Claudin‑9 is a novel prognostic biomarker for endometrial cancer

Abstract

The tight‑junction protein claudin‑9 (CLDN9) is barely distributed in normal adult tissues but is ectopically expressed in various cancer types. Although multiple databases indicated upregulation of CLDN9 in endometrial cancers at the mRNA level, its protein expression and biological roles remain obscure. In the present study, the prognostic significance of CLDN9 expression in endometrial cancer was evaluated by immunohistochemical staining and semi‑quantification using formalin‑fixed paraffin‑embedded specimens obtained from 248 endometrial carcinoma cases. A total of 43 cases (17.3%) had high CLDN9 expression, whereas 205 cases (82.7%) exhibited low CLDN9 expression. The 5‑year disease‑specific survival rates in the high and low CLDN9 expression groups were 62.8 and 87.8% (P<0.001), respectively. In addition, multivariate analysis revealed that high CLDN9 expression was an independent prognostic factor (hazard ratio, 4.99; 95% CI, 1.96‑12.70; P<0.001). Furthermore, CLDN9 expression was significantly correlated with the expression of CLDN6 (P<0.001), which is the closest CLDN member to CLDN9 and a poor prognostic factor for endometrial carcinoma. The 5‑year disease‑specific survival rate of cases with CLDN6‑high/CLDN9‑high, CLDN6‑high/CLDN9‑low and CLDN6‑low/CLDN9‑high status was 30.0, 37.5 and 72.7%, respectively, whereas that of CLDN6‑low/CLDN9‑low was 89.8% (P=0.004). In conclusion, aberrant CLDN9 expression is a predictor of poor prognosis for endometrial cancer and may be utilized in combination with CLDN6 to achieve higher sensitivity.

Keywords: biomarker; claudin; endometrial cancer; gynecological cancer; tight junction.

Figure 1

Establishment and characterization of a rat anti-human CLDN9 mAb. (A) Topology of CLDN9 and amino acid sequences of the C-terminal cytoplasmic domains of human CLDN9 and the corresponding regions of the closely related CLDNs. Conserved amino acids are displayed in red. The antigen region is highlighted in yellow. (B) Western blot and (C) immunohistochemical analyses indicating the specificity of the rat anti-human CLDN9 mAb. 293T cells were transfected with individual CLDN expression vectors and subjected to analyses (scale bar, 50 µm). (D) Localization of CLDN9 in Ishikawa cells co-transfected with CLDN9 and Lamin-RFP. Arrowheads indicate CLDN9-immunoreactive signals along a cell-cell boundary (scale bar, 20 µm). (E) Immunohistochemical staining of CLDN9 in the normal pituitary gland (scale bar, 200 µm). The square indicates the enlarged areas presented in the right panels. mAb, monoclonal antibody; CMV, cytomegalovirus promoter; LMNB1, Lamin B1; RFP, red fluorescent protein; CLDN, claudin; EF1a, human elongation factor 1-α promoter.

Figure 2

Immunohistochemical staining of CLDN9 in endometrial cancer tissues. Representative images show negative/weak/moderate/strong signal intensity of CLDN9 expression in endometrial cancer tissues (scale bar, 100 µm). The squares indicate the enlarged areas presented in the right panels. HE, hematoxylin-eosin; CLDN, claudin.

Figure 3

Association of high CLDN9 expression with poor outcomes in patients with endometrial cancer. Kaplan-Meier curves for (A) disease-specific and (B) disease-free survival for high and low expression of CLDN9 in endometrial cancer subjects. CLDN, claudin.

Figure 4

Association of CLDN6/9 and their expression in endometrial cancer tissues. (A) The location of CLDN9 and CLDN6 genes in the human genome. Their genes are located on chromosome 16 at p13.3 3014712-3020071 and p 13.3 3012923-3014505, respectively. (B) Sequence homology between human CLDN6 and CLDN9 protein. (C) Immunohistochemical staining of CLDN6 and CLDN9 in endometrial cancer tissues. A total of four representative patterns are presented with different expression patterns of CLDN6 and CLDN9 (scale bar, 100 µm). HE, hematoxylin-eosin; CLDN, claudin.

Figure 5

High CLDN9 expression is associated with poor prognosis in patients with endometrial cancer in the low CLDN6 expression group. Kaplan-Meier curves for (A) disease-specific and (B) disease-free survival are provided. CLDN, claudin.