Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

BACKGROUND: Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. METHODS: Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. RESULTS: Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts. CONCLUSIONS: Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors

Expression profiling of sodium butyrate (NaB)-treated cells: identification of regulation of genes related to cytokine signaling and cancer metastasis by NaB

Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (NaB), a short chain fatty acid, is a HDAC inhibitor and is produced in the colonic lumen as a consequence of microbial degradation of dietary fibers. In order to dissect out the mechanism of NaB-induced growth inhibition of cancer cells, we carried out expression profiling of a human lung carcinoma cell line H460) treated with NaB using a cDNA microarray. Of the total 1728 genes analysed, there were 32 genes with a mean expression value of 2.0-fold and higher and 66 genes with a mean expression value 3.0-fold and lower in NaBtreated cells. For a few selected genes, we demonstrate that their expression pattern by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis is matching with the results obtained by microarray analysis. Closer view at the expression profile of NaB treated cells revealed the downregulation of a total of 16 genes associated with cytokine signaling, in particular, interferon c (IFNc) pathway. In good correlation, NaBpretreated cells failed to induce interferon regulatory factor 1, an INFc target gene, efficiently upon IFNc addition. These results suggest that NaB inhibits proin- flammatory cytokine signaling pathway, thus providing proof of mechanism for its anti-inflammatory activity. We also found that NaB induced three genes, which are known metastatic suppressors, and downregulated 11 genes, which have been shown to promote metastasis. Upregulation of metastatic suppressor Kangai 1 (KAI1) by NaB in a time-dependent manner was confirmed by RT–PCR analysis. The differential regulation of metastasis-associated genes by NaB provides explanation for the antiinvasive properties of NaB. Therefore, our study presents new evidence for pathways regulated by NaB, thus providing evidence for the mechanism behind antiin flammatory and antimetastatic activities of NaB

Expression profiling of sodium butyrate (NaB)-treated cells: identification of regulation of genes related to cytokine signaling and cancer metastasis by NaB

Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (NaB), a short chain fatty acid, is a HDAC inhibitor and is produced in the colonic lumen as a consequence of microbial degradation of dietary fibers. In order to dissect out the mechanism of NaB-induced growth inhibition of cancer cells, we carried out expression profiling of a human lung carcinoma cell line (H460) treated with NaB using a cDNA microarray. Of the total 1728 genes analysed, there were 32 genes with a mean expression value of 2.0-fold and higher and 66 genes with a mean expression value 3.0-fold and lower in NaB-treated cells. For a few selected genes, we demonstrate that their expression pattern by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis is matching with the results obtained by microarray analysis. Closer view at the expression profile of NaB-treated cells revealed the downregulation of a total of 16 genes associated with cytokine signaling, in particular, interferon γ (IFNγ) pathway. In good correlation, NaB-pretreated cells failed to induce interferon regulatory factor 1, an INF γ target gene, efficiently upon IFN γ addition. These results suggest that NaB inhibits proinflammatory cytokine signaling pathway, thus providing proof of mechanism for its anti-inflammatory activity. We also found that NaB induced three genes, which are known metastatic suppressors, and downregulated 11 genes, which have been shown to promote metastasis. Upregulation of metastatic suppressor Kangai 1 (KAI1) by NaB in a time-dependent manner was confirmed by RT-PCR analysis. The differential regulation of metastasis-associated genes by NaB provides explanation for the anti-invasive properties of NaB. Therefore, our study presents new evidence for pathways regulated by NaB, thus providing evidence for the mechanism behind anti-inflammatory and antimetastatic activities of NaB

CD24 Induces Expression of the Oncomir miR-21 via Src, and CD24 and Src Are Both Post-Transcriptionally Downregulated by the Tumor Suppressor miR-34a

<div><p>Cancer is a complex disease process that evolves as a consequence of multiple malfunctions in key regulatory molecular networks. Understanding these networks will be essential to combat cancer. In this study, we focussed on central players in such networks. In a series of colon and breast cancer cell lines, we found that CD24 activates Src, and induces the activation of c-Jun and expression of c-Jun and c-Fos. Thereby CD24 increases the promoter activity and expression of miR-21, which in turn suppresses expression of Pdcd4 and PTEN. Co-transfection of a CD24 expression construct and an siRNA that silences Src showed that CD24-dependent upregulation of miR-21 is mediated by Src. Additionally, we found that miR-34a post-transcriptionally downregulates CD24 and Src expression, leading to the deactivation of c-Jun, reduced expression of c-Jun and c-Fos, inhibition of miR-21, and upregulation of Pdcd4 and PTEN. Furthermore, miR-34a-mediated inhibition of Src expression reduced migration and invasion of colorectal cancer cells. Resected tumor tissues from 26 colorectal patients showed significantly lower expression of Pdcd4 and miR-34a, and higher expression of CD24, Src and miR-21 compared to the corresponding normal tissues. Moreover, CD24 positively correlated with the amount of Src protein in tumor tissues, and a trend towards an inverse correlation between miR-34a and Src protein levels was also observed. Our results reveal essential players in the complex networks that regulate the progression of solid tumors such as colorectal cancer. These findings therefore identify novel therapeutic approaches for combating tumor growth and progression.</p> </div

Activated Src induces miR-21 expression.

