Expression profiling of Wilms tumors reveals new candidate genes for different clinical parameters

Wilms tumor is the most frequent renal neoplasm in children, but our understanding of its genetic basis is still limited. We performed cDNA microarray experiments using 63 primary Wilms tumors with the aim of detecting new candidate genes associated with malignancy grade and tumor progression. All tumors had received preoperative chemotherapy as mandated by the SIOP protocol, which sets this study apart from related approaches in the Unites States that are based on untreated samples. The stratification of expression data according to clinical criteria allowed a rather clear distinction between different subsets of Wilms tumors. Clear-cut differences in expression patterns were discovered between relapse-free as opposed to relapsed tumors and tumors with intermediate risk as opposed to high risk histology. Several differentially expressed genes, e.g.TRIM22, CENPF, MYCN, CTGF, RARRES3 and EZH2, were associated with Wilms tumor progression. For a subset of differentially expressed genes, microarray data were confirmed by real-time RT-PCR on the original set of tumors. Interestingly, we found the retinoic acid pathway to be deregulated at different levels in advanced tumors suggesting that treatment of these tumors with retinoic acid may represent a promising novel therapeutic approach.     

Expression profiling reveals hepsin overexpression in prostate cancer

Prostate cancer is the most commonly diagnosed noncutaneous cancer in men. Despite this fact, many of the genetic changes that coincide with prostate cancer progression remain enigmatic. We have addressed this problem by characterizing the expression profiles of several benign and malignant human prostate samples, and we have identified several genes that are differentially expressed between benign and malignant glands. One gene that was overexpressed encodes the serine protease hepsin. We used an independent sample set to confirm that hepsin is overexpressed in prostate tumors, and in situ hybridization demonstrates that hepsin is specifically overexpressed in the carcinoma cells themselves. These facts, together with the molecular properties of hepsin, make it an ideal target for prostate cancer therapy.     

NGS-based profiling reveals a critical contributing role of somatic D-loop mtDNA mutations in HBV-related hepatocarcinogenesis

BackgroundSomatic mutations of mitochondrial DNA (mtDNA) have been extensively identified mainly by traditional Sanger sequencing technology in various cancer types. However, low detection sensitivity of traditional methods greatly limits the comprehensive profiling of mtDNA somatic mutations in cancers, especially in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Moreover, the functional roles of mtDNA mutation in HBV-related hepatocarcinogenesis have not been systematically revealed.Patients and methodsNext-generation sequencing (NGS) platform was applied to profile the somatic mtDNA mutations of HCC and paired paratumor (non-HCC) tissues from a large cohort of 156 HBV-HCC patients.ResultsOur data revealed the common existence of mtDNA mutation in both inflammatory and cancer tissues with significantly different mutation pattern. The mutation density (mutation number/region length) of D-loop region was much higher than that of other regions in both HCC and non-HCC tissues. Unexpectedly, the average mutation number in D-loop region of HCC tissues was significantly less than that of non-HCC tissues. In contrast, the heteroplasmy level of D-loop region mutations was significantly increased in HCC tissues, implying that the D-loop mutations might be positively selected in HCC tissues. Furthermore, our results indicated that the patients with D-loop mutations had a significantly lower mtDNA copy number and were more likely to relapse. In vitro experiments demonstrated that proliferation, invasion and metastasis ability of HCC cells with D-loop region mutations were significantly higher than those without D-loop region mutations.ConclusionThese results emphasize the critical contributing role of somatic mtDNA D-loop mutations in HBV-related hepatocarcinogenesis.     

Methylation profiling of normal individuals reveals mosaic promoter methylation of cancer-associated genes

Epigenetic silencing by promoter methylation of genes associated with cancer initiation and progression is a hallmark of tumour cells. As a consequence, testing for DNA methylation biomarkers in plasma or other body fluids shows great promise for detection of malignancies at early stages and/or for monitoring response to treatment. However, DNA from normal leukocytes may contribute to the DNA in plasma and will affect biomarker specificity if there is any methylation in the leukocytes. DNA from 48 samples of normal peripheral blood mononuclear cells was evaluated for the presence of methylation of a panel of DNA methylation biomarkers that have been implicated in cancer. SMART-MSP, a methylation specific PCR (MSP) methodology based on real time PCR amplification, high-resolution melting and strategic primer design, enabled quantitative detection of low levels of methylated DNA. Methylation was observed in all tested mononuclear cell DNA samples for the CDH1 and HIC1 promoters and in majority of DNA samples for the TWIST1 and DAPK1 promoters. APC and RARB promoter methylation, at a lower average level, was also detected in a substantial proportion of DNA samples. We found no BRCA1, CDKN2A, GSTP1 and RASSF1A promoter methylation in this sample set. Several individuals had higher levels of methylation at several loci suggestive of a methylator phenotype. In conclusion, methylation of many potential DNA methylation biomarkers can be detected in normal peripheral blood mononuclear cells, and is likely to affect their specificity for detecting low level disease. However, we found no evidence of promoter methylation for other genes indicating that panels of analytically sensitive and specific methylation biomarkers in body fluids can be obtained.     

