Human Neuralized is a novel tumour suppressor targeting Wnt/ß-catenin signalling in colon cancer.

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/ß-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic ß-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic ß-catenin, and reducing its cytoplasmic levels in a GSK3ß- and ß-TrCP-independent manner, indicating that NEURL-ß-catenin interactions can lead to a disruption of the canonical Wnt/ß-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/ß-catenin signalling.

Comprehensive Analyses of Somatic Copy Number Alterations and Mutations Based on the Adenoma-Carcinoma Sequence.

AIMS: Identifying molecular alterations in the adenoma and carcinoma components within the same tumor would greatly contribute to understanding the neoplastic progression of early colorectal cancer. METHODS AND RESULTS: We examined somatic copy number alterations (SCNAs) and mutations involved in the adenoma and carcinoma components obtained from the same tumor in 46 cases of microsatellite-stable carcinoma in adenoma, using a genome-wide SNP array and gene mutation panel. In addition, we also performed hierarchical clustering to determine the SCNA frequencies in the tumors, resulting in stratification of the samples into two subgroups according to SCNA frequency. Subgroup 1 was characterized by multiple SCNAs and carcinoma components exclusively, while Subgroup 2 was characterized by a low frequency of SCNAs and both the adenoma and carcinoma components. The numbers of total genes and genes with gains were higher in the carcinoma than adenoma components. The three most frequent gains in both components were located at 1p36.33-1q44, 2p25.3-2q37.3, and 3p26.3-3q29. However, no candidate genes mapped to these regions. APC and KRAS mutations were common in both components, whereas the frequency of TP53 mutations was statistically higher in the carcinoma than adenoma component. However, TP53 mutations were not correlated with SCNA frequency. CONCLUSIONS: We suggest that considerable SCNAs and TP53 mutations are required for progression from adenoma to carcinoma within the same intramucosal neoplastic lesion.

Maternal diet during gestation affect prostatic tissue component in SHR/Izm offspring.

BACKGROUND: Numerous studies have investigated the associations between maternal nutritional status and various diseases, with the underlying mechanism often attributed to epigenetic changes. However, limited research has been conducted on the relationship between maternal nutrition and benign prostatic hyperplasia (BPH). In this study, we aimed to explore the potential association between maternal nutrition and BPH using an animal experiment and evaluating the findings through fluorescent immunostaining and genetic analysis. METHODS: Female spontaneously hypertensive rats (SHR/Izm) were randomly assigned to three groups at the start of pregnancy: a standard diet group (SD; 17% protein, 7% fat), a low-protein diet group (LPD; 6% protein, 7% fat), and a high-fat diet group (HFD; 22% protein, 35% fat). The diets were maintained throughout gestation. After giving birth, both the mothers and their pups were exclusively fed a standard diet. Male pups were euthanized at 48 weeks, and their prostates were removed. The composition of the ventral prostate (VP) was evaluated using fluorescent immunostaining with antibodies for cytokeratin, vimentin, and Ki-67. Microarray analysis, real-time RT-PCR, and DNA methylation analysis using pyrosequencing were performed. Statistical analysis was conducted using one-way ANOVA and Tukey's multiple comparison test, with a significance level set at p < 0.05. RESULTS: Pups in the LPD group exhibited significant underweight from birth (1 day; SD vs. LPD vs. HFD: 4.46 vs. 4.08 vs. 4.35, p = 0.04) until weaning (21 days; SD vs. LPD vs. HFD: 30.8 vs. 27.4 vs. 29.2, p = 0.03). However, they exhibited catch-up growth, and there was no significant difference at 48 weeks (p = 0.84). The epithelial area in the ventral prostate was significantly increased in the LPD group (SD vs. LPD vs. HFD: 39% vs. 48% vs. 37%, p = 0.01), while the stromal area was significantly increased in the HFD group (SD vs. LPD vs. HFD: 11% vs. 11% vs. 15%, p < 0.01). Gene ontology analysis of the gene expression microarray showed increased activity in developmental processes (SD vs. LPD: p = 6.3E-03, SD vs. HFD: p = 7.2E-03), anatomical structure development (SD vs. LPD: p = 6.3E-03, SD vs. HFD: p = 5.3E-03), and cell differentiation (SD vs. LPD: p = 0.018, SD vs. HFD: p = 0.041) in both the LPD and HFD groups. Real-time RT-PCR revealed high expression levels of the transcription factors NFκB (p < 0.01) and Smad3 (p < 0.01) in both the LPD and HFD groups. XIAP, an apoptosis inhibitor, was increased in the LPD group (p = 0.02). The TGF beta pathway, associated with epithelial mesenchymal transition (EMT), and vimentin (p < 0.01) were upregulated in the HFD group. Pyrosequencing DNA methylation analysis of the TGF beta pathway indicated hypomethylation of TGFb1, TGFbR1, and Smad3 in all groups, although there were no significant differences. CONCLUSIONS: Our findings suggest that both maternal undernutrition and obesity influence the prostatic development of offspring. Maternal consumption of a low protein diet promotes epithelial hyperplasia through the upregulation of apoptosis inhibitors, while a high fat diet leads to increased stromal growth through the induction of EMT.

