PlosOne. 2013; Sep 19;8(9):e74994.

Claudin-3 Overexpression Increases the Malignant Potential of Colorectal Cancer Cells: Roles of ERK1/2 and PI3K-Akt as Modulators of EGFR signaling.

Waldemir F. de Souza1, Natalia Fortunato-Miranda1, Bruno K. Robbs2, Wallace M. de Araujo1, Julio C. de-Freitas-Junior1, Lilian G. Bastos1, João P. B. Viola2, José A. Morgado-Díaz1*

1Structural Biology Group; 2Gene Regulation Group, Cell Biology Program – Research Center, Brazilian National Cancer Institute, Brazil



The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchorage-dependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways.

Key words: claudins; tight junctions; colorectal cancer; EGF; ERK1/2; PI3K/Akt



The cell-cell adhesion system is maintained by the apical junctional complex, which is constituted by the tight and adherent junctions that play important roles in epithelial homeostasis. Studies have reported that alterations in claudin expression, important proteins at the tight junctions, are associated with the tumorigenic process. In an early study we have shown that patients with colorectal cancer presented increased expression levels of claudins 1, 3 and 4, which altered the barrier function of tight junction (FEBS Letters, 2005, 579: 6179-85) . However, the molecular mechanisms that regulate the expression of claudin family members and the implications of claudin overexpression in colorectal cancer progression are still not well understood.

The results of our present study showed that: a) the activation of the MEK/ERK and PI3K-Akt signaling pathways Epidermal Growth Factor-induced increases the claudin-3 expression, and b) Claudin-3 overexpression induces alteration in barrier function of tight junctions and increase of the malignant potential in colorectal cancer cells. In summary, our results reveal that claudin-3 plays an important role as tumor promoter when its expression is imbalanced and implicate the ERK1/2 and PI3K-Akt signaling pathways as modulators of claudin-3 upregulation-related tumor progression in colorectal cancer cells. These findings contributes to a better understanding on the molecular mechanisms that regulate the expression of claudins and the relationship of these proteins with the malignant process. In addition, it is possible to suggest this protein as possible molecular targets for future applications in the treatment of colorectal cancer.

Jose Morgado-Diaz figure1

Acknowledgments: This study was sponsored by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Coordenação de Aperfeiçõamento de Pessoal de Nível Superior (CAPES), Ministério da Saúde – Brasil, Fundação Carlos Chagas Filho de Amparo á Pesquisa do Estado de Rio de Janeiro (FAPERJ) and Instituto nacional de Ciência e Tecnologia em Câncer (573806/2008-0 and 170.026/2008).

figure 2

Contact: José A. Morgado-Díaz, Research Center, Brazilian National Cancer Institute, 37 Andre Cavalcanti Street, 5th Floor, Centro, Rio de Janeiro, Brazil, Zip code: 20231-050, Tel: +55-21-32076533, Fax: +55-21-32076587, E-mail:

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