Marina Zemskova

The Pim protein kinases regulate energy metabolism and cell growth

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMP∶ATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5′-cap–dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.

Novel benzylidene-thiazolidine-2,4-diones inhibit Pim protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells

The Pim protein kinases play important roles in cancer development and progression, including prostate tumors and hematologic malignancies. To investigate the potential role of these enzymes as anticancer drug targets, we have synthesized novel benzylidene-thiazolidine-2,4-diones that function as potent Pim protein kinase inhibitors. With IC50 values in the nanomolar range, these compounds block the ability of Pim to phosphorylate peptides and proteins in vitro and, when added to DU145 prostate cancer cells overexpressing Pim, inhibit the ability of this enzyme to phosphorylate a known substrate, the BH3 protein BAD. When added to prostate cancer cell lines, including PC3, DU145, and CWR22Rv1, and human leukemic cells, MV4;11, K562, and U937 cells, these compounds induce G1-S cell cycle arrest and block the antiapoptotic effect of the Pim protein kinase. The cell cycle arrest induced by these compounds is associated with an inhibition of cyclin-dependent kinase 2 and activity and translocation of the Pim-1 substrate p27Kip1, a cyclin-dependent kinase 2 inhibitory protein, to the nucleus. Furthermore, when added to leukemic cells, these compounds synergize with the mammalian target of rapamycin inhibitor rapamycin to decrease the phosphorylation level of the translational repressor 4E-BP1 at sites phosphorylated by mammalian target of rapamycin. Combinations of rapamycin and the benzylidene-thiazolidine-2,4-diones synergistically block the growth of leukemic cells. Thus, these agents represent novel Pim inhibitors and point to an important role for the Pim protein kinases in cell cycle control in multiple types of cancer cells. [Mol Cancer Ther 2009;8(6):1473–83]

p53-Dependent Induction of Prostate Cancer Cell Senescence by the PIM1 Protein Kinase

The PIM family of serine threonine protein kinases plays an important role in regulating both the growth and transformation of malignant cells. However, in a cell line–dependent manner, overexpression of PIM1 can inhibit cell and tumor growth. In 22Rv1 human prostate cells, but not in Du145 or RWPE-2, PIM1 overexpression was associated with marked increases in cellular senescence, as shown by changes in the levels of β-galactosidase (SA-β-Gal), p21, interleukin (IL)-6 and IL-8 mRNA and protein. During early cell passages, PIM1 induced cellular polyploidy. As the passage number increased, markers of DNA damage, including the level of γH2AX and CHK2 phosphorylation, were seen. Coincident with these DNA damage markers, the level of p53 protein and genes transcriptionally activated by p53, such as p21, TP53INP1, and DDIT4, increased. In these 22Rv1 cells, the induction of p53 protein was associated not only with senescence but also with a significant level of apoptosis. The importance of the p53 pathway to PIM1-driven cellular senescence was further shown by the observation that expression of dominant-negative p53 or shRNA targeting p21 blocked the PIM1-induced changes in the DNA damage response and increases in SA-β-Gal activity. Likewise, in a subcutaneous tumor model, PIM1-induced senescence was rescued when the p53-p21 pathways are inactivated. Based on these results, PIM1 will have its most profound effects on tumorigenesis in situations where the senescence response is inactivated. Mol Cancer Res; 8(8); 1126–41. ©2010 AACR.

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCFSkp2 ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser64 and Ser72, we have identified Thr417 as a unique Pim-1 phosphorylation target. Phosphorylation of Thr417 controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.

Targeted glycoprotein enrichment and identification in stromal cell secretomes using azido sugar metabolic labeling

Effectively identifying the proteins present in the cellular secretome is complicated due to the presence of cellular protein leakage and serum protein supplements in culture media. A metabolic labeling and click chemistry capture method is described that facilitates the detection of lower abundance glycoproteins in the secretome, even in the presence of serum.

Enhanced chemokine-receptor expression, function, and signaling in healthy African American and scleroderma-patient monocytes are regulated by caveolin-1.

