Study On The Role And Mechanism Of Gap Junction Intercellular Communication In Malignant Progression Of Non-small Cell Lung Cancer Regulated By Cancer-Associated Fibroblasts

Objective: To study the role and mechanism of gap junction intercellular communication(GJIC)in malignant progression of non-small cell lung cancer(NSCLC)cells regulated by cancer-associated fibroblasts(CAFs)Methods: 1.The primary CAFs and paired NFs were isolated from tumoral and non-tumoral portions of resected lung tissue from primary NSCLCs;the protein expression of ?-SMA and FAP-? were identified by WB and Immunohistochemistry;the contractile ability of CAFs was identified by collagen gel contraction assay.2.NSCLC(A549 and H1299)were stained by GFP,and then the NSCLC-GEP and CAFs were cocultured with direct contact and separated by using Flow cytometer(FCM);those sorted cells(CAFs,NSCLC-GFP)were cultured for further study.3.The protein expression of E-cadherin and N-cadherin in sorted NSCLC were detected by WB;the migration and invasion in sorted NSCLC were detected by wound width and transwell assay.4.'Parachute' assay was performed to detecte the functional GJIC between CAFs and CAFs,A549 and A549,H1299 and H1299,CAFs and NSCLC;q RT-PCR and western blot analyses were performed to screen for the m RNA and protein expression of Cx26,Cx31.1,Cx32,and Cx43 in CAFs and NSCLC cells,which have been reported as the major connexins expressed in mammalian lung tissues or cultured cells;immunofluorescence was performed to detecte the subcellular location of Cx26,Cx31.1,Cx32,and Cx43 in CAFs and NSCLC cells.5.Vector(Ubi-MCS-EGFP-IRES-puromycin)that up-regulated Cx43 and vector(h U6-MCSubiquitin-EGFP-IRES-puromycin)down-regulated Cx43 were builded and transfected to CAFs and NSCLC cells;stable cell lines CAFs and NSCLC were screened by puromycin for two weeks;transfection efficiency was detected by WB.6.Inhibite or enhance of GJIC between CAFs and NSCLC cells by uasing 18?-glycyrrhetinic acid(18?-GA,4 ?M)or all-trans-retinoic acid(RA,1 ?M)for 48 h;the functional GJIC between the above CAFs and NSCLC cells were detected by 'Parachute' assay;compare the above functional GJIC with the GJIC of knockdown or overexpression of Cx43 in CAFs and/or NSCLC cells.7.The migration and invasion in sorted NSCLC were detected by wound width and transwell assay;the protein expression of epithelial cell marker protein E-cadherin,interstitial cell marker protein N-cadherin,Akt,p AKT,ERK and p ERK in sorted NSCLC were detected by western blot.8.Wound width and transwell assay was performed to detecte the migration and invasion of NSCLC cells that pretreatment with the PI3K/Akt inhibitor LY294002 or MAPK/ERK inhibitor U0126 alone or combination of LY294002 and U0126.Results: 1.CAFs displays spindle-shaped,flattened,and fibroblast-like morphology similar to NFs;western blot and immunofluorescence shows that the expression of ?-SMA and FAP-? was generally much stronger in CAFs than in matched NFs;collagen gel contraction assay shows that an increased contractile ability was observed in CAFs in when compared with their counterpart NFs.2.NSCLC cells lost their epithelial cobblestone appearance and became elongated and dispersed when co-cultured with CAFs;western blot shows that the protein expression of the epithelial marker E-cadherin was downregulated,whereas the protein expression of the mesenchymal marker N-cadherin was upregulated in co-cultured NSCLC cells;wound width and transwell assay shows that the mobility and invasive capability were significantly increased in co-cultured NSCLC cells compared with mono-cultured control cells.3.'Parachute' assay shows that CAFs,A549 cells,and H1299 cells efficiently transferred Calcein-AM to homologous adjacent cells with an average of 10.33 ± 1.53(SD),11.33 ± 3.06(SD),and 12.00 ± 3.61(SD)per labeled cell,respectively;moreover,CAFs showed a high capacity for GJIC with Calcein-AM transferred to heterogeneous neighboring A549 or H1299 cells with an average of 20.67 ± 3.229(SD)or 24.00 ± 2.65(SD)per labeled cell,respectively,while NSCLC cells displayed no detectable Calcein-AM transfer to CAFs.4.q RT-PCR and western blot analyses shows that Cx26 and Cx43 were the predominant connexin isoforms expressed in CAFs and NSCLC cell lines;furthermore,immunofluorescence clearly shows that Cx43 protein accumulated in the plasma membrane of CAFs and in the plasma membrane and cytoplasm of NSCLC cells,whereas Cx26,Cx32,and Cx31.1 protein accumulated in the cytoplasm of CAFs,and NSCLC cells.5.'Parachute' assay shows that knockdown of Cx43 expression in CAFs and/or NSCLC cells significantly abolished GJIC from CAFs and NSCLC cells,while overexpression of Cx43 in CAFs and/or NSCLC cells resulted in a significant increase in unidirectional GJIC;moreover,Cx43 overexpression in both CAFs and NSCLC cells caused a more significant increase in unidirectional GJIC than only overexpression of Cx43 in CAFs or in NSCLC cells.6.Western blot analyses,wound width and transwell assay shows that inhibition of GJIC by 4 ?M 18?-GA for 48 h(inhibition of GJIC but they did not change Cx43 protein levels)or sh Cx43(inhibition both GJIC and the expression of Cx43)could apparently abrogate the CAF-facilitated EMT,migration,and invasion of co-cultured NSCLC cells to a similar extent;on the other hand,enhancement of GJIC in these cells by 1 ?M RA for 48 h(enhance of GJIC but they did not change Cx43 protein levels)or lentiviral Cx43(enhance both GJIC and the expression of Cx43)could further strengthen the CAF-induced EMT,migration,and invasion of co-cultured NSCLC cells to a similar degree.7.Western blot analyses shows that CAF-induced increase in Akt and ERK activity in NSCLC cells;inhibition of GJIC reduced CAF-enhanced Akt and ERK activity in NSCLC cells,whereas enhancement of GJIC increased CAF-enhanced Akt and ERK activity in NSCLC cells.8.Wound width and transwell assay shows that pretreatment with the PI3K/Akt inhibitor LY294002 or MAPK/ERK inhibitor U0126 alone led to a similar decrease,and a combination of LY294002 and U0126 caused a synergetic decrease in PI3K/Akt and MAPK/ERK activity and the migration and invasion of NSCLC cells.Conclution: Cx43-formed unidirectional GJIC between CAFs and NSCLC cells contributes to NSCLC invasion and metastasis by inducing EMT mainly through the activiation of PI3K/Akt and MAPK/ERK signaling pathways.