additionally, 202 resected SCC specimens were also subjected to an immunohistochemical analysis. Patients with the high expression of SLC38A7 (alternative name is sodium-coupled amino acid transporters 7) protein showed significantly shorter OS in comparison to those with the low expression of SLC38A7 protein [median OS 3.9 years (95% confidence interval, 2.4-6.4 years) vs 2.2 years (95% confidence interval, 1.9-4.1 years); log rank test: P = 0.0021]. CONCLUSION: SLC38A7, which is the primary lysosomal glutamine transporter required for the extracellular protein-dependent growth of cancer cells, was identified as a candidate therapeutic target of SCC.

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PURPOSE: We sought to identify the symptoms and noninvasive test parameters associated with detrusor underactivity and develop a prediction model of detrusor underactivity. We analyzed clinical data on male patients with lower urinary tract symptoms who underwent pressure-flow studies. MATERIALS AND METHODS: Included in analysis were 909 men who met study criteria and whose data included the International Prostate Symptom Score, free uroflowmetry, post-void residual urine volume and prostate volume. Using these data we examined the significant symptoms and noninvasive test parameters associated with detrusor underactivity and developed a prediction model of detrusor underactivity. RESULTS: Of the 909 patients 454 (50%) were classified with detrusor underactivity. On logistic regression analysis older patient age, smaller prostate volume, a lower urgency symptom score, a higher weak stream symptom score and a lower maximum flow rate were selected as independent predictors of detrusor underactivity. The prediction model of detrusor underactivity consisting of these 5 factors showed satisfactory performance (C statistic 0.724). CONCLUSIONS: We developed a prediction model of detrusor underactivity in male patients with nonneurogenic lower urinary tract symptoms. The model was based on 5 predictive factors, namely older age, smaller prostate volume, 2 symptoms (less urgency and a weak stream) and a lower maximum flow rate. The model helps estimate the probability of detrusor underactivity in clinical practice without an invasive pressure-flow study.

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OBJECTIVE: To determine the factors predicting the subsequent development of pancreatic ductal adenocarcinoma in remnant pancreas (PDAC-RP) after partial pancreatectomy for PDAC. SUMMARY BACKGROUND DATA: PDAC-RP after partial pancreatectomy for PDAC is currently not so rare because of improved prognosis of PDAC patients due to recent advances in surgical techniques and adjuvant therapy. However, the predictive factors related to PDAC-RP remain unknown. METHODS: We retrospectively reviewed the clinicopathological data of a consecutive series of 379 patients with PDAC treated by partial pancreatectomy between 1992 and 2015; 14 patients (3.69%) had PDAC-RP. Clinicopathological variables were compared between PDAC-RP and non-PDAC-RP. RESULTS: In univariate analysis, concomitant intraductal papillary mucinous neoplasm (IPMN) (P = 0.0005), cancer location (body/tail) (P = 0.0060), and lower T factor in UICC (P = 0.0039) were correlated with PDAC-RP development. Multivariate analysis revealed concomitant IPMN (P = 0.0135) to be an independent predictive factor for PDAC-RP. PDAC concomitant with IPMN had higher cumulative incidence of PDAC-RP (47.5%/10 yrs) than PDAC without IPMN (9.96%/10 yrs) (P = 0.0071). Moreover, the density of pancreatic intraepithelial neoplasia lesions in the background pancreas of cases of PDAC concomitant with IPMN (1.86/cm) was higher than that of cases of PDAC without IPMN (0.91/cm) (P = 0.0007). CONCLUSIONS: Concomitant IPMN in PDAC is an independent predictive factor for the development of new PDAC in remnant pancreas. Cancer susceptibility of remnant pancreas after resection for PDAC concomitant with IPMN is probably due to an increased density of pancreatic intraepithelial neoplasia lesions.

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Human breast cancers comprise a complex and highly heterogeneous population of tumor cells. Intratumor heterogeneity is an underlying cause of resistance to effective therapies and disease recurrence. To explore prognostic factors based on intratumor heterogeneity, we analyzed genomic mutations in breast cancer patients registered in The Cancer Genome Atlas. We calculated the variant allele frequency (VAF) at each mutation site and evaluated the associations of VAFs with the prognosis of breast cancer. VAFs of HMCN1 correlated with the prognosis and lymph node status. Although the detailed function of HMCN1 remains unknown, it is located in extracellular matrix and the mutation in the gene might be associated with cancer cell invasion and metastasis. This finding suggests that HMCN1 is a potential metastatic factor and can be a candidate gene for targeted breast cancer therapy.

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Human cancers accumulate various mutations during development and consist of highly heterogeneous cell populations. This phenomenon is called intratumor heterogeneity (ITH). ITH is known to be involved in tumor growth, progression, invasion, and metastasis, presenting obstacles to accurate diagnoses and effective treatments. Numerous studies have explored the dynamics of ITH, including constructions of phylogenetic trees in cancer samples using multiregional ultradeep sequencing and simulations of evolution using statistical models. Although ITH is associated with prognosis, it is still challenging to use the characteristics of ITH as prognostic factors because of difficulties in quantifying ITH precisely. In this study, we analyzed the relationship between patient prognosis and the distribution of variant allele frequencies (VAFs) in cancer samples (n = 6,064) across 16 cancer types registered in The Cancer Genome Atlas. To measure VAF distributions multidimensionally, we adopted parameters that define the shape of VAF distributions and evaluated the relationships between these parameters and prognosis. In seven cancer types, we found significant relationships between prognosis and VAF distributions. Moreover, we observed that samples with a larger amount of mutations were not necessarily linked to worse prognosis. By evaluating the ITH from multidimensional viewpoints, it will be possible to provide a more accurate prediction of cancer prognosis.

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Epigenetic mechanisms such as DNA methylation or histone modifications are essential for the regulation of gene expression and development of tissues. Alteration of epigenetic modifications can be used as an epigenetic biomarker for diagnosis and as promising targets for epigenetic therapy. A recent study explored cancer-cell specific epigenetic biomarkers by examining different types of epigenetic modifications simultaneously. However, it was based on microarrays and reported biomarkers that were also present in normal cells at a low frequency. Here, we first analyzed multi-omics data (including ChIP-Seq data of six types of histone modifications: H3K27ac, H3K4me1, H3K9me3, H3K36me3, H3K27me3, and H3K4me3) obtained from 26 lung adenocarcinoma cell lines and a normal cell line. We identified six genes with both H3K27ac and H3K4me3 histone modifications in their promoter regions, which were not present in the normal cell line, but present in ≥85% (22 out of 26) and ≤96% (25 out of 26) of the lung adenocarcinoma cell lines. Of these genes, NUP210 (encoding a main component of the nuclear pore complex) was the only gene in which the two modifications were not detected in another normal cell line. RNA-Seq analysis revealed that NUP210 was aberrantly overexpressed among the 26 lung adenocarcinoma cell lines, although the frequency of NUP210 overexpression was lower (19.3%) in 57 lung adenocarcinoma tissue samples studied and stored in another database. This study provides a basis to discover epigenetic biomarkers highly specific to a certain cancer, based on multi-omics data at the cell population level.

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