Abstract
The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration.
Highlights
Renal cell carcinoma (RCC) was the sixth and eighth most common malignancy among men and women, respectively, in 2012, contributing to almost 14,000 deaths in the US (Siegel et al, 2012)
Anti-NFAT5 antibody, anti-Src antibody, anti-focal adhesion kinase (FAK) antibody, and anti-phospho-FAK antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-actin antibody was from Sigma; anti-p38, anti-phospho-p38, anti-ERK1/2, antiphospho-ERK1/2, anti-Jun-terminal kinase (JNK), anti-phospho-JNK, and horseradish peroxidase-conjugated anti-rabbit IgG were purchased from Cell Signaling (Beverly, MA, USA); anti-phospho–Src antibody was from Abgent (Suzhou, China); anti-S100A4 antibody was from Spring Bioscience (Pleasanton, CA, USA)
NFAT5 AND NFAT5 TARGET GENES ARE EXPRESSED ABUNDANTLY IN RENAL CARCINOMA CELLS The cell line CaKi-1 was used as model for metastatic clear cell renal cell carcinoma (RCC)
Summary
Renal cell carcinoma (RCC) was the sixth and eighth most common malignancy among men and women, respectively, in 2012, contributing to almost 14,000 deaths in the US (Siegel et al, 2012). Loss of VHL results in accumulation of HIF-α even under normoxic conditions and downstream induction of diverse growth and angiogenic factors that contribute to malignant transformation of tubular epithelial cells (Patel et al, 2006). Based on these observations, several new pharmaceutical agents that target the involved signaling pathways have been approved in recent years (Motzer, 2011). These agents increase the therapeutic options in the treatment of metastatic RCC, continuous research is necessary to gain a better insight to the biological basis of carcinogenesis and metastasis of renal tubular cells and to identify potential targets for new therapeutic strategies
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