Metastatic breast cancer is a highly lethal disease, and it is very important to evaluate the biomarkers associated with distant metastasis. However, molecular features of distant metastasis remain largely unknown in breast cancer. Estrogens play an important role in the progression of breast cancer and the majority of stage IV breast carcinomas express estrogen receptor (ER). Therefore, in this study, we examined molecular markers associated with distant metastasis in ER-positive breast carcinoma by microarray and immunohistochemistry. When we examined the gene expression profile of ER-positive stage IV breast carcinoma tissues (n = 7) comparing ER-positive stage I-III cases (n = 11) by microarray analysis, we newly identified OLFM4, LY6D and S100A7, which were closely associated with the distant metastasis. Subsequently, we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 168 ER-positive breast carcinomas. OLFM4, LY6D and S100A7 immunoreactivities were significantly associated with stage, pathological T factor, distant metastasis and Ki67 status in the ER-positive breast carcinomas. Moreover, these immunoreactivities were significantly associated with a worse prognostic factor for distant metastasis-free and breast cancer-specific survival in ER-positive stage I-III breast cancer patients. However, when we performed immunohistochemistry for OLFM4, LY6D and S100A7 in 40 ER-negative breast carcinomas, these immunoreactivities were not generally associated with the clinicopathological factors examined, including distant metastasis and prognosis of patients, in this study. These results suggest that OLFM4, LY6D and S100A7 immunoreactivity are associated with an aggressive phenotype of ER-positive breast carcinoma, and these are potent markers for distant metastasis of ER-positive breast cancer patients.

Concentration of 25-hydroxyvitamin D3 (25(OH)D3), the main circulating form of vitamin D, is inversely associated with incident, sporadic colorectal adenoma risk. We investigated whether this association differs by 2 functional variants in the vitamin D-binding protein (DBP) gene, group-specific component (GC), that encode for common protein isoforms Gc1s, Gc1f, and Gc2 linked to differences in vitamin D metabolism. We pooled data (418 patients with adenoma and 524 polyp-free control subjects) from 3 colonoscopy-based case-control studies (Minnesota, 1991-1994; North Carolina, 1994-1997; South Carolina, 2002). We estimated 25(OH)D3-adenoma associations, stratified by DBP isoforms, using multivariable logistic regression. Higher 25(OH)D3 concentrations were inversely associated with colorectal adenoma risk among those with the Gc2 isoform (per 10-ng/mL increase in 25(OH)D3, odds ratio = 0.71, 95% confidence interval: 0.56, 0.90), but not among those with only Gc1 isoforms (odds ratio = 1.07, 95% confidence interval: 0.87, 1.32; P for interaction = 0.03). Thus, the vitamin D-incident, sporadic colorectal adenoma association may differ by common DBP isoforms, and patients with the Gc2 isoform may particularly benefit from maintaining higher circulating 25(OH)D3 concentrations for adenoma prevention.

Evidence suggests that altered adipose tissue homeostasis may be an important contributor to the development and/or progression of prostate cancer. In this study, we investigated the adipose transcriptional profiles of low- and high-risk disease to determine both prognostic potential and possible biological drivers of aggressive disease. RNA was extracted from periprostatic adipose tissue from patients categorised as having prostate cancer with either a low or high risk of progression based on tumour characteristics at prostatectomy and profiled by RNA sequencing. The expression of selected genes was then quantified by qRT-PCR in a cross-validation cohort. In the first phase, a total of 677 differentially transcribed genes were identified, from which a subset of 14 genes was shortlisted. In the second phase, a 3 gene (

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (

Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan-fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events.

