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. 2014 Dec 30;5(24):12646-64.
doi: 10.18632/oncotarget.2653.

PRK1/PKN1 controls migration and metastasis of androgen-independent prostate cancer cells

Cordula A Jilg  1 , Anett Ketscher  2 , Eric Metzger  1 , Barbara Hummel  1 , Dominica Willmann  1 , Vanessa Rüsseler  3 , Vanessa Drendel  4 , Axel Imhof  5 , Manfred Jung  6 , Henriette Franz  1 , Stefanie Hölz  2 , Malte Krönig  1 , Judith M Müller  1 , Roland Schüle  7
Affiliations

PRK1/PKN1 controls migration and metastasis of androgen-independent prostate cancer cells

Cordula A Jilg et al. Oncotarget. .

Abstract

The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer.

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Figures

Figure 1
Figure 1. PRK1 controls migration and invasion of androgen-independent prostate cancer cell lines and determines development of metastases in vivo
Migration (A) and invasion (B) assays of PC-3M-luc2 cells treated with siRNA against PRK1 (siPRK1) or unrelated siRNA (siCtrl). Migration (D) and invasion (E) assay of PC-3M-luc2 stable overexpressing PRK1 or control transfected cells. (G) Migration of PC-3M-luc2 cells treated with PRK1-inhibitor Lestaurtinib (25 μM). (A, B, D, E, G) Cell indices and relative velocities are shown. (C, F, H) Levels of PRK1 were analyzed by Western blots decorated with the indicated antibodies. β-Actin was used as a loading control. n ≥ 3. (I) PRK1 knockdown abolished development of overall metastases in tail vein metastases assay. Bioluminescent signal of immunodeficient mice at day 35 after injection of PC-3M-luc2 cells either stably expressing miRNA against PRK1 (miR-PRK1) or control (miR-Ctrl). Statistics for comparing overall metastases (J) was done using Fisher′s exact test. Error bars represent ± SD or + SD. *** p ≤ 0.001.
Figure 2
Figure 2. PRK1 regulates transcription of genes determining migration and invasion
(A) MA-Plot representing the differentially regulated genes (red dots) in PC-3M-luc2 cells upon knock down of PRK1. (B) DAVID analysis for GO “cellular component” for differentially regulated genes with a p-value < 10−3. (C) Read coverage displaying the downregulation of NT5E, NEDD9, and PXN upon PRK1 knockdown. FC: Fold Change. (D, F) Migration assays of PC-3M-luc2 cells stable transfected with miRNA against NT5E (miR-NT5E), NEDD9 (miR-NEDD9), or unrelated control miRNA (miR-Ctrl). (E, G) Efficiency control of knockdown. mRNA levels of NT5E or NEDD9 were analyzed by qRT-PCR. (H) Migration assay of PC-3M-luc2 cells treated with siRNA against PXN (siPXN) or unrelated siRNA (siCtrl). (I) Efficiency control of knockdown. Protein expression of PXN was analyzed by Western blots. (D, F, H) Cell indices and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. *** p ≤ 0.001.
Figure 2
Figure 2. PRK1 regulates transcription of genes determining migration and invasion
(A) MA-Plot representing the differentially regulated genes (red dots) in PC-3M-luc2 cells upon knock down of PRK1. (B) DAVID analysis for GO “cellular component” for differentially regulated genes with a p-value < 10−3. (C) Read coverage displaying the downregulation of NT5E, NEDD9, and PXN upon PRK1 knockdown. FC: Fold Change. (D, F) Migration assays of PC-3M-luc2 cells stable transfected with miRNA against NT5E (miR-NT5E), NEDD9 (miR-NEDD9), or unrelated control miRNA (miR-Ctrl). (E, G) Efficiency control of knockdown. mRNA levels of NT5E or NEDD9 were analyzed by qRT-PCR. (H) Migration assay of PC-3M-luc2 cells treated with siRNA against PXN (siPXN) or unrelated siRNA (siCtrl). (I) Efficiency control of knockdown. Protein expression of PXN was analyzed by Western blots. (D, F, H) Cell indices and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. *** p ≤ 0.001.
Figure 3
Figure 3. PRK1 associates with SPAG9 in PC-3M-luc2 cells
