Glycerin-3-Phosphat-Acyltransferase-1 (GPAM) als Schlüsselfaktor der Tumorzellmigration
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Date
2015
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Abstract
Das Verhindern und die Behandlung von Metastasen stellen eine große Herausforderung der Krebstherapie dar, denn für die meisten Krebspatienten ist die Ausbildung von Metastasen todesursächlich. In früheren Studien konnte bei Patienten mit Endometriumkarzinom eine Assoziation zwischen hoher EDI3-Expression und einer geringeren tumorfreien Überlebensdauer beschrieben werden. In unserer Arbeitsgruppe wurde EDI3 als eine Glycerophosphocholin-Phosphodiesterase charakterisiert; sie katalysiert die Hydrolyse von Glycerophosphocholin (GPC) zu Glycerin-3-Phosphat (G3P) und Cholin (Cho). Durch Cholinkinasen wird Cho zu Phosphocholin (PCho) phosphoryliert. Sowohl die Expression von CHKA als auch die Konzentration von PCho ist in vielen Krebszelltypen erhöht. Das GPC/PCho-Verhältnis verringert sich bei Krebserkrankungen. Die enzymatische Reaktion von EDI3 könnte ebenfalls zu der beobachteten Verringerung des GPC/PCho-Verhältnisses beitragen und ist in vitro bereits nachgewiesen worden. Mit der hier vorliegenden Arbeit wurde diese enzymatische Funktion in Zellkultur bestätigt. Sie belegt darüber hinaus, dass EDI3 neben GPC auch Glycerophosphoethanolamin als Substrat verwenden kann. Weiterhin wurde in vorausgegangenen Arbeiten EDI3 eine entscheidende Rolle in der Migration zugeschrieben, ein für die Metastasierung bedeutender Prozess. In dieser Arbeit konnte nachwiesen werden, dass die Cholinmetaboliten GPC, Cho und Phosphocholin jedoch nicht die Ursache für die bei der Modulation von EDI3 beobachteten Änderungen der Migration sind, weil der Knockdown von Cholinkinase-α (CHKA) zu einer ähnlichen metabolischen Verschiebung der Cholinmetaboliten führt wie der EDI3-Knockdown. CHKA aber hat keinen Einfluss auf die Migration von in dieser Arbeit untersuchten humanen Tumorzellen. Die Ergebnisse der vergleichenden Untersuchungen der Migration bei Inhibition von EDI3, CHKA und GPAM validieren EDI3 als wichtigen Faktor in der Metastasierung und zeigen erstmals in vitro Ergebnisse zur Modulation von GPAM in einem zellulären Krebskontext.
Insgesamt gewährt diese Arbeit einen grundlegenden Einblick in die tumorbiologische Funktion des in diesem Zusammenhang noch uncharakterisierten Enzyms GPAM. Sie ist die erste umfassendere Studie über die metabolische Bedeutung von EDI3 und GPAM in humanen Mammakarzinom-Zellen. Die Beobachtung, dass neben EDI3 auch GPAM bei der Zellmigration mitwirkt, bildet den Grundstein zum genaueren Verständnis dafür, auf welche Weise EDI3 zur
Metastasierung von Tumorzellen beitragen kann. Das enzymatische Produkt G3P von EDI3, und nicht das Cholin mit seinen sekundären Metaboliten, ist für die Migration-stimulierende Wirkung von EDI3 verantwortlich. G3P wird durch die Glycerin-3-Phosphat-Acyltransferase GPAM zu Lysophosphatidsäure (LPA) verestert. LPA ist nicht nur Ausgangsstoff für die Triglycerid-Synthese, sondern auch als Ligand für Signalweg-gekoppelte Rezeptoren bekannt. Der GPAM- und der EDI3-Knockdown führen zu geringeren LPA-Konzentrationen in der Zelle. Die Resultate bestärken LPA als intrazellulären Regulator der Migration. Eine weitere Analyse der Wirkungsweise des GPAM-Produktes LPA könnte behilflich sein, neue Behandlungsmethoden gegen Metastasen zu entwickeln.
