Abstract
BackgroundProgression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved.MethodsWe used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets.ResultsHere we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens.ConclusionsBased on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer.Graphical abstract
Highlights
Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process
NFATC1 is endogenously expressed and constitutively active in PC-3 cells As we had previously shown both PIM kinases and NFATC1 to be essential for the motility of PC-3 prostate cancer cells [4], we decided to use these cells in order to investigate in more detail the functional interactions between PIM and NFATC1 proteins
Nuclear factor of activated T cells (NFAT)-dependent luciferase assays in turn revealed endogenous NFAT activity, which was dependent on the presence of NFAT binding sites (Fig. 1b), and which was enhanced by ectopic overexpression of NFATC1, but not by stimulation of cells with TPA and the calcium ionophore ionomycin (Fig. 1c)
Summary
Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. We have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. NFATC1 has been shown to support cell migration or invasion in multiple types of cancer, such as ovarian, breast and prostate cancer as well as glioblastoma [3,4,5,6,7]. It has been reported to support metastatic behavior of prostate or breast cancer cells via increased osteoclastogenesis [8, 9]
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