LKB1 and its importance for survival in EpCAM negative circulating tumor cells (CTC) in metastatic breast cancer.

Abstract

e23061 Background: To reach distant sites, CTC overcome metabolic stress and acquire a mesenchymal phenotype. So we assessed the pro survival function of liver kinase B1 (LKB1) a cellular metabolic stress controller, in combination with the EMT transcription factor Zinc finger E-box binding homeobox factor 1 (ZEB1) and stem cell markers e.g. CD44 in EpCAM negative CTC. Tumor spheroids and targeted si-RNA knockdown of LKB1 were analyzed in two breast cancer cell lines to support transcriptomic data. Methods: CTC were isolated from whole blood of 27 metastatic breast cancer patients. 17 age matched healthy donors were negative control. All patients were analyzed with CellSearch system to detect EpCAM positive CTC, which were eliminated by immunodepletion (AdnaTest BreastCancerSelect Kit, TATAA Biocenter) together with CD45 positive white blood cells (Miltenyi Biotec). RNA transcripts were quantified by qPCR (TaqMan Gene Expression Assay, ThermoFischer Scientific) and the relative expression was calculated by the 2-DCtmethod. EpCAM negative CTC and spheroids were analyzed by immunofluorescence. Data were analyzed by non-parametric statistical tests. Results: According to the CellSearch system, most patients (74%, n = 20) were CTC negative. 20% of this sub-group showed enhanced levels of ZEB1, 15% of LKB1, and 40% of stem cell markers. ZEB1 and LKB1 were never co-expressed, while there was an almost significant association between enhanced ZEB1 and stem cell marker expression (p = 0.053). Immunofluorescence analysis on single CTC confirmed the transcriptomic result. MDA-MB 231 cell lines, normally negative for LKB1 and unable to grow in suspension, showed the ability to generate spheroids only if LKB1 expression was activated. Targeted knockdown of LKB1 confirmed these results. Conclusions: CellSearch system negative patients present a heterogeneous CTC population with enhanced levels of ZEB1, CD44, or LKB1. While ZEB1 and CD44 might be highly expressed in cells that already have entered EMT, LKB1 overexpression could represent the response to metabolic stress induced in the early phase of intravasation Hence, LKB1 might represent a novel therapeutic target to chase CTC and prevent metastasis.