Identification of gene expression signature modulated by nicotinamide in a mouse bladder cancer model

Background

Urinary bladder cancer is often a result of exposure to chemical carcinogens such as cigarette smoking. Because of histological similarity, chemically-induced rodent cancer model was largely used for human bladder cancer studies. Previous investigations have suggested that nicotinamide, water-soluble vitamin B3, may play a key role in cancer prevention through its activities in cellular repair. However, to date, evidence towards identifying the genetic alterations of nicotinamide in cancer prevention has not been provided. Here, we search for the molecular signatures of cancer prevention by nicotinamide using a N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced urinary bladder cancer model in mice.

Methodology/Principal Findings

Via microarray gene expression profiling of 20 mice and 233 human bladder samples, we performed various statistical analyses and immunohistochemical staining for validation. The expression patterns of 893 genes associated with nicotinamide activity in cancer prevention were identified by microarray data analysis. Gene network analyses of these 893 genes revealed that the Myc and its associated genes may be the most important regulator of bladder cancer prevention, and the gene expression signature correlated well with protein expression data. Comparison of gene expression between human and mouse revealed that BBN-induced mouse bladder cancers exhibited gene expression profiles that were more similar to those of invasive human bladder cancers than to those of non-invasive human bladder cancers.

Conclusions/Significance

This study demonstrates that nicotinamide plays an important role as a chemo-preventive and therapeutic agent in bladder cancer through the regulation of the Myc oncogenic signature. Nicotinamide may represent a promising therapeutic modality in patients with muscle-invasive bladder cancer.     

cDNA microarray profiles of canine mammary tumour cell lines reveal deregulated pathways pertaining to their phenotype

Mammary cancer is the most common type of cancer in female dogs with a lifetime risk of over 24% when dogs are not spayed. The elucidation of the complete canine genome opens new areas for development of cancer therapies. These should be tested first by in vitro models such as cell lines. However, to date, no canine mammary cell lines have been characterized by expression profiling. In this study, canine mammary tumour cell lines with histologically distinct primary tumours of origin were characterized using a newly developed canine cDNA microarray. Comparisons of gene expression profiles showed enrichment for distinct biological pathways and were related to biological properties of the cell lines such as growth rate and in vitro tumourigenicity. Additionally, gene expression profiles of cell lines also showed correspondence to their tumour of origin. Major differences were found in Wnt, cell cycle, cytokine/Rho-GTPase, alternative complement and integrin signalling pathways. Because these pathways show an overlap at the molecular level with those found in human breast cancer, the expression profiling of spontaneous canine mammary cancer may also function as a biological sieve to identify conserved gene expression or pathway profiles of evolutionary significance that are involved in tumourigenesis. These results are the basis for further characterization of canine mammary carcinomas and development of new therapies directed towards specific pathways. In addition these cell lines can be used to further investigate identified deregulated pathways and characterize until now unannotated genes.     

Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia

Refined cancer models are required if researchers are to assess the burgeoning number of potential targets for cancer therapeutics in a clinically relevant context that allows a fast turnaround. Here we use tumor-associated genetic pathways to transform primary human epithelial cells from the epidermis, oropharynx, esophagus and cervix into genetically defined tumors in a human three-dimensional (3D) tissue environment that incorporates cell-populated stroma and intact basement membrane. These engineered organotypic tissues recapitulated natural features of tumor progression, including epithelial invasion through basement membrane, a complex process that is necessary for biological malignancy in 90% of human cancers. Invasion was rapid and was potentiated by stromal cells. Oncogenic signals in 3D tissue, but not 2D culture, resembled gene expression profiles from spontaneous human cancers. We screened 3D organotypic neoplasia with well-characterized signaling pathway inhibitors to distill a clinically faithful cancer gene signature. Multitissue 3D human tissue cancer models may provide an efficient and relevant complement to current approaches to characterizing cancer progression.     

