RNA expression of 6 genes from metastatic mucosal gastric cancer serves as the global prognostic marker for gastric cancer with functional validation.

BACKGROUND: Molecular analysis of advanced tumors can increase tumor heterogeneity and selection bias. We developed a robust prognostic signature for gastric cancer by comparing RNA expression between very rare early gastric cancers invading only mucosal layer (mEGCs) with lymph node metastasis (Npos) and those without metastasis (Nneg). METHODS: Out of 1003 mEGCs, all Npos were matched to Nneg using propensity scores. Machine learning approach comparing Npos and Nneg was used to develop prognostic signature. The function and robustness of prognostic signature was validated using cell lines and external datasets. RESULTS: Extensive machine learning with cross-validation identified the prognostic classifier consisting of four overexpressed genes (HDAC5, NPM1, DTX3, and PPP3R1) and two downregulated genes (MED12 and TP53), and enabled us to develop the risk score predicting poor prognosis. Cell lines engineered to high-risk score showed increased invasion, migration, and resistance to 5-FU and Oxaliplatin but maintained sensitivity to an HDAC inhibitor. Mouse models after tail vein injection of cell lines with high-risk score revealed increased metastasis. In three external cohorts, our risk score was identified as the independent prognostic factor for overall and recurrence-free survival. CONCLUSION: The risk score from the 6-gene classifier can successfully predict the prognosis of gastric cancer.

KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis.

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

Atopic dermatitis is associated with the clinical course of inflammatory bowel disease.

OBJECTIVES: There are a few studies about the relationship between inflammatory bowel disease (IBD) and atopic dermatitis (AD). It is implied that both diseases have common pathophysiologic mechanisms and can affect each other. However, little information is available on the effect of AD on the clinical course of patients with IBD. METHODS: This is a multi-center, retrospective, observational study. We define AD as a chronic eczematoid dermatosis diagnosed by dermatologists. Patients with concurrent IBD and AD were defined as a case group. Age, gender, and IBD subtype-matched patients without AD were included as a reference group. RESULTS: The numbers of patients in the case and reference groups were 61 and 122 respectively. There was a significantly shorter biologics-free survival in the case group than that in the reference group according to the multivariable-adjusted Cox regression analysis with the onset age, disease duration, smoking status, use of steroid, use of immunomodulator, initial C-reactive protein, initial erythrocyte sedimentation rate, presence of other allergic diseases and initial disease severity [hazard ratio (HR) 1.828, 95% confidence interval (CI) 1.022-3.271, p = .042]. The trend was consistent in the subgroup analysis with ulcerative colitis (HR 3.498, 95% CI 1.066-11.481, p = .039), but not with Crohn's disease (HR 1.542, 95% CI 0.720-3.301, p = .265). CONCLUSIONS: AD showed a significant effect on the biologics-free survival of patients with IBD and especially the UC subtype. Further mechanistic research is required to elucidate the pathogenesis of AD on the clinical course of IBD.

Association between severe hepatic steatosis examined by Fibroscan and the risk of high-risk colorectal neoplasia.

The prevalence of colorectal neoplasm in patients with non-alcoholic fatty liver disease has increased twice as high as that in the general population. FibroScan is a new modality for evaluating hepatic steatosis. This study aimed to investigate the relationship between the risk of high-risk colorectal neoplasia and hepatic steatosis examined using FibroScan. This was a cross sectional study of prospectively enrolled subjects who were scheduled to undergo index colonoscopy as a health screening between March 2018 and February 2019. The severity of steatosis was graded as normal, mild, moderate, or severe using FibroScan. A total of 140 consecutive subjects were enrolled and sequentially examined using FibroScan and colonoscopy. Subjects with hepatic steatosis had more high-risk colorectal neoplasia than those without hepatic steatosis. In addition, tumor size was larger in subjects with hepatic steatosis. In multivariable analysis, severe hepatic steatosis was an independent risk factor for high-risk colorectal neoplasia (adjusted odds ratio: 3.309, confidence interval: 1.043-10.498, p = 0.042). Alcohol consumption was also identified as a risk factor for high-risk colorectal neoplasia. In conclusion, severe hepatic steatosis on FibroScan is associated with the development of high-risk colorectal neoplasia. Thus, physicians should be aware of the association between colorectal neoplasia and hepatic steatosis assessed by FibroScan and its clinical implications.

