H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling.

BACKGROUND: Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated. METHODS: The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC. RESULTS: Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis. CONCLUSION: This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.

Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

Receptor tyrosine kinase fusions act as a significant alternative driver of the serrated pathway in colorectal cancer development.

Serrated polyps are a clinically and molecularly heterogeneous group of lesions that can contribute to the development of colorectal cancers (CRCs). However, the molecular mechanism underlying the development of serrated lesions is still not well understood. Here, we combined multiple approaches to analyze the genetic alterations in 86 colorectal adenomas (including 35 sessile serrated lesions, 15 traditional adenomas, and 36 conventional adenomatous polyps). We also investigated the in vitro and in vivo oncogenic properties of a novel variant of the NCOA4-RET fusion gene. Molecular profiling revealed that sessile serrated lesions and traditional serrated adenomas have distinct clinicopathological and molecular features. Moreover, we identified receptor tyrosine kinase translocations exclusively in sessile serrated lesions (17%), and the observation was validated in a separate cohort of 34 sessile serrated lesions (15%). The kinase fusions as well as the BRAF and KRAS mutations were mutually exclusive to each other. Ectopic expression of a novel variant of the NCOA4-RET fusion gene promoted cell proliferation in vitro and in vivo, and the proliferation was significantly suppressed by RET kinase inhibitors. All of these underscored the importance of mitogen-activated protein kinase (MAPK) pathway activation in the serrated pathway of colorectal tumorigenesis. In addition, we demonstrated that the kinase fusion may occur early in the precursor lesion and subsequent loss of TP53 may drives the transformation to carcinoma during serrated tumorigenesis. In conclusion, we identified kinase fusions as a significant alternative driver of the serrated pathway in colorectal cancer development, and detecting their presence may serve as a biomarker for the diagnosis of sessile serrated lesions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

WITER: a powerful method for estimation of cancer-driver genes using a weighted iterative regression modelling background mutation counts.

Genomic identification of driver mutations and genes in cancer cells are critical for precision medicine. Due to difficulty in modelling distribution of background mutation counts, existing statistical methods are often underpowered to discriminate cancer-driver genes from passenger genes. Here we propose a novel statistical approach, weighted iterative zero-truncated negative-binomial regression (WITER, http://grass.cgs.hku.hk/limx/witer or KGGSeq,http://grass.cgs.hku.hk/limx/kggseq/), to detect cancer-driver genes showing an excess of somatic mutations. By fitting the distribution of background mutation counts properly, this approach works well even in small or moderate samples. Compared to alternative methods, it detected more significant and cancer-consensus genes in most tested cancers. Applying this approach, we estimated 229 driver genes in 26 different types of cancers. In silico validation confirmed 78% of predicted genes as likely known drivers and many other genes as very likely new drivers for corresponding cancers. The technical advances of WITER enable the detection of driver genes in TCGA datasets as small as 30 subjects and rescue of more genes missed by alternative tools in moderate or small samples.

The Landscape of Actionable Molecular Alterations in Immunomarker-Defined Large-Cell Carcinoma of the Lung.

