Abstract 512: Pim kinases and Pim inhibitors in the regulation of prostate cancer cell migration and invasion
Authors: Eerola Sini , Santio Niina , Tuomela Johanna , Rainio Eeva-Marja , Moreau Pascale , Anizon Fabrice , Corthals Garry , Härkonen Pirkko , Koskinen Päivi 
Published: August 2015

Abstract
INTRODUCTION
PIM kinases (Proviral Integration Site for Moloney Murine Leukemia Virus) are well known oncoproteins which have been shown to regulate cell survival, proliferation and motility. In addition, their expression is often up-regulated in human hematopoietic malignancies and solid tumors. Since PIM kinases promote cancerous behavior of cells, they have become attractive targets for drug development. We have previously identified a novel PIM-selective kinase inhibitor 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9). Here we show the potential of DHPCC-9 to efficiently prevent PIM-dependent functions both in cell-based assays and in mouse models. To elucidate signaling mechanisms via which PIM kinases regulate cell motility, we studied the involvement of the previously known PIM substrate NFATC1 (Nuclear Factor of Activated T Cells). Here we show that NFATC1 may play a role as a downstream mediator of PIM1 signaling in promoting cancer cell migration.
METHODS
PIM inactivation by DHPCC-9 or PIM silencing was studied in wound healing and Boyden chamber assays in PC-3 prostate cancer cells. The effects of stable PIM overexpression on PC-3 cell tumor growth and metastasizing were studied in subcutanic and in orthotopic mouse models. Mice bearing PC-3/PIM3 cell-derived orthotopic tumors were treated daily by injecting 50 mg/kg of DHPCC-9. Three weeks later tumors were measured, and all the substantial organs were collected for further analysis. Phosphorylation of NFATC1 was studied by mass spectrometry and in vitro kinase assays while its transcriptional activity was measured by luciferase reporter assays. In addition, wound healing assays were performed to test the effect of NFATC1 on PC-3 prostate cancer cell migration.
RESULTS
Prostate cancer cell migration and invasion were efficiently decreased by either PIM inhibition by DHPCC-9 or silencing of both PIM1 and PIM2 by RNA interference. Stable PIM1 or PIM3 overexpression promoted prostate tumor growth, while PIM inhibitor DHPCC-9 treatment decreased tumor size. Stable PIM overexpression induced PC-3 cells to metastasize into the prostate- draining lymph nodes and lungs, and treatment with PIM inhibitor DHPCC-9 decreased the amount of both types of metastases. Mutagenesis of all PIM1 phosphorylation target sites results in decreased NFATC1 phosphorylation and transcriptional activity, along with a reduced potential of NFATC1 to promote prostate cancer cell migration.
CONCLUSIONS
Here we have shown that PIM kinases promote and PIM inhibition reduces tumor growth and the formation of metastases in vivo. These findings not only support the role of PIM kinases in the regulation of cancer cell invasiveness, but also reveal DHPCC-9 as a highly potential lead compound for developing selective PIM-targeted cancer therapy. In addition, we have discovered NFATC1 transcription factor as the first PIM substrate involved in the PIM-promoted cancer cell motility.
Citation Format: Sini Eerola, Niina Santio, Johanna Tuomela, Eeva-Marja Rainio, Pascale Moreau, Fabrice Anizon, Garry Corthals, Pirkko Härkonen, Päivi Koskinen. Pim kinases and Pim inhibitors in the regulation of prostate cancer cell migration and invasion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2015-512