Mice transgenic for human CTLA4-CD28 fusion gene show proliferation and transformation of ATLL-like and AITL-like T cells.

CTLA4-CD28 gene fusion has been reported to occur in diverse types of T cell lymphoma. The fusion event is expected to convert inhibitory signals to activating signals and promote proliferation and potentially transformation of T cells. To test the function of the CTLA4-CD28 fusion gene in vivo, we generated a murine model that expresses the gene in a T cell-specific manner. The transgenic mice have shorter life spans and display inflammatory responses including lymphadenopathy and splenomegaly. T cells in turn show higher levels of activation and infiltrate various organs including the lung and skin. T cells, in particular CD4+ helper T cells, were also readily transplantable to immunocompromised mice. Transcriptomic profiling revealed that the gene expression pattern in CD4 + T cells closely resembles that of adult T cell leukemia/lymphoma (ATLL) and that of angioimmunoblastic T cell lymphoma (AITL) tissues. Consistently, we detected supernumerary FOXP3+ cells and PD-1+ cells in transgenic and transplanted mice. This is the first report demonstrating the transforming activity of the CTLA4-CD28 fusion gene in vivo, and this murine model should be useful in dissecting the molecular events downstream to this mutation.

Comprehensive Analysis of Alternative Splicing in Gastric Cancer Identifies Epithelial-Mesenchymal Transition Subtypes Associated with Survival.

Alternatively spliced RNA isoforms are a hallmark of tumors, but their nature, prevalence, and clinical implications in gastric cancer have not been comprehensively characterized. We systematically profiled the splicing landscape of 83 gastric tumors and matched normal mucosa, identifying and experimentally validating eight splicing events that can classify all gastric cancers into three subtypes: epithelial-splicing (EpiS), mesenchymal-splicing (MesS), and hybrid-splicing. These subtypes were associated with distinct molecular signatures and epithelial-mesenchymal transition markers. Subtype-specific splicing events were enriched in motifs for splicing factors RBM24 and ESRP1, which were upregulated in MesS and EpiS tumors, respectively. A simple classifier based only on RNA levels of RBM24 and ESRP1, which can be readily implemented in the clinic, was sufficient to distinguish gastric cancer subtypes and predict patient survival in multiple independent patient cohorts. Overall, this study provides insights into alternative splicing in gastric cancer and the potential clinical utility of splicing-based patient classification. SIGNIFICANCE: This study presents a comprehensive analysis of alternative splicing in the context of patient classification, molecular mechanisms, and prognosis in gastric cancer.

Elucidating molecular mechanisms of acquired resistance to BRAF inhibitors in melanoma using a microfluidic device and deep sequencing.

BRAF inhibitors (e.g., vemurafenib) are widely used to treat metastatic melanoma with the BRAF V600E mutation. The initial response is often dramatic, but treatment resistance leads to disease progression in the majority of cases. Although secondary mutations in the mitogen-activated protein kinase signaling pathway are known to be responsible for this phenomenon, the molecular mechanisms governing acquired resistance are not known in more than half of patients. Here we report a genome- and transcriptome-wide study investigating the molecular mechanisms of acquired resistance to BRAF inhibitors. A microfluidic chip with a concentration gradient of vemurafenib was utilized to rapidly obtain therapy-resistant clones from two melanoma cell lines with the BRAF V600E mutation (A375 and SK-MEL-28). Exome and transcriptome data were produced from 13 resistant clones and analyzed to identify secondary mutations and gene expression changes. Various mechanisms, including phenotype switching and metabolic reprogramming, have been determined to contribute to resistance development differently for each clone. The roles of microphthalmia-associated transcription factor, the master transcription factor in melanocyte differentiation/dedifferentiation, were highlighted in terms of phenotype switching. Our study provides an omics-based comprehensive overview of the molecular mechanisms governing acquired resistance to BRAF inhibitor therapy.

FCN3 functions as a tumor suppressor of lung adenocarcinoma through induction of endoplasmic reticulum stress.