Downregulation of SMOC1 is associated with progression of colorectal traditional serrated adenomas.

BACKGROUND: Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions. METHODS: SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112). RESULTS: SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components. CONCLUSION: Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors.

The molecular profile of gastric intraepithelial foveolar type neoplasia based on somatic copy number alterations and multiple mutation analysis.

BACKGROUND: Gastric foveolar type neoplasia is a rare histological variant of gastric tumors. It is very difficult to differentiate between benign and malignant intraepithelial foveolar neoplasia (IFN). Although limited molecular alterations have been identified in IFNs, somatic copy number alterations (SCNAs), which are linked to tumor progression, have not been systematically evaluated in IFN. METHODS: The aim of the present study was to comprehensively examine SCNAs using a SNP array in 37 cases of IFN, compared with intestinal type dysplasia, including 39 low grade (LGD) and 32 high grade dysplasia (HGD) cases. In addition, gene mutations were evaluated using a gene panel. Finally, we attempted to determine molecular profiles using a hierarchical clustering analysis. RESULTS: Two patterns could be categorized according to the SCNAs in 108 tumors examined: high (subgroup 1) and low (subgroup 2) frequencies of SCNAs. Although IFN and LGD were associated with subgroup 2, HGD was found in both subgroups. The median numbers of total SCNAs and copy number gains were higher in IFN or HGD than in LGD. In addition, the IFN genotype was characterized by altered genes located at 4p13-4q35.2, including RAP1GDS1 and LEF1, which may be associated with IFN development. Finally, no significant mutations were found in IFNs using a gene panel. CONCLUSIONS: The current molecular profiles of IFN may help elucidate the mechanisms of IFN development.

Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

Differential Expression in the Tumor Microenvironment of mRNAs Closely Associated with Colorectal Cancer Metastasis.

BACKGROUND: Metastasis of colorectal cancer (CRC) is a major cause of CRC-related mortality. However, the detailed molecular mechanism of CRC metastasis remains unknown. A recent study showed that the tumor microenvironment, which includes cancer cells and the surrounding stromal cells, plays a major role in tumor invasion and metastasis. Identification of altered messenger RNA (mRNA) expression in the tumor microenvironment is essential to elucidation of the mechanisms responsible for tumor progression. This study investigated the mRNA expression of genes closely associated with metastatic CRC compared with non-metastatic CRC. METHODS: The samples examined were divided into cancer tissue and isolated cancer stromal tissue. The study examined altered mRNA expression in the cancer tissues using The Cancer Genome Atlas (TCGA) (377cases) and in 17 stromal tissues obtained from our laboratory via stromal isolation using an array-based analysis. In addition, 259 patients with CRC were enrolled to identify the association of the candidate markers identified with the prognosis of patients with stage 2 or 3 CRC. The study examined the enriched pathways identified by gene set enrichment analysis (GSEA) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) module in both the TCGA dataset and isolated stromal tissue. RESULTS: As a result, whereas tenascin-C, secreted phosphoprotein 1 and laminin were expressed in metastatic CRC cells, olfactory receptors (ORs) 11H1 and OR11H4 were expressed in stromal tissue cells isolated from metastatic CRC cases. Finally, upregulated expression of tenascin-C and OR11H4 was correlated with the outcome for CRC patients. CONCLUSION: The authors suggest that upregulated expression levels of tenascin-C and OR11H1 play an important role in CRC progression.

Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Serum amyloid A1 recruits neutrophils to the invasive front of T1 colorectal cancers.

BACKGROUND AND AIM: The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs. METHODS: Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n = 49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression. RESULTS: Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils. CONCLUSIONS: Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

Comprehensive Analysis of microRNA Expression During the Progression of Colorectal Tumors.

