The Effect And Mechanism Of Autophagy In Regulating The Chemoresistance Of Human Lung Adenocarcinoma Cells

Background and ObjectiveLung cancer is the leading cause of cancer-related mortality among malignancies worldwide and lung adenocarcinoma(LAD)is the most common histological type.Chemotherapy is a critical component of the current standard treatment for LAD patients,especially at an advanced stage.With the development of chemotherapeutics,in spite of the increased clinical response rate,the objective response rate(OR)of over half of LAD patients is still disappointed.Chemoresistance remains a major obstacle constraining the clinical application of this treatment.Therefore,identifying the underlying mechanisms of LAD chemoresistance and searching for potential therapeutic targets in order to guide accurate treatment is a major research direction.Autophagy,a regulated process occurring in all types of eukaryotic cells,is implicated in many physiological and pathological conditions.Accumulated evidence indicated that autophagy functions primarily as a pro-survival mechanaism,which led to chemoresistance.On the previous basis of docetaxel-resistant LAD cell lines establishment,our study aims to explore two signaling pathways at translation and transcription levels respectively,by which led to increased autophagic activity involved in LAD chemoresistance,one is HMGB1 in the regulation of the Beclinl-PI3KIII complex through the mitogen-activated protein kinase(MEK)-extracellular signal-regulated kinase(ERK)pathway,another is downregulated-miR-200b-dependent upregulation of ATG12.Together our data provide a novel strategy for individualized treatment and reversing chemoresistance in combating LAD.Materials and Methods1.The human LAD docetaxel-resistant SPC-A1and H1299 cell lines(SPC-A1/DTX and H1299/DTX)were established previously,and were resistant to multiple anti-tumor drugs,including docetaxel,paclitaxel,cisplatin,carboplatin,gemcitabine and vinorelbine.Transmission electron microscopy,Western blot and GFP-LC3 analysis were used to assess autophagic activity of LAD parental and docetaxel-resistant cells.The dose-dependent induction assay was designed to treat parental cells under different doses(0,5,10,20?g/L)of docetaxel for 24h;and the time-dependent induction assay was applied to treat parental cells at 10?g/L docetaxel for various time(0,6,12,24h).Western blot analysis was employed to identify the protein expression of LC3-II/I and P62.2.Inhibition autophagy of both parental and docetaxel-resistant LAD cells by autophagy inhibitor,3-methyladenine(3-MA)or transfected cells with small-interfering RNA(siRNA)specifically targeting the autophagy gene Atg5(ATG5 siRNA)before addition of docetaxel,Western blot analysis was used to assess the autophagic activity;and MTT assay,colony formation and flow cytometric analysis were applied to reflect chemosensitivity,cell proliferation ability and apoptosis level.3.The protein expression of High-mobility group box 1(HMGB1),nuclear-HMGB1and cytosolic-HMGB1 in parental and docetaxel-resistant LAD cells were examined by Western blot analysis,while extracellular HMGB1 levels were analyzed by enzyme-linked immune sorbent assay(ELISA).Same experiments were performed when exposing parental cells to 10?g/L docetaxel under different time periods(0,3,6,12,24,48h).4.Next step is aimed to investigate whether HMGB1 is a direct activator of autophagy.After limiting HMGB1 cytosolic translocation by ethyl pyruvate(EP),a pharmacological inhibitor of HMGB1 cytoplasmic translocation,or knockdown of HMGB1 by transfecting HMGB1-specific shRNA(HMGB1 shRNA),we evaluated the LC3-I to LC3-II conversion by Western blot analysis and LC3 punctate formation by fluorescent analysis as described above.Meanwhile,MTT assay,colony formation and flow cytometric analysis were applied to reflect chemosensitivity,cell proliferation ability and apoptosis level.Conversely,same experiments were conducted when overexpression of HMGB1 by transfection with cDNA encoding full-length human HMGB1(pcDNA3.1 HMGB1)combined treatment with 3-MA.5.Subcutaneous xenograft tumor model were established by SPC-A1/DTX cells stably transfected with HMGB1 shRNA or control shRNA.Docetaxel was administered via intraperitoneal injection at a dose of 1 mg/kg,and normal saline was administered to the mice of the control group in a similar manner After treated for 5 weeks,the mice were sacrificed and the tumor growth-curve of tumor volume was described according time in every group.Western blot was used to detect LC3-?