Nucleotide-binding domain, leucine-rich-repeat-containing proteins (NLRs) are intracellular innate immune sensors of pathogen-associated and damage-associated molecular patterns. NLRs regulate diverse biologic processes such as inflammatory responses, cell proliferation and death, and gut microbiota to attenuate tumorigenesis. In a recent publication in Nature, we identified NLRC3 as a negative regulator of PI3K-mTOR signaling and characterized its potential tumor suppressor function. Enterocytes lacking NLRC3 cannot control cellular proliferation because they are unable to suppress activation of PI3K-mTOR signaling pathways. In this Extra-View, we explore possible mechanisms through which NLRC3 regulates cellular proliferation and cell death. Besides interacting with PI3K, NLRC3 associates with TRAF6 and mTOR, confirming our recent finding that NLRC3 negatively regulates the PI3K-mTOR axis. Herein, we show that NLRC3 suppresses c-Myc expression and activation of PI3K-AKT targets FoxO3a and FoxO1 in the colon of Nlrc3

Our previous study showed olfactomedin 4 (OLFM4) suppressed triple-negative breast cancer cells migration, invasion and metastasis-associated protein MMP 9 expression. OLFM4 was identified as a potential target of miR-103 according to microRNA target databases and published studies. The aim of this study is to validate the relationship between miR-103 and OLFM4, and explore the function and clinical significance of miR-103 in triple-negative breast cancer patients. In our results, miR-103 negatively regulated OLFM4 expression by directly targeting its 3'-UTR. OLFM4 was a functional target of miR-103 to regulate triple-negative breast cancer cells migration, invasion and MMP 9 expression. Moreover, miR-103 overexpression was observed in triple-negative breast cancer tissues and cell lines, and associated with lymph node metastasis, distant metastasis and clinical stage. Univariate and multivariate analyses suggested that miR-103 overexpression was a poor independent prognostic factor for triple-negative breast cancer patients. In conclusion, miR-103 acts as an oncogene miRNA to promote triple-negative breast cancer cells migration and invasion through targeting OLFM4.

AIM: Anastomotic leak is associated with higher rates of recurrence after surgery for colorectal cancer. However, the mechanisms responsible are unknown. We hypothesized that the infection-induced inflammatory response may induce overexpression of tumour progression-related genes in immune cells. The aim was to investigate the effect of postoperative intra-abdominal infection on the gene expression patterns of peripheral blood leucocytes (PBL) after surgery for colorectal cancer.
METHOD: Prospective matched cohort study. Patients undergoing surgery for colorectal cancer were included. Patients who had anastomotic leak or intra-abdominal abscess were included in the infection group (n = 23) and matched with patients without complications for the control group (n = 23). PBL were isolated from postoperative blood samples. Total RNA was extracted and hybridized to the Affymetrix Human Gene 1.0 ST microarray.
RESULTS: Patients in the infection group displayed 162 upregulated genes and 146 downregulated genes with respect to the control group. Upregulated genes included examples coding for secreted cytokines involved in tumour growth and invasion (S100P, HGF, MMP8, MMP9, PDGFC, IL1R2). Infection also upregulated some proangiogenic genes (CEP55, TRPS1) and downregulated some inhibitors of angiogenesis (MME, ALOX15, CXCL10). Finally, some inhibitors (HP, ORM1, OLFM4, IRAK3) and activators (GNLY, PRF1, FGFBP2) of antitumour immunity were upregulated and downregulated, respectively, suggesting that the inflammatory environment caused by a postoperative infection favours immune evasion mechanisms of the tumour.
CONCLUSION: Analysis of PBL shows differential expression of certain tumour progression-related genes in colorectal cancer patients who have a postoperative intra-abdominal infection, which in turn may promote the growth of residual cancer cells to become recurrent tumours.

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (

MicroRNA-590 (miR-590) shows oncogenic functions in various tumor types, but little is known about biological functions of miR-590 in lung adenocarcinoma. In this study, we observe that miR-590 is not only overexpressed in lung adenocarcinoma tissues and metastatic lymph nodes, but also significantly increased in lung adenocarcinoma cell lines. Moreover, gain-of-function and loss-of-function studies show miR-590 serve as a tumor suppressor regulating lung adenocarcinoma cells migration and invasion. Furthermore, OLFM4 is proved to as a functional target for miR-590 to regulate lung adenocarcinoma cells migration and invasion. In conclusion, miR-590 regulates lung adenocarcinoma metastasis through directly modulating functional target OLFM4.