BACKGROUND: No effective early diagnostic biomarkers are available for colorectal cancer (CRC). Therefore, we sought to identify new biomarkers that could identify CRC from progression as a pre-cancerous lesion to its invasive form. Recent studies have shown that microRNAs (miRs) are associated with the onset of cancer invasion and progression. AIMS: We hypothesized that the identification of miRs associated with CRC might be useful to detect this disease at early stages. METHODS: We conducted an integrated analysis of 79 isolated colorectal tumor glands, including adenomas, intramucosal cancers, and invasive CRCs that showed a microsatellite stable phenotype using GeneChip miRNA 4.0 microarray assays. The colorectal tumors we examined were divided into 2 cohorts (42 in the first cohort and 37 in the second cohort). RESULTS: First, cluster analysis was performed to stratify expression patterns of multiple miRs that were pooled according to the following criteria: fold change in expression (< -2.0 or > 2.0), p < 0.05, and mature miRs. As a result, the expression patterns of pooled miRs were subdivided into 3 subgroups that were correlated with tumor grade. Each subgroup was characterized by specific miRs. In addition, we found that specific miRs, including miR-140-3p and miR-378i, were closely associated with cancer invasion. Finally, we analyzed paired dysregulated miRs between adenomatous and cancerous components present within the same tumor. DISCUSSION: We showed that several miRs were dysregulated during progression from adenoma to intramucosal cancer. Specific miRs may have key roles in progression from intramucosal tumor to invasive CRC.

Comprehensive analyses of microRNA and mRNA expression in colorectal serrated lesions and colorectal cancer with a microsatellite instability phenotype.

MicroRNA (miRNA) expression is dysregulated in human tumors, thereby contributing to tumorigenesis through altered expression of mRNA. Thus, identification of the relationships between miRNAs and mRNAs is important for evaluating the molecular mechanisms of tumors. In addition, elucidation of the molecular features of serrated lesions is essential in colorectal tumorigenesis. Here, we examined the relationships of miRNA and mRNA expressed in serrated lesions, including 26 sessile serrated lesions (SSLs), 12 traditional serrated adenomas (TSAs), and 11 colorectal cancers (CRCs) with a microsatellite instability (MSI) phenotype using crypt isolation. We divided the samples into the first and second cohorts for validation. Array-based expression analyses were used to evaluate miRNAs and mRNAs with opposite expression patterns in isolated tumor glands. In addition, we validated the relationships of miRNA/mRNA pairs in the second cohort using real-time polymerase chain reaction. We found that the expression of miRNA-5787 was correlated with reciprocal expression of two mRNAs, that is, SRRM2 and POLR2J3, in SSL samples. In TSA samples, two pairs of miRNAs/mRNAs showing opposite expression patterns, that is, miRNA-182-5p/ETF1 and miRNA-200b-3p/MYB, were identified. Ultimately, three pairs of miRNAs/mRNAs with opposite expression patterns, including miRNA-222-3p/SLC26A3, miRNA-6753-3p/FABP1, and miRNA-222-3p/OLFM4, were retained in CRC with an MSI phenotype. Finally, we performed transfection with an miR-222-3p mimic to confirm the expression of SLC26A3 and OLFM4; the results showed that ectopic expression of miR-222-3p moderately suppressed OLFM4 and downregulated SLC26A3 to some extent. Overall, our results provided basic insights into the evaluation of colorectal tumorigenesis of serrated lesions and CRC with an MSI phenotype.

Cribriform-type adenocarcinoma of the colorectum: comprehensive molecular analyses of a distinctive histologic subtype of colorectal cancer.

Colorectal adenocarcinoma (CRA) is characterized by marked heterogeneity and may be composed of an admixture of various histologic patterns, including well-formed gland and cribriform types. Although tumors displaying a prominent or predominant cribriform feature are frequently found in CRA, this type may contain specific histologic variants with a characteristic molecular alteration. We investigated the molecular features of 51 primary CRAs with a predominant cribriform histology using array-based analyses [somatic copy number alterations (SCNAs); mRNA expression]. Mutations (TP53, KRAS, PIK3CA and BRAF) and DNA methylation status were also analyzed. The crypt isolation method was used to obtain isolated tumor glands of each type separately. All patients were classified by their CRA histologic subtype into two groups: well-formed gland and cribriform. Next, we performed cluster analysis to stratify SCNA and mRNA expression patterns between the two subtypes. Two distinctive subgroups were stratified based on patterns of SCNA and mRNA expression and were correlated with each histologic subtype. The cribriform type was characterized by a high frequency of SCNA compared with that of the well-formed gland type and was closely associated with the expression of specific mRNAs. In addition, the frequency of KRAS mutation was significantly higher in the cribriform type than in the well-formed gland type. Finally, there was no difference in DNA methylation status between the two subtypes. Overall, these data suggest that the cribriform type provides important insights into colorectal carcinogenesis, suggesting specific potential histologic implications based on the molecular profile.

