Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

Abstract

Background: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.

Methodology/principal findings: A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration.

Conclusion/significance: Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.

Figure 1. CLDN7 expression in ovarian carcinoma.

A. The indicated ovarian carcinoma subtypes were tested for CLDN7 expression by RT-PCR and the levels are shown relative to HOSE-B cells (an ovarian surface epithelial cell line immortalized with E6 and E7 [52]). B. CLDN7 expression in normal tissues. The following tissues were analyzed: 1, Muscle; 2, Brain; 3, Heart; 4, Kidney; 5, Spleen; 6, Liver; 7, Colon; 8, Lung; 9, Small Intestine; 10, Stomach; 11,Testis; 12, Placenta; 13, Salivary; 14, Thyroid; 15, Adrenal Gland; 16, Pancreas; 17,Uterus; 18, Ovary; 19, Prostate; 20, Skin; 21, Plasma Blood Leukocytes; 22, Bone Marrow; 23, Fetal Brain; 24, Fetal Liver. The levels are shown relative to muscle and tissues with levels higher than 4-fold are indicated on the graph. C. Immunoblot analysis of claudin-7 expression in 2 normal ovarian tissues (N1 and N2), 7 serous (S1, S2, S3, S4, S5, S6, and S7), one clear cell (C1) and one endometrioid (E1) ovarian carcinomas. A total of 29 samples were tested by immunoblotting and representative examples are shown. D. Immunohistochemistry of serous ovarian carcinomas. Selected ovarian carcinomas (S3, S4, S5, S6) and a normal ovarian sample (N) are shown. Ovarian cancer samples stained with claudin-7 antibody (Cl-7) exhibit strong staining compared to negative controls staining lacking primary antibody (-).

Figure 2. CLDN7 expression in ovarian cancer cell lines.

A. Claudin-7 immunoblotting reveals high levels of expression in the indicated ovarian cancer cell lines. B. RT-PCR analysis of CLDN7 mRNA in cell lines. C. Immunofluorescence of selected cancer cell lines shows strong claudin-7 expression at the cell junction in BG-1, OVCA433, OVCA420 and OVCA432, as well as punctuate cytoplasmic staining in BG-1 and OVCA420. Claudin-7 was detected with an anti-claudin-7 antibody and visualized with secondary antibody conjugated to Alexa fluor. Nuclei of cells were counterstained with DAPI. Images were merged to determine correct localization.

Figure 3. CLDN7 knockdown and its effects on gene expression.

A. Immunoblot analysis of OVCAR-2 and OVCA420 cells following CLDN7 knockdown for 72 hrs. B. Effects of CLDN7 knockdown on TJ and permeability. Transepithelial resistance (TER) was measured at multiple time-points following CLDN7 knockdown. Permeability was measured by evaluating the ability of fluorescein-isothiocyanate-dextran (40 kDa) to cross the monolayer. Fluorescence measurements reflect monolayer permeability and the results mirror the TER measurements. Experiments were performed in triplicate and the results are shown as average +/− S.E.M. An asterisk (*) indicates a p-value<0.05, and a double asterisk (**), a p-value<0.01. C. Hierarchical clustering analysis of gene expression following CLDN7 knockdown in both cell lines. The green color represents down-regulation, while red represents up-regulation. A total of 20 clusters of genes exhibiting distinct patterns were identified.

Figure 4. Venn diagram showing the genes up-regulated and down-regulated in OVCAR-2 and OVCA420 following CLDN7 knockdown (absolute fold change>1.4).

In the Venn diagram, numbers in red represent genes up-regulated and numbers in green represent genes downregulated. The top 20 genes in each category are indicated in the table below the Venn diagram.

Figure 5. Validation of microarray data.

A. Fold changes of the various indicated genes in OVCA420 and OVCAR-2 cells following CLDN7 knockdown. B. RT-PCR validation of the genes shown in (A). C. Immunoblotting analysis of selected candidates at various time points following CLDN7 knockdown. GAPDH control was included to confirm similar loading in all the lanes.

Figure 6. Pathways affected by CLDN7 knockdown.

A. Pathway Studio analysis. Direct interactions of 33 significantly upregulated (red upward arrow) or downregulated (green downward arrows) genes are shown. The legend for the different types of interactions is shown below the map. B. Immunoblot analysis of Erk1/2, phospho-Erk1/2 (p-Erk) and Raf-1 and phospho-Raf (Ser-338) in OVCA420 at the indicated time points following CLDN7 knockdown.

Figure 7. Effects of CLDN7 on the migration and invasion of ovarian cancer cells.

A. Migration assays in OVCAR-2 and OVCA420 cells. The migration of cells with CLDN7 knocked down (dark bars) or control cells (white bars) was measured at different time point after the beginning of the assay. B. Boyden chamber invasion assay of OVCAR-2 and OVCA420 cells with and without CLDN7 knockdown. The number of invading cells was measured every hour for 6 hours. C. Invasion assays in a model of CLDN7 overexpression. Two stable lines, OV90-CLDN7 and OV90-CiNeo, as well as untransfected parental OV90 cells were used to test changes in invasion following CLDN7 overexpression. For all the results shown in this figure, the experiments were performed in triplicate and the results are shown as mean +/− S.E.M. An asterisk (*) indicated a p-value<0.05, and a double asterisk (**), a p-value<0.01.