Identifying Molecules Regulating The Formation Of Homotypic Cell-in-cell Structure And Their Primary Mechanistic Studies

Cell-in-cell structures,prevalent in a wide range of human tumors,refer to the presence of one or more living cells inside another cell.Formation of cell-in-cell structures generally leads to inner cell death in a non-autonomous manner.Therefore,cell-in-cell-mediated death was proposed as type IV cell death,which is different from the well-known cell-autonomous deaths,such as apoptosis,necrosis and autosis and the like.Based on the types of effect and target cells involved,cell-in-cell structures were roughly divided into two types: homotypic and heterotypic.This work focused on the homotypic cell-in-cell structure.During the past decade,considerable progress has been achieved in understanding the mechanisms underlying homotypic cell-in-cell formation.Matrix deadhesion,aberrant mitosis,and glucose starvation were identified as three major triggers.Successful structure formation required two core elements: the establishment of adherens junction and actomyosin contraction.Multiple essential molecules were identified as key players,such as the intercellular adhesion proteins E-cadherin and ?-catenin,the cytoskeletal proteins Actin,myosin,and signaling molecules RhoA and ROCK,etc.Besides,novel regulators(CDKN2A?MRTF?LPAR?PDZ-RhoGEF?Lgl2?TIP50 et al.)were continuously reported,suggesting homotypic cell-in-cell formation a complex cellular process.Accordingly,increasing efforts were endeavoured to deciphering its underlying molecular mechanisms.This study attempted to explore the molecular control of homotypic cell-in-cell formation by using two complementary strategies.Protocol 1: employing phenotype-based expression profiling to systemically screen genes that potentially participate in cell-in-cell regulation.Protocol 2: identifying membrane lipids that might affect homotypic cell-in-cell formation by the candidates-validation method.Two rounds of systematic screening were performed following protocol 1.In the first round of screening on 4 breast cancer cell lines,totally 62 genes were identified to be associated with homotypic cell-in-cell formation.Among them,IL-8 was selected for further validation,and capable of promoting homotypic cell-in-cell formation via increasing P-cadherin/?-catenin-mediated cell-cell adhesion.Next,to improve the efficacy and accuracy of screening,a second round of expression profiling was performed on 12 isogenic cell clones,who share similar genetic background.As a result,a group of cell-in-cell-associated genes were identified,of which ARHGAP36 was a promising candidate as its expression level was positively correlated with homotypic cell-in-cell frequency.Functional studies revealed that overexpression of ARHGAP36 in ARHGAP36-low cells could efficiently enhance cell internalization to form cell-in-cell structures.This function was fulfilled by promoting P-cadherin/?-catenin-mediated cell-cell adhesion via Arginine-rich motif but not RhoGAP domain within ARHGAP36.Meanwhile,we managed identifying membrane lipids that affect homotypic cell-in-cell formation by using candidate-validation method.We first identified Lipofectamine-2000 as a potent inhibitor from a set of commercially available liposomes,and the effect was tightly correlated with de-phosphorylation of myosin light chain.Then,via candidate analysis of liposome composition,four membrane lipids,including cholesterol,phosphatidylethanolamine,stearamide and hemolytic phosphatidic acid,were found to be able to inhibit homotypic cell-in-cell formation,respectively and/or synergistically.Similar to that of Lipofectamine-2000,cholesterol inhibited phosphorylation of myosin light chain and therefore actomyosin contraction to suppress the formation of homotypic cell-in-cell structures.In summary,we performed the first systemic screening of molecules that regulate homotypic cell-in-cell formation,and obtained a set of candidate genes and membrane lipids,followed by primary functional validation and mechanistic investigation.We identified IL-8 and ARHGAP36 as two regulators of homotypic cell-in-cell formation through enhancing P-cadherin/?-catenin or P-cadherin/?-catenin-mediated cell-cell adhesion.Whereas,membrane cholesterol could suppress the formation of homotypic cell-in-cell structures via promoting de-phosphorylation of myosin light chain.Our work provided novel targets for cell-in-cell based tumor therapy.