The Role Of Sema4D/plexinB1 In Vasculogenic Mimicry Formation In Non-small Cell Lung Cancer And The Underlying Mechanisms

Objectives:Vasculogenic mimicry is a special vasculature form in tumor,which is composed of highly malignant tumor cells.This tumor cell-mediated blood supply pattern is closely associated with poor prognosis in cancer patients.It could be particularly important to explore the molecular mechanism of vasculogenic mimicry and find strategies for it.It has been reported that tumor cells capable of forming vasculogenic mimcry is highly aggressive,and the plasticity and motility of these cells are closely related to their vasculogenic mimicry formation.The interaction of axon guidance factor sema4D and its high affinity receptor plexinBl could activate small GTPase RhoA and its downstream kinases;this process plays an important role in the migration of endothelial cells and tumor angiogenesis.Does this signaling pathway get the same role in tumor vasculogenic mimicry?Mounting evidence show that vasculogenic mimicry has been found in lung cancer,this paper aims to explore the role of sema4D/plexinB1 and its downstream RhoA/ROCK signaling pathway in vasculogenic mimicry of non-small cell lung cancer.Methods:(1)This study investigated the existence of VM channels and the expression of axon guidance factor sema4D in non-small cell lung carcinoma tissue samples through immunohistochemistry,and analyzed the relationship between them.(2)Knocking down the expression of sema4D in H1299 and HCC827 cells by constructing lenti-virus vector of RNA interference specific for it.Then we explored the effect of sema4D on the vasculogenic mimicry of H1299 and HCC827 cells in vitro and in vivo through various laboratory methods,including tube formation assay,western blot and animal model of implanted tumor.(3)Downregulating the expression of plexinBl in H1299 and HCC827 cells by siRNA expressing vector,inhibiting RhoA/ROCK signaling pathway using fasudil,then observing the tube formation ability by tube formation assay and detecting the expression of RhoA/ROCK related proteins by western blot.Furthermore,we accomplished Rhodamine-phalloidin immunofluorescence,Coomassie brilliant blue staining,wound healing assay,transwell migration assay on sema4D-downregualted cells to identify how sema4D affects the vasculogenic mimicry formation of H1299 and HCC827 cells.Results:(1)We confirmed the special way of vasculature form in non-small cell lung cancer tissues,and observed higher expression of sema4D in all lung cancer tissue samples compared with the corresponding normal tissues.The existence of vasculogenic mimicry channels in tumor tissues is correlated with sema4D expression.(2)Experiments in vitro and in vivo suggest that sema4D plays an important role in generating VM formation in NSCLC.Inhibition of sema4D can reduce VM formation and the expression of VM-related proteins in the lung cancer H1299 and HCC827 cells,at the same time,inhibition of sema4D can restrain the growth and vasculogenic mimicry formation of xenograft.(3)Sema4D may affect vasculogenic mimicry formation in H1299 and HCC827 cells through the regulation of the downstream RhoA/ROCK pathway.Inhibition of sema4D leads to less stress fibers and depleted motility of H1299 and HCC827 cellsConclusion:sema4D plays an important role in the process of VM formation in NSCLC through activation of RhoA/ROCK and regulating tumor cell plasticity and migration.Modulation the sema4D/plexinBland downstream RhoA/ROCK pathway may suppress the tumor plasticity and prevent blood supply of tumor through VM pattern,which may eventually halt the growth and metastasis of cancer.