Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion.

BACKGROUND: Progression 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. METHODS: We 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. RESULTS: Here 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. CONCLUSIONS: Based 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.

The PIM1 kinase promotes prostate cancer cell migration and adhesion via multiple signalling pathways.

The ability of cells to migrate and form metastases is one of the fatal hallmarks of cancer that can be conquered only with better understanding of the molecules and regulatory mechanisms involved. The oncogenic PIM kinases have been shown to support cancer cell survival and motility, but the PIM-regulated pathways stimulating cell migration and invasion are less well characterized than those affecting cell survival. Here we have identified the glycogen synthase kinase 3ß (GSK3B) and the forkhead box P3 (FOXP3) transcription factor as direct PIM targets, whose tumour-suppressive effects in prostate cancer cells are inhibited by PIM-induced phosphorylation, resulting in increased cell migration. Targeting GSK3B is also essential for the observed PIM-enhanced expression of the prostaglandin-endoperoxide synthase 2 (PTGS2), which is an important regulator of both cell migration and adhesion. Accordingly, selective inhibition of PIM activity not only reduces cell migration, but also affects integrin-mediated cell adhesion. Taken together, these data provide novel mechanistic insights on how and why patients with metastatic prostate cancer may benefit from therapies targeting PIM kinases, and how such approaches may also be applicable to inflammatory conditions.

Expression and ERG regulation of PIM kinases in prostate cancer.

The three oncogenic PIM family kinases have been implicated in the development of prostate cancer (PCa). The aim of this study was to examine the mRNA and protein expression levels of PIM1, PIM2, and PIM3 in PCa and their associations with the MYC and ERG oncogenes. We utilized prostate tissue specimens of normal, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), untreated PCa, and castration-resistant prostate cancer (CRPC) for immunohistochemical (IHC) analysis. In addition, we analyzed data from publicly available mRNA expression and chromatin immunoprecipitation sequencing (ChIP-Seq) datasets. Our data demonstrated that PIM expression levels are significantly elevated in PCa compared to benign samples. Strikingly, the expression of both PIM1 and PIM2 was further increased in CRPC compared to PCa. We also demonstrated a significant association between upregulated PIM family members and both the ERG and MYC oncoproteins. Interestingly, ERG directly binds to the regulatory regions of all PIM genes and upregulates their expression. Furthermore, ERG suppression with siRNA reduced the expression of PIM in PCa cells. These results provide evidence for cooperation of PIM and the MYC and ERG oncoproteins in PCa development and progression and may help to stratify suitable patients for PIM-targeted therapies.

Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts.

BACKGROUND AND METHODS: Pim family proteins are oncogenic kinases implicated in several types of cancer and involved in regulation of cell proliferation, survival as well as motility. Here we have investigated the ability of Pim kinases to promote metastatic growth of prostate cancer cells in two xenograft models for human prostate cancer. We have also evaluated the efficacy of Pim-selective inhibitors to antagonize these effects. RESULTS: We show here that tumorigenic growth of both subcutaneously and orthotopically inoculated prostate cancer xenografts is enhanced by stable overexpression of either Pim-1 or Pim-3. Moreover, Pim-overexpressing orthotopic prostate tumors are highly invasive and able to migrate not only to the nearby prostate-draining lymph nodes, but also into the lungs to form metastases. When the xenografted mice are daily treated with the Pim-selective inhibitor DHPCC-9, both the volumes as well as the metastatic capacity of the tumors are drastically decreased. Interestingly, the Pim-promoted metastatic growth of the orthotopic xenografts is associated with enhanced angiogenesis and lymphangiogenesis. Furthermore, forced Pim expression also increases phosphorylation of the CXCR4 chemokine receptor, which may enable the tumor cells to migrate towards tissues such as the lungs that express the CXCL12 chemokine ligand. CONCLUSIONS: Our results indicate that Pim overexpression enhances the invasive properties of prostate cancer cells in vivo. These effects can be reduced by the Pim-selective inhibitor DHPCC-9, which can reach tumor tissues without serious side effects. Thus, Pim-targeting therapies with DHPCC-9-like compounds may help to prevent progression of local prostate carcinomas to fatally metastatic malignancies.

Tricyclic Benzo[cd]azulenes selectively inhibit activities of Pim kinases and restrict growth of Epstein-Barr virus-transformed cells.

Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes.