Authors: Glotzbach, Annika
Title: Investigating a role for EDI3 in tumor growth and metastasis in breast cancer using a doxycycline-inducible knockdown system
Language (ISO): en
Abstract: Metastasis remains a major problem for tumor therapy. In endometrial and ovarian cancer, metastasis and worse survival was found to be associated with elevated EDI3 (GPCPD1; GDE5; GDPD6) expression in primary tumors. EDI3 is a glycerophosphodiesterase which cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P) and is therefore considered one of the key enzymes involved in choline metabolism. Altered choline metabolism is a recognized metabolic hallmark of cancer and was reported in breast, ovarian, and prostate cancers. Previously, in vitro studies revealed that silencing EDI3 transiently in various breast cancer cell lines resulted in altered choline metabolism and impaired cellular migration, attachment, and spreading. However, stable constitutive EDI3 knockdown led to compensation of metabolite levels over time, which was accompanied by a loss of the migration phenotype. Therefore, in the present work a doxycycline (Dox) inducible EDI3 knockdown system was established in luciferase-expressing ER-HER2+ breast cancer cells, which reduces compensatory effects and allows to investigate EDI3 in tumor growth and metastasis in vivo. To create cell lines in which EDI3 is inducibly silenced, stable luciferase-expressing HCC1954 cells were generated and subsequently transduced with lentiviral particles, which resulted in three different Dox-inducible EDI3 knockdown cell lines containing independent EDI3-targeting shRNA oligos. Dox treatment led to a time and dose dependent decrease in EDI3 RNA and protein expression. Mass spectrometry analyses revealed that induced EDI3 knockdown also led to dose dependent alterations in endogenous choline metabolites and phospholipid levels. Using various in vitro assays, it could be shown that EDI3 knockdown resulted in significant reduction in colony formation and proliferation, processes which are relevant in the formation of metastasis. Furthermore, EDI3 silencing rendered cells more susceptible towards anoikis. However, Dox-induced EDI3 knockdown had only little effect on adhesion and no effect on migration. To investigate EDI3’s role in tumor growth and metastasis in vivo, different tumor models were established in immunodeficient mice. The subcutaneous tumor model showed no significant effect on primary tumor growth. However, in a mouse model for peritoneal metastasis, luminescence imaging revealed lower signals indicative of less metastasis formation in the EDI3 knockdown condition. Furthermore, it could be shown that silencing EDI3 was associated with reduced tumor burden, less ascites fluid and longer survival time. Altogether, this thesis provides, for the first time, in vivo evidence that supports a role for EDI3 in metastasis formation, which further emphasizes the importance of choline and glycerophospholipid metabolism in this process.
Subject Headings: Breast cancer
CDX model
In vivo imaging
Subject Headings (RSWK): Brustkrebs
Issue Date: 2022
Appears in Collections:Chemische Biologie

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