Gsk-3β Effects Cell Migration And Autophagy By Mediating The Ampk Pathway In Breast Cancer

Background:Breast cancer（BC）is one of the most prevalent malignancies.Worldwide,it is the second largest contributor to mortality-linked malignancy in women^[26].It is particularly problematic as an early malignancy in Asia,where it displays a comparatively young median age upondiag-nosis in comparison with the West^[27,28].Research into the generation of BC helps determine innovative therapeutic targets and is promising to improve methods ofexamination,diagnosis,and early treatment^[29–32].Autophagy is triggered after stimulation of the ULK1/2（Unc-51-like kinase 1/2）complex,which regulates autophagosome generation in mammals^[33].As the dominant energy sensor,the function of AMP-activated protein kinase（AMPK）is promoted by increased concentration of AMP and decreased ATP concentration,upon nutrient or metabolic stress,including ischemia^[34,35].As a heterotrimeric complex,AMPK consists of a catalytic as well as two regulatory/subunits,which are encoded by different genes^[36].Phosphorylation of AMPKonhreonine 172（pT172）is crucial to complete stimulation^[37].As a persistently stimulated versatile kinase,glycogen synthase kinase-3（GSK-3）participates in various reactions determining cellular fate^[38].Two isoforms（αandβ）of GSK-3 exist in mammals.Previous work has indicated that suppression of GSK-3βfunction stimulates autophagic cellular reactions without serum,and this has also been seen in vivo in ischemic mice^[39,40].Nevertheless,understanding of the etiology of autophagy triggered via GSK-3βsuppression is insufficient.GSK-3 is a versatile protein kinase participating in many reactions.Currently,there is insufficient understanding of its influence on breast cancer（BC）.In order to explore its influence on migration and invasion in BC,we investigated its expression in BC cell lines using qRT-PCR and Western blot（WB）.Immunohisto chemistry（IHC）was used to examine the potential of GSK-3βto predict clinical outcome in BC patients.GSK-3βknockdown was achieved using an shRNA plasmid vector in T47D cells.Our research explored the biological reactions and downstream pathways involved.We found excessive GSK-3βexpression in BC tissues,which was correlated with worse clinico pathological parameters and clinical outcome.Progression of BC was suppressed by GSK-3βknockdown.Furthermore,suppression of GSK-3βfunctionled to a noticeable decrease in ATP generation,and this was a ssociated with stimulation of AMP-activated protein kinase（AMPK）in T47D cells.Activation of AMPK,a typical sign of autophagy stimulation,was triggered after suppression of GSK-3βfunction,in parallel with increased generation of LC3 II.Our findings therefore indicate that GSK-3βparticipates in regulation of migration as well as stimulation of autophagy via mediating activation of the AMPK pathway.This suggests that GSK-3 has potential as a predictor of clinical outcome and as a target for BC therapy.Methods:The first part:Clinical ResearchThe clinical specimens of the subject are from the first Affiliated Hospital of the Guangxi Traditional Chinese Medicine University.64 cases of breast cancer tissues and their corresponding adjacent（>5 cm）normal breast tissues were collected.In which the part is stored in a refrigerator at-80℃for retention,and the remaining paraffin sections are made.The translation of GSK-3βin 64 human BC tissues and matched non malignant tissues was examined.1.Immunohisto chemistry（IHC）and Tissue Microarrays（TMAs）TMAs were provided by Shanghai Biochip Co.Ltd.