Transcriptomic “portraits” of canine mammary cancer cell lines with various phenotypes

In light of the high incidence of mammary cancer in dogs and completion of the canine genome sequencing, the new possibilities of gene profiling by using DNA microarrays give hope to veterinary oncology. The cell lines isolated from mammary tumors are a valuable tool in developing and testing new pathway-specific cancer therapeutics. Differential cytometric analysis of 6 canine mammary cancer cell lines was performed. We divided cell lines into 3 groups based on their phenotype: 2 lines with high proliferative potential, 2 lines with high antiapoptotic potential, and 2 lines with high metastatic potential. DNA microarray analysis revealed common genes for cell lines of each group. We found that genes encoding the receptors for growth hormone and ghrelin are related to high proliferation rate, whileABR (active BCR-related) andTMD1 (TM2 domain containing 1) genes are related to a high antiapoptotic potential of the cancer cells. Metastatic properties of mammary cancer cells seem to be associated with elevated expression ofPGP (P glycoprotein),SEMA3B (semaphorin 3B), andSTIM1 (stromal interaction molecule 1).     

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.     

Independence and reproducibility across microarray platforms

Microarrays have been widely used for the analysis of gene expression, but the issue of reproducibility across platforms has yet to be fully resolved. To address this apparent problem, we compared gene expression between two microarray platforms: the short oligonucleotide Affymetrix Mouse Genome 430 2.0 GeneChip and a spotted cDNA array using a mouse model of angiotensin II-induced hypertension. RNA extracted from treated mice was analyzed using Affymetrix and cDNA platforms and then by quantitative RT-PCR (qRT-PCR) for validation of specific genes. For the 11,710 genes present on both arrays, we assessed the relative impact of experimental treatment and platform on measured expression and found that biological treatment had a far greater impact on measured expression than did platform for more than 90% of genes, a result validated by qRT-PCR. In the small number of cases in which platforms yielded discrepant results, qRT-PCR generally did not confirm either set of data, suggesting that sequence-specific effects may make expression predictions difficult to make using any technique.     

On predicting medulloblastoma metastasis by gene expression profiling

Accurately predicting clinical outcome or metastatic status from gene expression profiles remains one of the biggest hurdles facing the adoption of predictive medicine. Recently, MacDonald et al. (Nat. Genet. 2001, 29, 143-152) used gene expression profiles, from samples taken at diagnosis, to distinguish between clinically designated metastatic and nonmetastatic primary medulloblastomas, helping to elucidate the genetic mechanisms underlying metastasis and suggesting novel therapeutic targets. The obtained accuracy of predicting metastatic status does not, however, reach statistical significance on Fisher's exact test, although 22 training samples were used to make each prediction via leave-one-out testing. This paper introduces readily implemented nonlinear filters to transform sequences of gene expression levels into output signals that are significantly easier to classify and predict metastasis. It is shown that when only 3 exemplars each from the metastatic and nonmetastatic classes were assumed known, a predictor was constructed whose accuracy is statistically significant over the remaining profiles set aside as a test set. The predictor was as effective in recognizing metastatic as nonmetastatic medulloblastomas, and may be helpful in deciding which patients require more aggressive therapy. The same predictor was similarly effective on an independent set of 5 nonmetastatic tumors and 3 metastatic cell lines also used by MacDonald et al.     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

Integrated analysis of DNA methylation and gene expression profiles identified S100A9 as a potential biomarker in ulcerative colitis

Ulcerative colitis (UC) is a prevalent relapsing-remitting inflammatory bowel disease whose pathogenetic mechanisms remain elusive. In the present study, colonic biopsies samples from three UC patients treated in the Traditional Chinese Medicine Hospital and three healthy controls were obtained. The genome-wide mRNA and lncRNA expression of the samples were profiled through Agilent gene expression microarray. Moreover, the genome-wide DNA methylation dataset of normal and UC colon tissues was also downloaded from GEO for a collaborative analysis. Differential expression of lncRNA (DELs) and mRNAs (DEMs) in UC samples compared with healthy samples were identified by using limma Bioconductor package. Differentially methylated promoters (DMPs) in UC samples compared with controls were obtained through comparing the average methylation level of CpGs located at promoters by using t-test. Functional enrichment analysis was performed by the DAVID. STRING database was applied to the construction of gene functional interaction network. As a result, 2090 DEMs and 1242 DELs were screened out in UC samples that were closely associated with processes related to complement and coagulation cascades, osteoclast differentiation vaccinia, and hemorrhagic diseases. A total of 90 DEMs and 72 DELs were retained for the construction of functional network for the promoters of their corresponding genes were identified as DMPs. S100A9, HECW2, SOD3 and HIX0114733 showed high interaction degrees in the functional network, and expression of S100A9 was confirmed to be significantly elevated in colon tissues of UC patients compared with that of controls by qRT-PCR that was consistent with gene microarray analysis. These indicate that S100A9 could potentially be used as predictive biomarkers in UC.     

Is there a malignant progression associated with a linear change in protein expression levels from normal canine mammary gland to metastatic mammary tumors?

