The Impact Of Rapamycin On The Expression Of Programmed Cell Death4and Its Mechanisms In Breast Cancer Cells

Background and ObjectiveBreast cancer is one of the most common malignancies in women. According to the statistics of Guangzhou Center for Disease Control, breast cancer incidence ranks the first place of women malignancy in Guangzhou, in2012. Breast cancer is a heterogeneous disease. According to the hormone receptor expression status and epithelial cell growth factorreceptor2(Her2), it is classified as different subtypes, and these molecules are often used to predict the therapy responses.With the development of breast cancer research, more targeted drugs based on molecular mechanisms was found, such as trastuzumab which is targeted to HER2, and this target therapy has greatly improved the prognosis of breast cancer. However, there is still a considerable part of breast cancer recurrence and metastasis. Because the tumor is resistance to chemotherapy, endocrine therapy and targeted therapy. Therefore, we need to study its mechanism of tumor resistance so that we can overcome drug resistance and improve efficacy.Numerous studies have shown that there are many signaling pathways involved in the development ofbreast cancer, including the presence of high frequency offset of PI3K/Akt/mTORpathway which is up to70%. PI3K/Akt/mTORpathwayactivati onpromotethe growth ofbreast cancer cells, inhibit cancer cell death, and play an important role in the process of chemo-andendocrine-therapy resistance. It is also closely related with thetumor stage, histological grade andpoor prognosis.Programmed cell death4(PDCD4)wasrecently discoveredas a tumorsuppressor gene. Its downregulationhas now been foundinmany tumor tissuesand tumorcel lsincluding breast cancer. Jansen et al. detected PDCD4levels in60human tumor cell lines provided by the American Institute for Cancer Research by western-blot. Those cell lines are from10different parts of the body. In breast cancer cell lines, they found that PDCD4level was significantly downregulated in MDA-MB-231cell linescompared with MCF-7cells.Wen et al. detected the expression ofPDCD4in those relativelynormalbreast tissue, ductal carcinoma in situ(DCIS)andinvasive ductal carcinomaand theyfound that compared withnormal breast tissue, the expression level ofPDCD4in DCIS slightlydecreased, and get significantly decreased in invasive ductalcarcinoma, indicating an important rolein the regulation ofbreast cancerprogression. Missing orreducedexpression of PDCD4may be involved inthe process oftumorprogressionand maintain highlyinvasivecharacteristics. Thisis an independentprognostic factor forcancer. Currently the mechanism of PDCD4as a tumor suppressor gene is not very clear. Besides apoptosis induction, studies have suggested thatPDCD4plays amajorrole intranslational inhibition, with theeukaryotic translationinitiation complex, such as direct bindingofeIF4A, eIF4A’s closedhelicase activity; and the combination ofeIF4AwitheIF4Ginhibitcell proliferation,invasion andsynthesisoftranscription factors, which play itsanti-tumor effect. Another studyshowed thatPDCD4can promoteTIMP-2(Tissue Inhibitor of Metallopro teinases-2,a tumor suppressor gene) expression, thereby inhibit the invasive ability ofhuman breast cancerMCF-7cell line. PDCD4can alsoinducep21Wafl/Ciplex pressionafterreducingCDK4/6andCDK2, then cause cellcycle arrest, thereby stopping cell proliferation.According toliteraturereports, miR-21, TGF-β, mTOR, COX-2, and other factorsare involved intheregulation ofPDCD4. And mTOR/P70S6ksignaling pathwayplays an important rolein the regulation ofthe expressionofPDCD4. Dorrello et al. have foundthat S6K1can be activated by mitogenstimulation. The activationofS6Kl (ie P70S6k1) lead to phosphorylation of PDCD4atSer67siterapidly, followed by the binding of phosphorylatedPDCD4to ubiquitinligase, then they areubiquitinated, and finally degraded by the proteasome. ButSermut antvariantsPDCD4could not bephosphorylated.As ahomolog ofS6Kl, S6K2has