Variation in gene expression patterns in human gastric cancers

Gastric cancer is the world's second most common cause of cancer death. We analyzed gene expression patterns in 90 primary gastric cancers, 14 metastatic gastric cancers, and 22 nonneoplastic gastric tissues, using cDNA microarrays representing approximately 30,300 genes. Gastric cancers were distinguished from nonneoplastic gastric tissues by characteristic differences in their gene expression patterns. We found a diversity of gene expression patterns in gastric cancer, reflecting variation in intrinsic properties of tumor and normal cells and variation in the cellular composition of these complex tissues. We identified several genes whose expression levels were significantly correlated with patient survival. The variations in gene expression patterns among cancers in different patients suggest differences in pathogenetic pathways and potential therapeutic strategies.     

NMR-based metabolomics study of canine bladder cancer

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods. ¹H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Student's t-test. These metabolites were selected to build a separate partial least square‐discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved.     

Key pathways involved in prostate cancer based on gene set enrichment analysis and meta analysis

Prostate cancer is one of the most common male malignant neoplasms; however, its causes are not completely understood. A few recent studies have used gene expression profiling of prostate cancer to identify differentially expressed genes and possible relevant pathways. However, few studies have examined the genetic mechanics of prostate cancer at the pathway level to search for such pathways. We used gene set enrichment analysis and a meta-analysis of six independent studies after standardized microarray preprocessing, which increased concordance between these gene datasets. Based on gene set enrichment analysis, there were 12 down- and 25 up-regulated mixing pathways in more than two tissue datasets, while there were two down- and two up-regulated mixing pathways in three cell datasets. Based on the meta-analysis, there were 46 and nine common pathways in the tissue and cell datasets, respectively. Three up- and 10 down-regulated crossing pathways were detected with combined gene set enrichment analysis and meta-analysis. We found that genes with small changes are difficult to detect by classic univariate statistics; they can more easily be identified by pathway analysis. After standardized microarray preprocessing, we applied gene set enrichment analysis and a meta-analysis to increase the concordance in identifying biological mechanisms involved in prostate cancer. The gene pathways that we identified could provide insight concerning the development of prostate cancer.     

Disruption of chromosome 11 in canine fibrosarcomas highlights an unusual variability of CDKN2B in dogs

Background  

In dogs in the western world neoplasia constitutes the most frequently diagnosed cause of death. Although there appear to be similarities between canine and human cancers, rather little is known about the cytogenetic and molecular alterations in canine tumours. Different dog breeds are susceptible to different types of cancer, but the genetic basis of the great majority of these predispositions has yet to be discovered. In some retriever breeds there is a high incidence of soft tissue sarcomas and we have previously reported alterations of chromosomes 11 and 30 in two poorly differentiated fibrosarcomas. Here we extend our observations and present a case report on detail rearrangements on chromosome 11 as well as genetic variations in a tumour suppressor gene in normal dogs.     

Canine Spontaneous Head and Neck Squamous Cell Carcinomas Represent Their Human Counterparts at the Molecular Level

Spontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better understand and utilize this valuable resource, we performed a pilot study that represents its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers recapitulate many molecular features of human HNSCC. These include analogous genomic copy number abnormality landscapes and sequence mutation patterns, recurrent alteration of known HNSCC genes and pathways (e.g., cell cycle, PI3K/AKT signaling), and comparably extensive heterogeneity. Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial–mesenchymal transition genes TWIST1 and SNAI1 are also prominent in these canine tumors. This pilot study, along with a rapidly growing body of literature on canine cancer, reemphasizes the potential value of spontaneous canine cancers in HNSCC basic and translational research.     

Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.     