In this study, we report a novel function of FCN3 (Ficolin 3), a secreted lectin capable of activating the complement pathway, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of FCN3 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of FCN3 was shown to be significantly correlated with increased mortality among LUAD patients. Interestingly, while ectopic expression of FCN3 led to cell cycle arrest and apoptosis in A549 and H23 cells derived from LUAD, the secreted form of the protein had no effect on the cells. Rather, we found evidence indicating that activation of the unfolded protein response from endoplasmic reticulum (ER) stress is induced by ectopic expression of FCN3. Consistently, inhibition of ER stress response led to enhanced survival of the LUAD cells. Of note, the fibrinogen domain, which is not secreted, turned out to be both necessary and sufficient for induction of apoptosis when localized to ER, consistent with our proposed mechanism. Collectively, our data indicate that FCN3 is a tumor suppressor gene functioning through induction of ER stress.

Expectations and blind spots for structural variation detection from long-read assemblies and short-read genome sequencing technologies.

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and medical genetic initiatives are reliant upon short-read whole-genome sequencing (srWGS), which presents challenges for the detection of structural variants (SVs) relative to emerging long-read WGS (lrWGS) technologies. Given this ubiquity of srWGS in large-scale genomics initiatives, we sought to establish expectations for routine SV detection from this data type by comparison with lrWGS assembly, as well as to quantify the genomic properties and added value of SVs uniquely accessible to each technology. Analyses from the Human Genome Structural Variation Consortium (HGSVC) of three families captured ~11,000 SVs per genome from srWGS and ~25,000 SVs per genome from lrWGS assembly. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplication (SD) and simple repeat (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of reference sequence, we observed extremely high (93.8%) concordance between technologies for deletions in these datasets. In contrast, lrWGS was superior for detection of insertions across all genomic contexts. Given that non-SD/SR sequences encompass 95.9% of currently annotated disease-associated exons, improved sensitivity from lrWGS to discover novel pathogenic deletions in these currently interpretable genomic regions is likely to be incremental. However, these analyses highlight the considerable added value of assembly-based lrWGS to create new catalogs of insertions and transposable elements, as well as disease-associated repeat expansions in genomic sequences that were previously recalcitrant to routine assessment.

Genome-scale CRISPR screening identifies cell cycle and protein ubiquitination processes as druggable targets for erlotinib-resistant lung cancer.

Erlotinib is highly effective in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. However, despite initial favorable responses, most patients rapidly develop resistance to erlotinib soon after the initial treatment. This study aims to identify new genes and pathways associated with erlotinib resistance mechanisms in order to develop novel therapeutic strategies. Here, we induced knockout (KO) mutations in erlotinib-resistant human lung cancer cells (NCI-H820) using a genome-scale CRISPR-Cas9 sgRNA library to screen for genes involved in erlotinib susceptibility. The spectrum of sgRNAs incorporated among erlotinib-treated cells was substantially different to that of the untreated cells. Gene set analyses showed a significant depletion of 'cell cycle process' and 'protein ubiquitination pathway' genes among erlotinib-treated cells. Chemical inhibitors targeting genes in these two pathways, such as nutlin-3 and carfilzomib, increased cancer cell death when combined with erlotinib in both in vitro cell line and in vivo patient-derived xenograft experiments. Therefore, we propose that targeting cell cycle processes or protein ubiquitination pathways are promising treatment strategies for overcoming resistance to EGFR inhibitors in lung cancer.

iCSDB: an integrated database of CRISPR screens.

High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.

NuA3 HAT antagonizes the Rpd3S and Rpd3L HDACs to optimize mRNA and lncRNA expression dynamics.