Krüppel-like factor 5 (KLF5) transcriptionally controls the proliferation-differentiation balance of epithelium and is overexpressed in carcinomas. Although genomic region modifying KLF5 expression is widespread in different types of cells, the region that commonly regulates basal expression of the genes across cell-types is uncertain. In this study we determined the minimal essential region for the expression and its regulatory transcription factors using oral carcinoma cells. A reporter assay defined a 186bp region downstream of the transcription start site and a cluster of six GC boxes (GC1-GC6) as the minimal essential region. Mutation in the GC1 or GC6 regions but not other GC boxes significantly decreased the reporter expression. The decrease by the GC1 mutation was reproduced in the 2kbp full-length promoter, but not by the GC6 mutation. Additionally, specificity proteins (Sp) that can be expressed in epithelial cells and bind GC box, Sp3 co-localized with KLF5 in oral epithelium and carcinomas and chromatin immunoprecipitation analyses showed Sp3 as the prime GC1-binding protein. Inhibition of Sp-GC box binding by mithramycin A and knockdown of Sp3 by the short interfering RNA decreased expression of the reporter gene and endogenous KLF5. These data demonstrate that a 186bp region is the minimal essential region and that Sp3-GC1 binding is essential to the basal expression of KLF5.

Olfactomedin 4 (OLFM4) is an olfactomedin domain-containing glycoprotein. Multiple signaling pathways and factors, including NF-κB, Wnt, Notch, PU.1, retinoic acids, estrogen receptor, and miR-486, regulate its expression. OLFM4 interacts with several other proteins, such as gene associated with retinoic-interferon-induced mortality 19 (GRIM-19), cadherins, lectins, nucleotide oligomerization domain-1 (NOD1) and nucleotide oligomerization domain-2 (NOD2), and cathepsins C and D, known to regulate important cellular functions. Recent investigations using Olfm4-deficient mouse models have provided important clues about its in vivo biological functions. Olfm4 inhibited Helicobacter pylori-induced NF-κB pathway activity and inflammation and facilitated H. pylori colonization in the mouse stomach. Olfm4-deficient mice exhibited enhanced immunity against Escherichia coli and Staphylococcus aureus infection. Olfm4 deletion in a chronic granulomatous disease mouse model rescued them from S. aureus infection. Olfm4 deletion in mice treated with azoxymethane/dextran sodium sulfate led to robust intestinal inflammation and intestinal crypt hyperplasia. Olfm4 deletion in Apc (Min/+) mice promoted intestinal polyp formation as well as adenocarcinoma development in the distal colon. Further, Olfm4-deficient mice spontaneously developed prostatic epithelial lesions as they age. OLFM4 expression is correlated with cancer differentiation, stage, metastasis, and prognosis in a variety of cancers, suggesting its potential clinical value as an early-stage cancer marker or a therapeutic target. Collectively, these data suggest that OLFM4 plays important roles in innate immunity against bacterial infection, gastrointestinal inflammation, and cancer. In this review, we have summarized OLFM4's initial characterization, expression, regulation, protein interactions, and biological functions.

