Genomic editing to elucidate the effects of PIM kinases on cancer cell signalling

In humans, there are three PIM family genes, the two first of which were originally identified from mice as proviral integration sites for Moloney leukemia virus. They encode serine/threonine kinases that are aberrantly expressed in a variety of hematological malignancies and solid tumors. PIM kinases contribute to cell proliferation, cell survival and cell motility by phosphorylating multiple downstream substrates. PIM kinases have emerged as attractive anti-cancer drug targets, and their distinct structures enable the generation of selective inhibitors. The aim of this PhD study was to functionally validate three novel PIM targets that affect cancer cell signalling, namely actin capping proteins (CPs) that restrict elongation of actin fibers, liver kinase B1 (LKB1) that suppresses cell growth, and lactate dehydrogenase A (LDHA) that regulates cell metabolism. For this purpose, CRISPR/Cas9-based genome editing and structurally unrelated pharmacological PIM-selective inhibitors were used. PIM-targeted sites were identified by mass spectrometry and mutagenesis, after which the functional impacts of phosphorylation were studied in cultured cells or using a chick embryo xenograft model. In line with the pro-migratory function of PIM kinases, phosphorylation of CPs was shown to reduce their ability to bind to the plus ends of actin filaments and thereby to promote cell adhesion and migration. Catalytic activity of LKB1 towards its substrates such as AMPK was shown to be inhibited by PIM-dependent phosphorylation. These studies also indicated that the oncogenic effects of PIMs and the tumor-suppressive effects of LKB1 are tightly controlled at the cellular level. In the case of LDHA, PIM-dependent phosphorylation was observed to prevent nuclear LDHA from being degraded via K48-mediated ubiquitination, highlighting the connection between PIM kinases and the regulation of glycolytic enzymes. Altogether, the results from these studies help to better understand the mechanisms, through which PIM kinases stimulate cancer cell signalling.Genomin muokkaus PIM-kinaasien vaikutusten tutkimiseksi syöpäsoluissa Ihmisen PIM-geeniperheen kolmesta jäsenestä kaksi ensimmäistä löydettiin alun perin hiiristä Moloney-leukemiaviruksen integraatiokohtina. Niiden koodaamat seriini/treoniinikinaasit ilmentyvät poikkeavasti erilaisissa hematologisissa sekä kiinteissä kasvaimissa. PIM-kinaasit edistävät solujen lisääntymistä, eloonjäämistä sekä liikkuvuutta fosforyloimalla useita kohdeproteiinejaan. Kiinnostus PIMkinaasien toimintaa estäviä syöpälääkkeitä kohtaan on viime aikoina lisääntynyt, ja niiden poikkeava rakenne mahdollistaa valikoivien estolääkkeiden kehittämisen. Tämän väitöstutkimuksen tavoitteena oli toiminnallisesti tutkia kolmea uutta syöpäsolujen viestintään vaikuttavaa PIM-kohdeproteiinia: aktiinisäikeiden pitenemistä rajoittavia CP-proteiineja, solujen kasvua rajoittavaa LKB1-kinaasia ja aineenvaihduntaa säätelevää laktaattidehydrogenaasia A (LDHA). Tutkimuksissa käytettiin CRISPR/Cas9-pohjaista genominmuokkausta sekä rakenteellisesti erilaisia PIM-selektiivisiä estolääkkeitä. PIM-kinaasien fosforyloimat kohdat paikannettiin massaspektrometrian ja mutageneesin avulla, ja fosforylaation merkitystä tutkittiin soluviljelmissä tai kananmunan alkion siirrännäismallin avulla. CP-proteiinien fosforylaation osoitettiin vähentävän niiden kykyä sitoutua aktiinisäikeiden plus-päihin, ja siten edistävän solujen adheesiota ja migraatiota, selittäen PIM-kinaasien kykyä lisätä solujen liikkuvuutta. PIM-fosforylaatio heikensi LKB1:n katalyyttistä aktiivisuutta ja siten esti sitä fosforyloimasta omia kohdeproteiinejaan, kuten AMPK-kinaasia. Nämä tutkimukset myös osoittivat, että PIMkinaasien onkogeenisia ja LKB1:n vastakkaisia vaikutuksia säädellään ristiin solutasolla. LDHA:n tapauksessa fosforylaation havaittiin estävän LDHA-proteiinin hajoamista tumassa K48-välitteisen ubikitinaation kautta, korostaen PIM-kinaasien ja glykolyyttisten entsyymien säätelyn välistä yhteyttä. Kaiken kaikkiaan näiden tutkimusten tulokset auttavat paremmin ymmärtämään niitä mekanismeja, joilla PIM-kinaasit stimuloivat syöpäsolujen viestintää