基于双聚类挖掘癌症共享的基因功能模块

基因多效性是癌症遗传机制中的普遍现象, 但罕见系统性的分析。文章提出采用双聚类挖掘基因功能模块的新思路探索癌症的共享分子机制和不同癌症间的关系。获取20种癌症的基因表达数据, 应用改良t检验和倍数法筛选出至少在两种癌症中差异表达的基因, 得到10417×20的数据矩阵; 采用双聚类方法获得22个癌症共享的基因簇; 进一步富集分析得到17个基因功能模块(Bonferroni校正后P<0.05), 主要参与有丝分裂染色单体分离的调控、细胞分化、免疫和炎症反应、胶原纤维组织等生物过程; 主要执行ATP结合和微管活动、MHCⅡ类受体活性、肽链内切酶抑制活性等分子功能; 活动区域主要在细胞骨架、染色体、MHCⅡ蛋白质复合体、中间丝蛋白、胶原纤维等。基于模块构建癌症相关网络, 显示胃癌、卵巢腺癌、宫颈鳞癌和间皮瘤等之间相关程度较高, 而两种血液系统癌症(急性髓细胞性白血病与多发性骨髓瘤)分子机制与其他癌症存在较大差异。可见癌症共享的基因功能模块与多种生物机制有关, 癌症之间相似性可能与组织起源、共同的致癌机制等有关。文章提出的基因多效性分析方法有助于解释人类复杂性疾病的共享分子机制。     

Stem Cell-Associated Marker Expression in Canine Hair Follicles

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients.     

Comparative mRNA and microRNA expression profiling of three genitourinary cancers reveals common hallmarks and cancer-specific molecular events

Background

Genome-wide gene expression profile using deep sequencing technologies can drive the discovery of cancer biomarkers and therapeutic targets. Such efforts are often limited to profiling the expression signature of either mRNA or microRNA (miRNA) in a single type of cancer.

Methodology

Here we provided an integrated analysis of the genome-wide mRNA and miRNA expression profiles of three different genitourinary cancers: carcinomas of the bladder, kidney and testis.

Principal Findings

Our results highlight the general or cancer-specific roles of several genes and miRNAs that may serve as candidate oncogenes or suppressors of tumor development. Further comparative analyses at the systems level revealed that significant aberrations of the cell adhesion process, p53 signaling, calcium signaling, the ECM-receptor and cell cycle pathways, the DNA repair and replication processes and the immune and inflammatory response processes were the common hallmarks of human cancers. Gene sets showing testicular cancer-specific deregulation patterns were mainly implicated in processes related to male reproductive function, and general disruptions of multiple metabolic pathways and processes related to cell migration were the characteristic molecular events for renal and bladder cancer, respectively. Furthermore, we also demonstrated that tumors with the same histological origins and genes with similar functions tended to group together in a clustering analysis. By assessing the correlation between the expression of each miRNA and its targets, we determined that deregulation of ‘key’ miRNAs may result in the global aberration of one or more pathways or processes as a whole.

Conclusions

This systematic analysis deciphered the molecular phenotypes of three genitourinary cancers and investigated their variations at the miRNA level simultaneously. Our results provided a valuable source for future studies and highlighted some promising genes, miRNAs, pathways and processes that may be useful for diagnostic or therapeutic applications.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Latent Factor Analysis to Discover Pathway-Associated Putative Segmental Aneuploidies in Human Cancers