The molecular mechanisms of the development of canine mammary tumors are still incompletely understood. In the present study we hypothesized that there is a malignant progression from normal gland to malignant carcinomas that is associated with a linear change in protein expression. To this end, the proteome of canine normal mammary gland, adenomas, nonmetastatic carcinomas, and metastatic carcinomas was compared. Application of 2D-DIGE and MALDI-TOF-MS identified 48 proteins with significant changes (fold change >|1.5|; p < 0.05) in expression levels at the different stages of malignant progression. Forty-two of these followed three major stepwise but not linear expression patterns. Thirteen proteins showed the adenoma pattern characterized by a change in protein expression levels during progression from normal gland to adenomas which persisted on the same level at the subsequent stages of malignancy. Nine proteins followed the carcinoma pattern with an up- or down-regulation between adenomas and carcinomas. The majority of 20 proteins followed the metastasis pattern with a significant change of protein expression levels between nonmetastatic and metastatic carcinomas. The present study therefore shows that differences in malignancy are associated with a stepwise but not linear change in protein expression levels, which does not finally confirm or disapprove the existence of a malignant progression in canine mammary tumors. In addition, the acquisition of metastatic potential seems to be associated with the strongest changes in protein expression levels.     

Identification of a potential ovarian cancer stem cell gene expression profile from advanced stage papillary serous ovarian cancer

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.     

Clinical phenotype and gene expression profile in Crohn's disease

The clinical course varies significantly among patients with Crohn's disease (CD). This study investigated whether gene expression profiles generated by DNA microarray technology might predict disease progression. Biopsies from the descending colon were obtained colonoscopically from 40 CD patients. Gene profiling analyses were performed using a Human Genome U133 Plus 2.0 GeneChip Array, and summarization into a single expression measure for each probe set was performed using the robust multiple array procedure. Principal component analysis demonstrated that three components explain two-thirds of the total variation. The most important parameters for the determination of the colonic gene expression patterns were the presence of disease (CD) and presence of inflammation. Superimposition of clinical phenotype data revealed a grouping of the samples from patients with stenosis toward negative values on the axis of the second principal component. The functional annotation analysis suggested that the expression of genes involved in intracellular transport and cytoskeletal organization might influence the development of stenosis. In conclusion, even though most variation in the colonic gene expression patterns is due to presence or absence of CD and inflammation status, the development of stenosis is a parameter that affects colonic gene expression to some extent.     

Comparative Gene Expression Analyses Identify Luminal and Basal Subtypes of Canine Invasive Urothelial Carcinoma That Mimic Patterns in Human Invasive Bladder Cancer

More than 160,000 people are expected to die from invasive urothelial carcinoma (iUC) this year worldwide. Research in relevant animal models is essential to improving iUC management. Naturally-occurring canine iUC closely resembles human iUC in histopathology, metastatic behavior, and treatment response, and could provide a relevant model for human iUC. The molecular characterization of canine iUC, however, has been limited. Work was conducted to compare gene expression array results between tissue samples from iUC and normal bladder in dogs, with comparison to similar expression array data from human iUC and normal bladder in the literature. Considerable similarities between enrichment patterns of genes in canine and human iUC were observed. These included patterns mirroring basal and luminal subtypes initially observed in human breast cancer and more recently noted in human iUC. Canine iUC samples also exhibited enrichment for genes involved in P53 pathways, as has been reported in human iUC. This is particularly relevant as drugs targeting these genes/pathways in other cancers could be repurposed to treat iUC, with dogs providing a model to optimize therapy. As part of the validation of the results and proof of principal for evaluating individualized targeted therapy, the overexpression of EGFR in canine bladder iUC was confirmed. The similarities in gene expression patterns between dogs and humans add considerably to the value of naturally-occurring canine iUC as a relevant and much needed animal model for human iUC. Furthermore, the finding of expression patterns that cross different pathologically-defined cancers could allow studies of dogs with iUC to help optimize cancer management across multiple cancer types. The work is also expected to lead to a better understanding of the biological importance of the gene expression patterns, and the potential application of the cross-species comparisons approach to other cancer types as well.     

Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR

Gene expression analysis provides significant insight to understand regulatory mechanisms of biology, yet acquisition and reproduction of quality data, as well as data confirmation and verification remain challenging due to a lack of proper quality controls across different assay platforms. We present a set of six universal external RNA quality controls for microbial mRNA expression analysis that can be applied to both DNA oligo microarray and real-time qRT-PCR including using SYBR Green and TaqMan probe-based chemistry. This set of controls was applied for Saccharomyces cerevisiae and Pseudomonas fluorescens Pf-5 microarray assays and qRT-PCR for yeast gene expression analysis. Highly fitted linear relationships between detected signal intensity and mRNA input were described. Valid mRNA detection range, from 10 to 7000 pg and from 100 fg to 1000 pg were defined for microarray and qRT-PCR assay, respectively. Quantitative estimation of mRNA abundance was tested using randomly selected yeast ORF including function unknown genes using the same source of samples by the two assay platforms. Estimates of mRNA abundance by the two methods were similar and highly correlated in an overlapping detection range from 10 to 1000 pg. The universal external RNA controls provide a means to compare microbial gene expression data derived from different experiments and different platforms for verification and confirmation. Such quality controls ensure reliability and reproducibility of gene expression data, and provide unbiased normalization reference for validation, quantification, and estimate of variation of gene expression experiments. Application of these controls also improves efficiency and facilitates high throughput applications of gene expression analysis using the qRT-PCR assay.