also beenshown topromote thedegradation ofPDCD4by phosphorylation. SincePDCD4containstwositesof phosphorylation of AktSer67andSer457,and it is capable of specificallyphosphorylatedatSer67andSer457by Akt both in vivo and in vitro,Aktis also consideredto be involvedin PDCD4degradation directly. SinceS6klactivityis mainly affected bythe upstreamregulatorof cell signalingpathwaysPI3k/Akt/mTOR, so anyfactorcausingthe upstreamsignaling activitycan bealteredby affectingthe activity ofS6kl and thus indirectlyregulatethe expression ofPDCD4.S6Klis one of thedownstreamtargets ofmTORC1, rapamycin treatmentcan quicklymakeS6Kldephosphorylationandinactivation.BetweenmTORC1/S6K1and ERsignalpathway, there is a closeassociation:the endocrine therapy resistance of ER-positive breast cancer is associatedwiththe activation ofmTORsignaling pathway.S6K1can directly activateERa in Ser167site,resulting in increased transcription activity ofERa andpromotingER-dependent breast cancer cell proliferation. And ERa activationin turn leads toincreasedexpressionofS6Kl, then the positiveregulatorcircuitis formed. Studies have shown thatP-S6KlinER-positive breast cancer is associated withpoor prognosis.Overexpression ofS6K1can increa sethe sensitivity ofbreast cancercells torapamycin. S6K2is the homologousprotein of S6Kland is the downstreamtargets of mTORC1,it is alsooverexpressed in breast cancer. Studies have shown thatS6K2played a role in chemotherapy resistanceof small cell lung cancerinduced byFGF2andit also plays an importantrole in cell proliferationinresponseto mTORactivation. S6K2can alsomakeAktphosphorylationan dpromote the growth ofbreast cancer cellsRapamycin, also known assirolimus,is the firstmTORinhibitor.Inside the cell, rapamycin-FKBP12bindingcomplexesformed at the beginning, and then combinedwithmTORCl,inhibiting its activity, making cell cyclearrest inGlphase, then inducingapoptosis.Clinically,rapamycinand its analogshave beenused for the treatmentof solid tumors, including breast breast cancer. It canimprove the sensitivityof tumorcells tochemotherapy andendocrine therapy. Butin practice the Iimitedtherapeutic effectof rapamycin was verified, and it is also easy to producedrug resistance. However, the mechanismsofdrug resistanceis not very clear. Possibly, RAS/MEK/ERKsignaling pathwayandTGF-B/CTGF pathway arerelevant. According to literature, abnormal activation ofmTORwere present in both MCF-7cells andMDA-MB-231cells, and the level of Phosho-mTORwere basically the same, and phosho-Akt wereb not detectedin the two cell lines. The differencelies inthat the level of phosho-S6Klwere significantly higher in MCF-7cell lines thanthat in MDA-MB-231cell lines, andthe expresstion ofPDCD4was down regultated significantly in MDA-MB-231cell linescompared withMCF-7cell lines. Alsoasreported in other studies, MDA-MB-231cells are primarily resistant torapamycin; MCF-7cellscan begeneratedforacquired resistance torapamycin. Based on these findings, we assume thatPDCD4mayplay a role inthe resistance to rapamycin during the treatment ofbreast cancer.In our study, we found that, there arevarious degrees oflossof PDCD4expression in breast cancercompared with normalbreast tissue,the loss is also variant in different molecular subtypes. Intriple negative breast cancerand HER2-positive breast cancer,its expressiondeletion ratewas significantly higher thanluminal types, and is associated withhighhistological grade,lymph nodemetastasis.Patientswith PDCD4expression have higherdisease-freesurvival and overall survival rates. We also found thatrapamycincanincreasethe expression level ofPDCD4in MDA-MB-231cells. This process is achieved byS6K1andS6K2dephosphorylation. After PDCD4upregulation, cell proliferationwas inhibited. The effects of Rapamycin on PDCD4expression inMCF-7cells are totally different from MDA-MB-231, andPDCD4firstlyin creasesand thendecreases, andthe effectis throughphosphorylation ofS6K2. Downregulationof PDCD4resulted inenhancementof cellproliferation in MCF-7. The effects