Characterising a role for EDI3 in metastasis in vivo
Final Report Abstract
EDI3 (GPCPD1, GDE5, GDPD6) was first discovered by our group in a screen for potential markers of metastasis in endometrial cancer, where we showed that high EDI3 in primary tumours was associated with shorter metastasis-free survival. Our subsequent in vitro analyses to understand EDI3’s function indicated a role in cell migration, adhesion and viability – processes relevant in cancer. We also demonstrated that EDI3 hydrolyses glycerophosphocholine (GPC) to choline and glycerol-3-phosphate, key intermediates in choline, lipid, carbohydrate and glucose metabolism. In the present project, we aimed to understand whether EDI3 contributes to tumor development and metastasis in vivo. During the course of the project, we found that EDI3 is highly expressed in HER2+/ER- breast cancer. We therefore established HER2+/ER- cell lines where downregulation of EDI3 is controlled by the addition of doxycycline. These cells are also luminescent and fluorescent, and could therefore be detected and tracked in vivo. Decreasing EDI3 expression in the HCC1954 cells with doxycycline led to a dose dependent decrease in cell viability, attachment and colony formation, as well as metabolic changes, supporting our hypothesis that EDI3 enzymatic activity is important for its role in cellular processes. We could also show that HCC1954 cells produce xenografts and metastases in mice and that treating the mice with doxycycline, increases fluorescence, and decreases EDI3 expression and activity in the tumours. Initial results also suggest that inhibiting EDI3 influences tumor growth, which is currently being repeated. Experiments investigating EDI3’s role in tumor metastasis, as well as NMR and mass spectrometry for metabolic analyses are also ongoing. We are confident that the results will help us gain further insight into EDI3’s role in metabolism and tumor growth and metastasis. There were good and bad ‘surprises’ during the course of the project. A major challenge, which led to a delay in the project was the time spent trying to create an inducible EDI3 overexpressing cell line that fluoresced. After numerous cloning attempts, we concluded that EDI3 influences folding and thus function of tagged proteins, such as fluorophores. Furthermore, the choice of fluorophore also appears important. We were able to finally design a functioning vector with EDI3 linked to the very bright Turbo-RFP fluorophore by a 21 amino acid linker. Since we are still interested in creating an inducible EDI3 overexpressing cell line, one of our future plans is to clone the EDI3-TurboRFP sequence into a lentiviral vector with a tetracycline response element (TRE). A positive ‘finding’ that arose from the many attempts of creating and evaluating a fluorescent EDI3 expressing vector, as well as the mutant constructs, was the punctuated pattern seen for EDI3. EDI3 is one of three human proteins with a carbohydrate binding domain 20, which is reported to be important for glycogen binding and metabolism. Therefore, preliminary results in a new project support a role for EDI3 in muscle glycogen metabolism. Therefore, the tools we developed in the present project will continue help us characterize the seemingly multifunctional glycerophosphodiesterase, EDI3.
Publications
- Glycerol-3-phosphate Acyltransferase 1 Promotes Tumor Cell Migration and Poor Survival in Ovarian Carcinoma. Cancer Res. 2017 Sep 1;77(17):4589-4601
Marchan R, Büttner B, Lambert J, Edlund K, Glaeser I, Blaszkewicz M, Leonhardt G, Marienhoff L, Kaszta D, Anft M, Watzl C, Madjar K, Grinberg M, Rempel E, Hergenröder R, Selinski S, Rahnenführer J, Lesjak MS, Stewart JD, Cadenas C, Hengstler JG
(See online at https://doi.org/10.1158/0008-5472.can-16-2065)