In yeast, NuA3 histone acetyltransferase (NuA3 HAT) promotes acetylation of histone H3 lysine 14 (H3K14) and transcription of a subset of genes through interaction between the Yng1 plant homeodomain (PHD) finger and H3K4me3. Although NuA3 HAT has multiple chromatin binding modules with distinct specificities, their interdependence and combinatorial actions in chromatin binding and transcription remain unknown. Modified peptide pulldown assays reveal that the Yng1 N-terminal region is important for the integrity of NuA3 HAT by mediating the interaction between core subunits and two methyl-binding proteins, Yng1 and Pdp3. We further uncover that NuA3 HAT contributes to the regulation of mRNA and lncRNA expression dynamics by antagonizing the histone deacetylases (HDACs) Rpd3S and Rpd3L. The Yng1 N-terminal region, the Nto1 PHD finger and Pdp3 are important for optimal induction of mRNA and lncRNA transcription repressed by the Set2-Rpd3S HDAC pathway, whereas the Yng1 PHD finger-H3K4me3 interaction affects transcriptional repression memory regulated by Rpd3L HDAC. These findings suggest that NuA3 HAT uses distinct chromatin readers to compete with two Rpd3-containing HDACs to optimize mRNA and lncRNA expression dynamics.

Angioimmunoblastic T-cell lymphoma-like lymphadenopathy in mice transgenic for human RHOA with p.Gly17Val mutation.

A missense mutation in RHOA encoding p.Gly17 Val has been reported to occur frequently in angioimmunoblastic T-cell lymphoma (AITL). Here, we describe a murine model which expresses the human RHOA mutant gene product in a T-cell specific manner and develops AITL-like symptoms. Most transgenic mice feature with latency one or two enlarged lymph nodes characterized by aberrant lymph node architecture, extensive lymphocytic infiltration, extrafollicular meshwork of follicular dendritic cells (FDC) and arborized endothelial venules. Importantly, we provide evidence for expansion of PD-1+ follicular helper T (Tfh) cells which are the neoplastic cells of AITL. In addition, we saw proliferation of B-cells leading to hypergammaglobulinemia and the presence of dominant T cell clonal populations. Transplantation of lymph node cells to immunocompromised mice partly recreated lymphadenopathy after a long latency and with low penetrance suggesting that cells have undergone partial transformation to a premalignant state. Transcriptomic profiling revealed that the gene expression pattern within affected lymph nodes of the mice closely resembles that of AITL patients with the identical RHOA p.Gly17 Val mutation. The murine model should, therefore, be useful in dissecting pathogenesis of AITL at the molecular level particularly for the cases with the RHOA p.Gly17Val mutation.

Characterization of TNNC1 as a Novel Tumor Suppressor of Lung Adenocarcinoma.

In this study, we describe a novel function of TNNC1 (Troponin C1, Slow Skeletal and Cardiac Type), a component of actin-bound troponin, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of TNNC1 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of TNNC1 was shown to be strongly correlated with increased mortality among LUAD patients. Interestingly, TNNC1 expression was enhanced by suppression of KRAS, and ectopic expression of TNNC1 in turn inhibited KRASG12D-mediated anchorage independent growth of NIH3T3 cells. Consistently, activation of KRAS pathway in LUAD patients was shown to be strongly correlated with down-regulation of TNNC1. In addition, ectopic expression of TNNC1 inhibited colony formation of multiple LUAD cell lines and induced DNA damage, cell cycle arrest and ultimately apoptosis. We further examined potential correlations between expression levels of TNNC1 and various clinical parameters and found that low-level expression is significantly associated with invasiveness of the tumor. Indeed, RNA interference-mediated down-regulation of TNNC1 led to significant enhancement of invasiveness in vitro. Collectively, our data indicate that TNNC1 has a novel function as a tumor suppressor and is targeted for down-regulation by KRAS pathway during the carcinogenesis of LUAD.

Glutathione peroxidase-1 regulates adhesion and metastasis of triple-negative breast cancer cells via FAK signaling.

Triple-negative breast cancer (TNBC) cells, which do not express genes for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu, develop highly aggressive and metastatic tumors resistant to chemo- and hormonal therapies. We found that expression of glutathione peroxidase-1 (Gpx1) is silenced in the non-TNBC cells but significantly maintained in the TNBC cell lines. Such Gpx1 expression plays a vital role in the metastasis of TNBC cells by regulating cell adhesion. Transcriptomic and signaling pathway analyses demonstrate that depletion of Gpx1 essentially impairs cell adhesion/spreading by down-regulating FAK/c-Src activation. Mechanistically, Gpx1 interacts with FAK kinase and prevents the kinase inactivation by H2O2, not lipid hydroperoxide. As a result, depletion of Gpx1 suppresses lung metastasis of TNBC cells in vivo. Overall, our study identifies that Gpx1 is a redox safeguard of FAK kinase and its inhibition may provide an effective way to control the metastasis of deadly malignant TNBC.