Tumor microenvironmental stresses, such as hypoxia and lactic acidosis, play important roles in tumor progression. Although gene signatures reflecting the influence of these stresses are powerful approaches to link expression with phenotypes, they do not fully reflect the complexity of human cancers. Here, we describe the use of latent factor models to further dissect the stress gene signatures in a breast cancer expression dataset. The genes in these latent factors are coordinately expressed in tumors and depict distinct, interacting components of the biological processes. The genes in several latent factors are highly enriched in chromosomal locations. When these factors are analyzed in independent datasets with gene expression and array CGH data, the expression values of these factors are highly correlated with copy number alterations (CNAs) of the corresponding BAC clones in both the cell lines and tumors. Therefore, variation in the expression of these pathway-associated factors is at least partially caused by variation in gene dosage and CNAs among breast cancers. We have also found the expression of two latent factors without any chromosomal enrichment is highly associated with 12q CNA, likely an instance of “trans”-variations in which CNA leads to the variations in gene expression outside of the CNA region. In addition, we have found that factor 26 (1q CNA) is negatively correlated with HIF-1α protein and hypoxia pathways in breast tumors and cell lines. This agrees with, and for the first time links, known good prognosis associated with both a low hypoxia signature and the presence of CNA in this region. Taken together, these results suggest the possibility that tumor segmental aneuploidy makes significant contributions to variation in the lactic acidosis/hypoxia gene signatures in human cancers and demonstrate that latent factor analysis is a powerful means to uncover such a linkage.     

Assessment of the functionality of genome‐wide canine SNP arrays and implications for canine disease association studies

Domestic dogs share a wide range of important disease conditions with humans, including cancers, diabetes and epilepsy. Many of these conditions have similar or identical underlying pathologies to their human counterparts and thus dogs represent physiologically relevant natural models of human disorders. Comparative genomic approaches whereby disease genes can be identified in dog diseases and then mapped onto the human genome are now recognized as a valid method and are increasing in popularity. The majority of dog breeds have been created over the past few hundred years and, as a consequence, the dog genome is characterized by extensive linkage disequilibrium (LD), extending usually from hundreds of kilobases to several megabases within a breed, rather than tens of kilobases observed in the human genome. Genome‐wide canine SNP arrays have been developed, and increasing success of using these arrays to map disease loci in dogs is emerging. No equivalent of the human HapMap currently exists for different canine breeds, and the LD structure for such breeds is far less understood than for humans. This study is a dedicated large‐scale assessment of the functionalities (LD and SNP tagging performance) of canine genome‐wide SNP arrays in multiple domestic dog breeds. We have used genotype data from 18 breeds as well as wolves and coyotes genotyped by the Illumina 22K canine SNP array and Affymetrix 50K canine SNP array. As expected, high tagging performance was observed with most of the breeds using both Illumina and Affymetrix arrays when multi‐marker tagging was applied. In contrast, however, large differences in population structure, LD coverage and pairwise tagging performance were found between breeds, suggesting that study designs should be carefully assessed for individual breeds before undertaking genome‐wide association studies (GWAS).     

Comparison of the human and canine cytokines IL-1(alpha/beta) and TNF-alpha to orthologous other mammalians

The cytokines interleukin-1 (IL-1alpha and IL-1beta) and the tumor necrosis factor-alpha (TNF-alpha) both play a major role in the initiation and regulation of inflammation and immunity responses. Polymorphisms within the gene sequences of these cytokines IL-1 and TNF-alpha have been proposed to play an important role in the pathogenesis of certain diseases. Affecting nearly every organ, various diseases, including some cancers, are described to be associated with an increased level of IL-1 and TNF-alpha proteins, for example, solid tumors, hematologic malignancies, malignant histiocytosis, autoimmune disorders, Alzheimer's disease, Parkinson's disease, sepsis, and rheumatoid arthritis. Regarding genetic backgrounds and pathways, numerous canine diseases show close similarities to their human counterparts. As a genetic model, the dog could be used to unravel the genetic mechanisms, for example, in particular the predispositions, the development, and progression of cancer and metabolic diseases. The identity comparison of gene and protein sequences of different species could be used to elucidate the structure and function of the genes and proteins by identifying the evolutionary conserved regions and domains. Herein we analyzed in detail the mRNA and protein structures and identities of the present known mammalian (human, canine, murine, rat, ovine, equine, feline, porcine, and bovine) TNF-alpha, IL-1alpha, and IL-1beta mRNAs and proteins. Additionally, based on the canine genome sequence, we derived in silico the complete mRNA structures of the IL-1alpha and IL-1beta mRNAs.