Colon carcinogenesis is a multiple-step process involving the accumulation of a series of genetic and epigenetic alterations. The most commonly initiating event of intestinal carcinogenesis is mutation of the adenomatous polyposis coli (APC) gene, which leads to activation of the Wnt/β-catenin pathway. Olfactomedin 4 (OLFM4) has emerged as an intestinal stem-cell marker, but its biological function in the intestine remains to be determined. Here we show that Olfm4 deletion induced colon adenocarcinoma in the distal colon of Apc

Estrogen signaling influences the development and progression of ovarian tumors, but the underlying mechanisms are not well understood. In a previous study we demonstrated that impairment of estrogen receptor alpha (ERα)-mediated olfactomedin 4 (OLFM4) expression promotes the malignant progression of endometrioid adenocarcinoma, and we identified OLFM4 as a potential target of miR-486-5p. In this study we investigated the role of OLFM4 in ovarian serous adenocarcinoma. Ovarian serous adenocarcinoma tissues had reduced OLFM4 expression. Expression of OLFM4 was positively correlated with ERα expression, and estrogen (E2) treatment in ovarian cancer cells induced OLFM4 expression in an ERα-dependent manner. In contrast to ERα, miR-486-5p levels were inversely correlated with OLFM4 expression in ovarian serous adenocarcinoma. Ovarian cancer cells transfected with miR-486-5p mimics showed decreased OLFM4 mRNA expression, and ovarian cancer cells treated with E2 showed reduced cellular miR-486-5p levels. OLFM4 knockdown enhanced proliferation, migration, and invasion by ovarian cancer cells. Low expression of OLFM4 was also associated with high tumor FIGO stage and poor tumor differentiation. These results suggest OLFM4 is downregulated by miR-486-5p, which contributes to ovarian cancer tumorigenesis. Conversely, estrogen receptor signaling downregulates miR-486-5p and upregulates OLFM4 expression, slowing the development and progression of ovarian cancer.

Circulating tumor cells (CTC) may be defined as tumor- or metastasis-derived cells that are present in the bloodstream. The CTC pool in colorectal cancer (CRC) patients may include not only epithelial tumor cells, but also tumor cells undergoing epithelial-mesenchymal transition (EMT) and tumor stem cells. A significant number of patients diagnosed with early stage CRC subsequently relapse with recurrent or metastatic disease despite undergoing "curative" resection of their primary tumor. This suggests that an occult metastatic disease process was already underway, with viable tumor cells being shed from the primary tumor site, at least some of which have proliferative and metastatic potential and the ability to survive in the bloodstream. Such tumor cells are considered to be responsible for disease relapse in these patients. Their detection in peripheral blood at the time of diagnosis or after resection of the primary tumor may identify those early-stage patients who are at risk of developing recurrent or metastatic disease and who would benefit from adjuvant therapy. CTC may also be a useful adjunct to radiological assessment of tumor response to therapy. Over the last 20 years many approaches have been developed for the isolation and characterization of CTC. However, none of these methods can be considered the gold standard for detection of the entire pool of CTC. Recently our group has developed novel unbiased inertial microfluidics to enrich for CTC, followed by identification of CTC by imaging flow cytometry. Here, we provide a review of progress on CTC detection and clinical significance over the last 20 years.

Loss of olfactomedin 4 (OLFM4) gene expression is associated with the progression of human prostate cancer, but its role and the molecular mechanisms involved in this process have not been completely understood. In this study, we found that Olfm4-knockout mice developed prostatic intraepithelial neoplasia and prostatic adenocarcinoma. Importantly, we found that the hedgehog-signaling pathway was significantly upregulated in the Olfm4-knockout mouse model. We also found that restoration of OLFM4 in human prostate-cancer cells that lack OLFM4 expression significantly downregulated hedgehog signaling-pathway component expression. Furthermore, we demonstrated that the OLFM4 protein interacts with sonic hedgehog protein, as well as significantly inhibits GLI-reporter activity. Bioinformatic and immunohistochemistry analyses revealed that decreased OLFM4 and increased SHH expression was significantly associated with advanced human prostate cancer. Thus, olfactomedin 4 appears to play a critical role in regulating progression of prostate cancer, and has potential as a new biomarker for prostate cancer.

