CDKN2A Inhibits The Formation Of Homotypic Cell-in-cell Structure

Cell-in-cell structures are common in a variety of tumor cells,refer to a unique phenomenon with one or more viable cells present in the cytoplasm of another live cell.Cell-in-cell structures in human tumors are largely homotypic,and closely associated with clinical outcomes of cancer patients.Recently,chromosome aberrations,such as 9p21 chromosomal deletion frequently seen in clinics,were well accepted as one of the driving force for tumorigenesis.Actually,genomic changes(deletion,point mutation,promotor methylation)in 9p21 chromosomal fragment were detected in about one-third of human tumors,including mesothelioma,melanoma,glioma,lung cancer and breast cancer and the like.The p16INK4 a and p14 ARF,encoded by the CDKN2 A gene at 9p21,both act as tumor suppressors by regulating the cell cycle.Somatic mutations of CDKN2 A were found tightly associated with tumor development and progression.Interestingly,scientists found that mesothelioma cells positive for homozygous 9p21/CDKN2A/p16 deletion exhibited significantly more cell-in-cell structures than did CDKN2A/p16 deletion-negative mesothelial cells,suggesting a pivotal role for CDKN2 A deletion in inducing cell-in-cell formation.Accordingly,we attempt to explore the functional link between CDKN2 A deletion and cell-in-cell formation in this project,and decipher the molecular and cellular mechanisms underlying the effects of CDKN2 A on cell-in-cell formation.In this study,we first made cell lines for CDKN2 A genes(p16INK4a and p14ARF)in MCF7 cells,which were homozygously deleted in CDKN2 A gene.And the MCF7 cells expressing CDKN2 A genes exhibited increased cell size and prolonged contour lines while cell proliferation was inhibited as expected.Moreover,homotypic cell-in-cell formation was significantly suppressed,consistent with which,knocking down CDKN2 a expression by CRISPR/Cas9 technique promoted cell-in-cell formation in HEK293 cells.Mechanistically,CDKN2 a overexpression were found to be able to inhibit phosphorylation of myosin light chain(MLC),induce redistribution of E-cadherin to cytoplasmic vescical membrane and intracellular F-actin to cell periphery.In conclusion,we favour the idea that CDKN2 A inhibits the formation of homotypic cell-in-cell structure by inducing intracellular F-actin redistribution,inhibiting MLC phosphorylation and impairing intercellular junction formation.The lack of CDKN2 A expression may be one of the key factors that switch on formation of homotypic cell-in-cell structure in tumor cells and it helps us to gain a new perspective on studying cell-in-cell.