A method for finding putative causes of gene expression variation

The majority of microarray studies evaluate gene ex- pression differences between various specimens or con- ditions. However, the causes of this variability often re- main unknown. Our aim is to identify underlying causes of these patterns, a process that would eventually enable a mechanistic understanding of the deregulation of gene expression in cancer. The procedure consists of three phases: pre-processing, data integration and statistical analysis. We have applied the strategy to identify genes that are overexpressed due to amplification in breast cancer. The data were obtained from 14 breast cancer cell lines, which were subjected to cDNA microarray based copy number and expression experiments. The re- sult of the analysis was a list that consisted of 92 genes. This set includes several genes that are known to be both overexpressed and amplified in breast cancer. The com- plete study was published in Journal of the Franklin In- stitute 2004, and in this paper we focus on the main issues of the study

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

Peer reviewe

Deciphering downstream gene targets of PI3K/mTOR/p70S6K pathway in breast cancer

<p>Abstract</p> <p>Background</p> <p>The 70 kDa ribosomal protein S6 kinase (<it>RPS6KB1</it>), located at 17q23, is amplified and overexpressed in 10–30% of primary breast cancers and breast cancer cell lines. p70S6K is a serine/threonine kinase regulated by PI3K/mTOR pathway, which plays a crucial role in control of cell cycle, growth and survival. Our aim was to determine p70S6K and PI3K/mTOR/p70S6K pathway dependent gene expression profiles by microarrays using five breast cancer cell lines with predefined gene copy number and gene expression alterations. The p70S6K dependent profiles were determined by siRNA silencing of <it>RPS6KB1 </it>in two breast cancer cell lines overexpressing p70S6K. These profiles were further correlated with gene expression alterations caused by inhibition of PI3K/mTOR pathway with PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.</p> <p>Results</p> <p>Altogether, the silencing of p70S6K altered the expression of 109 and 173 genes in two breast cancer cell lines and 67 genes were altered in both cell lines in addition to <it>RPS6KB1</it>. Furthermore, 17 genes including <it>VTCN1 </it>and <it>CDKN2B </it>showed overlap with genes differentially expressed after PI3K or mTOR inhibition. The gene expression signatures responsive to both PI3K/mTOR pathway and p70S6K inhibitions revealed previously unidentified genes suggesting novel downstream targets for PI3K/mTOR/p70S6K pathway.</p> <p>Conclusion</p> <p>Since p70S6K overexpression is associated with aggressive disease and poor prognosis of breast cancer patients, the potential downstream targets of p70S6K and the whole PI3K/mTOR/p70S6K pathway identified in our study may have diagnostic value.</p

Detecting Activation of Ribosomal Protein S6 Kinase by Complementary DNA and Tissue Microarray Analysis

Background: Studies by comparative genomic hybridization (CGH) have shown that chromosomal region 17q23 is amplified in up to 20% of primary breast cancers. We used microarray analyses to measure the expression levels of genes in this region and to explore their prognostic importance. Methods: A microarray that contained 4209 complementary DNA (cDNA) clones was used to identify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal protein S6 kinase (S6K), and to localize it to the 17q23 region. Northern and western blot analyses were used to measure S6K gene and protein expression, and an enzymatic assay was used to measure S6K activity. Tumor tissue microarray analysis was used to study amplification of S6K and the HER-2 oncogene, another 17q-linked gene, and the relationship between amplification and prognosis was analyzed. The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are two-sided. Results: S6K was amplified and highly overexpressed in MCF-7 cells relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary breast tumors, and a statistically significant association between amplification and poor prognosis (P = .0021) was observed. Amplification of both S6K and HER-2 implied particularly poor survival (P = .0001). Conclusions: The combination of CGH information with cDNA and tissue microarray analyses can be used to identify amplified and overexpressed genes and to evaluate the clinical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and may adversely affect the prognosis of patients with this amplificatio

Analysis of BMP4 and BMP7 signaling in breast cancer cells unveils time-dependent transcription patterns and highlights a common synexpression group of genes

<p>Abstract</p> <p>Background</p> <p>Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors. They are known for their roles in regulation of osteogenesis and developmental processes and, in recent years, evidence has accumulated of their crucial functions in tumor biology. BMP4 and BMP7, in particular, have been implicated in breast cancer. However, little is known about BMP target genes in the context of tumor. We explored the effects of BMP4 and BMP7 treatment on global gene transcription in seven breast cancer cell lines during a 6-point time series, using a whole-genome oligo microarray. Data analysis included hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data.</p> <p>Results</p> <p>Both ligands had a strong effect on gene expression, although the response to BMP4 treatment was more pronounced. The cellular functions most strongly affected by BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, with regulation of gene expression and signal transduction leading to changes in metabolism and cell proliferation. Hierarchical clustering revealed distinct differences in the response of individual cell lines to BMPs, but also highlighted a synexpression group of genes for both ligands. Interestingly, the majority of the genes within these synexpression groups were shared by the two ligands, probably representing the core molecular responses common to BMP4 and BMP7 signaling pathways.</p> <p>Conclusions</p> <p>All in all, we show that BMP signaling has a remarkable effect on gene transcription in breast cancer cells and that the functions affected follow a logical temporal pattern. Our results also uncover components of the common cellular transcriptional response to BMP4 and BMP7. Most importantly, this study provides a list of potential novel BMP target genes relevant in breast cancer.</p

