Genome Wide Expression Analysis of Middle Eastern Colorectal Cancer Reveals FOXM1 as a Novel Target for Cancer Therapy
ABSTRACT: In order to identify potential genes that may play an important role in progression of colorectal carcinoma, we screened and validated the global gene expression using cDNA expression array on 36 CRC tissues and compared with 24 non-cancerous colorectal tissue. We investigated the differential expression levels using cDNA microarray technique in a series of 35 CRC and 24 normal samples. FoxM1 was identified as one of the dysregulated genes and was significantly overexpressed in the tumor samples in comparison to the normal samples. Overall design: 35 colorectal cancer samples versus 24 normal samples.
INSTRUMENT(S): [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array
Project description:In order to identify potential genes that may play an important role in progression of colorectal carcinoma, we screened and validated the global gene expression using cDNA expression array on 36 CRC tissues and compared with 24 non-cancerous colorectal tissue. We investigated the differential expression levels using cDNA microarray technique in a series of 35 CRC and 24 normal samples. FoxM1 was identified as one of the dysregulated genes and was significantly overexpressed in the tumor samples in comparison to the normal samples. 35 colorectal cancer samples versus 24 normal samples.
Project description:Experiment description To identify FoxM1-regulated genes, we carried out gene expression profile analysis after inducible activation of a FoxM1-ER fusion protein using high-density human cDNA microarrays. We constructed a stable line of U2OS cells expressing full-length HA-FoxM1 fused at its C-terminus to the ligand-binding domain of the estrogen receptor (ER), mutated such that it specifically responds to 4-hydroxytamoxifen (4-OHT) but not to estrogen. In the absence of 4-OHT the activity of the ER fusion protein is turned off, but can be rapidly induced when cells are exposed to 4-OHT. We isolated total RNA from untreated cultures and cultures treated with 4-OHT for 6 or 16 hr. As activation of FoxM1 results in accumulation of cells in G2/M, we additionally analyzed the induction of gene expression by FoxM1 in G1/S-synchronized cells by a thymidine block. Total RNA derived from untreated or stimulated FoxM1-ER cells was amplified, reverse transcribed, labeled and hybridized to cDNA microarrays containing 18,000 cDNAs and expressed sequence tags (ESTs). Bound cDNA was detected using Cy3 or Cy5 dyes. In each independent experiment we included reverted color controls (Cy3 (4-OHT treated); Cy5 (untreated) or Cy3 (untreated); Cy5 (treated)) and the expression profile was expressed as a Cy5:Cy3 ratio.
Project description:Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal polyps are recognised pre-cursors of CRC, however hyperplastic polyps lack malignant potential. The purpose of this study was to identify differences in gene expression between normal colonic mucosa, hyperplastic and adenomatous polyps from disease-free individuals. By comparing polyps believed to have malignant potential (adenomatous polyps) with hyperplastic polyps it is hoped that new insights into colorectal carcinogenesis can be achieved. 24 colonic samples comprising 8 normal colonic mucosa, 8 hyperplastic polyps and 8 adenomatous polyps.
Project description:Colorectal cancer (CRC) is a genetically heterogeneous disease with several distinct morphological growth patterns. This study was aimed to investigate genes differentially expressed between ulcerative and polypoid colorectal CRC. cDNA microarray was first employed to compare the gene expression profiling of ulcerative and polypoid CRC with paired normal mucosa. Potential candidates identified by data filtering were further validated using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Epigenetic regulation of gene expression was investigated using methylation-specific PCR (MSP). cDNA microarray identified 11 up-regulated and 14 down-regulated genes differentially expressed in both types of tumor compared to matched normal mucosa. Among them, S100P was the only upregulated gene preferentially associated with polypoid CRC (p = 0.032), whereas no genes demonstrated significantly differential association with ulcerative CRC. S100P protein and mRNA expression level of polypoid CRC was significantly higher than that of ulcerative CRC (p < 0.05, respectively). Immunoreactivity of S100P protein was localized predominantly in the nuclei and to a less extent in the cytoplasm. Overexpression of S100P occurred early in the adenoma stage. 80% (24/30) and 20% (6/30) polypoid CRC showed diffusely strong and moderate overexpression, respectively. In contrast, S100P was diffusely and strongly expressed in 15% (6/40) ulcerative CRC, with 52.5% (21/40) and 32.5% (13/40) tumors having moderate and weak overexpression, respectively. S100P overexpression was preferentially associated with polypoid CRC (p < 0.001). The relative methylation level determined by MSP was not statistically different between polypoid and ulcerative CRC (43.36% vs. 49.10%, p = 0.168), indicating that promoter hypomethylation was directly related to upregulation of S100P mRNA. The gene expression profiling of ulcerative and polypoid CRC with paired normal mucosa were compared. Dye swapping experiments with ulcerative CRC vs normal mucosa or polypoid CRC vs normal mucosa were performed and we averaged the log ratios of the duplicated spots on each slide.