Rpd3L HDAC links H3K4me3 to transcriptional repression memory.

Transcriptional memory is critical for the faster reactivation of necessary genes upon environmental changes and requires that the genes were previously in an active state. However, whether transcriptional repression also displays 'memory' of the prior transcriptionally inactive state remains unknown. In this study, we show that transcriptional repression of ∼540 genes in yeast occurs much more rapidly if the genes have been previously repressed during carbon source shifts. This novel transcriptional response has been termed transcriptional repression memory (TREM). Interestingly, Rpd3L histone deacetylase (HDAC), targeted to active promoters induces TREM. Mutants for Rpd3L exhibit increased acetylation at active promoters and delay TREM significantly. Surprisingly, the interaction between H3K4me3 and Rpd3L via the Pho23 PHD finger is critical to promote histone deacetylation and TREM by Rpd3L. Therefore, we propose that an active mark, H3K4me3 enriched at active promoters, instructs Rpd3L HDAC to induce histone deacetylation and TREM.

Integrative Profiling of Alternative Splicing Induced by U2AF1 S34F Mutation in Lung Adenocarcinoma Reveals a Mechanistic Link to Mitotic Stress.

Mutations in spliceosome components have been implicated in carcinogenesis of various types of cancer. One of the most frequently found is U2AF1 S34F missense mutation. Functional analyses of this mutation have been largely limited to hematological malignancies although the mutation is also frequently seen in other cancer types including lung adenocarcinoma (LUAD). We examined the impact of knockdown (KD) of wild type (wt) U2AF1 and ectopic expression of two splice variant S34F mutant proteins in terms of alternative splicing (AS) pattern and cell cycle progression in A549 lung cancer cells. We demonstrate that induction of distinct AS events and disruption of mitosis at distinct sub-stages result from KD and ectopic expression of the mutant proteins. Importantly, when compared with the splicing pattern seen in LUAD patients with U2AF1 S34F mutation, ectopic expression of S34F mutants but not KD was shown to result in common AS events in several genes involved in cell cycle progression. Our study thus points to an active role of U2AF1 S34F mutant protein in inducing cell cycle dysregulation and mitotic stress. In addition, alternatively spliced genes which we describe here may represent novel potential markers of lung cancer development.

GEMiCCL: mining genotype and expression data of cancer cell lines with elaborate visualization.

Database URL: GEMiCCL is available at https://www.kobic.kr/GEMICCL/.

Silencing of peroxiredoxin II by promoter methylation is necessary for the survival and migration of gastric cancer cells.

Peroxiredoxin (Prx), a family of ubiquitous thiol peroxidases, functions as a redox signaling regulator that controls cellular H2O2 in mammalian cells and has recently received attention for being overexpressed in various cancer types. In this study, we show that Prx type II (PrxII) is rather silenced in gastric cancer cells. PrxII expression is severely downregulated in 9 out of the 28 gastric cancer cell lines. Strikingly, PrxII expression is completely lost in three cell lines, MKN28, MKN74 and SNU484. Loss of PrxII expression is due to DNA methyltransferase 1-dependent methylation at the promoter region of the PrxII gene. Restoration of PrxII expression using a retroviral system markedly reduces the colony-forming ability and migratory activity of both MKN28 and SNU484 cells by inhibiting Src kinase. Mechanistically, PrxII peroxidase activity is essential for regulating gastric cancer cell migration. Bioinformatics analysis from The Cancer Genome Atlas stomach cancer data (STAD) revealed significantly low PrxII expression in gastric cancer patients and a negative correlation between PrxII expression and methylation levels. More importantly, low PrxII expression also strongly correlates with poor survival in cancer patients. Thus our study suggests that PrxII may be the first thiol peroxidase that simultaneously regulates both survival and metastasis in gastric cancer cells with high clinical relevance.