Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma

<p>Abstract</p> <p>Background</p> <p>Human Disabled-2 (DAB2), is a multi-function signalling molecule that it is frequently down-regulated in human cancers. We aimed to investigate the possible tumour suppressor effect of DAB2 in nasopharyngeal carcinoma (NPC).</p> <p>Methods</p> <p>We studied the expression of DAB2 in NPC cell lines, xenografts and primary tumour samples. The status of promoter methylation was assessed by methylation specific PCR and bisulfite sequencing. The functional role of DAB2 in NPC was investigated by re-introducing DAB2 expression into NPC cell line C666-1.</p> <p>Results</p> <p>Decrease or absent of <it>DAB2 </it>transcript was observed in NPC cell lines and xenografts. Loss of DAB2 protein expression was seen in 72% (33/46) of primary NPC as demonstrated by immunohistochemistry. Aberrant <it>DAB2 </it>promoter methylation was detected in 65.2% (30/46) of primary NPC samples by methylation specific PCR. Treatment of the DAB2 negative NPC cell line C666-1 with 5-aza-2'-deoxycytidine resulted in restoration of DAB2 expression in a dose-dependent manner. Overexpression of DAB2 in NPC cell line C666-1 resulted in reduced growth rate and 35% reduction in anchorage-dependent colony formation, and inhibition of serum-induced c-Fos expression compared to vector-transfected controls. Over expression of DAB2 resulted in alterations of multiple pathways as demonstrated by expression profiling and functional network analysis, which confirmed the role of DAB2 as an adaptor molecule involved in multiple receptor-mediated signalling pathways.</p> <p>Conclusions</p> <p>We report the frequent down regulation of DAB2 in NPC and the promoter hypermethylation contributes to the loss of expression of DAB2. This is the first study demonstrating frequent DAB2 promoter hypermethylation in human cancer. Our functional studies support the putative tumour suppressor effect of DAB2 in NPC cells.</p

Stathmin1 plays oncogenic role and is a target of microRNA-223 in gastric cancer.

Stathmin1 (STMN1) is a candidate oncoprotein and prognosis marker in several kinds of cancers. This study was aimed to analyze its expression and biological functions in gastric cancer. The expression of STMN1 was evaluated by qRT-PCR, western blot and immunohistochemistry. The biological function of STMN1 was determined by MTT proliferation assays, monolayer colony formation and cell invasion assays using small interference RNA technique in gastric cancer cell lines. We also explored the regulation of STMN1 expression by microRNA-223. STMN1 was upregulated in gastric cancer cell lines and primary gastric adenocarcinomas. STMN1-positive tumors were more likely to be found in old age group and associated with p53 nuclear expression. In diffuse type gastric adenocarcinomas, STMN1 expression was correlated with age (p = 0.043), T stage (p = 0.004) and lymph node metastasis (p = 0.046). Expression of STMN1 in diffuse type gastric adenocarcinoma was associated with poor disease specific survival by univariate analysis (p = 0.01). STMN1 knockdown in AGS and MKN7 cell lines suppressed proliferation (p<0.001), reduced monolayer colony formation (p<0.001), inhibited cell invasion and migration ability (p<0.001) and induced G1 phase arrest. siSTMN1 could also suppress cell growth in vivo (p<0. 01). We finally confirmed that STMN1 is a putative downstream target of miR-223 in gastric cancer. Our findings supported an oncogenic role of STMN1 in gastric cancer. STMN1 might serve as a prognostic marker and a potential therapeutic target for gastric cancer

Clinical significance of STMN1 overexpression in gastric adenocarcinoma.

<p>(A) Representative photos of STMN1 immunohistochemistry in gastric cancer, case 37, intestinal type and case 112, diffuse type (original magnification ×100, insertion ×400). (B) Kaplan-Meier plot of disease-specific survival according to STMN1 expression status in diffuse type gastric adenocarcinoma.</p

STMN1 is a putative downstream target of miR-223 in gastric cancer.

<p>(A) MiR-223 expression level in 9 gastric cancer cell lines as determined by qRT-PCR. A borderline correlation was observed between STMN1 protein level and reduced miR-223 expression (<i>p</i> = 0.05). (B) MiR-223 expression in 31 primary gastric adenocarcinomas stratified by STMN1 protein level. (C) MiR-223 down-regulated endogenous STMN1 mRNA and protein expression (**, <i>p</i><0.001) in AGS and MKN7 cells. (D) Down-regulation of STMN1 protein expression by miR-223 was alleviated by miR-223 blocker in MKN7. (E) Luciferase reporter assays suggested STMN1 was a putative target of miR-223 (**, <i>p</i><0.001). Wildtype: Luciferase construct containing wild type STMN1 3′UTR seed sequence; Mutant 1: the seed sequence was deleted; Mutant 2: 4-nucleotide mutations were introduced to the seed sequence.</p

<i>P</i>-value of univariate and multivariate analysis of the association between clinicopathologic features and disease specific survival in patients with gastric adenocarcinoma.

<p>(NS, not significant; NA, not available).</p

Correlation of STMN1 expression with other clinicopathologic features.

<p>(NS, not significant; NA, not available).</p

STMN1 upregulation in gastric cancer cell lines and primary gastric tumors.

<p>(A) STMN1 mRNA expression in gastric cancer cell lines compared with normal gastric mRNA commercially available from Ambion (AM7996). (B) STMN1 protein expression was assessed by Western blot in gastric cancer cell lines and normal gastric mucosa from patients underwent weight reduction gastric surgery. (C) Western blot of STMN1 in paired gastric cancer (T) and adjacent non-tumorous mucosal tissues (N). (D) STMN1 mRNA expression in 50 pairs of gastric adenocarcinoma and adjacent non-tumorous mucosa (<i>p</i> = 0.040).</p

Knockdown of STMN1 by siRNA in gastric cancer cell lines AGS and MKN7.

<p>(A) Transfection of STMN1 siRNA successfully reduced STMN1 mRNA and protein expression in AGS and MKN7 cells (**, <i>p</i><0.001). (B) MTT assays suggested knockdown STMN1 significantly suppressed proliferation in AGS and MKN7 (**, <i>p</i><0.001). (C) Monolayer colony formation assays suggested transfection with siSTMN1 could reduce anchorage-dependent colony formation in AGS and MKN7 cell (**, <i>p</i><0.001). All the experiments were performed in triplicate and the error bars represent standard deviations. (D) Representative images of cells invaded through the Matrigel-coated membrane to the underside of the micropores are shown. Significant reduction in the invasive ability was shown on STMN1 knockdown (**, <i>p</i><0.001). The cell number was counted in 5 random view fields and the error bars represented standard deviations.</p

siSTMN1 induces G1 phase arrest in gastric cancer cells and inhibits cell growth <i>in vivo</i>.

<p>(A) Flow cytometric analysis revealed the accumulation of cells in G1 phase 24 hours after siSTMN1 treatment. Representative data from two independent experiments was shown. (B) Western blot analysis showed p-Rb (S807/811) reduction and increase of cleaved-PARP after STMN1 knockdown. p-AKT (S473) and p-Stat3 (T705) showed no difference. (C) siSTMN1-SGC7901 formed smaller xenograft tumors than siScramble-SGC7901 3 weeks after injection (<i>p</i><0.01).</p