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. 2018 Feb 5;15(4):339-351.
doi: 10.7150/ijms.22927. eCollection 2018.

Claudin-3 Inhibits Lung Squamous Cell Carcinoma Cell Epithelial-mesenchymal Transition and Invasion via Suppression of the Wnt/β-catenin Signaling Pathway

Juanjuan Che  1 , Dongsheng Yue  2 , Bin Zhang  2 , Hua Zhang  2 , Yansong Huo  2 , Liuwei Gao  2 , Hongchao Zhen  1 , Yan Yang  3 , Bangwei Cao  1
Affiliations

Claudin-3 Inhibits Lung Squamous Cell Carcinoma Cell Epithelial-mesenchymal Transition and Invasion via Suppression of the Wnt/β-catenin Signaling Pathway

Juanjuan Che et al. Int J Med Sci. .

Abstract

Altered expression of claudin-3 (CLDN3), a key cytoskeletal structural protein of the tight junctions in the epithelium, is associated with the development and metastasis of various human cancers. CLDN3 expression has been shown to be significantly associated with the prognosis of lung squamous cell carcinoma (SqCC). This study investigated the role of CLDN3 in inhibiting lung SqCC cell migration and invasion as well as the underlying molecular mechanisms. The CLDN3 levels were assessed between 20 paired lung SqCC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The ectopic CLDN3 overexpression or knockdown was generated by using a plasmid carrying CLDN3 cDNA or shRNA, respectively. CLDN3 expression was significantly reduced in lung SqCC tissues vs. the adjacent normal tissues. The ectopic CLDN3 overexpression markedly inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of lung cancer H520 cells, whereas CLDN3 knockdown had an inverse effect on SK-MES-1 cells. However, cell viability and plate colony formation assays showed that both CLDN3 knockdown and overexpression did not affect SqCC cell proliferation. Both tissue and cell data revealed that CLDN3 expression was significantly associated with the expression of the EMT biomarkers E-cadherin and Vimentin. Furthermore, CLDN3-modulated EMT and expression of the EMT markers were through regulation of the Wnt/β-catenin signaling pathway. In conclusion, this study identified reduced CLDN3 expression in lung SqCC tissues, which was associated with the progression and metastasis of lung SqCC and was attributed to EMT by activation of the Wnt pathway. Thus, CLDN3 could be further evaluated as a novel biomarker for predicting the prognosis of lung SqCC and as a target for the treatment of lung SqCC in the future.

Keywords: EMT; Lung squamous cell carcinoma; Wnt; claudin-3; metastasis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Reduced CLDN3 expression in lung SqCC tissues and association with the expression of EMT-related genes. A. Western blot. Expression of CLDN3, E-cadherin, and Vimentin proteins was detected in lung SqCC and adjacent normal lung tissues from four patients. Total tissue lysates were prepared from matched frozen normal and cancer lung tissues and subjected to western blotting. Tubulin was used as a loading control. (N, adjacent normal tissue; T, tumor tissue). The results showed that CLDN3 expression was downregulated in lung SqCC tissues, compared with normal tissues, and was associated with decreased E-cadherin and increased Vimentin expression. B. qRT-PCR. The mRNA levels of CLDN3, E-cadherin, and Vimentin in 20 paired lung SqCC and adjacent normal lung samples were determined by qRT-PCR; β-actin was used as an internal control. C. Association of CLDN3 with E-cadherin protein expression (p < 0.001) and CLDN3 with Vimentin protein expression (p < 0.01).
Figure 2
Figure 2
Effects of CLDN3 overexpression or knockdown on the regulation of lung SqCC cell wound healing and Transwell migration. A and B. Transwell migration assay. Ectopic CLDN3 expression significantly inhibited the H520 cell Transwell migration capacity, whereas knockdown of CLDN3 expression significantly enhanced SK-MES1 cell migration. C and D. Quantified data of A and B, respectively (**p < 0.01 using the Student's t-test). E and F. Wound healing assay. Ectopic CLDN3 expression significantly inhibited the H520 cell wound healing capacity, whereas knockdown of CLDN3 expression significantly enhanced SK-MES1 cell wound healing. G and H. Quantified data of E and F, respectively (**p < 0.01 using the Student's t-test).
Figure 3
Figure 3
Effects of CLDN3 overexpression or knockdown on the regulation of lung SqCC cell proliferation and colony formation. A and B. Colony formation assay. Representative photographs of anchorage-dependent colonies that were stained with crystal violet. The bar graphs show that the number of cell colonies formed was not significantly different between H520-PCDH and H520-CLDN3 cells, or between SK-MES1-CON and SK-MES1-shRNA cells (p > 0.05 using the Student's t-test). C. Cell viability MTT assay. CLDN3 overexpression and knockdown in H520 and SK-MES1 cells, respectively, did not have any visible effects on cell viability.
Figure 4
Figure 4
Effects of CLDN3 overexpression or knockdown on the expression of EMT-related biomarkers. A. Western blot. CLDN3 expression was detected in lung SqCC cell lines, i.e., SK-MES1 cells expressed the highest CLDN3 level, H1703 and H2170 cells expressed moderate levels of CLDN3, and H520 cells expressed the lowest level of CLDN3. B. Western blot. CLDN3 overexpression altered the expression of the EMT markers, including E-cadherin and Vimentin in H520 cells, whereas CLDN3 knockdown promoted Vimentin expression but decreased E-cadherin expression.
Figure 5
Figure 5
Effects of CLDN3 overexpression or knockdown on the regulation of lung SqCC cell EMT proteins. A. Immunofluorescence staining. Both CLDN3 and E-cadherin proteins appeared in red in H520 cells stably infected with CLDN3 cDNA or control virus. B. Immunofluorescence staining. Both CLDN3 and E-cadherin proteins appeared in red in SK-MES-1 cells stably infected with control shRNA or CLDN3 shRNA. Two representative fields are shown; the transfected cells had dramatic co-upregulation of CLDN3 and membrane E-cadherin, compared to control cells. DAPI (blue) was used to stain the nuclei of the cells shown in panels A and B.
Figure 6
Figure 6
Effects of CLDN3 overexpression or knockdown on the regulation of the Wnt/β-catenin pathway activity. A. Western blot. Lentivirus-carrying CLDN3 cDNA or control-infected H520 cells showed that CLDN3 overexpression significantly inhibited the expression of the Wnt/β-catenin pathway proteins. B. Western blot. Lentivirus-carrying CLDN3 shRNA or negative control shRNA-infected cells significantly promoted the expression of the Wnt/β-catenin pathway proteins.
Figure 7
Figure 7
Illustration of CLDN3 signaling in cells. CLDN3 is a tetraspanin transmembrane protein with both the N- and C-terminal domains oriented toward the cytoplasm. The two extracellular loop (EL) domains are shown across the cell membrane. CLDN3 combines with the PDZ-binding motif of ZO-1/ZO-2 through the “COOH-YV” sequence. CLDN3 is linked via the scaffolding ZO-1 to proteins of the cytoskeleton such as actin. ZO-1 could connect with ZO-2 through other PDZ-binding motifs.
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