BackgroundA major health disparity suffered by African Americans (AA) is a predisposition toward fibrotic diseases of the skin, lung, and other organs. We previously showed that healthy AA and scleroderma (systemic sclerosis (SSc)) patient monocytes share biochemical and functional differences from control Caucasian (C) monocytes that may predispose AA to SSc. The central difference is a decrease in caveolin-1. Low caveolin-1 levels promote monocyte migration, their differentiation into fibrocytes, and fibrocyte recruitment into fibrotic tissues. Here we have greatly expanded our studies on the mechanism of action in fibrosis of caveolin-1 in AA and SSc monocytes.ResultsExpression of chemokine receptors (CCR1, CCR2, CCR3) is enhanced in healthy AA monocytes compared to healthy C monocytes and further increased in SSc monocytes. A parallel increase in function occurs assessed by migration toward chemokines MCP-1 and MCP-3. Chemokine-receptor expression and function are inhibited by the caveolin-1 scaffolding domain peptide (CSD) via its action as a surrogate for caveolin-1. Cells bearing chemokine receptors accumulate to high levels in fibrotic lung and skin tissue from SSc patients and from mice treated with bleomycin. This accumulation is almost completely blocked in mice treated with CSD. In signaling studies, Src activation is enhanced in AA monocytes compared to C monocytes and further increased in SSc monocytes. Lyn is also highly activated in SSc monocytes. Src and Lyn activation are inhibited by CSD. Src and Lyn’s roles in monocyte migration were demonstrated using specific inhibitors.ConclusionsTo the best of our knowledge, this is the first report that the expression and function of CCR1, CCR2, and CCR3 are upregulated in monocytes from healthy AA and from SSc patients via molecular mechanisms involving caveolin-1, Src/Lyn, and MEK/ERK. The results suggest that the migration/recruitment of monocytes and fibrocytes into fibrotic tissues, mediated at least in part by CCR1, CCR2, and CCR3, plays a major role in the progression of lung and skin fibrosis and in the predisposition of AA to fibrotic diseases. Our findings further suggest that chemokine receptors and signaling molecules, particularly caveolin-1, that control their expression/function are promising targets for treating fibrotic diseases.

Deficient Adipogenesis of Scleroderma Patient and Healthy African American Monocytes

Monocytes from systemic sclerosis (SSc, scleroderma) patients and healthy African Americans (AA) are deficient in the regulatory protein caveolin-1 leading to enhanced migration toward chemokines and fibrogenic differentiation. While dermal fibrosis is the hallmark of SSc, loss of subcutaneous adipose tissue is a lesser-known feature. To better understand the etiology of SSc and the predisposition of AA to SSc, we studied the adipogenic potential of SSc and healthy AA monocytes. The ability of SSc and healthy AA monocytes to differentiate into adipocyte-like cells (ALC) is inhibited compared to healthy Caucasian (C) monocytes. We validated that monocyte-derived ALCs are distinct from macrophages by flow cytometry and immunocytochemistry. Like their enhanced fibrogenic differentiation, their inhibited adipogenic differentiation is reversed by the caveolin-1 scaffolding domain peptide (CSD, a surrogate for caveolin-1). The altered differentiation of SSc and healthy AA monocytes is additionally regulated by peroxisome proliferator-activated receptor γ (PPARγ) which is also present at reduced levels in these cells. In vivo studies further support the importance of caveolin-1 and PPARγ in fibrogenesis and adipogenesis. In SSc patients, healthy AA, and mice treated systemically with bleomycin, adipocytes lose caveolin-1 and PPARγ and the subcutaneous adipose layer is diminished. CSD treatment of these mice leads to a reappearance of the caveolin-1+/PPARγ+/FABP4+ subcutaneous adipose layer. Moreover, many of these adipocytes are CD45+, suggesting they are monocyte derived. Tracing experiments with injected EGFP+ monocytes confirm that monocytes contribute to the repair of the adipose layer when it is damaged by bleomycin treatment. Our observations strongly suggest that caveolin-1 and PPARγ work together to maintain a balance between the fibrogenic and adipogenic differentiation of monocytes, that this balance is altered in SSc and in healthy AA, and that monocytes make a major contribution to the repair of the adipose layer.

Abstract 3596: The role of PIM1 kinase in the prostate tumor microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in men. The prostate is a tubulo-alveolar gland composed of epithelial tissues embedded in stromal components. PCa includes reactive stroma which consists of myofibroblasts and extracellular matrix (ECM) remodeling. Increasing evidence suggests that myofibroblasts may refine the prognostic assessment of tumors. The PIM1 serine/threonine protein kinase is a true oncogene and has been implicated in the initiation and progression of multiple cancer types including PCa. The level of PIM1 correlates with the disease state and Gleason grade and has been shown to stimulate growth of murine prostate cancer. The role of PIM1 in prostate stromal cells has never been investigated. Using human prostate tissue microarray analysis we now demonstrate that (1) the PIM1 protein is detected in human stroma adjacent to cancer tissue; (2) stromal cells isolated from PIM1 knockout mice contain a decreased number of myofibroblasts and expose slow growth rate when compared with wild type control, suggesting that kinase expression can alter stromal cell composition and proliferation; (3) elevated level of PIM1 kinase in human benign prostate stromal cells increases migration and growth of prostate epithelial cells and this effect can be reversed by PIM inhibitors; (4) PIM1 over-expressing stromal cells increase epithelial cell growth in tissue regeneration model; (5) stromal cells with elevated level of PIM1 expose increased number of myofibroblasts when co-cultivated with prostate epithelial cells. These data suggest that PIM1 kinase may contribute in formation of a “reactive stroma” phenotype. We used the conditioning media analysis to decipher the PIM1 specific signal transduction pathways that may lead to stroma activation and drive prostate cancer growth. Using cytokine/chemokine arrays and ECM protein analysis we found that overexpression of PIM1 kinase in stromal cells is associated with elevated level of CCL5 chemokine and increased secretion of collagen1. Examination of the pathways involved in formation of myofibroblasts revealed that PIM1 can modulate activation of PDGFRβ leading to the increased CCL5 expression. We are suggesting that PIM1 is a modulator of stromal cells response to the stimuli produced by prostate epithelial cells through increase of matrix stiffness, receptor activation and accumulation of myofibroblasts which induce further ECM remodeling and expression of the growth factors leading to epithelial cells transformation and cancer propagation. Citation Format: Marina Zemskova, Andrew S. Kraft. The role of PIM1 kinase in the prostate tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3596. doi:10.1158/1538-7445.AM2014-3596