Project description:FOXM1 plays a key role in M phase in normal cells and is overexpressed in human glioma. We found that FOXM1 deprivation could sensitize the glioma cells to TMZ chemotherapy. To find out the mechanistic regulation of FOXM1 in chemo-resistant genes, we used microarrays to select the potential genes regulated by FOXM1 which dominates in glioma chemo-resistance. U87 glioma cells were transiently transfected with none-target siRNA or FOXM1 siRNA. Total RNA were extracted after 48 hours and subjected to the microarray.
Project description:Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal polyps are recognised pre-cursors of CRC, however hyperplastic polyps lack malignant potential. The purpose of this study was to identify differences in gene expression between normal colonic mucosa, hyperplastic and adenomatous polyps from disease-free individuals. By comparing polyps believed to have malignant potential (adenomatous polyps) with hyperplastic polyps it is hoped that new insights into colorectal carcinogenesis can be achieved. Overall design: 24 colonic samples comprising 8 normal colonic mucosa, 8 hyperplastic polyps and 8 adenomatous polyps.
Project description:The Forkhead Box m1 (Foxm1 or Foxm1b) protein (previously called HFH-11B, Trident, Win, or MPP2) is abundantly expressed in human non-small cell lung cancers where it transcriptionally induces expression of genes essential for proliferation of tumor cells. In this study, we used Rosa26-Foxm1 transgenic mice, in which the Rosa26 promoter drives ubiquitous expression of Foxm1 transgene, to identify new signaling pathways regulated by Foxm1. Lung tumors in Rosa26-Foxm1 mice were induced using the 3-methylcholanthrene (MCA)/ butylated hydroxytoluene (BHT) lung tumor initiation/ promotion protocol. MCA/BHT-treated Rosa26-Foxm1 mice displayed a significant increase in the proliferation of lung tumor cells as well as in the number and size of lung adenomas. Elevated tumor formation in Rosa26-Foxm1 transgenic lungs was associated with persistent pulmonary inflammation, macrophage infiltration and increased expression of Cdc25C phosphatase, cyclin E2, chemokine ligands Cxcl5, Cxcl1 and Ccl3, cathepsins, and Matrix metalloprotease 12, all of which stimulate signaling pathways essential for cellular proliferation, pulmonary inflammation and remodeling. Lung tumors from Rosa26-Foxm1 mice displayed increased expression of Cyclooxygenase-2 (Cox-2), whereas diminished Cox-2 levels were observed in Foxm1-deficient lung tumors from Mx-Cre Foxm1 fl/fl mice. Cell culture experiments with A549 human lung adenocarcinoma cells demonstrated that depletion of Foxm1 by either siRNA transfection or treatment with Foxm1-inhibiting ARF 26-44 peptide significantly reduced Cox-2 expression. Foxm1 protein bound to the –3479/–3461 and –7826/–7795 bp regions of the human Cox-2 promoter, indicating that the human Cox-2 gene is a direct transcriptional target of Foxm1 in lung tumor cells. Keywords: Influence of genetic modification to the tumor development Overall design: Rosa26-Foxm1 TG mice and wild type FVB/N mice were injected intraperitoneally (i.p.) with one dose of 3-methylcholanthrene (MCA; 15 µg/g mouse weight), followed by six weekly i.p. injections of butylated hydroxytoluene (BHT; the first dose was 150 µg/g mouse weight, and subsequent doses were 200 µg/g mouse weight). Seven mice from each group were sacrificed at 12 weeks and 24 weeks after MCA injection and examined for lung tumors using a dissecting microscope. Lung tissues were used to prepare total RNA with RNA-STAT-60 (Tel-Test "B" Inc. Friendswood, TX).
