Claudin-4 overexpression in epithelial ovarian cancer is associated with hypomethylation and is a potential target for modulation of tight junction barrier function using a C-terminal fragment of Clostridium perfringens enterotoxin - PubMed (original) (raw)

Comparative Study

doi: 10.1593/neo.07118.

Joseph Kwong, Chun-Min Lo, James G Smedley 3rd, Bruce A McClane, Margarita Aponte, Zhijian Gao, Jennifer L Sarno, Jennifer Hinners, William R Welch, Ross S Berkowitz, Samuel C Mok, Elizabeth I O Garner

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Comparative Study

Claudin-4 overexpression in epithelial ovarian cancer is associated with hypomethylation and is a potential target for modulation of tight junction barrier function using a C-terminal fragment of Clostridium perfringens enterotoxin

Babak Litkouhi et al. Neoplasia. 2007 Apr.

Abstract

Background: Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ.

Methods: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment.

Results: Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner.

Conclusions: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies.

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Figures

Figure 1

Figure 1

(A) qRT-PCR was used to compare claudin-4 expression in 79 serous ovarian cancer tissue samples of various grades to HOSE tissue. Claudin-4 expression was significantly higher in borderline, low-grade, and high-grade cancers when compared to HOSE. *P = .003, **P = .02, ***P < .001; (B) expression was not significantly different between high-grade and borderline or low-grade cancers (P = .14). Table: Using IHC, 76 ovarian cancer tissue samples of various histologic types were stained for claudin-4 and compared to nine HOSE and six endosalpingiosis samples. Slides were scored as follows: 0, absent/trace staining; 1, minimal staining; 2, moderate staining; 3, strong staining. Samples were categorized into “claudin-4-negative” (absent/trace staining, score 0) and “claudin-4-positive” (any degree of staining, scores 1–3) groups for comparison and statistical analysis.

Figure 2

Figure 2

Representative claudin-4-immunostained IHC slides from various histologic types of ovarian tissue. (A) Normal ovarian surface epithelium, (B) endosalpingiosis, (C and D) borderline serous carcinoma, (E and F) high-grade serous carcinoma, (G) clear cell carcinoma, (H) endometrioid carcinoma, and (I) mucinous carcinoma. Scale bar = 20 µm.

Figure 3

Figure 3

(A) qRT-PCR was used to compare claudin-4 expression in 20 ovarian cancer cell lines to eight HOSE and six immortalized HOSE cell lines. *P < .001, **P = .04. (B) Claudin-4 expression, as determined by Western blot, in ovarian cancer and HOSE cell lines. β-Actin was used as a loading control. (C) Claudin-4 expression, as determined by qRT-PCR, was compared in a subset of high-grade advanced-stage tissue samples. There was no statistically significant difference between paired primary and recurrent tumors, platinum-sensitive and platinum-resistant tumors, or short-term (less than 24 months) and long-term (60 months or more) survivors.

Figure 4

Figure 4

MSPCR was performed and average of three to four times on genomic DNA obtained from (A) nine claudin-4-expressing ovarian cancer tissue samples (393, 358, 377, 317, 351, 345, 312, 380, 412), (B) 11 ovarian cancer cell lines (OVCAR3, OVCA429, OVCA433, DOV13, PEO4, ES2, CAOV3, TOV112D, MCAS, RMUG-L, OVCA432) and three HOSE cell lines (HOSE2166, HOSE2170, HOSE2177). Also shown in (B), the methylation status of CLDN4 was compared to its protein expression in the cell lines. β-Actin was used as a loading control. Me, methylated primer; U-Me, unmethylated primer; WB, Western blot.

Figure 5

Figure 5

Ovarian cancer cell lines DOV13 and TOV112D, neither of which expressed claudin-4, were treated with 5-AZA (1 µmol/l), an inhibitor of DNA methylation. Treatment with 5-AZA resulted in the appearance of new unmethylated MSPCR products (inset) and corresponded with increased expression of claudin-4, as assessed by qRT-PCR (bar graph). The experiments were performed in duplicate. Also shown, MSPCR resulted in a methylated product for the methylated control DNA; PCR products were absent for unmodified DNA (which had not undergone bisulfite treatment). Me, methylated primer; U-Me, unmethylated primer.

Figure 6

Figure 6

(A) The schematic diagram represents claudin-4 mRNA: +1, transcription initiation site; | (vertical marks), CpG dinucleotide; blue band, CpG island identified using CpG Island Searcher software (

http://ccnt.hsc.usc.edu/cpgislands/

) and the displayed criteria; → (arrowhead), forward (BSF, bp 156–180) and reverse (BSR, bp 468–491) primer locations for the bisulfite sequencing; ▸ (arrows), forward (MSPF, bp 233–256) and reverse (MSPR, bp 432–452) primer locations for MSPCR. (B) The ovarian cancer cell line OVCA429, which exhibited a hypomethylated CLDN4 allele only (per Figure 4_B_), underwent bisulfite sequencing. Complete hypomethylation was demonstrated at all 21 CpG dinucleotides (circled and numbered) in the sequenced fragment.

Figure 7

Figure 7

(A) Claudin-4 Western blot for SKOV3 WT, a negative control, and SKOV3 siRNA (claudin-4 silenced). β-Actin was used as a loading control. (B) Junctional (paracellular) resistance (Rb) was compared between SKOV3 WT (wild type) and SKOV3 siRNA (siRNA claudin-4 silenced) cell lines over 7 days in cell culture medium, *P < .05. Cell lines (C) OVCA429 and (D) SKOV3 were treated with increasing amounts of C-CPE (µg/µl), resulting in a dose-dependent decrease in Rb compared to control (cell culture medium only, C-CPE concentration of 0 µg/µl). Bars represent means ± SD. Measurements were repeated four to six times, *P < .05.

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