The Effect Of CEMIP On Promoting Migration And Invasion Of Small Cell Lung Cancer Through Microenvironment Degradation Product LMW-HA And Its Molecular Mechanism

BACKGROUND AND OBJECTIVESmall cell lung cancer(SCLC)ranks the most malignant type of lung cancer.Metastasis occurs on multiple organs in early stage is the hallmark feature of SCLC and attribute to the main reason for the failure of medical intervention.Discovering and elucidating the vital targets and molecular mechanisms that regulate metastasis is the fundamental basis for the development of new targeted drugs and therapeutic strategy in aspect of blocking its metastasis.The tumor microenvironment contains a large amount of extracellular matrix(ECM),of which 85%belongs to hyaluronic acid(HA),wherein composit of the high molecule weigh HA(HMW-HA).Under pathological conditions such as tumor and inflammation,enhanced the decomposition of HMW-HA leads to a accumulation of LMW-HA(Low molecule weigh HA).Studies have shown that LMW-HA was essential to promote tumor invasion and metastasis,but the specific mechanism remains unknown.Cell migration inducing hyaluronan binding protein(CEMIP)is a new type of hyaluronidase that could bind HA and depolymerize it.In normal condition,CEMIP mainly expresses in brain,lung,pancreas,testis and ovary tissues.Recent studies have found that CEMIP was highly expressed in a variety of solid tumors,and the expression level was positively correlated to the poor prognosis of patients.Also it was a key factor in promoting tumor growth and metastasis.However,whether CEMIP promotes SCLC invasion and metastasis through degrading HA from microenvironment still require further elucidation.Therefore,this dissertation systematically aims at indicating the role of CEMIP in degradation of microenvironmental HA towards regulating SCLC invasion and metastasis,in aspect of clinical specimens,cells and animal model,as well as discovering the underlying molecular mechanism.This study roots in clarifying new regulatory factors and mechanisms of action that affect the metastasis of refractory SCLC.So that providing theoretical basis in order to formulate new anti-metastasis treatment and to improve the prognosis of SCLC patients in clinical treatment.METHOD1.Immunofluorescence was conducted for detecting the expression and distribution of CEMIP and HA in the tissues slides of SCLC patients.Various statistic approaches were used for analyzing the correlation between the expressions of the CEMIP and HA,and analyzing the relationship between their expression and clinicopathological characteristics.2.q PCR and Western Blot were used for detecting the expression level of CEMIP in human normal lung epithelial cells(BEAS-2B)and human SCLC cells(H446,H1688,H209,H196)derived from four different metastatic sites and their supernatants.Lentiviral vector LV-sh CEMIP or GV146-CEMIP plasmid were used to establishing SCLC cells(H446 sh CEMIP,H1688 OE)containing stably silencing CEMIP or transited overexpressing CEMIP.q PCR and Western Blot used to verify the efficacy of silencing or overexpression.Wound healing assays,transwell migration and invasion assays were used to detect the changes of migration and invasion ability on SCLC cells after silencing or overexpressing CEMIP.MTT and flow cytometry assays were used to detect the changes of proliferation and apoptosis on SCLC cells after silencing or overexpressing CEMIP.3.TMT(Tandem Mass Tags,TMT)labeled quantitative proteomics sequence detects the protein expression profiles of SCLC cells after silencing CEMIP expression,and bioinformatics methods were applied to identified differential expressed proteins(DEPs)and analyzed their enriched cellular biological functions,signal pathway and protein interaction network.q PCR and Western Blot were used for verifying the changes of DEP expression in in vitro model after silencing or overexpressing CEMIP.4.ELISA and agarose gel electrophoresis assays were used to detect the concentration and distribution of LMW-HA in the SCLC supernatant after silencing or overexpressing CEMIP,respectively.Western blot and q PCR were used to detect the expression of HA-related receptors.Immunofluorescence staining was used to observe the binding of HA,CEMIP and TLR2 with or without LMW-HA or C29 treatment.HE staining assays were used to observe the morphological change of SCLC cells when treating with different inhibitors or HA.Wound healing assays,Transwell-migration and invasion assays were used for detecting the change of migrated and invasive ability on SCLC cells with silencing or overexpressing CEMIP,when treating with LMW-HA alone or in combination of TLR inhibitor C29,MAPK inhibitor PD98059(MEK/ERK inhibitor)and F-actin inhibitor Latruculin A.Immunofluorescence was conducted for observation the rearrangement effect of filamentous actin(Filamentous actin,F-actin)when over-expressing CEMIP or adding LMW-HA.Co-IP and Western Blot assays were used for detecting the binding,expression and activation of TLR2,SRC and ERK1/2.5.H446 sh NC cells or H446 sh CEMIP cells was using to mimic the in vivo process of SCLC brain colonization model in nude mouse by intracranial injection.HE staining was applied to evaluate the formation of metastases,immunofluorescence immunohistochemistry and Western Blot assays were used to detecting the expression of CEMIP,HA and receptor TLR2,p-ERK1/2 and PCNA in metastases.RESULTS1.In the tumor tissues of SCLC patients,the expressions of CEMIP and HA were significantly higher than those of adjacent tissues(P<0.05),and the expressions of these two factors were positively correlated(r=0.51,r~2=0.26,P<0.001).CEMIP was mainly expressed in the cytoplasm of tumor cells and ECM,and HA was mainly expressed in the ECM.Both of them were co-localized in the cytoplasm