PIM kinases phosphorylate lactate dehydrogenase A at serine 161 and suppress its nuclear ubiquitination

Lactate dehydrogenase A (LDHA) is a glycolytic enzyme catalysing the reversible conversion of pyruvate to lactate. It has been implicated as a substrate for PIM kinases, yet the relevant target sites and functional consequences of phosphorylation have remained unknown. Here, we show that all three PIM family members can phosphorylate LDHA at serine 161. When we investigated the physiological consequences of this phosphorylation in PC3 prostate cancer and MCF7 breast cancer cells, we noticed that it suppressed ubiquitin-mediated degradation of nuclear LDHA and promoted interactions between LDHA and 14-3-3 proteins. By contrast, in CRISPR/Cas9-edited knock-out cells lacking all three PIM family members, ubiquitination of nuclear LDHA was dramatically increased followed by its decreased expression. Our data suggest that PIM kinases support nuclear LDHA expression and activities by promoting phosphorylation-dependent interactions of LDHA with 14-3-3 epsilon, which shields nuclear LDHA from ubiquitin-mediated degradation

PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1

BackgroundThe oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells.MethodsWe first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model.ResultsWe provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors.ConclusionPIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors.</p

PIM1 accelerates prostate cancer cell motility by phosphorylating actin capping proteins

Background: The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them. Methods: Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays. Results: We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly. </p

Regulation of glycogen phosphorylase in hypoxic cancer cells

Compared to normal cells, many tumor cells have to subsist in a hypoxic intratumoral environment that has an unstable supply of oxygen and nutrients including glucose. How tumor cells may survive the metabolic stress arising from tumor hypoxia is not yet fully understood. Recent studies revealed that tumor cells are able to accumulate large quantities of intracellular glycogen. Whether glycogen would serve as fuel reserve in hypoxic tumor cells is presently not clear. This question is being addressed in this study. When HeLa, HT29, HEK293 and HepG2 cells were incubated under hypoxic condition in the absence of glucose, the steady state intracellular glycogen level dropped by more than 50% in 3 hours. The specific pharmacological inhibition of the liver isoform glycogen phosphorylase (PYGL) (CAS 648926-15-2) partially inhibited hypoxia-induced glycogen degradation. More complete inhibition was achieved by combined incubation using the pharmacological inhibitor and 2-deoxyglucose. Inhibition of glycogen degradation resulted in decrease in hypoxia-induced lactate formation, supporting the idea that glycogen serves as a fuel reserve in hypoxic cancer cells. Inhibition of autophagy or alpha-glucosidase failed to prevent glycogen degradation in hypoxic condition, suggesting that cytosolic glycogen phosphorylase is the major enzyme involved in glycogen degradation. The mRNA, protein and phosphorylation levels of glycogen phosphorylase were unaltered by hypoxia. The siRNA-mediated knockdown of the brain form of glycogen phosphorylase (PYGB) resulted in markedly greater inhibition of glycogen degradation than did the knockdown of PYGL. Whereas the enzyme activity of PYGB can be markedly stimulated by AMP, the activity of PYGL is only slightly stimulated in the presence of AMP. The relative proportion of AMP-sensitive and AMP-insensitive GP activity is little affected by acute hypoxia. In conclusion, direct evidence is provided in this study that glycogen may serve as an intracellular fuel reserve in tumor cells. The involvement of the brain form of glycogen phosphorylase is for the first time demonstrated to be involved in the mobilization of this fuel reserve in tumor cells.published_or_final_versionBiochemistryMasterMaster of Philosoph

PIM1 accelerates prostate cancer cell motility by phosphorylating actin capping proteins

Background: The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them. Methods: Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays. Results: We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly. Conclusions: Our data suggest that PIM kinases are able to induce changes in actin dynamics to support cell adhesion and movement. Thus, we have identified a novel mechanism through which PIM kinases enhance motility and metastatic behaviour of cancer cells.Peer reviewe

Additional file 3: of Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning

Figure S3. Negative controls for neural marker immunocytochemistry. Immunocytochemistry performed on 3T3 cells and human BMSCs as negative controls against the neuronal marker Tuj-1 (A, B) and glial markers GFAP (C, D), p75 (E, F), and S100β (G, H) demonstrated an absence of expression. (PDF 478 kb

Additional file 2: of Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning

Figure S2. Isotype controls for immunocytochemistry. (A, B) Mouse and rabbit isotype control antibody-stained neurospheres derived following normoxic and hypoxic treatment, respectively. (C, D) Mouse and rabbit isotype control antibody-stained SCLCs derived from normoxia- and hypoxia-treated BMSCs, respectively. (E, F) Mouse and rabbit isotype control antibody-stained fate-committed Schwann cells generated from normoxia- and hypoxia-treated BMSCs, respectively. (PDF 211 kb