INTRODUCTION: Patients with pulmonary large-cell carcinoma (LCC) have poor prognosis and limited treatment options. The identification of clinically actionable molecular alterations helps to guide personalized cancer treatment decisions. PATIENTS AND METHODS: A consecutive cohort of 789 resected NSCLC cases were reviewed. Fifty-nine NSCLC cases lacking morphologic differentiation, accounting for 7.5% of all resected NSCLCs, were identified and further characterized by immunohistochemistry according to the 2015 WHO lung tumor classification. Molecular alterations were investigated by multiple technologies including target capture sequencing, immunohistochemistry, and fluorescence in situ hybridizations. RESULTS: Of 59 NSCLC cases lacking morphologic differentiation, 20 (33.9%) were reclassified as adenocarcinoma (LCC-AD), 14 (23.7%) as squamous cell carcinoma (LCC-SqCC), and 25 (42.4%) as LCC-Null. Approximately 92% of LCC-Null, 95% of LCC-AD, and 86% of LCC-SqCC harbored clinically relevant alterations. Alterations characteristic of adenocarcinoma (EGFR, KRAS, ALK receptor tyrosine kinase [ALK], ROS1, and serine/threonine kinase 11 [STK11]) were detected in the LCC-AD subgroup but not in LCC-SqCC, whereas squamous-lineage alterations (phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha [PIK3CA], SRY-box 2 [SOX2], fibroblast growth factor receptor 1 [FGFR1], and AKT1) were detected in the LCC-SqCC subgroup but not in the LCC-AD group. Although some LCC-Null tumors displayed a genetic profile similar to either adenocarcinoma or squamous-cell carcinoma, more than half of the LCC-Null group were completely devoid of recognizable lineage-specific genetic profiles. High programmed death ligand 1 expression and high frequency of cell cycle regulatory gene alterations were found in the LCC-Null group offering alternative options of targeted therapy. CONCLUSIONS: This comprehensive molecular study provided further insight into the genetic architecture of LCC. The presence of clinically actionable alterations in a majority of the tumors allowed personalized treatment to emerge.

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.

Assessment of programmed cell death ligand-1 expression by 4 diagnostic assays and its clinicopathological correlation in a large cohort of surgical resected non-small cell lung carcinoma.

Immune checkpoint blockade targeting the PD-1/PD-L1 axis has recently demonstrated efficacy and promise in cancer treatment. Appropriate biomarker selection is therefore essential for improving treatment efficacy. However, the establishment of PD-L1 assay in pathology laboratories is complicated by the presence of multiple testing platforms using different scoring systems. Here we assessed the PD-L1 expression in 713 consecutive non-small cell lung carcinomas by four commercially available PD-L1 immunohistochemical assays, namely, 22C3, 28-8, SP142 and SP263. The analytical performances of the four assays and diagnostic performances across clinically relevant cutoffs were evaluated. The prevalence of PD-L1 (22C3) expression was 21% with a ≥50% cutoff and 56% with a ≥1% cutoff. High PD-L1 expression (using a ≥50% cutoff) was significantly associated with male sex (P = 0.001), ever smoking history (P < 0.001), squamous cell carcinoma (P = 0.001), large cell carcinoma (P < 0.001), lymphoepithelioma-like carcinoma (P = 0.006), sarcomatoid carcinoma (P < 0.001), mutant KRAS (P = 0.005) and wild-type EGFR (P = 0.003). Elevated PD-L1 expression was also significantly associated with shorter survival in patients with adenocarcinoma (log-rank P = 0.026) and remained an independent prognostic factor by multivariable analysis. Among the four assays, 22C3, 28-8 and SP263 were highly concordant for tumor cell scoring. With a cutoff of ≥50% (i.e., the threshold for first-line patient selection), inter-rater agreement was high among the three assays with percentage agreement >97%. In conclusion, three PD-L1 assays showed good analytical performance and a high agreement with each other, but not all cases were correctly classified using the same clinical cutoff. Further studies comparing the predictive value of these assays are required to address the interchangeability of these assays for clinical use.

PIEZO1 functions as a potential oncogene by promoting cell proliferation and migration in gastric carcinogenesis.