(A) Coomassie-staining of SDS-page after immunoprecipitation with anti-PRK1-antibody. (B) PRK1 interacting proteins identified by mass spectrometry. (C, D) Western blots showing coimmunoprecipitation of endogenous PRK1 with SPAG9. (E) PRK1 in complex with SPAG9 verified by sucrose gradient centrifugation. Western blot were decorated with the indicated antibodies. (F) Migration assays of PC-3M-luc2 cells stable expressing miRNA against SPAG9 (miR-SPAG9) or control (miR-Ctrl). (G) Levels of SPAG9 and PRK1 were analyzed by Western blot. (H) Efficiency control of SPAG knockdown and expression of “signature genes”. mRNA levels of SPAG9, PRK1, NT5E, NEDD9, and PXN were analyzed by qRT-PCR. (F) Cell index and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. *** p ≤ 0.001.
Figure 3
Figure 3. PRK1 associates with SPAG9 in PC-3M-luc2 cells
(A) Coomassie-staining of SDS-page after immunoprecipitation with anti-PRK1-antibody. (B) PRK1 interacting proteins identified by mass spectrometry. (C, D) Western blots showing coimmunoprecipitation of endogenous PRK1 with SPAG9. (E) PRK1 in complex with SPAG9 verified by sucrose gradient centrifugation. Western blot were decorated with the indicated antibodies. (F) Migration assays of PC-3M-luc2 cells stable expressing miRNA against SPAG9 (miR-SPAG9) or control (miR-Ctrl). (G) Levels of SPAG9 and PRK1 were analyzed by Western blot. (H) Efficiency control of SPAG knockdown and expression of “signature genes”. mRNA levels of SPAG9, PRK1, NT5E, NEDD9, and PXN were analyzed by qRT-PCR. (F) Cell index and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. *** p ≤ 0.001.
Figure 4
Figure 4. PRK1 and SPAG9 determine phosphorylation status of p38
(A, B) Western blots showing levels of phospho-p38 (ph-p38) upon knockdown of PRK1 or SPAG9. (C) Level of phospho-p38 was analyzed in Western blots with the indicated antibodies upon overexpression of PRK1 in PC-3M-luc2 cells. Total amount of p38 protein is shown as control. (D) Migration assay of PC-3M-luc2 cells treated with either TGFβ1 (2ng/ml), or the p38 inhibitor SB203580 (20 μM) or both compared to control vehicle. (E) mRNA levels of PRK1, NT5E, NEDD9, and PXN upon treatment of PC-3M-luc2 with TGFβ1 (2 ng/ml) were analyzed by qRT-PCR. (F) Migration assay of PC-3M-luc2 cells treated with TGFβ1 or vehicle after knockdown of PRK1 (siPRK1) or control (siCtrl). (G) Migration assay of PC-3M-luc2 cells treated with TGFβ1 or vehicle after miRNA-mediated knockdown of SPAG9 (miR-SPAG9) versus control (miR-Ctrl). (H) Migration assay of PC-3M-luc2 treated either with siRNA against p38 (sip38) or unrelated control siRNA (siCtrl). (I) Efficiency control of p38 knockdown was performed by Western blot analysis. (J) mRNA levels of PRK1, SPAG9, NT5E, NEDD9, PXN, and p38 upon knockdown of p38 in PC-3M-luc2 cells analyzed by qRT-PCR. (D, F, G, H) Cell indices and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. ** p ≤ 0.01, *** p ≤ 0.001.
Figure 4
Figure 4. PRK1 and SPAG9 determine phosphorylation status of p38
(A, B) Western blots showing levels of phospho-p38 (ph-p38) upon knockdown of PRK1 or SPAG9. (C) Level of phospho-p38 was analyzed in Western blots with the indicated antibodies upon overexpression of PRK1 in PC-3M-luc2 cells. Total amount of p38 protein is shown as control. (D) Migration assay of PC-3M-luc2 cells treated with either TGFβ1 (2ng/ml), or the p38 inhibitor SB203580 (20 μM) or both compared to control vehicle. (E) mRNA levels of PRK1, NT5E, NEDD9, and PXN upon treatment of PC-3M-luc2 with TGFβ1 (2 ng/ml) were analyzed by qRT-PCR. (F) Migration assay of PC-3M-luc2 cells treated with TGFβ1 or vehicle after knockdown of PRK1 (siPRK1) or control (siCtrl). (G) Migration assay of PC-3M-luc2 cells treated with TGFβ1 or vehicle after miRNA-mediated knockdown of SPAG9 (miR-SPAG9) versus control (miR-Ctrl). (H) Migration assay of PC-3M-luc2 treated either with siRNA against p38 (sip38) or unrelated control siRNA (siCtrl). (I) Efficiency control of p38 knockdown was performed by Western blot analysis. (J) mRNA levels of PRK1, SPAG9, NT5E, NEDD9, PXN, and p38 upon knockdown of p38 in PC-3M-luc2 cells analyzed by qRT-PCR. (D, F, G, H) Cell indices and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. ** p ≤ 0.01, *** p ≤ 0.001.