Glycerol-3-Phosphate-Acyltransferase-1 (GPAM) is a key factor in tumour cell migration A major challenge in cancer therapy remains the prevention and/or treatment of metastasis, which is usually the main cause of death in cancer patients. Previous studies in our group reported an association between high expression of Endometrial Differential Cancer-3 (EDI3) expression in the primary tumour of ovarian and endometrial cancer patients and decreased survival. In addition, work in our group showed that EDI3 is a glycerophosphodiesterase which catalyses the hydrolysis of glycerophosphocholine (GPC) to glycerol-3-phosphate (G3P) and choline (Cho). Choline is phosphorylated by choline kinase (CHKA) to phosphocholine (PCho), and both enzyme and product are elevated in many cancer cell types. The GPC/PCho ratio has also been reported to decrease in different types of cancer. Via its enzymatic reactivity, EDI3 was also shown to contribute to the observed decrease in GPC/PCho ratio in different types of cancer cells. EDI3’s enzymatic function and its role in influencing the GPC/PCho ratio were confirmed in the present work. In addition, EDI3 was also shown to hydrolyze another glycerophosphodiester, glycerophosphoethanolamine. Previous work also described an important role for EDI3 in cell migration, a process important for metastasis. However, the present study indicated that EDI3’s role in migration appears to not be via its contribution to choline metabolic pathways, as knockdown of choline kinase-α (CHKA) did not inhibit migration, despite similar metabolic alterations as observed after EDI3 knock down. Comparative knockdown of EDI3, CHKA and GPAM confirmed EDI3’s importance in migration and showed for the first time the contribution of a GPAT enzyme to a process linked to cancer. Overall, this work provides further insight into the turmorigenesis process by describing a new role for GPAM. This is the first comprehensive study of the metabolic importance of both EDI3 and GPAM in human breast cancer cells. The results showing that GPAM, in addition to EDI3, participates in cell migration provides additional insight as to how EDI3 contributes to metastasis, and suggests that G3P and not choline and its secondary metabolites is responsible for EDI3’s role in migration. G3P is esterified by glycerol-3-phosphate acyltransferase GPAM to lysophosphatidic acid (LPA). LPA is not only a key precursor for triglyceride synthesis, but also a ligand for G-protein-coupled receptors. The GPAM- and EDI3 knockdown resulted in lower concentrations of LPA in the cell, implying that LPA also functions as an intracellular regulator of migration. Further understanding of the mechanism underlying intracellular LPA’s role in migration could help in the development of new treatments against metastasis.
Glycerol-3-Phosphate-Acyltransferase-1 (GPAM) is a key factor in tumour cell migration A major challenge in cancer therapy remains the prevention and/or treatment of metastasis, which is usually the main cause of death in cancer patients. Previous studies in our group reported an association between high expression of Endometrial Differential Cancer-3 (EDI3) expression in the primary tumour of ovarian and endometrial cancer patients and decreased survival. In addition, work in our group showed that EDI3 is a glycerophosphodiesterase which catalyses the hydrolysis of glycerophosphocholine (GPC) to glycerol-3-phosphate (G3P) and choline (Cho). Choline is phosphorylated by choline kinase (CHKA) to phosphocholine (PCho), and both enzyme and product are elevated in many cancer cell types. The GPC/PCho ratio has also been reported to decrease in different types of cancer. Via its enzymatic reactivity, EDI3 was also shown to contribute to the observed decrease in GPC/PCho ratio in different types of cancer cells. EDI3’s enzymatic function and its role in influencing the GPC/PCho ratio were confirmed in the present work. In addition, EDI3 was also shown to hydrolyze another glycerophosphodiester, glycerophosphoethanolamine. Previous work also described an important role for EDI3 in cell migration, a process important for metastasis. However, the present study indicated that EDI3’s role in migration appears to not be via its contribution to choline metabolic pathways, as knockdown of choline kinase-α (CHKA) did not inhibit migration, despite similar metabolic alterations as observed after EDI3 knock down. Comparative knockdown of EDI3, CHKA and GPAM confirmed EDI3’s importance in migration and showed for the first time the contribution of a GPAT enzyme to a process linked to cancer. Overall, this work provides further insight into the turmorigenesis process by describing a new role for GPAM. This is the first comprehensive study of the metabolic importance of both EDI3 and GPAM in human breast cancer cells. The results showing that GPAM, in addition to EDI3, participates in cell migration provides additional insight as to how EDI3 contributes to metastasis, and suggests that G3P and not choline and its secondary metabolites is responsible for EDI3’s role in migration. G3P is esterified by glycerol-3-phosphate acyltransferase GPAM to lysophosphatidic acid (LPA). LPA is not only a key precursor for triglyceride synthesis, but also a ligand for G-protein-coupled receptors. The GPAM- and EDI3 knockdown resulted in lower concentrations of LPA in the cell, implying that LPA also functions as an intracellular regulator of migration. Further understanding of the mechanism underlying intracellular LPA’s role in migration could help in the development of new treatments against metastasis.
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Keywords
GPAT, EDI3, GDE5, Migration, Metabolismus, LC-MSIMS, NMR-Sprektroskopie, LPA, GPC