Abstract
The potassium chloride cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. Here, we investigate if KCC is involved in the regulation of epithelial-mesenchymal transition (EMT), a critical cellular event of malignancy. E-cadherin and beta-catenin colocalize in the cell-cell junctions, which becomes more obvious in a time-dependent manner by blockade of KCC activity in cervical cancer SiHa and CaSki cells. Real-time reverse transcription-PCR on the samples collected from the laser microdissection indicates that KCC3 is the most abundant KCC isoform in cervical carcinoma. The characteristics of EMT appear in KCC3-overexpressed, but not in KCC1- or KCC4-overexpressed cervical cancer cells, including the elongated cell shape, increased scattering, down-regulated epithelial markers (E-cadherin and beta-catenin), and up-regulated mesenchymal marker (vimentin). Some cellular functions are enhanced by KCC3 overexpression, such as increased invasiveness and proliferation, and weakened cell-cell association. KCC3 overexpression decreases mRNA level of E-cadherin. The promoter activity assays of various regulatory sequences confirm that KCC3 expression is a potent negative regulator for human E-cadherin gene expression. The proteosome inhibitor restores the decreased protein abundance of beta-catenin by KCC3 overexpression. In the surgical specimens of cervical carcinoma, the decreased E-cadherin amount was accompanied by the increased KCC3 abundance. Vimentin begins to appear at the invasive front and becomes significantly expressed in the tumor nest. In conclusion, KCC3 down-regulates E-cadherin/beta-catenin complex formation by inhibiting transcription of E-cadherin gene and accelerating proteosome-dependent degradation of beta-catenin protein. The disruption of E-cadherin/beta-catenin complex formation promotes EMT, thereby stimulating tumor progression.
Highlights
The activity of potassium chloride cotransporter (KCC) plays an important role in several cellular functions, such as cell volumeNote: Supplementary data for this article are available at Cancer Research Online.Requests for reprints: Meng-Ru Shen, Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan 704, Taiwan
Because epithelial-mesenchymal transition (EMT) has been characterized as a hallmark of cellular event for cancer invasion and metastasis, we investigate if KCC activity is involved in the regulation of EMT by the model of cervical carcinoma
The immunofluorescent images of two different cervical cancer cell lines show that E-cadherin and h-catenin colocalized in the cell-cell junctions, which became more obvious upon the inhibition of KCC activity (Fig. 1C and D)
Summary
The activity of potassium chloride cotransporter (KCC) plays an important role in several cellular functions, such as cell volume. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Doi:10.1158/0008-5472.CAN-07-2443 regulation, epithelial ion transport, and osmotic homeostasis [1]. KCC is defined as the ClÀ-dependent bidirectional K+ transport measured in the presence of ouabain and bumetanide to inhibit Na+-K+ pump and Na+K+2ClÀ cotransporter, respectively [2]. The activities of KCC1, KCC3, and KCC4 are osmotically sensitive and involved in cell volume regulation [3]. KCC3 plays an important role in the regulation of cell proliferation [8]. Deafness and renal tubular acidosis were noted in mice lacking KCC4 [9]
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