Project description:Genomic abnormalities leading to colorectal cancer (CRC) include somatic events causing copy number aberrations (CNAs) as well as copy neutral manifestations such as loss of heterozygosity (LOH) and uniparental disomy (UPD). We studied the causal effect of these events by analyzing high resolution cytogenetic microarray data of 15 tumor-normal paired samples. We detected 144 genes affected by CNAs. A subset of 91 genes are known to be CRC related yet high GISTIC scores indicate 24 genes on chromosomes 7, 8, 18 and 20 to be strongly relevant. Combining GISTIC ranking with functional analyses and degree of loss/gain we identify three genes in regions of significant loss (ATP8B1, NARS, and ATP5A1) and eight in regions of gain (CTCFL, SPO11, ZNF217, PLEKHA8, HOXA3, GPNMB, IGF2BP3 and PCAT1) as novel in their association with CRC. Pathway analysis indicates TGF-β signaling pathway to be affected by the cytogenetic events causing CRC as evidenced by the action of genes impacted by CNAs on SMAD family of proteins. Finally, LOH and UPD collectively affected nine cancer related genes. Transcription factor binding sites on regions of >35% copy number loss/gain influenced 16 CRC genes. Our analysis shows patient specific CRC manifestations at the genomic level and that these different events affect individual CRC patients differently. Copy number analysis of Affymetrix CytoScanHD arrays was performed for 15 Tumors and 15 Adjacent Normals from Colorectal Tissue was used as reference for the study.
Project description:The Forkhead transcription factor, FOXM1, is a key regulator of the cell cycle and is over-expressed in most types of cancer. FOXM1, similar to other Forkhead (FKH) factors binds to a canonical FKH motif in vitro. However, genome-wide mapping studies in different cell lines have shown a lack of enrichment of the FKH motif at FOXM1 binding sites suggesting an alternative mode of chromatin recruitment. We have investigated the role of direct versus indirect DNA binding in FOXM1 recruitment by performing ChIP-seq with WT and DNA binding deficient FOXM1. An in vitro fluorescence polarization (FP) assay was used to identify point mutations in the DNA binding domain (DBD) of FOXM1 that inhibit binding to a FKH consensus sequence. Stable cell lines expressing either WT or DBD mutant green fluorescence protein (GFP)-tagged FOXM1 were utilized for genome-wide mapping studies comparing the distribution of the DBD mutant protein to the WT. This showed that interaction of the DBD of FOXM1 with target DNA is essential for recruitment. Moreover, analysis of protein interactome of the WT versus DBD mutant FOXM1 showed that the reduced recruitment is not due to the mutation inhibiting protein-protein interactions. This study showed that a functional DBD domain is essential for FOXM1 chromatin recruitment. Even in FOXM1 mutants that show almost complete loss of binding the protein-protein interactome and pattern of phosphorylation are largely unaffected. These results strongly support a model whereby FOXM1 is specifically recruited to chromatin through co-factor interactions by binding directly to both canonical and non-canonical DNA sequences. Overall design: 15 samples, 40 bp single-ended ChIP-Seq libraries from cell lines with antibody for FOXM1 or expressing GFP-tagged FOXM1 (GFP-FOXM1), either wild type (GFP-FOXM1 WT) and with 2 different point mutations (GFP-FOXM1-HA and GFP-FOXM1-RA respectively). FOXM1: 2 replicates, samples labelled FOXM1_GTX; FOXM1-GFP WT: 5 replicates, samples labelled GFP and WT_GFP_FOXM1; FOXM1-GFP HA: 3 replicates, samples labelled GFP_FOXM1_HA; FOXM1-GFP RA: 2 replicates, samples labelled GFP_FOXM1_RA; input libraries: 3 different inputs for the respective experiments.
Project description:Cross-platform target gene screening in colorectal cancer (CRC): we have compared 25 tumoural CRC biopsies against their normal counterpart in 30 hybridizations with a home-made cDNA array (CNIO oncochip) and 16 hybridisations with a custom oligoarray (Agilent Technologies).