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CLDN3 inhibits cancer aggressiveness via Wnt-EMT signaling and is a potential prognostic biomarker for hepatocellular carcinoma.

Jiang L, Yang YD, Fu L, Xu W, Liu D, Liang Q, Zhang X, Xu L, Guan XY, Wu B, Sung JJ, Yu J - Oncotarget (2014)

Bottom Line: Absent expression of CLDN3 was also detected in 67% of HCC cell lines, which was significantly associated with its promoter hypermethylation.Mechanistic investigations suggested through downregulation of GSK3B, CTNNB1, SNAI2, and CDH2, CLDN3 could significantly suppress metastasis by inactivating the Wnt/β-catenin-epithelial mesenchymal transition (EMT) axis in HCC cells.A better understanding of the molecular mechanism of CLDN3 in inhibiting liver cancer cell metastasis may lead to a more effective management of HCC patients with the inactivation of CLDN3.

View Article: PubMed Central - PubMed

Affiliation: Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, CUHK Shenzhen Research Institute, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Contributed equally to this work.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies but the molecular genetic basis of this disease remains unclear. By using genome-wide methylation profiling analysis, we identified CLDN3 as an epigenetically regulated gene in cancer. Here, we investigated its function and clinical relevance in human HCC. CLDN3 downregulation occurred in 87/114 (76.3%) of primary HCCs, where it was correlated significantly with shorter survival of HCC patients (P=0.021). Moreover, multivariate cyclooxygenase regression analysis showed that CLDN3 was an independent prognostic factor for overall survival (P=0.014). Absent expression of CLDN3 was also detected in 67% of HCC cell lines, which was significantly associated with its promoter hypermethylation. Ectopic expression of CLDN3 in HCC cells could inhibit cell motility, cell invasiveness, and tumor formation in nude mice. Mechanistic investigations suggested through downregulation of GSK3B, CTNNB1, SNAI2, and CDH2, CLDN3 could significantly suppress metastasis by inactivating the Wnt/β-catenin-epithelial mesenchymal transition (EMT) axis in HCC cells. Collectively, our findings demonstrated that CLDN3 is an epigenetically silenced metastasis suppressor gene in HCC. A better understanding of the molecular mechanism of CLDN3 in inhibiting liver cancer cell metastasis may lead to a more effective management of HCC patients with the inactivation of CLDN3.

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Morphological change and foci inhibition role of CLDN3 in HCC cellsEctopic expression of CLDN3 in HCC cell lines (HepG2 and Huh7) was confirmed by RT-PCR (A) and western blot (B). (C) Representatives of cell morphology of CLDN3-expressing cells (HepG2-CLDN3/Huh7-CLDN3) and control cells (upper, original magnification ×100; lower, ×200). (D) Representative of foci formation in monolayer culture. Quantitative analyses of foci numbers were shown in the right panel. Values were the mean ± SD of at least three independent experiments. *P<0.05; independent Student's t-test.
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Figure 3: Morphological change and foci inhibition role of CLDN3 in HCC cellsEctopic expression of CLDN3 in HCC cell lines (HepG2 and Huh7) was confirmed by RT-PCR (A) and western blot (B). (C) Representatives of cell morphology of CLDN3-expressing cells (HepG2-CLDN3/Huh7-CLDN3) and control cells (upper, original magnification ×100; lower, ×200). (D) Representative of foci formation in monolayer culture. Quantitative analyses of foci numbers were shown in the right panel. Values were the mean ± SD of at least three independent experiments. *P<0.05; independent Student's t-test.

Mentions: To determine if CLDN3 has tumor-suppressive function, stably CLDN3-expressing clones were established from HepG2 and Huh7 cells. CLDN3 gene and protein expression in these clones were confirmed by RT-PCR and Western blot analyses (Fig. 3A,B). The CLDN3-transfected HCC cells displayed an obvious morphological change compared with control cells. As shown in Fig. 3C, empty vector-transfected HCC cells grew in a normal state of monolayer culture while CLDN3-transfected cells formed cobblestone-like colonies (Fig. 3C). Ectopic expression of CLDN3 in these HCC cells also caused a significant decrease in cell foci formation. The number of foci formed in CLDN3-transfected cells were significantly reduced than those in empty vector-transfected cells (down to 48%-63% of vector control, P<0.01, Fig. 3D). However, no obvious difference was observed between CLDN3- and empty vector-transfected HepG2 and Huh7 cells by cell proliferation, cell cycle and cell apoptosis analyses (Fig. S1, P>0.05).


