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A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival.

ABSTRACT: Choline kinase alpha (ChoK?) is regarded as an attractive cancer target. The enzyme catalyses the formation of phosphocholine(PCho), an important precursor in the generation of phospholipids essential for cell growth. ChoK? has oncogenic properties and is critical for the survival of cancer cells. Overexpression of the ChoK? protein can transform noncancer cells into cells with a cancerous phenotype, and depletion of the ChoK? protein can result in cancer cell death. However, the mechanisms underlying the tumourigenic properties of ChoK? are not fully understood. ChoK? was recently demonstrated to associate with other oncogenic proteins, raising the possibility that a non-catalytic protein scaffolding function drives the tumourigenic properties of ChoK? rather than a catalytic function. In order to differentiate these two roles, we compared the impact on cancer cell survival using two tools specific for ChoK?: (1) small interfering RNA (siRNA) to knockdown the ChoK? protein levels; and (2) compound V-11-0711, a novel potent and selective ChoK? inhibitor (ChoK? IC50 20 nM), to impede the catalytic activity. Both treatments targeted the endogenous ChoK? protein in HeLa cells, as demonstrated by a substantial reduction in the PCho levels. siRNA knockdown of the ChoK? protein in HeLa cells resulted in significant cell death through apoptosis. In contrast, compound V-11-0711 caused a reversible growth arrest. This suggests that inhibition of ChoK? catalytic activity alone is not sufficient to kill cancer cells, and leads us to conclude that there is a role for the ChoK? protein in promoting cancer cell survival that is independent of its catalytic activity.

SUBMITTER: Falcon SC

PROVIDER: S-EPMC3641355 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival.

Falcon S C SC Hudson C S CS Huang Y Y Mortimore M M Golec J M JM Charlton P A PA Weber P P Sundaram H H

Oncogenesis 20130101

Choline kinase alpha (ChoKα) is regarded as an attractive cancer target. The enzyme catalyses the formation of phosphocholine(PCho), an important precursor in the generation of phospholipids essential for cell growth. ChoKα has oncogenic properties and is critical for the survival of cancer cells. Overexpression of the ChoKα protein can transform noncancer cells into cells with a cancerous phenotype, and depletion of the ChoKα protein can result in cancer cell death. However, the mechanisms unde ...[more]

PMID: 25522435

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Project description:Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell-derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non-coding RNA, microRNA-24 (miR-24), as a pro-survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR-24 via viral transduction or chemical transfection, where the observed protective role of miR-24 in cardiomyocytes might have partially resulted from its effect on non-cardiomyocyte cells. To elucidate the cardiomyocyte-specific effects of miR-24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR-24 expression is driven by the cardiac-specific Myh6 promoter. The Myh6-miR-24 transgenic mice did not exhibit apparent difference from their wild-type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post-MI compared to their wild-type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non-genetic approach, raising the possibility that non-genetic approaches of introducing miR-24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte-specific role of miR-24 in the adult heart, and may provide insight to strategy design when considering miRNA-based therapies for cardiovascular diseases.

Project description:BackgroundCholine kinase-? (ChoK?/CHKA) overexpression and hyper-activation sustain altered choline metabolism conferring the cholinic phenotype to epithelial ovarian cancer (OC), the most lethal gynecological tumor. We previously proved that CHKA down-modulation reduced OC cell aggressiveness and increased sensitivity to in vitro chemotherapeutics' treatment also affecting intracellular content of one-carbon metabolites. In tumor types other than ovary, methionine decrease was shown to increase sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 triggering. These effects were suggestive of a potential role for ChoK? in regulating susceptibility to TRAIL cytokine.MethodsThe relationship between ChoK?/CHKA and TRAIL-receptor 2 (TRAIL-R2) expression was investigated in silico in OC patients' GEO datasets and in vitro in a panel of OC cell lines upon transient CHKA silencing (siCHKA). The effect of siCHKA on metabolites content was assessed by LC-MS. The triggered apoptotic signalling was studied following soluble-TRAIL or anti-TRAIL-R2 agonist antibody treatment. Lipid rafts were isolated by Triton X-100 fractionation. Preclinical ex vivo studies were performed in OC cells derived from patients' ascites using autologous PBLs as effectors and a bispecific anti-TRAIL-R2/anti-CD3 antibody as triggering agent.ResultsHere we demonstrate that siCHKA specifically overcomes resistance to TRAIL-mediated apoptosis in OC cells. Upon siCHKA we detected: a significant sensitization to caspase-dependent apoptosis triggered by both soluble TRAIL and anti-TRAIL-R2 agonist antibody, a specific increase of TRAIL-R2 expression and TRAIL-R2 relocation into lipid rafts. In siCHKA-OC cells the acquired TRAIL sensitivity was completely reverted upon recovery of ChoK? expression but, at variance of other tumor cell types, TRAIL sensitivity was not efficiently phenocopied by methionine deprivation. Of note, we were also able to show that siCHKA sensitized tumor cells derived ex vivo from OC patients' ascites to the cytotoxic activity of autologous lymphocytes redirected by a bispecific anti-TRAIL-R2/anti-CD3 antibody.ConclusionsOur findings suggest that ChoK?/CHKA impairment, by restoring drug-induced or receptor-mediated cell death, could be a suitable therapeutic strategy to be used in combination with chemotherapeutics or immunomodulators to improve OC patients' outcome.

Project description:BackgroundAberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha).MethodsTo assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments.ResultsIn silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by (1)H-magnetic spectroscopy analysis; (III) a 35-36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context.ConclusionWe showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

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A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival.

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A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival.

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