/? and HMGB1 protein expressions of transplantation tumors.Tissue biopsies from tumors were observed under microscope with H&E staining,immunohistochemistry(IHC)analysis and TUNEL staining.6.Western blot was employed to assess the molecular activity of Akt/mTOR and MEK/ERK1/2/Beclin-1-PI3K? signaling pathway,and co-immunoprecipitation assay(IP)was used to evaluate the binding capacity of Bcelinl to PI3K? when altering the expression level of HMGB1.7.Based on the microarrays of SPC-A1/DTX cells compared with parental SPC-A1 cells,we transfected sequence-specific inhibitors of upregulated miRNAs(AmiR-192,AmiR-424,AmiR-98)or precursors of downregulated miRNAs(PmiR-200b,PmiR-194,PmiR-212)into SPC-A1/DTX cells.Real-time PCR analysis was used to verify the mRNA levels of miRNAs,while Western blot was employed to detect the ratio of LC3-? to LC3-I.Our date indicated that miR-200b mimics(PmiR-200b)exhibited maximum inhibitory potency against autophagy activity.8.After transfection of PmiR-200b into docetaxel-resistant LAD cells or transfection of miR-200b inhibitor(AmiR-200b)into parental LAD cells,we assessed autophagic activity by Western blot and GFP-LC3 analysis as described above.9.Three different online miRNA database(Microran.org,DIANA-microT v3.0 and TargetScanHuman 6.0)were applied for predicting putative targets of miR-200b.Dual-luciferase reporter gene assay was employed to further confirm the result.Taken together these data demonstrated that ATG12 is a direct target of miR-200b.10.Western blot and Real-time PCR analysis were used to assess basal ATG12 mRNA and protein levels of parental and docetaxel-resistant LAD cells.After transfection of ATG12 siRNA into docetaxel-resistant cells or transfection of pDsRedl ATG12 plasmid into parental cells,Western blot analysis was performed to detect ATG12 protein expression and LC3-?/? ratio;MTT assay,colony formation and flow cytometric analysis were applied to reflect chemosensitivity,cell proliferation ability and apoptosis level.Rescue experiments were further performed by co-transfection of PmiR-200b and plasmid carried ATG12 gene(pDsRedl ATG12)into docetaxel-resistant cells.11.SPC-A1/DTX cells stably transfected with Pri-miR-200b gene expression vector(pPG/miR-200b)or pDsRedl ATG12 plasmid or both of them were identified and used to establish the subcutaneous tumor model in nude mice.Docetaxel was administered via intraperitoneal injection at a dose of 1 mg/kg,and normal saline was administered to the mice of the control group in a similar manner.Draw the tumor growth-curve of tumor volume according time in every group and all mice were sacrificed 5weeks after treatment.Ultrastructure changes of transplantation tumor cells were observed under microscope with H&E staining,IHC analysis and TUNEL staining.12.A total of 60 cases of clinical LAD tissues were obtained from patients of progressive stage.Tumor response was examined by computed tomography and evaluated according to the Response Evaluation Criteria in Solid Tumors(RECIST)as complete response(CR),partial response(PR),stable disease(SD),or progressive disease(PD).Based on the above response evaluation criterion,tumor samples were divided into "sensitive"(CR or PR)and "insensitive"(SD or PD)groups.MiR-200b and ATG12 mRNA levels of tumor tissues were detected by Real-time PCR and the linear relationship between them was described by linear regression analysis.The correlation between miR-200b and clinicopathological factors or prognosis was determined using Chi-square test and Kaplan-Meier analyses,respectively.Results1.The baseline autophagic activity was higher in the docetaxel-resistant cells than in the parental cells.Docetaxel treatment led to a dose-and time-dependent increase in the level of LC3-? and decrease in the amount of p62 in parental cells.2.Both 3-MA and ATG5 siRNA efficiently blocked activation of autophagy,which caused an increase of cytotoxicity and apoptosis and decrease in the proliferation rate of SPC-A1 and H1299 cells.3.Docetaxel-resistant LAD cells showed relatively higher total level of HMGB1 which included a higher cytosolic level and a lower nuclear level under basal conditions compared to parental cells and showed no significant difference in the extracellular environment.Docetaxel markedly enhanced levels of cytosolic-HMGB1 and LC3-?/? ratio,and reduced levels of nuclear-HMGB1 in both parental cells in a time-dependent manner.4.Inhibition of HMGB1 cytosolic translocation with EP or silencing HMGB1 in SPC-A1/DTX and H1299/DTX cells suppressed autophagy,enhanced the cellular response to docetaxel,inhibited cell proliferation and induced apoptotic cell death to a great extent.In contrast,overexpression of HMGB1 significantly induced autophagy in parental LAD cells,as shown by an elevated conversion of LC3-I to LC3-II and increased formation of GFP-LC3 punctate staining,which were abolished by combined treatment with 3-MA.5.In vivo experiments,tumors derived from HMGB1 shRNA-transfected SPC-A1/DTX cells grew more slowly compared with those derived from control shRNA-transfected cells after being treated with docetaxel.Lower levels of LC3-? and PCNA expression coupled with higher apoptosis rate were observed in HMGB1 shRNA-transfected tumors in response to docetaxel in comparison with control shRNA-transfected tumors.6.In the study about whether HMGB1 could regulate docetaxel-induced autophagy through the Akt/mTOR or MEK/ERK1/2 pathway in LAD cells,we found that changes in levels of HMGB1 showed no obvious effect on the phosphorylation of Akt and mTOR.However,transfection of SPC-A1 cells with an HMGB1 cDNA plasmid