A genome-wide study of the relationship between chromosomal abnormalities and gene expression in colorectal tumors.

The role of somatic copy number alterations (SCNAs) that occur in colorectal tumors is poorly understood. SCNAs are correlated with corresponding gene expression changes that may contribute to neoplastic progression. Thus, we examined SCNAs and the expression of messenger RNAs (mRNAs) located at corresponding loci in colorectal neoplasia, a progression model of human neoplasm. We used 42 colorectal neoplastic samples, including adenomas, intramucosal cancers (IMC) and invasive colorectal cancers (CRC) that were microsatellite stable (MSS) using a genome-wide SNP array and gene expression array (first cohort). In addition, validation analyses were examined (37 colorectal neoplasias). None of the mRNAs with a corresponding SCNA was found in the adenomas. However, three mRNAs, including ARFGEF2 at 20q13.13, N4BP2L2 at 13q13.1 and OLFM4 at 13q14.3 with a copy number (CN) gain at the corresponding locus were upregulated in IMCs of the first cohort. Moreover, upregulated expression of ARFGEF2 and OLFM4 was upregulated in the validation analysis. Finally, 28 mRNAs with gains of corresponding loci were pooled in invasive CRC of the first cohort. The mRNAs, including ACSS2 (20q11.22), DDX27 (20q13.13), MAPRE1 (20q11.21), OSBPL2 (20q11.22) and PHF20 (20q11.22-q11.23) with CN gains of the corresponding loci were identified in 28 mRNAs. Four of these mRNAs (DDX27, MAPRE1, OSBPL2 and PHF20) were upregulated in the invasive CRC in the validation analysis. We conclude that specific 13q and 22q CN gains with gene expression changes in the corresponding loci may play an important role in IMC cells' progression into invasive CRC.

Activated macrophages promote invasion by early colorectal cancer via an interleukin 1ß-serum amyloid A1 axis.

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA-sequencing (RNA-seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP-1 cells suggested that interleukin 1ß (IL-1ß) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL-1ß. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1-like/M2-like macrophages at SAA1-positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase-9 in macrophages. Our data suggest that tumor-associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target.

Regulatory roles of claudin-1 in cell adhesion and microvilli formation.

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.

Integrated genetic and epigenetic analysis of cancer-related genes in non-ampullary duodenal adenomas and intramucosal adenocarcinomas.

The molecular and clinical characteristics of non-ampullary duodenal adenomas and intramucosal adenocarcinomas are not fully understood because they are rare. To clarify these characteristics, we performed genetic and epigenetic analysis of cancer-related genes in these lesions. One hundred and seven non-ampullary duodenal adenomas and intramucosal adenocarcinomas, including 100 small intestinal-type tumors (90 adenomas and 10 intramucosal adenocarcinomas) and 7 gastric-type tumors (2 pyloric gland adenomas and 5 intramucosal adenocarcinomas), were investigated. Using bisulfite pyrosequencing, we assessed the methylation status of CpG island methylator phenotype (CIMP) markers and MLH1. Then using next-generation sequencing, we performed targeted exome sequence analysis within 75 cancer-related genes in 102 lesions. There were significant differences in the clinicopathological and molecular variables between small intestinal- and gastric-type tumors, which suggests the presence of at least two separate carcinogenic pathways in non-ampullary duodenal adenocarcinomas. The prevalence of CIMP-positive lesions was higher in intramucosal adenocarcinomas than in adenomas. Thus, concurrent hypermethylation of multiple CpG islands is likely associated with development of non-ampullary duodenal intramucosal adenocarcinomas. Mutation analysis showed that APC was the most frequently mutated gene in these lesions (56/102; 55%), followed by KRAS (13/102; 13%), LRP1B (10/102; 10%), GNAS (8/102; 8%), ERBB3 (7/102; 7%), and RNF43 (6/102; 6%). Additionally, the high prevalence of diffuse or focal nuclear ß-catenin accumulation (87/102; 85%) as well as mutations of WNT pathway components (60/102; 59%) indicates the importance of WNT signaling to the initiation of duodenal adenomas. The higher than previously reported frequency of APC gene mutations in small bowel adenocarcinomas as well as the difference in the APC mutation distributions between small intestinal-type adenomas and intramucosal adenocarcinomas may indicate that the adenoma-carcinoma sequence has only limited involvement in duodenal carcinogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

A genome-wide analysis of the molecular alterations occurring in the adenomatous and carcinomatous components of the same tumor based on the adenoma-carcinoma sequence.

