Epigenetic Downregulation Of RUNX3by DNA Methylation Induces Docetaxel Chemoresistance In Human Lung Adenocarcinoma Cells By Activation Of The AKT Pathway

Lung cancer is one of the leading cause of all cancer-related deaths worldwide.Approximately75-85%of lung cancers are classified as non-small cell lung cancer(NSCLC), which includes squamous cell carcinoma, adenocarcinoma and large cellcarcinoma. The risk of disease recurrence is high, even for patients treated by surgery inthe early stages, and continuous efforts are being made to identify molecular markers thatpredict prognosis and response to additional therapy. Docetaxel, a semi-synthetic analog ofpaclitaxel, is one of several first-line chemotherapy agents used to treat advanced NSCLC,with genotoxic effects attributed to induction of apoptosis via microtubule bundling andstabilization and inhibition of Bcl-2. Unfortunately, there are several problems associatedwith docetaxel-based chemotherapy, with many patients non-responsive to docetaxel oracquiring resistance during treatment. To gain further insight into the molecularmechanisms underlying docetaxel resistance and explore novel potential therapeutic targetsfor reversing this resistance in LAD, we established two LAD cell lines with docetaxelresistance (SPC-A1/DTX and H1299/DTX) by continuous exposure to increasingconcentrations of docetaxel. To date, there have been few studies focused on themechanisms of docetaxel resistance in human lung adenocarcinomas (LADs). To gainfurther insight into the molecular mechanisms underlying docetaxel resistance and explorenovel potential therapeutic targets for reversing this resistance in LAD, we established twoLAD cell lines with docetaxel resistance (SPC-A1/DTX and H1299/DTX) by continuousexposure to increasing concentrations of docetaxel. Compared with parental LAD cells,resistant cells are larger in size and have irregular distributions before cell fusion. Colonyformation assay revealed significant enhancement of proliferating ability of resistant cells.Predominant accumulation in S phase was observed in SPC-A1/DTX cells compared withSPC-A1cells by flow cytometric analysis, while no significant deviation in apoptosiswas observed. Global array analysis of DNA methylation and gene expression identifiedepigenetic downregulation of human runt-related transcription factor3(RUNX3),suggesting an important role for this gene in docetaxel resistance of SPC-A1/DTX cells. Inresistant LAD cells, ectopic RUNX3expression led to significant resensitization todocetaxel), suppression of the in vitro proliferating ability, a dramatic up-regulation of apoptosis rate, as well as an increased percentage of cells in G1phase and a decreasedpopulation in G2/M phase. In contrast, inhibited expression of RUNX3brought significantresistance to docetaxel to parental LAD cells, and a mild enhancement of the in vitroproliferating ability to parental cells.Tumors derived from RUNX3-eGFP-transfectedresistance LAD cells grew substantially more slowly in comparison with the NC onesunder the presure of docetaxel, indicating that ectopic RUNX3expression in resistancecells could significantly enhance the in vivo response to docetaxel. We found thatinactivation of RUNX3may relieve the transcriptional repression of Akt1, thuscontributing to docetaxel resistance. In this study, we show that RUNX3downregulation ispreferentially observed in tumor tissue from docetaxel insensitive patients, and isassociated with worse outcome in patients who received docetaxel-based adjuvantchemotherapy. Therefore, increasing RUNX3represents a promising strategy for reversingdocetaxel-resistant LAD patients. Taken together, our study shows that re-expression ofRUNX3may increase docetaxel chemosensitivity in human LAD cells by inactivation ofthe AKT pathway and indicates that targeting RUNX3may represent a novel strategy forreversing docetaxel-resistance in LAD patients in the future.Part I Microarray-based gene expression and DNAmethylation analysis between parental LAD cells anddocetaxel resistant LAD cellsObjective: To investigate the differences of gene expression and methylation in CpGinland between parental LAD cells and docetaxel resistant LAD cells, and provide aguidance for further in-depth study of the target gene therapy in docetaxel resistant LADpatients.Methods: DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA)according to the manufacturer’s instructions. After quality assessment, bisulfite conversionof genomic DNA was performed with the EZ DNA methylation Kit (Zymo Research,Orange, CA) in accordance with the manufacturer’s instructions, with modifications for theIllumina Infinium Methylation Assay. Bisulfite-converted