Expression profiling reveals genes associated with transendothelial migration of tumor cells: a functional role for alphavbeta3 integrin

Transendothelial migration is a key step in the extravasation of tumor cells during metastasis formation. Here, we have classified 45 human tumor cell lines derived from various tissues according to their capacity for transendothelial migration in vitro. We could distinguish cell lines showing strong transmigration (TEM+ cell lines) from others that did not transmigrate (TEM- cell lines). By DNA microarray analysis we could cluster TEM+ and TEM- cell lines according to their gene expression pattern and identify genes differentially expressed between the 2 groups. Among these we found the integrin beta3 subunit to be highly expressed in TEM+ cell lines as compared to TEM- cell lines. Cell surface localization of alphavbeta3 integrin receptors was exclusively found in TEM+ cell lines. Transendothelial migration of TEM+ cells but not their adhesion to the endothelial cells, or invasion into collagen gels could be blocked with an antibody against alphavbeta3 integrin and by RNAi mediated knock-down of the integrin beta3 subunit. These data establishes alphavbeta3 integrin as one key component of the transendothelial migration process of tumor cells, and as a potential target for anti-metastatic therapy. Our gene expression analysis of a defined collection of tumor cell lines can be used as a starting point to identify further genes functionally involved in transendothelial migration.     

Sequential analysis of multistage hepatocarcinogenesis reveals that miR-100 and PLK1 dysregulation is an early event maintained along tumor progression

MicroRNAs (miRNAs) have an important role in a wide range of physiological and pathological processes, and their dysregulation has been reported to affect the development and progression of cancers, including hepatocellular carcinoma (HCC). However, in the plethora of dysregulated miRNAs, it is largely unknown which of them have a causative role in the hepatocarcinogenic process. In the present study, we first aimed to determine changes in the expression profile of miRNAs in human HCCs and to compare them with liver tumors generated in a rat model of chemically induced HCC. We found that members of the miR-100 family (miR-100, miR-99a) were downregulated in human HCCs; a similar downregulation was also observed in rat HCCs. Their reduction was paralleled by an increased expression of polo like kinase 1 (PLK1), a target of these miRNAs. The introduction of miR-100 in HCC cells impaired their growth ability and their capability to form colonies in soft agar. Next, we aimed at investigating, in the same animal model, if dysregulation of miR-100 and PLK1 is an early or late event along the multistep process of hepatocarcinogenesis. The obtained results showed that miR-100 downregulation (i) is already evident in very early preneoplastic lesions generated 9 weeks after carcinogenic treatment; (ii) is also observed in adenomas and early HCCs; and (iii) is not simply a marker of proliferating hepatocytes. To our knowledge, this is the first work unveiling the role of a miRNA family along HCC progression.     

Expression profiling of CC531 colon carcinoma cells reveals similar regulation of beta-catenin target genes by both butyrate and aspirin

The CC531 cell line has been widely used to study different aspects of tumor growth and metastasis and provides an excellent experimental platform to develop novel antitumor strategies. To characterize the CC531 model at the molecular level, we screened for mutations in genes covering important signal-transduction pathways that are known to play major roles during colon carcinogenesis, the wnt and the ki-ras signaling pathways. We found both a prototypic beta-catenin (Ctnnb1) mutation (Thr(41)Ile) and a ki-ras (G12D) mutation, providing unambiguous evidence for the constitutive activation of these pathways in CC531 cells. We further established comprehensive gene expression profiles of CC531 cells and investigated the molecular response to 2 antitumor drugs, butyrate and aspirin. Using oligonucleotide microarrays, we screened the expression levels of 7,700 genes and identified a total of 398 genes whose expression was significantly changed upon treatment with butyrate. When using aspirin, 121 genes were significantly altered. Interestingly, 36 genes were regulated by both butyrate and aspirin and 35 of them were regulated in the same direction. We found 7 differentially expressed genes, cyclin D1, cyclin E, c-myc, Fosl1, c-fos, Cd44 and follistatin, which are known targets of the beta-catenin and/or the ras pathway. In all cases, butyrate and aspirin reversed the changes in expression normally found in response to active signaling of these oncogenic pathways. The microarray data are available (http://ncbi.nlm.nih.gov/geo/).     

