VEGF-C Mediates Tumor Growth And Metastasis Through Mediating Crosstalk Between EMT And Epithelial Breast Cancer Cells

Objective: It is well established that a subset of cells within primary breast cancers can undergo an epithelial to mesenchymal transition(EMT),although the role of EMT in metastasis remains controversial.We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via paracrine-mediated activation of non-canonical GLI activity that is dependent on Six1 expression in the EMT cancer cells.However,the mechanism by which these Six1-expressing EMT cells activate GLI,remained unclear.Method: In this study we first compared the expression levels of VEGF-C between EMT cells and non-EMT cells using q RT-PCR,western blot analysis,and ELISA.Then we test the malignant properties,including proliferation assays,migration assays,invasion assays and ICC,and Gli signal activity in non-EMT cells treated with EMT cells' conditioned medium with/without VEGF-C neutralizing antibody.We also analyzed the relationship between VEGFC and GLI pathway signal with TCGA and GEO datasets.In addition,we knocked down NRP2 in non-EMT cells,then treated the non-EMT cells with EMT cells' CM with/without VEGF-C antibody and retested the GLI signal activity.In order to establish the xenograft models,we labeled the non-EMT cells with F-luc and knocked down NRP2 and VEGF-C in EMT cells.We mixed the labeled non-EMT cells(SCR control and sh NRP2 groups)with non-labeled nonEMT cells and EMT cells(SCR control and sh VEGF-C groups)and injected the mixed cells into mice and tracked the luciferase signal weekly.Results: In this study,we demonstrate a novel mechanism for activation of non-canonical GLImediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C.We show that VEGF-C,secreted by breast cancer cells that have undergone an EMT,promotes paracrine-mediated increases in proliferation,migration and invasion of epithelial breast cancer cells,via non-canonical activation of GLI-signaling.We further show that the increasingly aggressive phenotypes imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2,which can interact with VEGF-C,in neighboring epithelial cancer cells both in vitro and in vivo.Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer,as VEGF-C and GLI pathway genes significantly positively correlate.Conclusion: Our study suggests that the VEGF-C/NRP2/GLI axis may be a novel and conserved paracrine means by which EMT cells enhance metastasis and provides potential targets for therapeutic intervention in this heterogeneous disease.