CLDN3 inhibits cancer aggressiveness via Wnt-EMT signaling and is a potential prognostic biomarker for hepatocellular carcinoma.

Jiang L, Yang YD, Fu L, Xu W, Liu D, Liang Q, Zhang X, Xu L, Guan XY, Wu B, Sung JJ, Yu J - Oncotarget (2014)

Morphological change and foci inhibition role of CLDN3 in HCC cellsEctopic expression of CLDN3 in HCC cell lines (HepG2 and Huh7) was confirmed by RT-PCR (A) and western blot (B). (C) Representatives of cell morphology of CLDN3-expressing cells (HepG2-CLDN3/Huh7-CLDN3) and control cells (upper, original magnification ×100; lower, ×200). (D) Representative of foci formation in monolayer culture. Quantitative analyses of foci numbers were shown in the right panel. Values were the mean ± SD of at least three independent experiments. *P<0.05; independent Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202152&req=5

Figure 3: Morphological change and foci inhibition role of CLDN3 in HCC cellsEctopic expression of CLDN3 in HCC cell lines (HepG2 and Huh7) was confirmed by RT-PCR (A) and western blot (B). (C) Representatives of cell morphology of CLDN3-expressing cells (HepG2-CLDN3/Huh7-CLDN3) and control cells (upper, original magnification ×100; lower, ×200). (D) Representative of foci formation in monolayer culture. Quantitative analyses of foci numbers were shown in the right panel. Values were the mean ± SD of at least three independent experiments. *P<0.05; independent Student's t-test.
Mentions: To determine if CLDN3 has tumor-suppressive function, stably CLDN3-expressing clones were established from HepG2 and Huh7 cells. CLDN3 gene and protein expression in these clones were confirmed by RT-PCR and Western blot analyses (Fig. 3A,B). The CLDN3-transfected HCC cells displayed an obvious morphological change compared with control cells. As shown in Fig. 3C, empty vector-transfected HCC cells grew in a normal state of monolayer culture while CLDN3-transfected cells formed cobblestone-like colonies (Fig. 3C). Ectopic expression of CLDN3 in these HCC cells also caused a significant decrease in cell foci formation. The number of foci formed in CLDN3-transfected cells were significantly reduced than those in empty vector-transfected cells (down to 48%-63% of vector control, P<0.01, Fig. 3D). However, no obvious difference was observed between CLDN3- and empty vector-transfected HepG2 and Huh7 cells by cell proliferation, cell cycle and cell apoptosis analyses (Fig. S1, P>0.05).

Bottom Line: Absent expression of CLDN3 was also detected in 67% of HCC cell lines, which was significantly associated with its promoter hypermethylation.Mechanistic investigations suggested through downregulation of GSK3B, CTNNB1, SNAI2, and CDH2, CLDN3 could significantly suppress metastasis by inactivating the Wnt/β-catenin-epithelial mesenchymal transition (EMT) axis in HCC cells.A better understanding of the molecular mechanism of CLDN3 in inhibiting liver cancer cell metastasis may lead to a more effective management of HCC patients with the inactivation of CLDN3.

View Article: PubMed Central - PubMed

Affiliation: Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, CUHK Shenzhen Research Institute, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Contributed equally to this work.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies but the molecular genetic basis of this disease remains unclear. By using genome-wide methylation profiling analysis, we identified CLDN3 as an epigenetically regulated gene in cancer. Here, we investigated its function and clinical relevance in human HCC. CLDN3 downregulation occurred in 87/114 (76.3%) of primary HCCs, where it was correlated significantly with shorter survival of HCC patients (P=0.021). Moreover, multivariate cyclooxygenase regression analysis showed that CLDN3 was an independent prognostic factor for overall survival (P=0.014). Absent expression of CLDN3 was also detected in 67% of HCC cell lines, which was significantly associated with its promoter hypermethylation. Ectopic expression of CLDN3 in HCC cells could inhibit cell motility, cell invasiveness, and tumor formation in nude mice. Mechanistic investigations suggested through downregulation of GSK3B, CTNNB1, SNAI2, and CDH2, CLDN3 could significantly suppress metastasis by inactivating the Wnt/β-catenin-epithelial mesenchymal transition (EMT) axis in HCC cells. Collectively, our findings demonstrated that CLDN3 is an epigenetically silenced metastasis suppressor gene in HCC. A better understanding of the molecular mechanism of CLDN3 in inhibiting liver cancer cell metastasis may lead to a more effective management of HCC patients with the inactivation of CLDN3.

Show MeSH
Related in: MedlinePlus