genomic DNA was analyzedusing Illumina’s Infinium Human Methylation27Beadchip Kit (WG-311-1202; performedby LC Sciences, Houston, Texas). MSP was used to verify the results.The quality and quantity of the RNA samples were assessed by standard electrophoresis andspectrophotometry methods. Complementary DNA (cDNA) microarray analysis wasperformed with reagents and according to protocols provided by Affymetrix (Santa Clara,CA, USA). Briefly, we prepared double-stranded cDNA using the One-Cycle cDNAsynthesis kit. The GeneChip IVT labeling kit was then used to synthesize biotinlabeledcRNA, which was then fragmented prior to hybridization.Results: The microarray data showed that a total of2332genes were differentiallyexpressed between the SPC-A1and SPC-A1/DTX cell lines. By the fold-change analysis,we found that338or31of the29,187flagged cDNAs in SPC-A1/DTX cells showed atleast a4.0-or15.0-fold change in expression level compared with parental SPC-A1cells.Then, a real-time quantitative reverse transcription (RT)-PCR assay was employed tovalidate the top13downregulated genes Compared with SPC-A1cell: SERPINB5(60.9fold change)，LUM(57.3fold change)，ING4(39.4fold change)，CYP1A1(33.8foldchange)，RASIP1(24.5fold change)，RNASEK(22.1fold change)，TMPRSS11F(20.4foldchange)，GALR2(18.7fold change)，KRT15(18.5fold change) RUNX3(17.8foldchange)，ABCG2(17.1fold change)，ABCC3(16.5fold change). The methylation chip datashowed that the top16genes with hypermethylated CpG islands in the promoterregions(_beta=0.801): FOXO1A(0.952)，ADAM33（0.915），CEBPA（0.897），SFRP1（0.882），FLNC（0.873），DAPK1（0.862），SNX9（0.842），MATN2（0.831），NAP1L5（0.819），STAP2（0.816），ADAM12（0.807），HOXD11（0.806），F2R（0.804），TAPBPL（0.803），MMP14（0.802），RUNX3（0.801）. Furthermore, we focused on RUNX3,one of the top16genes with hypermethylated CpG islands in the promoter regions and oneof the most highly downregulated genes in SPC-A1/DTX cells. We hypothesize thatepigenetic inactivation of RUNX3may contribute to docetaxel resistance.Conclusion: Compared with parental cells (SPC-A1), RUNX3was found to bedownregulated by hypermethylation in docetaxel-resistant LAD cells (SPC-A1/DTX),which may induce docetaxel resistance in LAD cells. Increasing RUNX3may represent apromising strategy for reversing docetaxel resistance in LAD patients in the future. Part II The Analysis of function of RUNX3In LADObjective: To investigate the impact of demethylation drug5-Aza-dc on RUNX3genemethylation，gene expression and docetaxel resistance of LAD cell lines．To explorewhether the RUNX3promoter methylation correlate with the doxetaxel-resistance in LADcell lines.Methods: MSP for detection of RUNX3methylation status in docetaxel-resistant LADcell lines and its parental LAD cell lines．MTT method and flow cytometry (FCM) for testof docetaxel resistance index in resistant LAD cells，and the IC50for docetaxel of resistantLAD cells48hours after different concentration of5-Aza-dc(0uM,5uM,10uM,15Um,20uM,25uM,30uM) treatment．RUNX3-gene-expressing plasmid(GFP-RUNX3), the negative control plasmid (GFP-NC), and RUNX3inhibitor plasmid(pGPU-shRUNX3) were introduced into human lung adenoma SPC-A1,H1299orSPC-A1/DTX, H1299/DTX cells through transient transfection. MTT assay was performedto detect the IC50values for docetaxel. Colony formation assay was performed to detectthe in vitro cell proliferating abilities. Flow cytometric analysis was performed to detectthe apoptosis status and cell cycle distribution. GFP-RUNX3and GFP-NC were introducedinto SPC-A1/DTX or H1299/DTX cells through transient transfection and subcutaneouslyinto nude mice. Tumor growth was examined every other day. When the average tumorsize reached about50mm3, docetaxel was given through intraperitoneal injection. After2weeks, all mice were sacrificed. Transplanted tumors were excised and tumor tissues wereused to perform hematoxylin and eosin (H&E) staining,proliferating cell nuclear antigen(PCNA), TdT-mediated dUTP nick-end labeling (TURNEL),RUNX3immunostaininganalysis.Results: In both SPC-A1/DTX and H1299/DTX cells, ectopic RUNX3expression led tosignificant resensitization to docetaxel, suppression of the in vitro proliferating ability, adramatic up-regulation of apoptosis rate, as well as an increased percentage of cells in G1phase and a decreased population in G2/M phase(p<0.01). In contrast, inhibited expressionof RUNX3brought significant resistance to docetaxel to both SPC-A1and H1299cells(p<0.05), and a mild enhancement of the in vitro proliferating ability to SPC-A1cells(p<0.05). Tumors derived from eGFP-RUNX3-transfected SPC-A1/DTX or H1299/DTX cells grew substantially more slowly in comparison with the NC ones under the presure ofdocetaxel, indicating that ectopic