of Rapamycin on theexpressionofPDCD4intriple-negativebreast cancer cellsMDA-MB-231andER-positive breast cancer MCF-7cells are completely different,probably this is one of thereasonsforrapamycinresistance. Therefore, the present studyisto furtherstudythe mechanismof rapamycinresistancein breast cancertreatment, intends to provides anew experimentalbasis, andto overcomethis drug resistance. Ourstudy is divided intotwo parts, which are summarized as follows:Part I:PDCD4andp70S6K expression and their significance in different subtypes of breast cancerObjective: Tostudytheexpressionof PDCD4andp70S6K in different molecularsubtypes ofbreast cancertissuesand its relationship withclinicopathol ogicalfeaturesand prognosisMethods:1.SP immunohistochemistry was usedto show P70S6K and PDCD4expression in differentmolecularsubtypes ofbreast cancer. Samples:198cases ofbreast cancer of Luminal type,64cases of HER2-positive typeand76cases of triple-negativetype.2.Compare expression levels ofPDCD4andP70S6K in differentmolecules subtypes ofbreast cancer and analyze their relationshipwithclinicopathological features.3. UseKaplan-Meier statistic method to analyze the disease-free survival and overallsurvival ofpatients in each group.Log-ranktest is introduced to analyzes urvivalcurvesofdifferent groups. Cox regressionmodel is used for multivariateanalysis of disease-freesurvival and overallsurvival and to identifyprognostic factors.Results:1.PDCD4proteinexpressionindifferent molecularsubtypes ofbreast cancer: in allbreast cancer, the total rate of PDCD4proteinexpression was58.28%, and in luminaltypes, Her-2positiveandtriple negativetypes, PDCD4proteinexpression ratewere74.36%、33.33%及36.84%respectively. The expression rate in Luminal type was significantly higher thanthat in Her2positive andtriple negative types, and the difference was statistically significant (X2=35.197,P<0.001,X2=33.620,P<0.001). In Her2-positive andtriple negative breast cancer, there was no significant difference (X2=0.186, p=0.666) about the expression rate. In adjacent normalbreast tissue,PDCD4protein expression rate was96.44%, much higher than the threesubtypes ofbreast cancerand thedifferencewas statistically significant (X2=39.206, P<0.001; X2=60.398, P <0.001; X2=65.185, P<0.001).2. p70S6k expression indifferent molecularsubtypesofbreast cancer: there was no expressionof p70S6K in normal breast tissue. For allbreast cancer, the expression rate was23.96%, in which the luminaltype, Her-2positiveandtriple negative, the expression rates were12.06%,38.09%and36.84%, respectively. The expression rates inHer2-positive andtriple-negativegroupswere significantly higher thaninluminal group (X2=21.675,P<0.001, X2=22.027,P<0.001). There was no significant differencebetween Her2positive andtriple-negative group (X2=0.023, P=0.879) 3. The effects of PDCD4expression onbreast cancerprognosis in differentmolecular subtypes:Univariate analysisshowed that in338breast cancer cases,PDCD4expression is positively related withdisease-free suuvial and overall survival (X2=71.403, P<0.001; X2=34.919, P<0.001);ineachmolecular subtypeof breast cancer, data also showsPDCD4expression is positively related with disease-free survival and overall survival (X2=50.997, P<0.001).Multivariate analysisshowed thatthe tumorTstage, N stageandPDCD4expression were independentfactors for the disease-free survivalin breast cancer (P<0.01, P<0.001,P <0.001);and histological grade of tumor, Tstage, N stageandPDCD4expression were the independentfactorsfor the overall survival in breast cancer (P<0.01, P<0.01,P <0.001,P<0.01)Conclusions:1.There were various degree of loss of PDCD4protein expressionin breast cancer, whichis the mostobvious in Her2-positive andtriple-negativebreast cancer2.P70S6K expressionincreased significantly in breast cancer tissuecompared tonormal breast tissue,whichHer2-positive andtriple-negativebreast cancer P30S6Kincreasedthe mostobviously.3.In breast cancerthe lack of PDCD4expression is associated with high histological