Figure 4
Figure 4. PRK1 and SPAG9 determine phosphorylation status of p38
(A, B) Western blots showing levels of phospho-p38 (ph-p38) upon knockdown of PRK1 or SPAG9. (C) Level of phospho-p38 was analyzed in Western blots with the indicated antibodies upon overexpression of PRK1 in PC-3M-luc2 cells. Total amount of p38 protein is shown as control. (D) Migration assay of PC-3M-luc2 cells treated with either TGFβ1 (2ng/ml), or the p38 inhibitor SB203580 (20 μM) or both compared to control vehicle. (E) mRNA levels of PRK1, NT5E, NEDD9, and PXN upon treatment of PC-3M-luc2 with TGFβ1 (2 ng/ml) were analyzed by qRT-PCR. (F) Migration assay of PC-3M-luc2 cells treated with TGFβ1 or vehicle after knockdown of PRK1 (siPRK1) or control (siCtrl). (G) Migration assay of PC-3M-luc2 cells treated with TGFβ1 or vehicle after miRNA-mediated knockdown of SPAG9 (miR-SPAG9) versus control (miR-Ctrl). (H) Migration assay of PC-3M-luc2 treated either with siRNA against p38 (sip38) or unrelated control siRNA (siCtrl). (I) Efficiency control of p38 knockdown was performed by Western blot analysis. (J) mRNA levels of PRK1, SPAG9, NT5E, NEDD9, PXN, and p38 upon knockdown of p38 in PC-3M-luc2 cells analyzed by qRT-PCR. (D, F, G, H) Cell indices and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. ** p ≤ 0.01, *** p ≤ 0.001.
Figure 5
Figure 5. ELK1 regulates migration and occupies PRK1-regulated genes
(A) Migration assay of PC-3M-luc2 cells treated either with siRNA against ELK1 (siELK1) or unrelated control siRNA (siCtrl). (B) Verification of ELK1 knockdown by Western blot analysis. (C) mRNA levels of ELK1, NT5E, NEDD9, and PXN after knockdown of ELK1 (siELK1) or control siRNA (siCtrl) in PC- 3M-luc2 analyzed by qRT-PCR. (D) Migration assay of PC-3M-luc2 overexpressing PRK1 or control treated with either siRNA against ElK1 (siELK1) or unrelated control siRNA (siCtrl). (E) PRK1 overexpression and ELK1 knockdown was verified by Western blot analysis with the indicated antibodies. β-Actin was used as loading control. (A, D) Cell indices and relative velocities are shown. n ≥ 3. Error bars represent ± SD or + SD. ** p ≤ 0.01, *** p ≤ 0.001.
Figure 6
Figure 6. PRK1 and SPAG9 are overexpressed in human prostate cancer tissue
(A) Analysis of gene expression level of PRK1 and SPAG9 in different human tissue samples (normal prostate n = 18, normal tissue adjacent to tumor n = 63, prostate tumor n = 65, metastases n = 25. Raw data provided by Chandran et al. [29] and Yu et al. [28]. (B) Overlap of PRK1 and SPAG9 in immunhistochemistry shown in benign prostate tissue (white triangle), prostate cancer (predominantly Gleason 4 pattern) (black triangle) or affected lymph node metastases.
Figure 7
Figure 7. PRK1 controls metastases in an orthotopic prostate mouse model
(A) Macroscopic view of primary tumour and lymph node metastases of immunodeficient mice injected either with PRK1 depleted PC-3M-luc2 cells (miR-PRK1) or control transfect cells (miR-Ctrl) into the dorsal prostate lobe. (B, C) Number of developed lymph node metastasis (B) and primary prostate tumor volume (C) is shown 28 days after injection. (D) Macroscopic view (mice, primary tumors, lymph node metastasis) from orthotopic prostate metastases assay with wildtype PC-3M-luc2 cells and subsequent daily treatment of mice with PRK1 inhibitor Lestaurtinib or vehicle. (E, F) Number of developed lymph node metastasis (E) and primary prostate tumor volume (F) is shown from both groups 28 days after injection. Statistics were done using Fisher′s exact test, error bars represent + SD. ** p ≤ 0.01, * p ≤ 0.05.
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