（Shanghai,P.R.China）.Histopathological diagnosis was made in conformity with World Health Organization（WHO）criteria.Overall survival（OS）referred to the period between operation and mortality.Data from deceased patients were examined on the day of mortality,while data from living patients were examined at the end of the follow-up period.Paraffin sections were taken for IHC,which was conducted on slides with thickness of 4μm.Malignancy was verified with the help of hematoxylin and eosin（H&E）staining.Slices underwent overnight incubation at 4℃with primary antibodies following antigen retrieval.They were then incubated for 30 min with secondary antibodies conjugated with HRP at 37℃.Diaminobenzidine was applied for visualization.2.Assessment of IHC VariablesGSK-3βprotein was found in the cytoplasm as well as the nuclei of cells and was stained as brown granules.GSK-3βexpression was evaluated with a 4-point scale（0–4）based on quantity of positive cells.The scores for the proportion of positive cells were categorized as follows:5%（0）,5%–25%（1）,25%–50%（2）,50%–75%（3）,or 75%（4）.Staining intensity was scored as follows:no staining（0）,light brown（1）,brown（2）,and dark brown（3）.A threshold score of 5 was chosen to delineate high from low expression.Patients with high or low expression were designated GSK-3β^highor GSK-3β^low,respectively.The second part:Cell CultureHuman BC cells（T47D）were bought from the American Type Culture Collection（ATCC）（Manassas,VA,USA）.Cells underwent cultivation in DMEM media（Gibco,Gaithersburg,MD,USA）including 10%newborn bovine serum deactivated by heat,100μg/ml streptomycin,and 100 U/ml penicillin（Invitrogen,Carlsbad,CA,USA）.1.Transduction and Clone SelectionWe bought shRNA targeting GSK-3βgenerated in p LKO.1-puro from Santa Cruz Bio technology（Dallas,TX,USA）.Steady knockdown（KD）clones were created.Immunoblotting of GSK-3βwas applied to verification of the clones,which received steady transduction.2.Transwell AssayApproximately 1×10⁵cells were suspended in 200μl of media without serum,before being added to chamberson the top of a Transwell（eight micromoles for 24-wellplates）.Full media（600μl）were supplemented to the chambers below.After24h,cells underwent fixing with formalin and staining with 0.1%crystal violet.3.In Vitro Proliferation and Colony Generation AssayWe used 96-well platesto plant the cell satadensity of 500 cells per well.Cells underwent overnight cultivation and were then supplemented with cell countingkit-8（CCK-8）（Promega,Madison,WI,USA）.They were then incubated for 2 hours at 37°C.Cell quantification was performed with a microplate spectro photometer（Bio Tek,Winooski,VT,USA）at an absorbance of 450 nm.The reaction was allowed to continue for 5 days,and cellular growth curves were plotted.Procedures were carried out in triplicate.In order to investigate effects over longer time periods,1,000 cells were planted in six-well plates with complete media and incubated at 37°C for 14 days.Cells underwent staining with 0.1%crystal violet.4.Real-Time Reverse Transcription Polymerase Chain Reaction（RT-PCR）We isolated total RNA using TRIzol reagent.An equivalent quantity of RNA underwent RT to obtain c DNA（Applied Biosystems,Foster City,CA,USA）.β-Actin served as internal reference.An ABI Prism 7900 HT system was used to quantify transcription.RT underwent assessment according to Ct values,which were normalized toβ-actin using the comparative Ct method.5.Western Blot AssayWestern blot（WB）was carried out as previously described^41,42,with antibodies for GSK-3β,β-actin（Santa Cruz Biotechnology）,N-cadherin,E-cadherin,vimentin,ATG5,LC3 Ⅰ/Ⅱ,p-AMPK,or AMPK（Cell Signaling Technology,Danvers,MA,USA）.6.ATP-Dependent Luciferase Assay,AMP/ATP Detection,and Glycolysis Assay Using High-Performance Liquid Chromatography（HPLC）Concentration of ATP in treated cells was measure d using the ATP Lite assay kit by Perkin Elmer（Waltham,MA,USA）.Cultivation media were acquired in preparation for detection of glycolysis function using a cell-based assay kit for quantifying L-lactate,the terminal product generated by cellular glycolysis.Prior to HPLC,cells were washed and resuspended in phosphate-buffered saline（PBS）.Nucleotides（ATP and AMP）were isolated via fast lysing the cells with 0.05 M KOH solution,which