Identification of molecular alterations occurring in the adenomatous and carcinomatous components within the same tumor would greatly enhance understanding of the neoplastic progression of colorectal cancer. We examined somatic copy number alterations (SCNAs) and mRNA expression at the corresponding loci involved in the adenoma-carcinoma sequence in the isolated adenomatous and cancer glands of the same tumor in 15 cases of microsatellite-stable "carcinoma in adenoma," using genome-wide SNP and global gene expression arrays. Multiple copy-neutral loss of heterozygosity events were detected at 4q13.2, 15q15.1, and 14q24.3 in the adenomatous component and at 4q13.2, 15q15.1, and 14q24.3 in the carcinomatous component. There were significant differences in the copy number (CN) gain frequencies at 20q11.21-q13.33, 8q13.3, 8p23.1, and 8q21.2-q22.2 between the adenomatous and carcinomatous components. Finally, we found a high frequency of five genotypes involving CN gain with upregulated expression of the corresponding gene (RPS21, MIR3654, RSP20, SNORD54, or ASPH) in the carcinomatous component, whereas none of these genotypes were detected in the adenomatous component. This finding is interesting in that CN gain with upregulated gene expression may enhance gene function and play a crucial role in the progression of an adenoma into a carcinomatous lesion.

Upregulation of adipocyte enhancer-binding protein 1 in endothelial cells promotes tumor angiogenesis in colorectal cancer.

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT-PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target.

Hypermethylation of CSF3R is a novel cisplatin resistance marker and predictor of response to postoperative chemotherapy in hepatoblastoma.

AIM: Most hepatoblastoma patients undergo pre/postoperative cisplatin treatment. Approximately 20% patients are cisplatin resistant, and show poor prognosis and high recurrence rates. However, some cisplatin-sensitive patients show early recurrence. We consider that a small population of cisplatin-resistant cells may remain after preoperative chemotherapy. Previous studies showed a correlation between DNA hypermethylation and hepatoblastoma progression. Here, we examined whether DNA hypermethylation was related to cisplatin resistance and could be a potential indicator for cisplatin as postoperative chemotherapy. METHODS: We extracted DNA from 43 resected hepatoblastoma tumors. Methylation array analyses were performed in 11 samples, including six cisplatin-sensitive and five cisplatin-resistant samples. We also performed cDNA microarray analysis in parental and cisplatin-resistant HuH6 cells. Through comparison of the datasets, we selected the strongest correlated cisplatin-resistant candidate gene. Using bisulfite pyrosequencing, the candidate gene methylation level was assessed in 38 cisplatin-sensitive patients after checking its usefulness as a substitute modality of methylation array. Correlations between the methylation status and clinical data were analyzed. RESULTS: CSF3R was the strongest correlated variable. Bisulfite pyrosequencing analysis also confirmed CSF3R was significantly hypermethylated in cisplatin-resistant patients. Among the 38 cisplatin-sensitive patients, recurrence curves showed that the CSF3R high methylation patients had significantly higher recurrence than CSF3R low methylation patients. The recurrence curve of methylation high patients was similar to that of cisplatin-resistant patients. CONCLUSIONS: Our findings suggested that CSF3R hypermethylation was related to cisplatin resistance in HB patients and could be a predictor of postoperative chemotherapy, and indicate that CSF3R high methylation patients should be treated with non-CDDP regimens.

Differential expression of microRNAs in colorectal cancer: Different patterns between isolated cancer gland and stromal cells.

Although microRNAs (miRNAs) play an important role in invasive tumor lesions, which involve cancer tissues mixed with stromal tissues, the differences in miRNA expression between cancer and stromal cells remain unclear. We selected 13 miRNAs and examined their differential expression patterns in cancer gland cells and surrounding stromal cells isolated from 24 colorectal cancer (CRC) specimens using a crypt isolation method. Although six miRNAs were upregulated in gland cells, only three were upregulated in the corresponding stromal cells, in the cancer compared with non-cancer specimens. Next, we examined the differences in miRNA expression between isolated cancer gland and stromal cells. Five miRNAs showed statistical differences in their cancer-related differential expression patterns between isolated cancer gland and stromal cells. We then compared these miRNA expression patterns in isolated cancer gland and stromal cells with those in fresh intact tumor tissues, consisting of cancer nests and stromal tissue, obtained from the 24 CRCs. The expression patterns of three miRNAs in the intact cancer tissue samples did not correspond with those in the isolated components. Identification of the expression patterns of miRNAs in both the cancer gland and stromal cell components of the tumor microenvironment greatly contributes to evaluating epigenetic regulation in CRC.