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Pim1 Kinase Is Required to Maintain Tumorigenicity in MYC-Expressing Prostate Cancer Cells

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Pim1 Kinase Is Required to Maintain Tumorigenicity in MYC-Expressing Prostate Cancer Cells Oncogene (2012) 31, 1794–1803 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Pim1 kinase is required to maintain tumorigenicity in MYC-expressing prostate cancer cells J Wang1, PD Anderson2, W Luo3, D Gius1,4,5, M Roh2 and SA Abdulkadir1,2 1Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA; 2Department of Pathology, Vanderbilt University Medical Center, Nashville, TN, USA; 3Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA; 4Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA and 5Department of Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN, USA PIM1 kinase and MYC are commonly co-expressed in ment in mouse models (Verbeek et al.,1991;Wanget al., human prostate cancer and synergize to induce rapidly 2010). Whereas mice with homozygous deficiency in Myc progressing prostate cancer in mouse models. Deficiency of uniformly exhibit widespread developmental defects the Pim kinase genes is well tolerated in vivo, suggesting and embryonic lethality (Davis et al., 1993), mice with that PIM1 inhibition might offer an attractive therapeutic homozygous deficiency in all three Pim kinases are viable, modality for prostate cancer, particularly for MYC- fertile and exhibit only minor phenotypic abnormalities expressing tumors. Here we examine the molecular (Mikkers et al., 2004). These observations show that consequences of Pim1 and MYC overexpression in the inhibition of PIM kinase activity is well tolerated in vivo, prostate as well as the effects of depleting Pim1 in prostate and suggest that drugs targeting PIM kinases might be carcinoma cells with high levels of MYC. Overexpression of better tolerated than those targeting MYC. Pim1 in the mouse prostate induces several pro-tumorigenic Using a prostate regeneration system, we have recently genetic programs including cell cycle genes and Myc- shown that Pim1 potently interacts with MYC to regulated genes before the induction of any discernible accelerate prostate cancer progression, although Pim1 pathology. Pim1 depletion by RNA interference in mouse alone did not lead to significant pathology (Wang et al., and human prostate cancer cells decreased cellular pro- 2010). MYC/Pim1 expression in this model led to the liferation, survival, Erk signaling and tumorigenicity even development of neuroendocrine prostate cancer, appar- when MYC levels were not significantly altered. These ently by differentiation via an adenocarcinoma inter- results indicate that PIM1 may be necessary to maintain mediate. The mechanisms underlying cooperativity tumorigenicity, and further support efforts aimed at between Pim1 and Myc in prostate tumorigenesis in vivo developing PIM1 inhibitors for prostate cancer therapy. have not been clearly elucidated. Studies have suggested Oncogene (2012) 31, 1794–1803; doi:10.1038/onc.2011.371; that Pim1 may enhance Myc tumorigenesis by increasing published online 22 August 2011 Myc protein stability or transcriptional activity (Chen et al., 2005; Zippo et al., 2007; Zhang et al., 2008; Kim Keywords: Pim1 kinase; MYC; gene expression; Erk; et al., 2010). We have reported that Pim1 overexpression tumorigenicity in the prostate in vivo produced minimal morphological alterationsby6weeks(Wanget al., 2010). These results prompted us to test whether we could discern molecular alterations induced by Pim1 overexpression in the absence Introduction of concurrent overexpression of Myc. In this report, we used gene expression profiling to investigate the molecular The PIM kinase family consists of three constitutively pathways modulated by expression of Pim1 and MYC active serine/threonine kinases, PIM1, PIM2 and PIM3, alone or together in the mouse prostate. We also knocked which have been implicated in diverse human malignan- down Pim1 expression using RNA interference to test cies including leukemias and lymphomas, as well whether Pim1 is necessary to maintain the malignancy of as pancreatic, hepatic, oral and prostate cancers MYC-expressing prostate cancer cells. Our results show (Shah et al., 2008; Magnuson et al., 2010). PIM1 kinase that Pim1 knock-down resulted in decreased proliferation, is an attractive molecular target for cancer pharma- cell survival, Erk signaling and tumorigenicity even when cotherapy because it enhances the transforming potential MYC levels were not significantly altered. of oncogenes. For instance, Pim1 accelerates Myc- initiated lymphomagenesis and prostate cancer develop- Results Correspondence: Dr SA Abdulkadir, Department of Pathology, Vanderbilt University Medical Center, B33321A MCN, Nashville, Gene expression