was adjusted to pH6and was used to conduct reverse phase chromatography.The mobile phase（pH6）included0.1 M KH₂PO₄,0.008 M tetrabuty lammonium hydrogen sulfate,and acetonitrile（containing 30%solvent B and 2%solvent A）.Empower II software（Waters,Milford,MA,USA）was used for analyses and instrument control.Statistical AnalysisResults are presented in the form of mean standard deviation（SD）.SPSS 19.0 for Macintosh（SPSS Inc.,Chicago,IL,USA）was used for analyses.Significance was inferred with a value of two-tailed p<0.05.Results:The first part:Detection of GSK-3βtranslation by IHC was carried out in 64 human BC tissues and paired nonma-lignant tissues.A typical IHC outcome is presented in Figure.We found that GSK-3βprotein displayed elevated IHC scores in BC specimens in comparison with surrounding normal tissues.Kaplan–Meier analysis was used to examine the correlation between GSK-3βexpression and prognosis.We discovered that increased GSK-3βexpression was correlated with shortened OS.Furthermore,survival of GSK-3β^lowpatients was significantly higher than that of GSK-3β^highpatients.The second part:1.GSK-3 KD Suppressed BC ProgressionIn order to investigate the effects of GSK-3βactivity,we used shRNA to produce GSK-3β-KD cells.GSK-3βshRNA supplement brought about a noticeable decline in translation as well as transcription of GSK-3β.We initially investigated the influence of GSK-3βdown regulation once proliferation in the T47D cell line.Colony generation assays showed greater quantities of T47D-NC clones in comparison with the T47D/sh GSK-3βgroup.It was also discovered that the volume of clones generated in the T47D/sh GSK-3βgroup was suppressed compared with that in the control group.Our proliferation assay showed inhibition of T47D proliferation following GSK-3βshRNA supplement.These results therefore indicate that GSK-3βenhances proliferation of BC cells.Furthermore,in vitro migration assays showed that quantity of migrated cells was clearly higher for T47D-NC than for T47D/sh GSK-3β.As it is widely accepted that epithelial–mesenchymal transition（EMT）is the transition from polarized epithelial cancer cells to motile and contractile mesenchymal cells during the progression and metastasis of malignancy,we aimed to investigate the influence of GSK-3βon EMT development.Using WB and quantitative RT-PCR（qRT-PCR）,we evaluated the expression of representative biomarkers linked with EMT in T47D cells.Following GSK-3βshRNA supplement,expression of E-cadherin was noticeably promoted.However,expression of the mesenchymal biomarkers N-cadherin and vimentin was remarkably suppressed.2.GSK-3βDownregulation Suppressed Autophagy in BCPrevious research has shown that GSK-3βinhibits autophagy and enhances radiosensitivity in non-small cell lung cancer.Moreover,KD of GSK-3βincreases basal autophagy in nutrient-laden human aortic endothelial cells.We therefore examined whether GSK-3β-KD affected autophagy of BC cells.WB was carried out to evaluate expression of the autophagy markers ATG5 and LC3 Ⅰ/Ⅱ in T47D cells with and without GSK-3β-KD.GSK-3β-KD promoted expression of ATG5 and LC3 Ⅰ/Ⅱ conversion.However,addition of 200μmol of 3-MA（an autophagy inhibitor）for 24 h significantly inhibited GSK-3β-KD stimulated excessive expression of ATG5 and LC3 Ⅰ/Ⅱ conversion.3.GSK-3βSuppression Decreased ATP GenerationBecause energy crisis can participate in the stimulation of autophagy,we next checked whether GSK-3βsuppression affected ATP concentration.T47D cells wereXIIIstarved of serum for 24 hours and then supplemented with GSK-3βinhibitor.ATP concentration moderately decreased with time subsequent to serum deprivation,based on an ATP-dependent luciferase assay.Treatment with GSK-3βinhibitor TWS119brought about a noticeable reduction of ATP concentration.This indicated that GSK-3βsuppression suppressed ATP generation after starvation of serum.4.GSK-3βInhibition Promotes AMPK ActivationAs