Abstract
The emergence of multidrug resistance is always associated with more rapid tumor recurrence and metastasis. P-glycoprotein (P-gp), which is a well-known multidrug-efflux transporter, confers enhanced invasion ability in drug-resistant cells. Previous studies have shown that P-gp probably exerts its tumor-promoting function via protein-protein interaction. These interactions were implicated in the activation of intracellular signal transduction. We previously showed that P-gp binds to Anxa2 and promotes the invasiveness of multidrug-resistant (MDR) breast cancer cells through regulation of Anxa2 phosphorylation. However, the accurate mechanism remains unclear. In the present study, a co-immunoprecipitation coupled with liquid chromatography tandem mass spectrometry-based interactomic approach was performed to screen P-gp binding proteins. We identified Rack1 as a novel P-gp binding protein. Knockdown of Rack1 significantly inhibited proliferation and invasion of MDR cancer cells. Mechanistic studies demonstrated that Rack1 functioned as a scaffold protein that mediated the binding of P-gp to Anxa2 and Src. We showed that Rack1 regulated P-gp activity, which was necessary for adriamycin-induced P-gp-mediated phosphorylation of Anxa2 and Erk1/2. Overall, the findings in this study augment novel insights to the understanding of the mechanism employed by P-gp for promoting migration and invasion of MDR cancer cells.
Highlights
Chemotherapy is one of the major methods for treating late-stage malignant tumors [1,2]
We previously showed that Rack1 was a binding protein of Anxa2 [32] and that Rack1 was a known substrate of tyrosine protein kinase Src [33]
Our results showed that Rack1 interacted with P-gp, Anxa2 and Src in the endogenous condition in drug-resistant cells
Summary
Chemotherapy is one of the major methods for treating late-stage malignant tumors [1,2]. Enhanced invasive and metastatic ability is associated with upregulated expression of metastasis-related genes [10,11] and epithelial to mesenchymal transition-associated proteins in MDR cancer cells [6,8,12] These phenomena suggest a functional link between drug resistance and cancer cell invasion and metastasis [5]. P-gp activity was associated with angiogenesis [15,16], cancer stem cells [5,17,18], cell migration and invasion [7,19,20,21,22], which are essential for carcinogenesis and cancer progression These findings suggest that upregulated P-gp may confer enhanced cell invasion ability in drug-resistant cells. FigFuigreur1e. 1I.dIednetnifiticfiactaitoionnoof fPP-g-glylyccoopprrootteeiinn ((PP--ggpp)) bbiinnddiinngg pprrootteeiinnssuussininggccoo-i-mimmmunuonporperceipciitpaittiaotnion (co(-ciop-)i-pc)o-cuopulpedledliqliuqiudidchcrhormomataotoggrarapphhyytatannddeemmmmaassss ssppeecctromeettrryy ((LLCC--MMSS//MMS)S-b)-absaesdedinitnertaecratocmtoeme analnyasliyss.i(sA. ()AW) eWsteesrtnerbnlobtltointtginagnalnyasliyssoisf oPf-gPp-gapndanFdlaFglaegxperxepsrseiossnioin HinEHKE2K932T93cTellcsetllrsantrsafnecstfedctewdith Flawg-itahgFgleadg-Pta-ggpgepdlaPsm-gipd oprlaesmmpidtyovrecetmorp.tCyevllelcytosar.teCs efrllomlysPa-tgeps efrxopmresPs-ignpg MexCpFre-s7s/iAngDMR (CAFd-7r/iAamDyRcin Res(Aisdtarniacme)ycceilnlsRweesrisetaunsceed) acseallspowseitrieveucsoedntraosl;a(Bp)oCsiotinvfeocaolnitmroml; u(Bno) flCuoonrfeosccaelnicme mmuicnrofslcuoopryesacnenaclyesis shomwicerdostchoaptyPa-ngaplywsiasssehxopwreedsstehdatnPo-rgmpawllyasinextphreescseeldl mnoermmbarlalynein(Rtheed:ceFllamg,emGrbereane: P(R-gepd;: YFelalglo, w: MeGrgreee;n6:0P0×-gpm; aYgenlliofiwca: tMioenr)g; e(C; 6)0F0l×owmcahganritfiocfatsiaomn)p; l(eCp) rFelpowarcahtiaornt oafnsdampalses pspreepcatrroagtiroanphanicdamnaalsyssis in sthpiesctsrtougdryap; h(Dic)aSnoadlyisuims indtohdisesctyuldsyu;l(fDat)eSpoodliyuamcrdyoldameciydlesuglefaltelpecotlryoapcrhyolaremsids e(SgDelSe-lPeActGroEp)haonreasliyssis of (PS-DgpS-PbiAnGdEin)gapnraolytesins so. fThPe-gPp-gbpinbdinindginpgroptreointesi.nTs hweerPe-gspepbairnadteindgbyprSoDteSin-PsAwGeEreansdepsatraaitneedd bwyith CoSoDmSa-sPsAieGbErilalniadntstbaliuneedR-w25it0h; (CEo) oRmeparsessieenbtraitlilviaenmt balsusespRe-c2t5r0u;m(Eo)fRPe-pgrpesinentetraaticvtienmg pasrsotsepinesctirduemntiofifed byPM-gSp/iMntSeraancatilnygsips.roteins identified by MS/MS analysis
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