The consistent presence of Epstein-Barr virus (EBV) in malignant cells of EBV-associated gastric carcinoma (EBVaGC) suggests it plays an important role during the development of EBVaGC. However, the entire genomic sequence of EBV from EBVaGC has yet to be determined. This study first determined, annotated, and analyzed the full genomic sequence of EBV from the naturally infected gastric carcinoma cell line SNU-719 using next-generation sequencing and comparative analyses. In consistent with the notion that EBV sequence isolates better reflect their geographic area than tissue origin, the SNU-719 EBV (named as GC1) was categorized as an East Asian type I EBV. Compared with the prototype B95.8 sequence, SNU-719 EBV contained 1372 variations, with 937 and 435 within coding and non-coding regions, respectively. Of the 937 variations, 465 were non-synonymous changes, while 472 synonymous changes included partial internal deletions in the coding regions of LMP1 and gp350. The RNAseq transcriptome revealed that multiple BART transcripts comprised the majority of EBV RNA reads. The SNU-719 EBV expressed high levels of BART, LF3, BHLF1, and BNLF2. Evidence of RNA editing at multiple sites in the host chromosome was found; however, no evidence of genome integration was seen. The annotated SNU-719 EBV sequence will be a useful reference in future EBVaGC studies.

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 μg/ml, mean 0.028 μg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 μg/ml, mean 15 μg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

Gastric cancer (GC) is now one of the most common malignancies with a relatively high incidence and high mortality rate. The prognosis is closely related to the degree of tumor metastasis. The mechanism of metastasis is still unclear. Proteomics analysis is a powerful tool to study and evaluate protein expression in tumor tissues. In the present study, we collected 15 gastric cancer and adjacent normal gastric tissues and used the isobaric tags for relative and absolute quantitation (iTRAQ) method to identify differentially expressed proteins. A total of 134 proteins were differentially expressed between the cancerous and non-cancerous samples. Azurocidin 1 (AZU1), CPVL, olfactomedin 4 (OLFM4) and Villin 1 (VIL1) were upregulated and confirmed by western blot analysis, real-time quantitative PCR and immunohistochemical analyses. These results were in accordance with iTRAQ. Furthermore, silencing the OLFM4 expression suppressed the migration, invasion and proliferation of the GC cells in vitro. The present study represents a successful application of the iTRAQ method in analyzing the expression levels of thousands of proteins. Overexpression of OLFM4 in gastric cancer may induce the development of gastric cancer. Overall, suppression of OLFM4 expression may be a promising strategy in the development of novel cancer therapeutic drugs.

Downregulation of olfactomedin-4 (OLFM4) is associated with tumor progression, lymph node invasion and metastases. However, whether or not downregulation of OLFM4 is associated with epigenetic silencing remains unknown. In this study, we investigate the role of OLFM4 in gastric cancer cell invasion. We confirm the previous result that OLFM4 expression is increased in gastric cancer tissues and decreases with an increasing number of metastatic lymph nodes, which are associated with OLFM4 promoter hypermethylation. Overexpression of OLFM4 in gastric cancer cells had an inhibitory effect on cell invasion. Furthermore, we found that focal adhesion kinase (FAK) was negatively correlated with OLFM4 in regards to lymph node metastasis in gastric cancer tissues. Also, inhibition of FAK induced by OLFM4 knockdown resulted in a decrease in cell invasion. Thus, our study demonstrates that epigenetic silencing of OLFM4 enhances gastric cancer cell invasion via activation of FAK signaling.