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma.Peer reviewe

Novel ZNF414 activity characterized by integrative analysis of ChIP-exo, ATAC-seq and RNA-seq data

Transcription factor binding to DNA is a central mechanism regulating gene expression. Thus, thorough characterization of this process is essential for understanding cellular biology in both health and disease. We combined data from three sequencing-based methods to unravel the DNA binding function of the novel ZNF414 protein in cells representing two tumor types. ChIP-exo served to map protein binding sites, ATAC-seq allowed identification of open chromatin, and RNA-seq examined the transcriptome. We show that ZNF414 is a DNAbinding protein that both induces and represses gene expression. This transcriptional response has an impact on cellular processes related to proliferation and other malignancy-associated functions, such as cell migration and DNA repair. Approximately 20% of the differentially expressed genes harbored ZNF414 binding sites in their promoters in accessible chromatin, likely representing direct targets of ZNF414. De novo motif discovery revealed several putative ZNF414 binding sequences, one of which was validated using EMSA. In conclusion, this study illustrates a highly efficient integrative approach for the characterization of the DNA binding and transcriptional activity of transcription factors.Peer reviewe

RAD51B in Familial Breast Cancer

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk.Public Library of Science open acces

PLA2G7 associates with hormone receptor negativity in clinical breast cancer samples and regulates epithelial-mesenchymal transition in cultured breast cancer cells

Breast cancer is the leading cause of cancer-related deaths in women due to distinct cancer subtypes associated with early recurrence and aggressive metastatic progression. High lipoprotein-associated phospholipase A2 (PLA2G7) expression has previously been associated with aggressive disease and metastasis in prostate cancer. Here, we explore the expression pattern and functional role of PLA2G7 in breast cancer. First, a bioinformatic analysis of genome-wide gene expression data from 970 breast samples was carried out to evaluate the expression pattern of PLA2G7 mRNA in breast cancer. Second, the expression profile of PLA2G7 was studied in 1042 breast cancer samples including 89 matched lymph node metastasis samples using immunohistochemistry. Third, the effect of PLA2G7 silencing on genome-wide gene expression profile was studied and validated in cultured breast cancer cells expressing PLA2G7 at high level. Last, the expression pattern of PLA2G7 mRNA was investigated in 24 nonmalignant tissue samples and 65 primary and 7 metastatic tumour samples derived from various organs using qRT-PCR. The results from clinical breast cancer samples indicated that PLA2G7 is overexpressed in a subset of breast cancer samples compared to its expression in benign breast tissue samples and that high PLA2G7 expression associated with hormone receptor negativity as well as with poor prognosis in a subset of breast cancer samples. In vitro functional studies highlighted the putative role of PLA2G7 in the regulation of epithelial-mesenchymal transition (EMT)-related signalling pathways, vimentin and E-cadherin protein expression as well as cell migration in cultured breast cancer cells. Furthermore, supporting the findings in breast and prostate cancer, high PLA2G7 mRNA expression was associated with metastatic cancer in four additional organs of origin. In conclusion, our results indicate that PLA2G7 is highly expressed in a subset of metastatic and aggressive breast cancers and in metastatic samples of various tissues of origin and promotes EMT and migration in cultured breast cancer cells

Identification of fusion genes in breast cancer by paired-end RNA-sequencing

Background Until recently, chromosomal translocations and fusion genes have been an underappreciated class of mutations in solid tumors. Next-generation sequencing technologies provide an opportunity for systematic characterization of cancer cell transcriptomes, including the discovery of expressed fusion genes resulting from underlying genomic rearrangements. Results We applied paired-end RNA-seq to identify 24 novel and 3 previously known fusion genes in breast cancer cells. Supported by an improved bioinformatic approach, we had a 95% success rate of validating gene fusions initially detected by RNA-seq. Fusion partner genes were found to contribute promoters (5' UTR), coding sequences and 3' UTRs. Most fusion genes were associated with copy number transitions and were particularly common in high-level DNA amplifications. This suggests that fusion events may contribute to the selective advantage provided by DNA amplifications and deletions. Some of the fusion partner genes, such as GSDMB in the TATDN1-GSDMB fusion and IKZF3 in the VAPB-IKZF3 fusion, were only detected as a fusion transcript, indicating activation of a dormant gene by the fusion event. A number of fusion gene partners have either been previously observed in oncogenic gene fusions, mostly in leukemias, or otherwise reported to be oncogenic. RNA interference-mediated knock-down of the VAPB-IKZF3 fusion gene indicated that it may be necessary for cancer cell growth and survival. Conclusions In summary, using RNA-sequencing and improved bioinformatic stratification, we have discovered a number of novel fusion genes in breast cancer, and identified VAPB-IKZF3 as a potential fusion gene with importance for the growth and survival of breast cancer cells