and ECM of tumor cells.In addition,the results of the chi-square analysis indicated that the high expression of CEMIP positive related to the high level of serum neuron-specific enolase(NSE)(P=0.027),higher tumor stage(T stage)(P=0.033)and higher lymph node staging(N staging)(P=0.033).High HA level related to higher lymph node staging(N staging)(P=0.046),and high expression of CEMIP became a risk factor for lymph node metastasis(P=0.007,95%CI=3.090-1450.577,HR=66.946).2.In human SCLC cells(H446,H1688,H209,H196)derived from four different metastatic sites,the m RNA and protein expression of CEMIP were higher than those in BEAS-2B counterpart(all P<0.05).The m RNA and protein expression levels were significantly down-regulated or up-regulated(P<0.01 or P<0.05)after stably silencing CEMIP with LV-sh CEMIP on H446 cells or transited over-expressing CEMIP with GV146-plasmid on H1688 cells,respectively.After silencing with CEMIP,the migration,invasion,proliferation and cloning ability of H446 cells were significantly reduced.On the contrary,overexpressing CEMIP on H1688 lead to increased cellular ability towards migration,invasion,and proliferation(all P<0.05).In addition,silencing CEMIP on H446 or overexpressing CEMIP on H1688 has no significant effect on the apoptosis.3.A total of 215 differentially expressed proteins(DEPs)were identified in H446 cells after interference with CEMIP expression(109 up-regulated and 106down-regulated).DEPs were enriched in cell or tissue migration processes,such as epithelial cell migration,epithelium migration,and tissue migration;participated in regulating protein activity,such as secondary active transmembrane transporter activity,calcium ion binding,and organic anion exchanger activity;distributed in caveolae,cornified envelope and intermediate filament and other cell components.The KEGG pathway annotation of DEPs found that the signal pathways such as MAPK signaling pathway,GABAergic synapse and TGF-beta signaling pathway have undergone significant changes.Pathway verification results found that the expression of CEMIP was positively correlated with the activation of MAPK pathway.4.After silencing with CEMIP,the level and distribbution of LMW-HA in supernatant of H446 sh CEMIP cells was decreased while after overexpressing CEMIP on H1688,the level and distribution of LMW-HA in supernatant was increased.After interference or overexpression of CEMIP,the m RNA and protein expression levels of HA receptors TLR2 and CD44 in SCLC cells were decreased or increased accordingly(P<0.05).In addition,overexpression of CEMIP induced the rearrangement effects on F-actin of H1688 cells such as,edge aggregation,alone with the lamellipodia and filopodia increased significantly at the edge,while the arrangement of the center of the cell decreased.Similar to the overexpression of CEMIP,the addition of LMW-HA treatment could also enhance the migration,invasion ability and F-actin rearrangement effects on H1688 cells,and could rescue the migration and invasion ability of H446 caused by silencing CEMIP(P<0.05).Additionally,treatment of TLR2 inhibitors C29,or PD98059(MEK/ERK inhibitor),and Latrunculin A(F-actin inhibitor)on SCLC cells that interfered with or overexpressd CEMIP could effectively block the enhanced effects from overexpression of CEMIP or the addition of LMW-HA,which induced promotion of migration and invasion and F-actin rearrangement effects(all P<0.05).After silencing the expression of CEMIP in H446 cell,the binding affinity of ERK1/2 among SRC and TLR2 was reduced,and the expression of p-ERK1/2 also showed decrease.LMW-HA treatment could significantly enhance the binding affinity of ERK1/2 among SRC and TLR2,as well as up-regulated the expression of p-ERK1/2.Meanwhile,the overexpression of CEMIP or treating LMW-HA on H1688 cell could obviously enhance the binding affinity of ERK1/2 among SRC and TLR2,also increased the expression of p-ERK1/2.5.The colonization ability of H446 sh CEMIP cells in liver,brain,and lung tissues was slightly reduced(P>0.05),when compared with H446 sh NC group.Tissue immunofluorescence results showed that there was no significant difference between the two groups in brain metastases(P>0.05).In liver and lung metastases,the expression levels of CEMIP and HA in the H446 sh CEMIP group were significantly lower than those in the control group H446 sh NC(both P<0.05).In brain metastases,the expression of CEMIP and PCNA in H446sh CEMIP group was not significantly different with control group H446 sh NC(P>0.05),but the expression of TLR2 was enhanced(P<0.05).In liver and lung metastases,the protein expression of CEMIP,TLR2,p-ERK1/2 and PCNA were all lower in the H446 sh CEMIP group than those of in the H446 sh NC group(all P<0.05).CONCLUSION1.CEMIP and HA are highly expressed in SCLC,and are positively correlated.The high expression of CEMIP is positively correlated with high NSE levels,high T and N stages,and is a risk factor for lymphatic metastasis.2.CEMIP enhances the ability of SCLC cell migration,invasion and proliferation,but has no significant effect on SCLC apoptosis.3.Down-regulation of CEMIP expression mainly affects the biological processes related to epithelial cell migration,molecular functions such as the activity of active transmembrane transporters,cell location such as caveola,and signaling pathways such as MAPK.4.CEMIP promotes HA catabolism and causes the accumulation of LMW-HA.LMW-HA induces F-actin rearrangement by activating the TLR2/SRC/ERK1/2signaling axis thereby enhancing the migration and invasion of SCLC cells.5.Donw-regulating the expression of CEMIP can reduce the occurrence of liver and lung metastases to a certain extent,but diffenrent expression of CEMIP has no significant effect on the colonization of SCLC brain metastases.