Expression profiling of small cellular samples in cancer: less is more

Expression profiling of tumours from cancer patients has uncovered several genes that are critically important in the progression of a normal cell to an oncogenic phenotype. Leading the way in these discoveries is the use of microarrays, a technology that is currently in transition from basic science applications to use in the clinic. Microarrays can determine the global gene regulation of an individual cancer, which may be useful in formulating an individualised therapy for the patient. Currently, cells used in breast cancer microarray studies often come from either homogenous cultures or heterogeneous biopsy samples. Both cell sources are at a disadvantage in determining the most accurate gene profile of cancer, which often consists of multiple subspecies of cancerous cells within a background of normal cells. Therefore, acquisition of small, but highly specific biopsies for analysis may be required for an accurate expression analysis of the disease. Amplification methods, such as polymerase chain reaction (PCR) and amplified antisense RNA (aRNA) amplification, have been used to amplify the mRNA signal from very small samples, which can then be used for microarray analysis. In this study, we describe the acquisition, amplification, and analysis of very small samples (<10000 cells) for expression analysis and demonstrate that the ultimate resolution of cancer expression analysis, one cell, is both feasible and practical.     

Expression profiling and identification of novel genes in hepatocellular carcinomas.

Liver cancer is the fifth most common cancer worldwide and unlike certain other cancers, such as colon cancer, a mutational model has not yet been developed. We have performed gene expression profiling of normal and neoplastic livers in C3H/HeJ mice treated with diethylnitrosamine. Using oligonucleotide microarrays, we compared gene expression in liver tumors to three different states of the normal liver: quiescent adult, regenerating adult, and newborn. Although each comparison revealed hundreds of differentially expressed genes, only 22 genes were found to be deregulated in the tumors in all three comparisons. Three of these genes were examined in human hepatocellular carcinomas and were found to be upregulated. As a second method of analysis, we used Representational Difference Analysis (RDA) to clone mRNA fragments differentially expressed in liver tumors versus regenerating livers. We cloned several novel mRNAs that are differentially regulated in murine liver tumors. Here we report the sequence of a novel cDNA whose expression is upregulated in both murine and human hepatocellular carcinomas. Our results suggest that DEN-treated mice provide an excellent model for human hepatocellular carcinomas.     

High-resolution genomic and expression profiling reveals 105 putative amplification target genes in pancreatic cancer

Comparative genomic hybridization (CGH) studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4), which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.     

Expression profiling identifies genes involved in neoplastic transformation of serous ovarian cancer

Background  

The malignant potential of serous ovarian tumors, the most common ovarian tumor subtype, varies from benign to low malignant potential (LMP) tumors to frankly invasive cancers. Given the uncertainty about the relationship between these different forms, we compared their patterns of gene expression.     

New cellular tools reveal complex epithelial-mesenchymal interactions in hepatocarcinogenesis

To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.     

Large‐scale profiling of metabolic dysregulation in ovarian cancer

Ovarian cancer is the leading cause of death in gynecologic malignancies. Profiling of endogenous metabolites has potential to identify changes caused by cancer and provide inspiring insights into cancer metabolism. To systematically investigate ovarian cancer metabolism, we performed metabolic profiling of 448 plasma samples related to epithelial ovarian cancer (EOC) based on ultra‐performance liquid chromatography mass spectrometry in both positive and negative modes. These unbiased metabolomic profiles could well distinguish EOC from benign ovarian tumor (BOT) and uterine fibroid (UF). Fifty‐three metabolites were identified as specific biomarkers for EOC, and this is the first report of piperine, 3‐indolepropionic acid, 5‐hydroxyindoleacetaldehyde and hydroxyphenyllactate as metabolic biomarkers of EOC. The AUC values of these metabolites for discriminating EOC from BOT/UF and early‐stage EOC from BOT/UF were 0.9100/0.9428 and 0.8385/0.8624, respectively. Meanwhile, our metabolites were able to distinguish early‐stage EOC from late‐stage EOC with an AUC of 0.8801. Importantly, analysis of dysregulated metabolic pathways extends our current understanding of EOC metabolism. Metabolic pathways in EOC patients are mainly characterized by abnormal phospholipid metabolism, altered l ‐tryptophan catabolism, aggressive fatty acid β‐oxidation and aberrant metabolism of piperidine derivatives. Together, these metabolic pathways provide a foundation to support cancer development and progression. In conclusion, our large‐scale plasma metabolomics study yielded fundamental insights into dysregulated metabolism in ovarian cancer, which could facilitate clinical diagnosis, therapy, prognosis and shed new lights on ovarian cancer pathogenesis.     

Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times

Background  

Gene expression profiling of spontaneous tumors in the dog offers a unique translational opportunity to identify prognostic biomarkers and signaling pathways that are common to both canine and human. Osteosarcoma (OS) accounts for approximately 80% of all malignant bone tumors in the dog. Canine OS are highly comparable with their human counterpart with respect to histology, high metastatic rate and poor long-term survival. This study investigates the prognostic gene profile among thirty-two primary canine OS using canine specific cDNA microarrays representing 20,313 genes to identify genes and cellular signaling pathways associated with survival. This, the first report of its kind in dogs with OS, also demonstrates the advantages of cross-species comparison with human OS.     

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer

Background:

Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples.

Methods:

In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs).

Results:

We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system.

Conclusion:

This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.