Decreased expression of gastrokine 1 and the trefoil factor interacting protein TFIZ1/GKN2 in gastric cancer: influence of tumor histology and relationship to prognosis

Abstract

Purpose: Transcriptional profiling showed decreased expression of gastrokine 1 (GKN1) and the related trefoil factor interacting protein (TFIZ1/GKN2) in Helicobacter pylori infection. Decreased GKN1 and GKN2 mRNA expression has been reported in gastric adenocarcinoma. We have examined GKN1 and GKN2 protein expression in a large gastric cancer series, correlated expression with tumor subtype, and evaluated their utility as prognostic biomarkers.

Experimental design: GKN1, GKN2, and the trefoil factors TFF1 and TFF3 were examined in tissue microarrays from 155 distal gastric adenocarcinomas. Immunohistochemical expression was correlated with clinical outcome. GKN1 and GKN2 expression was measured by real-time PCR and Western analysis in samples of gastric cancer and adjacent nonneoplastic mucosa.

Results: GKN1 was lost in 78% of diffuse and 42% of intestinal cancers (P < 0.0001, diffuse versus intestinal). GKN2 expression was lost in 85% of diffuse and 54% of intestinal type cancers (P < 0.002). GKN1 and GKN2 down-regulation were confirmed by Western and real-time PCR analysis. Loss of either protein was associated with significantly worse outcome in intestinal-type tumors by univariate analysis; and GKN2 loss remained a predictor of poor outcome in multivariate analysis (P < 0.033). TFF1 was lost in >70%, and TFF3 was expressed in approximately 50% of gastric cancers.

Conclusions: Loss of GKN1 and GKN2 expression occurs frequently in gastric adenocarcinomas, especially in the diffuse subtype. GKN1 and GKN2 loss are associated with shorter overall survival in the intestinal subtype.

Fig 1

Representative photomicrographs of immunohistochemical staining of gastric tissue, demonstrating expression of GKN1 (a,c,e,g) and GKN2 (b,d,f,h) in gastric epithelial cells. a,b: normal mucosa; c,d: intestinal metaplasia; e,f: gastric cancer (intestinal type); g,h: gastric cancer (diffuse type). All original magnifications × 200.

Fig 2

Summary of immunostaining of gastric cancer cases, grouped into (a) those cancers staining negative (score 0–3) or positive (score 4–9) for GKN1 or GKN2, and (b) those staining negative (score 0–3) or positive (score 4–9) for TFF1 or TFF3. P values refer to difference between proportions of the staining expression of these markers in the intestinal and diffuse cancer types.

Fig 3

Western blot of tissue lysates from 4 cases of diffuse type gastric cancer (Ca) with adjacent non-neoplastic mucosa (N) showing marked loss/absence of GKN1 and GKN2 in the cancers. GAPDH expression (bottom panel) serves as a control, demonstrating equal protein loading across all lanes.

Fig 4

Expression of GKN1 and GKN2 mRNA in tumors compared with non-neoplastic resection margins. Data expressed as mean fold decrease in the tumor epithelium relative to non-neoplastic gastric tissue. * p< 0.004, ** p<0.007.

Fig 5

Kaplan-Meier plots of immunohistochemical scores of GKN1 (a, b) and GKN2 (c,d) expression in intestinal-type (a,c) and diffuse type (b,d) gastric cancers, demonstrating that intestinal tumors with low GKN1 and GKN2 expression were associated with significantly worse prognosis. Univariate analyses.

Fig 6

Kaplan-Meier plots of immunohistochemical scores of TFF1 (a, b) and TFF3 (c,d) expression in intestinal type (a,c) and diffuse type (b,d) gastric cancers, demonstrating that intestinal tumors with high TFF3 expression were associated with significantly worse prognosis. Univariate analyses.