Study On The Mechanism Of CPLA2α Reversibly Regulating Different Subsets Of Cancer Stem Cells Transformation In Cervical Cancer

Cervical carcinoma(CC)is one of the most common types of gynecological malignancy worldwide and is the second leading cause of cancer death in young women aged 20 to 39 years,especially in developing countries.Despite the increases in screening programs and uptake of prophylactic human papillomavirus(HPV)vaccination,the current standard treatments for CC—radiotherapy and chemotherapy—are less than satisfactory due to chemo-/radioresistance,lymph node metastasis and pelvic recurrence.Cancer stem cells(CSCs),as the driving force of chemo-/radioresistance and metastasis,may be the major causes of this treatment status.Cancer stem cells have the ability of self-renewal and differentiation,which allow them to transform between different states to adapt to the environment.An increasing number of studies have also found that CSCs are heterogeneous in phenotype and function,which leads to the failure of killing CSCs completely by traditional radiotherapy and chemotherapy,eventually resulting in tumor recurrence and metastasis.Therefore,the understanding of CSCs markers and regulatory mechanism of different phenotypes of CSCs are crucial to optimize treatment strategies and improve the prognosis of patients.cPLA2αis a common cytoplasmic phospholipase,which plays an important role in tumorigenesis and development.Our previous work found that cPLA2αaffect the invasion and metastasis of tumor cells by epithelial-mesenchymal transition(EMT).In this study,we found that the cervical cancer stem cells(CCSCs)with different levels of cPLA2αshowed distinct morphology through tumor sphere formation assay in vitro.The cells with overexpression(OE)of cPLA2αformed loose grape-shaped clusters,while spheres enriched by knockdown(KD)cPLA2αwere tight clustes.RT-PCR and Western blotting analysis further showed that mesenchymal associated genes were enriched in cPLA2αOE groups,whereas epithelial genes were enriched in cPLA2αKD spheres.Through flow sorting,Smart-seq amplification and characterization of stemness markers in cervical cancer cells and tissues,CCSCs existed in two biologically distinct phenotypes characterized by different levels of cPLA2α.And they had different stemness surface markers.The epithelial CCSCs with cPLA2αdownregulation expressed CD133,whereas CCSCs with overexpression of cPLA2αwere characterized by the expression pattern of CD44~+CD24~-.Transcriptome microarray analysis further found that the the significant enrichment of differential genes between two CCSC subpopulations was closely related to tumor cell functions including proliferation,colony formation,cell cycle progression,adhesion ability and tumor cells migration and invasion.The CCSCs with low expression of cPLA2αshowed relatively quiescent epithelial characteristics with arrested G0/G1 phase,increased the cell-cell adhesion ability and lower proliferation,invasion and migration abilities.The CCSCs with high expression of cPLA2αlocated at the edge of tumor associated with mesenchymal traits,including increased adhesion to cell matrix and higher proliferation,colony formation,invasive and migration abilities in vitro and high tumorigenicity in vivo.The microarray analysis,Tet-on system and mass spectrometry analysis were performed to study the regulatory mechanism of cPLA2αon different CCSC subpopulations.We revealed that cPLA2αregulated the reversible transition between mesenchymal and epithelial CCSC states through phosphorylation of PKCζ,which in turn feeds positively to Wnt/β-catenin signal pathway,further inhibitingβ-catenin-E-cadherin interaction in membrane and promotingβ-catenin translocation into the nucleus to affect the transcriptional regulation of stemness signals.β-catenin can induce the upregulation of mesenchymal gene signals and the activation of EMT program,which promote epithelial quiescent CCSCs transform into malignant mesenchymal states.Further analysis of clinicopathology and immunohistochemical staining demonstrated that elevated cPLA2αexpression was associated with cervical cancer metastasis and poor survival.These suggested that the existence and plasticity of CCSCs regulated by cPLA2αmay be the cause of leading to cells exist from dormancy or latent period,finally resulting in tumor metastasis and recurrence.Targeting cPLA2αmay provide a new potential therapeutic target for stem cell targeted therapy of cervical cancer,and provide a new theoretical basis for eradicating different states of CCSCs to eliminate tumors more effectively and reducing the risk of metastasis.