activated the MEK-ERK1/2 pathway by increasing the phosphorylation of ERK1/2,and enhanced the formation of the Beelin-1-PI3K? complex in order.Disruption of this pathway reduced HMGB1-induced LC3-II and Beclin-1-PI3K? complex formation.Conversely,transfection of the active MEK construct,which conferred resistance to the suppression of MEK-ERK1/2 pathway phosphorylation,eliminated the negative control of Beclin-1-PI3K-?complex formation by HMGB1 silencing7.Using microarrays,we previously showed that 6 of 52 miRNAs were differentially expressed more than two fold in docetaxel-resistant SPC-A1/DTX cells compared with parental SPC-A1 cells.In SPC-A1/DTX cells transfected with sequence-specific inhibitors of upregulated miRNA(AmiR-192,AmiR-424,AmiR-98r)or precursors of downregulated miRNAs(PmiR-200b,PmiR-194,PmiR-212),PmiR-200b exhibited maximum inhibitory potency against autophagy activity in comparision with a nonspecific mimic or inhibitor-negative control,as demonstrated by the significant decrease in the conversion of LC3-? to LC3-?.8.H1299/DTX cells exhibited a 2.81-fold downregulation in miR-200b expression compared with parental H1299 cells.Forced expression of miR-200b limited autophagy activity in both SPC-A1/DTX and H1299/DTX cells even when treated with docetaxel or cisplatin.By contrast,miR-200b silencing led to the activation of autophagy in both parental cells,which was futher enhanced when exposed to these antitumor drugs.9.The dual-luciferase reporter system demonstrated ATG12 is a direct target of miR-200b.Docetaxel-resistant cells,including SPC-A1/DTX and H1299/DTX cells,had higher basal ATG12 mRNA and protein levels compared with their parental cells.Overexpression of miR-200b decreased ATG12 mRNA and protein levels in both SPC-A1/DTX and H1299/DTX cells,whereas silencing endogenous miR-200b increased ATG12 expression in parental cells.10.Restoration of miR-200b or ATG12 knockdown caused antophagy inhibition,which led to cell death promotion,growth suppression and increased apoptosis of docetaxel-resistant cells when exposed to cytotoxic agents.Notably,a gain-of-function study verified that PmiR-200b-dependent cytotoxic effects and cell proliferation of antitumor agents were eliminated by ectopic expression of ATG12.Conversely,AmiR-200b or ATG12 overexpression limited the cell killing efficiency,promoted colony-formation efficiency and largely abrogated both docetaxel and cisplatin-induced apoptotic cell death due to activation of autophagy.11.Tumors derived from pPG/miR-200b-transfected SPC-A1/DTX cells were prevented when treated with docetaxel in animal experiments.IHC analysis and TUNEL staining showed a decreased level of ATG12 and PCNA and an elevated level of apoptosis in pPG/miR-200b-transfected tumors under docetaxel treatment when compared with pPG/miR-NC-transfected tumors.However,overexpression of ATG12 promoted tumor growth,increased levels of ATG12 and PCNA and inhibited apoptosis in a miR-200b-overexpressed genetic background under treatment with docetaxel.12.MiR-200b mRNA expression was markedly decreased in the "insensitive" group compared with the"sensitive" group,and vice versa in the case of ATG12 mRNA expression.Linear regression analysis indicated the inverse association between miR-200b and ATG12 expression(rho=-0.126,p=0.032).Patients with a low miR-200b expression had a shorter progression free survival(PFS)than those patients with high miR-200b expression(median:3.4 months vs.4.4 months,p=0.021).Conclusions1.Activation of autophagy contributed to chemoresistance of LAD cells.Blockade of autophagy led to enhanced cytotoxicity,decreased proliferation ability and increased apoptotic cell death.2.HMGB1 translocated from the nucleus to the cytoplasm in response to docetaxel in LAD cells.Suppressing HMGB1 cytosolic translocation or knockdown of HMGB1 caused autophagy disruption both in vitro and in vivo,which resulted in increased cytotoxicity,elevated susceptibility to apoptosis and reduced proliferation rate when treated with docetaxel.HMGB1 promoted the formation of Beclin-l-PI3K-III complex through activating the MEK/ERK1/2 signaling pathway,thereby regulating autophagosome formation and contributing to chemoresistant phenotype in human lung adenocarcinoma.3.MiR-200b regulated autophagy in LAD cells.Specifically,we reported for the first time that miR-200b in regulation of ATG12 expression by base paring with 3'-untranslated regions(3'-UTR)of mRNA,which blocked autophagy and resensitized LAD cells to chemotherapy both in vitro and in vivo.4.Our study explored two signaling pathways at translation and transcription levels respectively for the first time:one is HMGB1 in the regulation of the Beclinl-PI3K? complex through the mitogen-activated protein kinase(MEK)-extracellular signal-regulated kinase(ERK)pathway,another is downregulated-miR-200b-dependent upregulation of ATG12,by which led to increased autophagic activity involved in chemoresistance of LAD cells.Together our data provide a novel strategy for individualized treatment and reversing chemoresistance in combating LAD.