Metabolomics reveals the Glycerophosphodiester Phosphodiesterase GDPD5 to regulate an invasive phenotype in lung cancer cells and potentially constitute a novel drug target

Abstract

Study ObjectiveEpithelial to Mesenchymal Transition (EMT) is a process by which epithelial cells lose their cell polarity and cell‐cell adhesion, and gain migratory and invasive properties required for invasion and metastasis. The discovery of new targets affecting this process opens up for novel cancer therapies.MethodsWe explored the metabolic profiles of A549 lung cancer cells undergoing EMT induced by TGF‐β in a time series using LC‐MS based metabolomics. Follow up on the gene and protein leads suggested by changes in the metabolic profiles included gene expression measurement with qPCR, gene knockdown (lentiviral siRNA), gene over expression (V5 vector), protein analysis using FACS and western blotting. Phenotypic evaluation was performed by transwell invasion assay and zymography. The publically available gene expression database Kaplan‐Meier Plotter was queried for survival analysis correlating with GDPD5 gene expression.ResultsIntracellular glycerophosphocholine (GPC) levels were increased 3‐fold within 24 hours after TGF‐β application. GPC is an intermediate in the phosphocholine (PC) metabolism and is the substrate for a family of phosphodiesterase's. Administration of TGF‐β together with the phosphodiesterase inhibitor dipyridamole increased the GPC levels but not N‐cadherin, a marker for EMT, suggesting that GPC itself is no driver of EMT. Genes in the Glycerophosphodiesterase family were downregulated when treated with TGF‐β, supporting accumulation of GPC. One member, Glycerophosphodiester phosphodiesterase 2, GDE2 (GDPD5) displayed a pattern of increased mRNA and protein expression upon TGF‐β administration. Knockdown of GDE2 using siRNA decreased the invasive phenotype and the amount of protease excreted into the media whereas stable overproduction of GDE2 increased the invasive phenotype and protease excretion. Interestingly the phosphodiesterase inhibitor dipyridamole, an approved drug for blood vessel dilation and inhibition of blood clot formation, also reduced protease excretion in GDE2 over producing cells. Finally, querying a public gene expression database gave that high expression of GDE2 is associated with decreased overall survival in lung, breast and gastric cancers.ConclusionsThese data suggests a central role of phosphodiesterase's and GDE2 in particular for the invasion and metastasis process and identifies a previously unrecognized EMT signaling mechanism which potentially could constitute a therapeutic target.