RUNX3expression in SPC-A1/DTX or H1299/DTXcells could significantly enhance the in vivo response to docetaxel(p<0.01).Immunostaining analysis showed that the positive rate of proliferating cell nuclear antigen(PCNA) of tumors from eGFP-RUNX3-transfected SPCA-1/DTX or H1299/DTX cellswas decreased and increased in comparison with that of the NC ones, indicating adown-regulation of the in vivo cell proliferating ability and a up-regulation of the in vivoapoptosis attributed to RUNX3.Conclusion: Low dose5-Aza-dc cause no significant growth inhibition and apoptosis todoxetaxel resistant LAD cells，but it can partly reverse the chemoresistance throughremoval of RUNX3methylation and increased expression of RUNX3gene．Our dataindicate that RUNX3could function as a restorer of docetaxel chemosensitivity in humanlung adenocarcinoma cells both in vitro and in vivo, causing cell proliferation suppression,apoptosis enhancement and G1arrest in cell cycle progress. Part III The function of RUNX3in its mediated AKTsignaling pathwayObjective:To further investigate the roles of RUNX3in Aberrant activation of theAkt1/GSK3β/β-catenin signaling pathway in acquiring docetaxel resistance duringtreatment. To gain further insights into a novel strategy for reversing docetaxel-resistancein LAD patients in the future.Methods: A customized PCR array from CT Bioscience (Changzhou, China)[20]was usedto compare the expression profile of a selected group of genes in RUNX3-transfectedversus NC-transfected SPC-A1/DTX cells. Target mRNAs included88potential moleculesinvolved in cancer signaling pathways. We found that Akt1and β-catenin were remarkablydecreased in RUNX3-overexpressing docetaxel-resistant LAD cells, suggesting thatRUNX3may resensitize docetaxel-resistant LAD cells to docetaxel through Akt1signaling.Since Akt1/GSK3β/β-catenin was chosen as a preferred candidate RUNX3-mediated signaling pathway, a constitutively active form of Akt (Akt-myr) or AKT inhibitor(MK-2206) were introduced into RUNX3-transfected resistant LAD cells or parentalLAD ones.Wstern blot was to detect the proteins in the pathway. MTT assay wasperformed to detect the IC50values for docetaxel. Colony formation assay was performedto detect the in vitro cell proliferating abilities. Flow cytometric analysis was performed todetect the apoptosis status and cell cycle distribution. A total of39lung adenocarcinomatissues were collected from patients with advanced lung adenocarcinoma who havereceived surgical resection following by adjuvant chemotherapy. Immunohistochemicalstaining of RUNX3(mouse monoclonal antibody, R&D Systems, USA) and Akt1protein(rabbit mAb, CellSignal Technology, USA) was described previously.Results: Aberrant activation of the Akt1/GSK3β/β-catenin signaling pathway wasconfirmed by western blot in SPC-A1/DTX cells compared with SPC-A1cells. Weobserved a significant induction of Akt1, p-AKT, p-GSK3β, β-catenin and bcl-xl anddecrease in p21and Bax protein expression in SPC-A1/DTX cells. Conversely,overexpression of RUNX3in SPC-A1/DTX cells led to a significant decrease in Akt1,p-AKT, p-GSK3β, β-catenin, CyclinD1, bcl-2and bcl-xl protein expression, andupregulation of p21and Bax levels, indicating that restoration of RUNX3may induce theinactivation of AKT signaling in SPC-A1/DTX cells. Both decreased CyclinD1andincreased p21may induce G1phase arrest, while the increased ratio of Bax/bcl-2orBax/bcl-xl induced final apoptosis enhancement, which might contribute to resensitizingSPC-A1/DTX cells to docetaxel. Furthermore, co-transfection of RUNX3and Akt-Myr, aconstitutively active form of Akt, was capable of reversing the above-mentioned effects ofRUNX3overexpression co-transfection of RUNX3and Akt-Myr, a constitutively activeform of Akt, was capable of reversing the above-mentioned effects of RUNX3overexpression. Moreover, AKT inhibition by MK-2206treatment notably decreasedproliferation ability (P<0.05), induced G1phase arrest, decreased G2/M phase percentages(P<0.01), and enhanced apoptosis in SPC-A1/DTX cells (P<0.01). Immunohistochemicalanalysis revealed that RUNX3was downregulated or inactive (low expression, score≤2) in39cases (57.4%), and low RUNX3expression was correlated with high expression ofAkt1(Spearman rank test rho=-0.625, P<0.01). DFS in patients with low RUNX3expression (n=21) was inferior to those with high expression (n=18)(mean of17.0months(95%CI:14.424–19.576) vs21.3months (95%CI:19.492–23.175), P=0.022). Conclusion: Re-expression of RUNX3may increase docetaxel chemosensitivity in humanLAD cells by inactivation of the AKT pathway and indicates that targeting RUNX3mayrepresent a novel strategy for reversing docetaxel-resistance in LAD patients in the future.