ABSTRACT
BACKGROUND: Despite progress understanding the mechanisms underlying tumor spread, metastasis remains a clinical challenge. We identified the choline-producing glycerophosphodiesterase, EDI3 and reported its association with metastasis-free survival in endometrial cancer. We also observed that silencing EDI3 slowed cell migration and other cancer-relevant phenotypes in vitro. Recent work demonstrated high EDI3 expression in ER-HER2+ breast cancer compared to the other molecular subtypes. Silencing EDI3 in ER-HER2+ cells significantly reduced cell survival in vitro and decreased tumor growth in vivo. However, a role for EDI3 in tumor metastasis in this breast cancer subtype was not explored. Therefore, in the present work we investigate whether silencing EDI3 in ER-HER2+ breast cancer cell lines alters phenotypes linked to metastasis in vitro, and metastasis formation in vivo using mouse models of experimental metastasis. METHODS: To inducibly silence EDI3, luciferase-expressing HCC1954 cells were transduced with lentiviral particles containing shRNA oligos targeting EDI3 under the control of doxycycline. The effect on cell migration, adhesion, colony formation and anoikis was determined in vitro, and significant findings were confirmed in a second ER-HER2+ cell line, SUM190PT. Doxycycline-induced HCC1954-luc shEDI3 cells were injected into the tail vein or peritoneum of immunodeficient mice to generate lung and peritoneal metastases, respectively and monitored using non-invasive bioluminescence imaging. Metabolite levels in cells and tumor tissue were analyzed using targeted mass spectrometry and MALDI mass spectrometry imaging (MALDI-MSI), respectively. RESULTS: Inducibly silencing EDI3 reduced cell adhesion and colony formation, as well as increased susceptibility to anoikis in HCC1954-luc cells, which was confirmed in SUM190PT cells. No influence on cell migration was observed. Reduced luminescence was seen in lungs and peritoneum of mice injected with cells expressing less EDI3 after tail vein and intraperitoneal injection, respectively, indicative of reduced metastasis. Importantly, mice injected with EDI3-silenced cells survived longer. Closer analysis of the peritoneal organs revealed that silencing EDI3 had no effect on metastatic organotropism but instead reduced metastatic burden. Finally, metabolic analyses revealed significant changes in choline and glycerophospholipid metabolites in cells and in pancreatic metastases in vivo. CONCLUSIONS: Reduced metastasis upon silencing supports EDI3's potential as a treatment target in metastasizing ER-HER2+ breast cancer.
Subject(s)
Breast Neoplasms , Receptor, ErbB-2 , Receptors, Estrogen , Animals , Female , Humans , Mice , Cell Line, Tumor , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/genetics , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Receptors, Estrogen/metabolism , Disease Models, Animal , Cell Movement , Gene Knockdown Techniques , Tumor Burden , Neoplasm Metastasis , Lung Neoplasms/secondary , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Cell ProliferationABSTRACT
BACKGROUND: Intrinsic or acquired resistance to HER2-targeted therapy is often a problem when small molecule tyrosine kinase inhibitors or antibodies are used to treat patients with HER2 positive breast cancer. Therefore, the identification of new targets and therapies for this patient group is warranted. Activated choline metabolism, characterized by elevated levels of choline-containing compounds, has been previously reported in breast cancer. The glycerophosphodiesterase EDI3 (GPCPD1), which hydrolyses glycerophosphocholine to choline and glycerol-3-phosphate, directly influences choline and phospholipid metabolism, and has been linked to cancer-relevant phenotypes in vitro. While the importance of choline metabolism has been addressed in breast cancer, the role of EDI3 in this cancer type has not been explored. METHODS: EDI3 mRNA and protein expression in human breast cancer tissue were investigated using publicly-available Affymetrix gene expression microarray datasets (n = 540) and with immunohistochemistry on a tissue microarray (n = 265), respectively. A panel of breast cancer cell lines of different molecular subtypes were used to investigate expression and activity of EDI3 in vitro. To determine whether EDI3 expression is regulated by HER2 signalling, the effect of pharmacological inhibition and siRNA silencing of HER2, as well as the influence of inhibiting key components of signalling cascades downstream of HER2 were studied. Finally, the influence of silencing and pharmacologically inhibiting EDI3 on viability was investigated in vitro and on tumour growth in vivo. RESULTS: In the present study, we show that EDI3 expression is highest in ER-HER2 + human breast tumours, and both expression and activity were also highest in ER-HER2 + breast cancer cell lines. Silencing HER2 using siRNA, as well as inhibiting HER2 signalling with lapatinib decreased EDI3 expression. Pathways downstream of PI3K/Akt/mTOR and GSK3ß, and transcription factors, including HIF1α, CREB and STAT3 were identified as relevant in regulating EDI3 expression. Silencing EDI3 preferentially decreased cell viability in the ER-HER2 + cells. Furthermore, silencing or pharmacologically inhibiting EDI3 using dipyridamole in ER-HER2 + cells resistant to HER2-targeted therapy decreased cell viability in vitro and tumour growth in vivo. CONCLUSIONS: Our results indicate that EDI3 may be a potential novel therapeutic target in patients with HER2-targeted therapy-resistant ER-HER2 + breast cancer that should be further explored.
