Dataset Information

Choline kinase alpha impairment overcomes TRAIL resistance in ovarian cancer cells.

ABSTRACT:

Background

Choline 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.

Methods

The 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.

Results

Here 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.

Conclusions

Our 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.

SUBMITTER: Rizzo A

PROVIDER: S-EPMC7780408 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Choline kinase alpha impairment overcomes TRAIL resistance in ovarian cancer cells.

Rizzo Andrea A Satta Alessandro A Garrone Giulia G Cavalleri Adalberto A Napoli Alessandra A Raspagliesi Francesco F Figini Mariangela M De Cecco Loris L Iorio Egidio E Tomassetti Antonella A Mezzanzanica Delia D Bagnoli Marina M

Journal of experimental & clinical cancer research : CR 20210104 1

<h4>Background</h4>Choline kinase-α (ChoKα/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 ...[more]

PMID: 33390181

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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.

Project description:Detection of the abnormal phosphatidylcholine (PtdCho) metabolism in EOC by analysis of magnetic resonance profile (MRS), showed a significant increase of PCho content in EOC cells as compared with non tumoral counterparts associated with activation of choline kinase (ChoK), the enzyme responsible for PCho production following choline phosphorylation in the PtdCho biosynthetic pathway (Kennedy’s pathway). The α-isoform of ChoK has an essential role in growth control and signal transduction and it has been implicated in the carcinogenic process. Aim of the study is the evaluation of the biological relevance of ChoK expression and activity to define its possible role as a non-invasively detectable EOC biomarker and druggable target. We specifically silenced ChoKα expression by transient RNA interference in two different EOC cell lines and evaluated the biological effects related to metabolic profiles, cell proliferation, cell cycle regulation and alteration of global gene expression to possibly identify new pathways implicated in altered choline metabolism. Following ChoKα silencing we observed a 70% reduction of mRNA and protein expression with a similar reduction in PCho accumulation as assessed by HMRS analysis. The ChoKα silencing was accompanied by 20% inhibition of cell growth and similar percentage of cells blocked in the G1-phase of cell cycle. No alteration in cell morphology and modulation of the main survival signaling pathways (PI3K and MAPK-related signaling) were observed. By gene expression analysis we found 476 differentially expressed genes (p< 0.01; FDR 25%) in silenced cells. By transcriptome analysis of CHKA silenced cells as compared to controls, among the most relevant co-repressed genes we found CyclinA, related to regulation of cell cycle progression, and cytokines genes (IL-6 and IL-8) related to inflammation and EOC aggressiveness. Acyl-CoA synthetase medium-chain family member 3 (ACSM3), phosphatidic acid phosphatase type 2 (PPAP2A) and Osteoprotegerin were among the most up-regulated genes. All co-modulated genes have been validated by RTqPCR also in independent biological replicates. Ingenuity System Pathways Analysis (IPA) identified a network of molecules most significantly affected by CHKA silencing whose main functions is related to cell morphology cellular assembly and organization, cellular function and maintenance. Also, the cellular functions predicted to be mostly affected by silencing were cellular movement and cell death that are expected to be respectively decreased and increased in silenced cells. a total of 12 samples were analyzed: Two EOC cell line SKOV3 and INTOV11 were transiently silenced with unrelated or CHKA-specific siRNA pool. Three independent experiment were run for each cell line.

Dataset Information

Choline kinase alpha impairment overcomes TRAIL resistance in ovarian cancer cells.

Background

Methods

Results

Conclusions

Publications

Choline kinase alpha impairment overcomes TRAIL resistance in ovarian cancer cells.

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