Gastric cancer (GC), one of the most common human cancers, can be classified into gastric or intestinal phenotype according to mucin expression. TP53 mutation, allelic deletion of the APC gene and nuclear staining of β-catenin are frequently detected in the intestinal phenotype of GC, whereas CDH1 gene mutation, microsatellite instability and DNA hypermethylation of MLH1 are common events in the gastric phenotype of GC. Our Serial Analysis of Gene Expression (SAGE) and Escherichia coli ampicillin secretion trap (CAST) analyses revealed that CDH17, REG4, OLFM4, HOXA10, DSC2, TSPAN8 and TM9SF3 are upregulated in GC and that CLDN18 is downregulated in GC. Expression of CDH17, REG4, HOXA10 and DSC2 and downregulation of CLDN18 are observed in the intestinal phenotype of GC. In contrast, OLFM4 is expressed in the gastric phenotype of GC. Expression of TSPAN8, TM9SF3 and HER2 are not associated with either gastric or intestinal phenotypes. Ectopic CDX2 expression plays a key function in the GC intestinal phenotype. MUC2, CDH17, REG4, DSC2 and ABCB1 are direct targets of CDX2. Importantly, these genes encode transmembrane/secretory proteins, indicating that the microenvironment as well as cancer cells are also different between gastric and intestinal phenotypes of GC.

PURPOSE: To investigate the expression of GW112 and GRIM-19 in colorectal cancer tissues.
METHODS: Immunohistochemistry and semi-quantitative PCR were used to simultaneously detect the levels of expression of GW112 and GRIM-19 in colorectal cancer tissues and normal colorectal tissues in 39 cases.
RESULTS: Expression of GW112 protein and mRNA were significantly higher in colorectal cancer tissues than in normal tissues (p<0.05). Expression of GRIM-19 protein and mRNA were significantly lower in colorectal cancer tissues than in normal tissues (p<0.05). GW112 gene mRNA copy number(GAPDH gene mRNA copy number were 0.53 ± 0.21 and 1.81 ± 0.65 in normal colorectal tissues and colorectal cancer tissues respectively, and GRIM-19 gene mRNA copy number/GAPDH gene mRNA copy number were 1.15 ± 0.29 and 1.74 ± 0.0.44 in colorectal cancer tissues and normal colorectal tissues, respectively. Expression of GW112 gene mRNA was significantly higher in colorectal cancer tissues than in normal tissues (p<0.05), and expression of GRIM- 19 gene mRNA was significantly lower in colorectal cancer tissues than in normal tissues (p<0.05).
CONCLUSION: High expression of GW112 in colorectal cancer tissues and reduced expression of GRIM-19 in colorectal cancer tissues may be associated with abnormal proliferation of cancer cells and are possibly one of the reasons for development of colorectal cancer, which can provide effective targets for clinical treatment of this disease.

Gastric intestinal metaplasia (IM) is a highly prevalent preneoplastic lesion; however, the molecular mechanisms regulating its development remain unclear. We have previously shown that a population of cells expressing the intestinal stem cell (ISC) marker LGR5 increases remarkably in IM. In this study, we further investigated the molecular characteristics of these LGR5+ cells in IM by examining the expression profile of several ISC markers. Notably, we found that ISC markers-including OLFM4 and EPHB2-are positively associated with the CDX2 expression in non-tumorous gastric tissues. This finding was confirmed in stomach lesions with or without metaplasia, which demonstrated that OLFM4 and EPHB2 expression gradually increased with metaplastic progression. Moreover, RNA in situ hybridization revealed that LGR5+ cells coexpress several ISC markers and remained confined to the base of metaplastic glands, reminiscent to that of normal intestinal crypts, whereas those in normal antral glands expressed none of these markers. Furthermore, a large number of ISC marker-expressing cells were diffusely distributed in gastric adenomas, suggesting that these markers may facilitate gastric tumorigenesis. In addition, Barrett's esophagus (BE)-which is histologically similar to intestinal metaplasia-exhibited a similar distribution of ISC markers, indicating the presence of a stem cell population with intestinal differentiation potential. In conclusion, we identified that LGR5+ cells in gastric IM and BE coexpress ISC markers, and exhibit the same expression profile as those found in normal intestinal crypts. Taken together, these results implicate an intestinal-like stem cell population in the pathogenesis of IM, and provide an important basis for understanding the development and maintenance of this disease.

