Leibniz Research Centre for Working Environment and Human Factors at the TU Dortmund (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
Department of Biology, Science Faculty, Dicle University, Diyarbakir, Turkey.
Breast Cancer Res. 2024 May 30;26(1):87. doi: 10.1186/s13058-024-01849-y.
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.
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.
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.
Reduced metastasis upon silencing supports EDI3's potential as a treatment target in metastasizing ER-HER2+ breast cancer.
尽管在理解肿瘤扩散机制方面取得了进展，但转移仍然是一个临床挑战。我们鉴定了产生胆碱的甘油磷酸二酯酶 EDI3，并报告了其与子宫内膜癌无转移生存的关联。我们还观察到沉默 EDI3 可减缓体外细胞迁移和其他癌症相关表型。最近的工作表明，在 ER-HER2+ 乳腺癌中，EDI3 的表达高于其他分子亚型。在 ER-HER2+ 细胞中沉默 EDI3 可显著降低体外细胞存活率，并减少体内肿瘤生长。然而，在这种乳腺癌亚型中，EDI3 在肿瘤转移中的作用尚未得到探索。因此，在本研究中，我们使用实验性转移的小鼠模型，研究在 ER-HER2+ 乳腺癌细胞系中沉默 EDI3 是否会改变体外与转移相关的表型，并在体内形成转移。
为了诱导沉默 EDI3，用含有 EDI3 靶向 shRNA 寡核苷酸的慢病毒颗粒转导表达荧光素酶的 HCC1954 细胞，这些寡核苷酸受多西环素的控制。在体外测定细胞迁移、黏附、集落形成和失巢凋亡的影响，并在第二种 ER-HER2+ 细胞系 SUM190PT 中确认了显著发现。将诱导性沉默 EDI3 的 HCC1954-luc shEDI3 细胞注入免疫缺陷小鼠的尾静脉或腹腔，分别产生肺和腹膜转移，并使用非侵入性生物发光成像进行监测。使用靶向质谱法和 MALDI 质谱成像（MALDI-MSI）分别分析细胞和肿瘤组织中的代谢物水平。
在 HCC1954-luc 细胞中，可诱导沉默 EDI3 可降低细胞黏附性和集落形成，并增加对失巢凋亡的敏感性，这在 SUM190PT 细胞中得到了证实。未观察到细胞迁移的影响。与尾静脉和腹腔内注射后表达 EDI3 减少的细胞相比，注射细胞的小鼠肺部和腹膜中的发光减少，表明转移减少。重要的是，注射 EDI3 沉默细胞的小鼠存活时间更长。对腹膜器官的更仔细分析表明，沉默 EDI3 对转移的器官嗜性没有影响，而是减少了转移负担。最后，代谢分析显示细胞内胆碱和甘油磷脂代谢物以及体内胰腺转移有显著变化。
沉默 EDI3 后转移减少支持 EDI3 作为转移 ER-HER2+ 乳腺癌治疗靶点的潜力。
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