Abstract 1258: Regulation of energy metabolism and protein synthesis by the Pim protein kinases

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked mTORC1 activity by stimulating the phosphorylation, and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases (TKO MEFs) demonstrated activated AMPK driven by elevated ratios of AMP:ATP relative to wild type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5′-cap dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and PGC-1α, enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. These results demonstrate the Pim kinase -mediated control of energy metabolism through regulation of AMPK activity, and identify a new and important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth. In prostate cancer cell lines we find that the Pim-3 protein kinase is elevated in those cells that are highly metastatic including PC3-LN4, ARCaPM and M12 when compared to PC-3, ARCaPE and p69 and the amount of Pim-3 correlates with c-Myc levels. Further experiments will be needed to determine whether the Pim-3 protein kinase expression regulates the metastatic potential of prostate cancer cells through control of energy metabolism and protein synthesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1258. doi:10.1158/1538-7445.AM2011-1258

Abstract C64: Treatment with the Pim protein kinase inhibitor SMI‐4a enhances AMPK phosphorylation, decreases Raptor levels, and blocks mTORC1 activity

We have identified a class of small molecule inhibitors of the Pim protein kinases, benzylidene thiazolidine‐2‐4 diones (J. Med. Chem. (2009) 52:74) with the most potent members having IC50s of 13 nM for Pim‐1 and 2.3 µM for Pim‐2. Compounds in this chemotype demonstrated selectivity of more than 2500‐fold and 400‐fold for Pim‐1 or Pim‐2 respectively while other congeners had equivalent potency towards both isozymes. In vivo, these molecules inhibited Pim kinase autophosphorylation and in a murine model inhibited the growth of subcutaneously implanted murine adenocarcinoma JC cells. One of the members of this chemotype, SMI‐4a, has been shown to block the phosphorylation of the mTOR regulatory protein PRAS40 and subsequently the activity of the mTOR pathway (Mol. Cancer. Ther. (2009) 8: 1473; Cancer Biol. Ther. (2009) 8: 846). Now we show that the addition of SMI‐4a to malignant cells increases the phosphorylation of AMPKα on Thr 172 in a LKB1‐dependent manner, induces the phosphorylation of Raptor on Ser792, decreases the levels of Raptor protein, and inhibits mTORC1 activity. Immunoprecipitation of mTOR from SMI‐4a treated cells consistently showed lower levels of bound Raptor and in vitro mTOR kinase assays from treated cells demonstrated a decreased ability to phosphorylate 4E‐BP1. Knockdown of PIM‐1 via siRNA in K562 leukemic cells showed increased AMPK phosphorylation and decreased Raptor protein levels, further demonstrating an important role for Pim kinase in regulating AMPK phosphorylation and Raptor levels. Additionally, mouse embryo fibroblasts (MEFs) deficient for Pim‐1, Pim‐2 and Pim‐3 kinase (TKO MEFs) showed a significantly increased level of AMPK phosphorylation compared to wild type MEFs, which correlated with decreased mTORC1 activity and increased binding of 4E‐BP1 with eIF‐4E. The TKO MEFs grew significantly more slowly than wild type. The decreased mTORC1 activity correlated with an increase in the cellular level of AMP in TKO MEFs. Furthermore, the correlation between increased AMPK phosphorylation and a lower level of Raptor protein observed with SMI‐4a treatment was also observed in TKO MEFs. Infection of TKO MEFs with lentiviruses expressing Pim 1 or Pim2 was able to reverse these effects, decreasing AMPK phosphorylation, and increasing Raptor protein levels. The cellular activity of mTORC1 was difficult to assess in TKO MEFs as we found substantially lower protein levels of the mTORC1 substrates 4E‐BP1 and p70S6K. Akt was readily phosphorylated upon serum stimulation of TKO MEFs and mTORC2 activity was unchanged. Given the role of Pim kinase in regulating mTORC1 activity, we have combined SMI‐4a and the mTOR inhibitor rapamycin inducing synergistic blockade of this pathway. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C64.