programs regulated by Pim1 and TN 37232, USA. E-mail: [email protected] c-MYC in prostate cells in vivo Received 22 February 2011; revised and accepted 19 July 2011; published To characterize the effects of Pim1 and MYC on online 22 August 2011 prostate tumorigenesis, we used a tissue recombination Pim1 in prostate cancer J Wang et al 1795 model in which lentiviral vectors overexpressing MYC, model of adipocyte differentiation of 3T3L1 cells Pim1, or MYC and Pim1 were used to infect normal induced by insulin, dexamethasone treatment (Burton mouse prostate cells. Cells were implanted under the et al., 2004). Many of the genes in this cluster are also renal capsule of SCID mice hosts, and regenerated related to the cell cycle as differentiation is associated prostate tissue grafts were harvested after 6 weeks. We with exit from the cell cycle. Another notable gene set have previously shown that at this time point, Pim1 identified by Gene Set Enrichment Analysis analysis grafts were morphologically indistinguishable from consists of genes upregulated in doxorubicin-resistant control grafts, consisting of benign prostatic glands gastric cancer cells. Therefore, the gene sets identified as (Wang et al., 2010). MYC grafts, on the other hand, enriched in Pim1, MYC or MYC/Pim1 grafts consist of contained multiple foci of high-grade PIN (HGPIN) genes involved in cell cycle progression, differentiation lesions, (Wang et al., 2010). However, MYC/Pim1 grafts and drug resistance. The differential expression had the most severe pathology, consisting of poorly of several selected genes identified by this analysis was differentiated cancer with evidence of neuroendocrine confirmed by quantitative real-time PCR (Supplemen- differentiation. These observations indicate that Pim1 tary Figure S1B). Although observing these gene may be dependent on collaborating mutations in genes expression changes in MYC- or MYC/Pim1-expressign such as in Myc before it can manifest its tumorigenic neoplastic prostate grafts is not surprising, it is effects. unexpected that grafts expressing Pim1 alone also show To gain insight into the oncogenic functions of Pim1 such gene expression changes, considering that Pim1 in prostate cells in vivo, we compared the gene grafts at 6 weeks show no discernible morphological expression profiles of these regenerated mouse prostate abnormalities (Wang et al., 2010). Thus, Pim1 may grafts overexpressing human MYC, mouse Pim1, both perturb the expression of cancer-relevant genes before Pim1 and MYC or control lentivirus (Wang et al., 2010) the appearance of any discernible histological abnorm- using Affymetrix microarray analysis. By employing alities. Significance Analysis of Microarrays (SAM) analysis, The enrichment of these gene set modules in the we were able to identify 4172 probes (corresponding to prostate-regenerated grafts is not due to the presence of 2770 unique genes) that were differentially expressed a common set of genes in all modules, as leading edge between control, Pim1, MYC or MYC/Pim1 grafts analysis showed little overlap among them (Figure 1b; (Supplementary Table S1). To identify subgroups of Supplementary Figure S2). The ‘leading edge’ in a gene genes with similar expression profiles, we performed set represents the individual genes that are primarily hierarchical clustering (Supplementary Figure S1) for responsible for the observed association. Examination the genes deemed significant in the microarray analysis. of the ‘leading edge’ could provide important insights The most extreme gene expression changes were into the biological character of the gene set. The observed in three MYC/Pim1 grafts and revealed several similarity between modules enriched in Pim1-expressing distinct clusters of genes with altered expression. Gene grafts to those in MYC-expressing grafts (Figures 1a expression differences among samples are a probably a and c) is consistent with reports in the literature reflection of the stages of tumorigenesis in each of the that Pim1 enhances MYC transcriptional activity (Chen grafts. For example, one MYC/Pim1 array clustered et al., 2005; Zippo et al., 2007; Zhang et al., 2008; Kim with the MYC arrays (Supplementary Figure S1), et al., 2010). Thus, our gene expression analysis supports suggesting that RNA isolated from this sample might the hypothesis that overexpression of Pim1 stimulates have come from tissue consisting of mostly HGPIN. the transcriptional activity of endogenous Myc present Although single analysis of differentially expressed in the cells, resulting in the activation of gene programs genes can provide insight into the underlying biological regulated by Myc. differences between samples, it can miss modest but coordinated changes in groups of genes (such as those belonging to the same
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