Gastric cancer (GC) is one of the most common malignancies in Asian areas. PIEZO1 has been implied to regulate epithelial homeostasis to play an important role in physiological processes. It is also related to tumor initiation and progression. However, the role of PIEZO1 has not yet been explored in GC. The expression of PIEZO1 in GC cell lines and primary samples was determined by qRT-PCR and Western blot. The clinical correlation of PIEZO1 in GC was evaluated by immunohistochemistry on tissue microarray. The oncogenic role of PIEZO1 was demonstrated in gastric tumorigenesis through a series of functional assays, including cell proliferation, cell invasion, and flow cytometry analysis. Drug sensitivity was also assessed by PIEZO1 knockdown experiment. PIEZO1 exhibited an upregulation in most of the GC cell lines and primary samples compared with non-tumorous gastric epithelial tissues. Increase of PIEZO1 was associated with poor disease specific survival. PIEZO1 knockdown led to inhibitory effect by suppressing cell proliferation and invasion and inhibiting xenograft formation. Decreased PIEZO1 enhanced the sensitivity of Cisplatin or 5-FU treatment. Morphological alteration was also observed in siPIEZO1 treated cells. GTP-Rac1 showed accumulated activated form, while total-RhoA was decreased accompanied with PIEZO1 knockdown. In the present study, PIEZO1 is required for cell proliferation, migration and invasion to promote GC progression. PIEZO1 also maintains cellular morphology related to GC cell motility by regulating the activity of Rho GTPase family members. Our data not only suggested a novel prognostic marker, but also provided a useful clinical therapeutic target for GC.

SRGAP1, a crucial target of miR-340 and miR-124, functions as a potential oncogene in gastric tumorigenesis.

Slit-Robo GTPase-activating protein 1 (SRGAP1) functions as a GAP for Rho-family GTPases and downstream of Slit-Robo signaling. We aim to investigate the biological function of SRGAP1 and reveal its regulation by deregulated microRNAs (miRNAs) in gastric cancer (GC). mRNA and protein expression of SRGAP1 were examined by quantitative reverse transcription PCR (qRT-PCR) and western blot. The biological role of SRGAP1 was demonstrated through siRNA-mediated knockdown experiments. The regulation of SRGAP1 by miR-340 and miR-124 was confirmed by western blot, dual luciferase activity assays and rescue experiments. SRGAP1 is overexpressed in 9 out of 12 (75.0%) GC cell lines. In primary GC samples from TCGA cohort, SRGAP1 shows gene amplification in 5/258 (1.9%) of cases and its mRNA expression demonstrates a positive correlation with copy number gain. Knockdown of SRGAP1 in GC cells suppressed cell proliferation, reduced colony formation, and significantly inhibited cell invasion and migration. Luciferase reporter assays revealed that SRGAP1 knockdown significantly inhibited Wnt/ß-catenin pathway. In addition, SRGAP1 was found to be a direct target of two tumor-suppressive miRNAs, miR-340 and miR-124. Concordantly, these two miRNAs were downregulated in primary gastric tumors and these decreasing levels w5ere associated with poor outcomes. Expression of miR-340 and SRGAP1 displayed a reverse relationship in primary samples and re-expressed SRGAP1, rescued the anti-cancer effects of miR-340. Taken together, these data strongly suggest that, apart from gene amplification and mutation, the activation of SRGAP1 in GC is partly due to the downregulation of tumor-suppressive miRNAs, miR-340 and miR-124. Thus SRGAP1 is overexpressed in gastric carcinogenesis and plays an oncogenic role through activating Wnt/ß-catenin pathway.

EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Meta-analysis of genome-wide association studies identifies two loci associated with circulating osteoprotegerin levels.

Osteoprotegerin (OPG) is involved in bone homeostasis and tumor cell survival. Circulating OPG levels are also important biomarkers of various clinical traits, such as cancers and atherosclerosis. OPG levels were measured in serum or in plasma. In a meta-analysis of genome-wide association studies in up to 10 336 individuals from European and Asian origin, we discovered that variants >100 kb upstream of the TNFRSF11B gene encoding OPG and another new locus on chromosome 17q11.2 were significantly associated with OPG variation. We also identified a suggestive locus on chromosome 14q21.2 associated with the trait. Moreover, we estimated that over half of the heritability of OPG levels could be explained by all variants examined in our study. Our findings provide further insight into the genetic regulation of circulating OPG levels.

Predicting mendelian disease-causing non-synonymous single nucleotide variants in exome sequencing studies.