Olfactomedin 4 (OLFM4) has been demonstrated to serve an important function in tumor progression. This study aims to analyze the correlation between OLFM4 expression and clinicopathological features and the prognostic significance of OLFM4 in the context of smoking status of non-small cell lung cancer (NSCLC) patients. A total of 218 NSCLC patients, who were histopathologically diagnosed from 2001 to 2013, were reviewed in the study. OLFM4 expression was analyzed by immunohistochemical staining of tissue samples. The association of OLFM4 with clinicopathological parameters was evaluated. Overall survival and disease-specific survival were evaluated by Kaplan-Meier survival analysis. Immunohistochemical analyses showed that OLFM4 was highly expressed in 64.2% of NSCLC patients. OLFM4 expression level in NSCLC lesions was strongly correlated with pathologic grade (P = .017), lymph node metastasis (P = .012), peritumor intravascular cancer emboli (P = .03), and smoking status (P < .001). Kaplan-Meier survival curves showed that, among smoking patients, those with low OLFM4 expression had shorter survival time (overall survival and disease-specific survival) than those with high OLFM4 (P < .05). Conclusively, although low OLFM4 expression is not an independent prognostic biomarker, it might indicate worse prognosis for smoking patients with NSCLC, thereby identifying patients who might benefit from targeting OLFM4 therapy.

The aim of this study was to clarify the significance of DNA methylation alterations during gastric carcinogenesis. Single-CpG resolution genome-wide DNA methylation analysis using the Infinium assay was performed on 109 samples of non-cancerous gastric mucosa (N) and 105 samples of tumorous tissue (T). DNA methylation alterations in T samples relative to N samples were evident for 3861 probes. Since N can be at the precancerous stage according to the field cancerization concept, unsupervised hierarchical clustering based on DNA methylation levels was performed on N samples (βN) using the 3861 probes. This divided the 109 patients into three clusters: A (n = 20), B1 (n = 20), and B2 (n = 69). Gastric carcinomas belonging to Cluster B1 showed tumor aggressiveness more frequently than those belonging to Clusters A and B2. The recurrence-free and overall survival rates of patients in Cluster B1 were lower than those of patients in Clusters A and B2. Sixty hallmark genes for which βN characterized the epigenetic clustering were identified. We then focused on DNA methylation levels in T samples (βT) of the 60 hallmark genes. In 48 of them, including the ADAM23, OLFM4, AMER2, GPSM1, CCL28, DTX1 and COL23A1 genes, βT was again significantly correlated with tumor aggressiveness, and the recurrence-free and/or overall survival rates. Multivariate analyses revealed that βT was a significant prognostic factor, being independent of clinicopathological parameters. These data indicate that DNA methylation profiles at the precancerous stage may be inherited by gastric carcinomas themselves, thus determining tumor aggressiveness and patient outcome.

Olfactomedin-4 (OLFM4, OLM4) is a 72 kDa secreted glycoprotein belonging to the olfactomedin family. The OLFM4 gene expression is regulated by the transcription factors NF-kappa B and AP-1, and the OLM4 functions are poorly understood. OLM4 has been described as being able to interact with cell surface proteins such as lectins and concanavalin-A suggesting that one function of OLM4 is to regulate cell adhesion and migration. OLM4 is a marker for intestinal stem cells and is expressed at the bottom of the intestinal crypts. Expression of OLM4 during tumor development showed that OLM4 expression is increased in the early stages of tumor initiation. As OLM4 is a secreted protein, it is a prime candidate for biomarker research for tumor detection or progression. Levels of circulating OLM4 were significantly higher in patients with gastric, colorectal, and pancreatic cancers than in healthy subjects.