<p>(<b>a</b>) Western blot analysis of phosphorylated Src, Src, phosphorylated c-jun, c-Jun, c-Fos, was performed 48 h post transfection. Transfection of Rko and HCT-116 cells either with vector control or with a constitutively active Src expression construct (A-Src) is shown in the left panel. Transfection of HT-29 and Geo cell lines with either negative control siRNA (NC) or an siRNA against Src (si-Src) is shown in the right panel. β-Actin served as an internal control. (<b>b</b>) Luciferase reporter assays of the miR-21 promoter co-transfected either with A-Src in Rko and HCT-116 cells (left panel) or with si-Src in HT29 and Geo cells (right panel) along with respective controls. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.05; HCT116: p = 0.005; HT29: p = 0.001; Geo: p<0.001). (<b>c</b>) miR-21 expression levels were evaluated by RT-PCR 48 h post transfection with A-Src in Rko and HCT-116 cells, or with si-Src in HT29 and Geo cells. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.05; HCT116: p = 0.005; HT29: p = 0.02; Geo: p = 0.02). Specific p-c-Jun band intensities were normalized relative to β-actin and are represented as fold change in comparison to the control.</p

Endogenous expression of Pdcd4, CD24, Src, miR-21 and miR-34a in resected colorectal tissues.

<p>(<b>a</b>) Western blot analysis was performed for Pdcd4, CD24 and Src in colorectal tumors (Tumor) and corresponding normal tissues (Normal) taken from a series of 26 patients. β-Actin served as internal control. Relative mean protein amounts (Fold change comparative to normal tissue expression) of Pdcd4, CD24 and Src obtained by densitometry analysis are represented as bar graphs. Specific Pdcd4, CD24 or Src band intersities were normalized with β-actin. Pdcd4 was downregulated, CD24 and Src were upregulated significantly in the tumor tissues (p = 0.003, p = 0.05 and p = 0.001, respectively) (<b>b</b>) Real-time PCR results of miR-21 and miR-34a in the same colorectal tumor (Tumor) and normal tissue (Normal) samples. Mean relative expression (fold change compared to expression in normal tissue) of miR-21 and miR-34a is represented as bar graphs. miR-21 was upregulated and miR-34a was downregulated significantly in the tumor tissues. (p = 0.002, p = 0.05, respectively) (<b>c</b>) Lysates from 7 representative normal tissue (N) and colorectal tumor (T) samples were subjected to Western blotting and probed for the expression of Pdcd4, CD24 and Src and represented. β-Actin served as a loading control (<b>d</b>) Schematic representation of the functional network between CD24, Src, AP-1, miR-21, Pdcd4 and miR-34a.</p

miR-34a targets the CD24- and Src-3′-UTR and regulates their expression.

<p>(<b>a,b</b>) Luciferase assays using the CD24− and Src-3′-UTR or mutant reporter constructs transfected into HT-29 and Geo cells together with either control-miRNA (NC) or PM-34a. Percent luciferase activity was calculated either with the CD24− or Src-3′-UTR or control-miR samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three technical replicates. (<b>c</b>) Geo, Rko, HT-29 and MDA-MB-231 cells were transfected either with control miRNA or PM-34a, and 48 h later protein was isolated and western blot analysis for CD24 and Src was performed (left panel). Rko and MDA-MB-231 cells were transfected either with control miRNA or AM-34a, and 48 h later protein was isolated and western blot analysis for CD24 and Src was performed (right panel).</p

miR-34a downregulates miR-21 by targeting the CD24/Src mediated pathway.

<p>(<b>a</b>) Western blot analysis of phosphorylated Src (p-Src), Src, CD24, phosphorylated c-jun (p-c-jun), c-Jun, c-Fos, Pdcd4 and PTEN was performed 48 h post transfection. Rko (left panel) and Geo cells (right panel) were transfected with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. β-actin served as an internal control. (<b>b</b>) Luciferase reporter assays in Rko and Geo cells of the miR-21 promoter co-transfected with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>c</b>) miR-21 expression levels were evaluated by RT-PCR 48 h post transfection with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (p = <0.05). (<b>d</b>) Luciferase assays in Rko and Geo cells transfected with the Pdcd4-3′-UTR reporter construct together with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. Percent luciferase activity was calculated either with the Pdcd4-3′-UTR or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates. (<b>e</b>) The <i>in vivo</i> association of phosphorylated c-jun with the miR-21 promoter was evaluated with a ChIP assay in Rko cells after 48 h of transfection with a constitutively active Src expression construct (A-Src), empty vector (Vector), PM-34a or negative control (NC) as indicated. DNA immunoprecipitated with the p-c-jun antibody or an isotype IgG control antibody was amplified by real time PCR. Specific p-c-Jun band intensities were normalized relative to β-actin and are represented as fold change in comparison to the control.</p

Overexpression of CD24 activates Src and induces miR-21 expression.

<p>(<b>a</b>) Western blot analysis of CD24, p-Src, Src, phosphorylated c-jun, c-Jun and c-Fos was performed 48 h post transfection. Rko and HCT-116 cells transfected with either with vector control or with a CD24 expression construct is shown in the left panel. Transfection of HT-29 and Geo cell lines either with negative control siRNA (NC) or siRNA against CD24 (si-CD24) is shown in the right panel). β-Actin served as an internal control. (<b>b</b>) Luciferase reporter assays of the miR-21 promoter co-transfected either with a CD24 expression construct in Rko and HCT-116 (left panel) or with siRNA against CD24 (si-CD24) in HT29 and Geo cells (right panel) along with respective controls. Percent luciferase activity was calculated either with the miR-21 promoter or control samples set as 100%. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.01; HCT116: p = 0.02). (<b>c</b>) miR-21 expression levels were evaluated by RT-PCR 48 h post transfection upon overexpression or knock-down of CD24 in Rko, HCT-116 or HT29, Geo cell lines, respectively. The data are presented as the mean ± S.D. Each bar represents the mean value of three biological replicates (Rko: p = 0.009; HCT116: p = 0.01; HT29: p<0.001; Geo: p = 0.02). Specific p-c-Jun band intensities were normalized relative to β-actin and are represented as fold change in comparison to the control.</p