Exome sequencing is becoming a standard tool for mapping Mendelian disease-causing (or pathogenic) non-synonymous single nucleotide variants (nsSNVs). Minor allele frequency (MAF) filtering approach and functional prediction methods are commonly used to identify candidate pathogenic mutations in these studies. Combining multiple functional prediction methods may increase accuracy in prediction. Here, we propose to use a logit model to combine multiple prediction methods and compute an unbiased probability of a rare variant being pathogenic. Also, for the first time we assess the predictive power of seven prediction methods (including SIFT, PolyPhen2, CONDEL, and logit) in predicting pathogenic nsSNVs from other rare variants, which reflects the situation after MAF filtering is done in exome-sequencing studies. We found that a logit model combining all or some original prediction methods outperforms other methods examined, but is unable to discriminate between autosomal dominant and autosomal recessive disease mutations. Finally, based on the predictions of the logit model, we estimate that an individual has around 5% of rare nsSNVs that are pathogenic and carries ~22 pathogenic derived alleles at least, which if made homozygous by consanguineous marriages may lead to recessive diseases.

Heritability of serum osteoprotegerin.

Serum osteoprotegerin (OPG) level is a key biomarker for numerous traits of clinical importance like diabetes, coronary artery disease, blood pressure, lipid profile, and cancers, but its genetic basis remains poorly understood. We estimated the heritability (h(2)) of serum OPG level in 1442 southern Chinese subjects from 306 families. The h(2) for unadjusted OPG was 0.62 for females and 0.17 for males; and for age-adjusted OPG, 0.75 for females and 0.37 for males. Adjustment for lifestyle factors including calcium and phytoestrogen intake, exercise, smoking, and alcohol consumption exerted only a modest effect on the h(2). In conclusion, we confirmed that circulating OPG is a heritable trait and there is a significant difference in heritability between sexes.

Risk prediction of complex diseases from family history and known susceptibility loci, with applications for cancer screening.

Risk prediction based on genomic profiles has raised a lot of attention recently. However, family history is usually ignored in genetic risk prediction. In this study we proposed a statistical framework for risk prediction given an individual's genotype profile and family history. Genotype information about the relatives can also be incorporated. We allow risk prediction given the current age and follow-up period and consider competing risks of mortality. The framework allows easy extension to any family size and structure. In addition, the predicted risk at any percentile and the risk distribution graphs can be computed analytically. We applied the method to risk prediction for breast and prostate cancers by using known susceptibility loci from genome-wide association studies. For breast cancer, in the population the 10-year risk at age 50 ranged from 1.1% at the 5th percentile to 4.7% at the 95th percentile. If we consider the average 10-year risk at age 50 (2.39%) as the threshold for screening, the screening age ranged from 62 at the 20th percentile to 38 at the 95th percentile (and some never reach the threshold). For women with one affected first-degree relative, the 10-year risks ranged from 2.6% (at the 5th percentile) to 8.1% (at the 95th percentile). For prostate cancer, the corresponding 10-year risks at age 60 varied from 1.8% to 14.9% in the population and from 4.2% to 23.2% in those with an affected first-degree relative. We suggest that for some diseases genetic testing that incorporates family history can stratify people into diverse risk categories and might be useful in targeted prevention and screening.

GATES: a rapid and powerful gene-based association test using extended Simes procedure.

The gene has been proposed as an attractive unit of analysis for association studies, but a simple yet valid, powerful, and sufficiently fast method of evaluating the statistical significance of all genes in large, genome-wide datasets has been lacking. Here we propose the use of an extended Simes test that integrates functional information and association evidence to combine the p values of the single nucleotide polymorphisms within a gene to obtain an overall p value for the association of the entire gene. Our computer simulations demonstrate that this test is more powerful than the SNP-based test, offers effective control of the type 1 error rate regardless of gene size and linkage-disequilibrium pattern among markers, and does not need permutation or simulation to evaluate empirical significance. Its statistical power in simulated data is at least comparable, and often superior, to that of several alternative gene-based tests. When applied to real genome-wide association study (GWAS) datasets on Crohn disease, the test detected more significant genes than SNP-based tests and alternative gene-based tests. The proposed test, implemented in an open-source package, has the potential to identify additional novel disease-susceptibility genes for complex diseases from large GWAS datasets.