gradeand lymph node metastasis.4.Regardless ofmolecular subtypes, patients with positive PDCD4expression have betterdisease-free survival and overall survival.Part Ⅱ The impact of rapamycin on PDCD4expression in triple negative breast cancer cell line MDA-MB-231and ER positive breast cancer cell line MCF-7 Objective:To study the changes of PDCD4expression in triple negative breast cancer cell line MDA-MB-231and ER positive breast cancer cell line MCF-7treated by rapamycinMethods:1. MDA-MB-231and MCF-7cell line grows in complete medium and in its Logarithmic growth phase, cells were treated by100nM rapamycin.2. Cells were harvested after24or48hours treatment, then PDCD4expression was analyzed by western blotting.3. Similarly, P70S6K1and P70S6K2expression were analyzed respectively by western blotting in MDA-MB-231cells and MCF-7cells treated by rapamycin for24or48hours. The mechanism of rapamycin on PDCD4expression in two breast cancer cells was further studied.4. After rapamycin treatment, cell proliferation was determined, and the significance of rapamycin effects on the PDCD4expression was explored.5. All data were statistically analyzed using SPSS17.0software package, western-blot results were recorded as mean±standard deviation, one-sample t test or independent sample t test was used for statistical analysis, P<0.05was considered statistically significant.Results:1.MDA-MB-231cellswereincubated with100nMrapamycin for24h,48h. Western blottingshowed increased PDCD4expression.(P<0.01, n=3, VS Ohgroup);MCF-7cells were treated by100nMrapamycin for24h,48h respectively. Western blotting data showed increased PDCD4expression after24h incubation, but after longer time (48h), PDCD4expression decreased significantly(P<0.01, n=3, VS Ohgroup).2.MDA-MB-231cellswereincubated with100nMrapamycin for24h. Western blotting showed that together with increased PDCD4expression,the expression ofp-S6Klandp-S6K2inMDA-MB-231cells decreased.(P<0.05, P<0.01, P<0.01, n=3, VS control); MCF-7cells were treated by100nMrapamycin for24hs. Western blotting was used to detect PDCD4, p-S6Klandp-S6K2expressionin cells. Data showed that the expression ofp-S6Klislowered, but p-S6K2expression appeared no significant change compared tothe controlandDMSO group.(P=0.839, n=3, VS control).3. MDA-MB-231cellswereincubated with100nMrapamycin for48hs. Western blotting showed that together with increased PDCD4expression,the expression ofp-S6Klandp-S6K2inMDA-MB-231cellsalso decreased.(P<0.05, P<0.01, P<0.01, n=3, VS control); MCF-7cells were treated by100nMrapamycin for48hs. Western blotting was used to detect PDCD4, p-S6Klandp-S6K2expressionin cells. Data showed that the expression ofp-S6K2isupregulated, but p-S6K1expression appeared no significant change compared tothe controlandDMSO group.(P=0.769, n=3, VS control).4. After rapamycin treatment, the proliferation of MDA-MB-231cells was significantly inhibited; MCF-7cell growth was inhibited firstly, but then its proliferation was promoted.Conclusions:1.Rapamycin canincreasePDCD4expression inMDA-MB-231cell lines; Rapamycin treatment firstly up-regulated and then down-regulated PDCD4expression in MCF-7cell lines.2. The up-regulation effects of rapamycin on PDCD4expression in MDA-MB-231cell linesmay be due to the downregulation of both P-S6KlandP-S6K2. The down-regulation effects of rapamycin on PDCD4expression in MCF-7cell lines was also related to P-S6KlandP-S6K2.PDCD4expression was up regulated by P-S6K1in early stage of stimulation by rapamycin and the following downregulation of PDCD4might be achievedby up-regulatingP-S6K2.3.Rapamycin could inhibit the cell proliferation in MDA-MB-231by up regulateing PDCD4expression,and thusconfront the resistance to rapamycin; and in MCF-7cells, the expression of PDCD4was increased firstly and then decreased by rapamycin treatment correspondingly, and the cell growth was inhibited firstly and then disinhibited. These data suggestied that PDCD4downregulationmay result in the rapamycinresistance in MCF-7cell lines.