22 research outputs found
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
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.Peer reviewe
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.Peer reviewe
Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells
Peer reviewe
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
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.publishedVersionPeer reviewe
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.publishedVersionPeer reviewe
Correction: Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells
Pim-1 and Pim-3 enhance CXCR4 phosphorylation and cell surface expression in prostate cancer cells.
<p>Phosphorylation of CXCR4 at S339 as well as Pim levels were analysed by western blotting in the stable Pim-1 (P1), Pim-3 (P3) or control vector (C) overexpressing PC-3 cells or the parental PC-3 cell line treated with 0.1% DMSO or 10 ÎĽM DHPCC-9. Shown are results from one representative experiment with loading controls and molecular weight (kDa) markers (A-B). The ability of Pim family members to phosphorylate CXCR4 <i>in vitro</i> was analysed by incubating GST-tagged Pim-1 (P1), Pim-2 (P2) or Pim-3 (P3) proteins with GST-tagged fragments of wild-type (WT) or Ser339>Ala (SA) mutant human CXCR4. Phosphorylated CXCR4 was detected by phospho(Ser339)-CXCR4 antibody and protein loading by Ponceau S staining. Shown are results from one representative experiment (C). Localization and signal intensity of phosphorylated versus overall CXCR4 expression was analysed by immunofluorescent (IF) staining of stably transfected cells treated with either 0.1% DMSO or 10 ÎĽM DHPCC-9. The experiment was controlled by parallel samples stained only with the secondary antibody (-ab). Stainings were repeated twice and stacks of images were taken by confocal microscopy from at least 30 cells per sample per experiment. Shown are the signal intensities of phospho-CXCR4 stainings compared to overall CXCR4 levels along with representative images from phospho-CXCR4 and CXCR4 stainings (D-E). Phosphorylation and localization of CXCR4 was also analysed by immunohistochemical staining of the paraffin-embedded tissue sections from orthopic prostate tumors. Shown is the relative increase in the amount of phospho-CXCR4-positive cells versus overall CXCR4 expression measured by whole tumor scanning. PBS instead of the primary antibody was used as a negative control. Representative images were taken to visualize the differences in phospho-CXCR4 (dark brown) stainings (F).</p
Pim inhibition by DHPCC-9 reduces the number of metastases in orthotopic prostate tumors overexpressing Pim-3.
<p>Different organs were collected from mice with orthotopic prostate tumor xenografts formed by PC-3 cells stably overexpressing an empty vector (C), Pim-1 (P1) or Pim-3 (P3). Paraffin-embedded tissue sections were stained with hematoxylin and eosin and analysed for the presence of metastases. Shown are representative images (A) from lymph node and lung sections (tumor cells indicated by arrows). The metastatic properties of xenografts from mice treated with DHPCC-9, BA-1a or their solvents were also analysed. Shown are percentages of mice positive for either lymph node metastases (B) or lung metastases (C) in each group.</p