Colorectal cancer is one of the leading causes of death due to cancer worldwide. Therefore, the identification of high-specificity and -sensitivity biomarkers for the early detection of colorectal cancer is urgently needed. Post-translational modifications, such as glycosylation, are known to play an important role in cancer progression. In the present work, we used a quantitative proteomic technique based on (18)O stable isotope labeling to identify differentially expressed N-linked glycoproteins in colorectal cancer tissue samples compared with healthy colorectal tissue from 19 patients undergoing colorectal cancer surgery. We identified 54 up-regulated glycoproteins in colorectal cancer samples, therefore potentially involved in the biological processes of tumorigenesis. In particular, nine of these (PLOD2, DPEP1, SE1L1, CD82, PAR1, PLOD3, S12A2, LAMP3, OLFM4) were found to be up-regulated in the great majority of the cohort, and, interestingly, the association with colorectal cancer of four (PLOD2, S12A2, PLOD3, CD82) has not been hitherto described.

UNLABELLED: Systemic mining of cancer exoproteome/secretome has emerged as a pivotal strategy for delineation of molecular pathways with mechanistic importance in cancer development, as well as the discovery of diagnostic/prognostic biomarkers. Although major advances in diagnostic and therapeutic management of colorectal cancer have been underscored in the last decade, this cancer still remains the second leading cause of cancer-related deaths in the developed world. Despite previous studies on deciphering the colorectal cancer exoproteome, such studies lack adequate depth and robustness due to technological limitations. Here, using a well-established LC-MS/MS method on an LTQ-Orbitrap mass spectrometer, we extensively delineated the exoproteome of 12 colon cancer cell lines. In total, 2979 non-redundant proteins were identified with a minimum of two peptides, of which ~62% were extracellular or cell membrane-bound, based on prediction software. To further characterize this dataset and identify clinical opportunities, first, we investigated overrepresented molecular concepts of interest via enrichment map analysis and second, we demonstrated translational importance of certain proteins, such as olfactomedin-4 and kallikrein-related peptidases-6 and -10, by investigating their expression levels in patient tissues and/or fluids. Overall, the present study details a comprehensive colorectal cancer exoproteome dataset, and may be used as future platform for biomarker discovery, and hypothesis-generating studies.
BIOLOGICAL SIGNIFICANCE: This article represents one of the most extensive and comprehensive proteomic datasets regarding the secreted/extracellular proteome of colorectal cancer cell lines. The reported datasets may form a platform for a plethora of future, discovery-based or hypothesis-generating studies, attempting to either delineate putative cancer biomarkers for CRC, or elucidate questions of mechanistic importance (e.g. investigation of deregulated pathways for CRC progression).

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3β (GSK3β) inhibitor, induced β-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.

Endometrial adenocarcinoma is the most common tumour of the female genital tract in developed countries, and oestrogen receptor (ER) signalling plays a pivotal role in its pathogenesis. When we used bioinformatics tools to search for the genes contributing to gynecological cancers, the expression of Olfactomedin 4 (OLFM4) was found by digital differential display to be associated with differentiation of endometrial adenocarcinoma. Aberrant expression of OLFM4 has been primarily reported in tumours of the digestive system. The mechanism of OLFM4 in tumuorigenesis is elusive. We investigated OLFM4 expression in endometrium, analysed the association of OLFM4 with ER signalling in endometrial adenocarcinoma, and examined the roles of OLFM4 in endometrial adenocarcinoma. Expression of OLFM4 was increased during endometrial carcinogenesis, linked to the differentiation of endometrioid adenocarcinoma, and positively related to the expression of oestrogen receptor-α (ERα) and progesterone receptor. Moreover, ERα-mediated signalling regulated expression of OLFM4, and knockdown of OLFM4 enhanced proliferation, migration and invasion of endometrial carcinoma cells. Down-regulation of OLFM4 was associated with decreased cumulative survival rate of patients with endometrioid adenocarcinoma. Our data suggested that impairment of ERα signal-mediated OLFM4 expression promoted the malignant progression of endometrioid adenocarcinoma, which may have significance for the therapy of this carcinoma.