the dominant sensor of energy,AMPK is stimulated immediately after energy crisis.As GSK-3βsuppression impaired ATP generation,we asked whether AMPK was activated parallel to autophagy stimulated by GSK-3βsuppression subsequent to starvation of serum.We used TWS119 supplementation to investigate AMPK activation via assessment of its phosphorylation.Four hours after TWS119 supplementation,AMPK pT172 concentration was noticeably promoted in comparison with solvent supplement.Findings were similar when GSK-3β-KD was achieved using specific siRNA.These findings suggested that generation of LC3 II was triggered via serum deprivation.Its processing was promoted subsequent to GSK-3βsuppression,in parallel with the stimulation of AMPK.Conclusions:GSK-3β,aserine/threonine protein kinase,is a complex regulator of numerous cellular functions.GSK-3βis a unique kinase that is constitutive lyactivein resting and nonstimulated cells^[45].GSK-3βhas been implicated in a wide range of diseases including neuro degeneration,inflammation and fibrosis,noninsulin-dependent diabetes mellitus,and cancer^[46].Elevated GSK-3βexpression in BC tissues has been shown previously^[47].We investigated effects of GSK-3β-KD using invitro and invivo assays.Our findings revealed that GSK-3βshRNA supplement suppressed proliferation and migration of BC cells in vitro and limited metastasis as well as growth of malignancy invivo.Expression of GSK-3βin BC tissues was negatively correlated with OS of patients.We therefore conclude that GSK-3βcan be an essential oncogene crucial to modulation of malignancy growth and metastasis in BC.The influence of GSK-3βon metabolism of malignancy has been shown inprevious research,but knowledge of its metabolic activities remains unclear^[42,48,49].Emerging evidence reveal selevated expression of GSK-3βin multiple malignancies^[50-52].Nevertheless,understanding of the link between GSK-3βand BC is insufficient.Autophagy is initiated after activation of the ULKⅠ/Ⅱ complex that controls the nucleation step of autophagosome formation in mammalian cells^[53].As the major energy sensor,the enzyme AMPK activity increases once cellular AMP levels elevate or ATP levels decrease under nutrient/metabolic stress such as ischemia^[54].AMPK is a heterotrimeric complex with one catalytic and two regulatory/subunits encoded by distinct genes,and AMPK phosphorylation on threonine 172（pT172）is required for full activation^[55].Several protein kinases are capable of phosphorylating this site,including the ubiquitously expressed liver kinase B-1（LKB1）and Ca²⁺/calmodulin-dependent protein kinase（Ca MKK）^[56].Currently,it is conceivable that among several cellular signal pathways that regulate autophagy response under physiological or pathological conditions,the LKB1–AMPK pathway was demonstrated to directly modulate autophagy activity by suppressing the protein translation activator mammalian target of rapamycin（m TOR）path-way and by activating autophagy initiator ULK1^[57].We showed that suppression of GSK-3βfunction brought about clear suppression of ATP generation and stimulation of AMPK.These results provide insight into our finding that GSK-3βsuppression stimulates a strong autophagic reaction and subsequent necrotic apoptosis without serum.It has been regarded as a promising therapeutic target for neuronal degenerative diseases and type II diabetes,as well as malignancies linked with metabolic reprogramming and autophagy.In summary,our results show that GSK-3βKD is able to suppress proliferation as well as migration of BC cells in vivo and invitro.It can also enhance autophagy mediated by the AMPK pathway.Moreover,expression of GSK-3βcould serve as an innovative predictor of clinical outcome in BC patients who underwent curative operation.In general,our research throws light upon the influence of GSK-3βon BC development,indicating that GSK-3βcan be a promising target to treat BC.