mTORC1 inhibition and ECM–cell adhesion-independent drug resistance via PI3K–AKT and PI3K–RAS–MAPK feedback loops

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Tumor BiologyTumor Markers, Tumor Targeting andTranslational Cancer Research ISSN 1010-4283 Tumor Biol.DOI 10.1007/s13277-011-0315-x

mTORC1 inhibition and ECM–celladhesion-independent drug resistancevia PI3K–AKT and PI3K–RAS–MAPKfeedback loops

Karina Galoian, H. T. Temple & ArmenGaloyan

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RESEARCH ARTICLE

mTORC1 inhibition and ECM–cell adhesion-independentdrug resistance via PI3K–AKT and PI3K–RAS–MAPKfeedback loops

Karina Galoian & H. T. Temple & Armen Galoyan

Received: 13 December 2011 /Accepted: 29 December 2011# International Society of Oncology and BioMarkers (ISOBM) 2012

Abstract Mammalian target of rapamycin (mTOR) serinethreonine kinase is the enzyme that regulates cancer cellgrowth by altering nutrient supplies to cancer cells. Theneuropeptide (proline-rich peptide 1 (PRP-1)), galarmin,produced by the brain neurosecretory cells is a mTOR kinaseinhibitor with powerful 80% antiproliferative cytostatic effectin a high-grade chondosarcoma and other mesenchymaltumors. However, the negative feedback loop of phosphatidy-linositol 3 kinase–Protein kinase B (PKB), PI3K–AKT andPI3K–rat sarcoma (RAS)–mitogen-activated protein kinase(MAPK) activation is well documented for mTOR inhibitors.This study explored the involvement of those loops in drugresistance after the treatment with mTOR complex 1(mTORC1) inhibitor, PRP-1. Multidrug resistance assay(MDR) demonstrated that this cytokine did not inhibit perme-ability glycoprotein-mediated MDR in chondrosarcoma.Phospho-MAPK array in human chondrosarcoma cell linetreated with galarmin (10 μg/ml,) showed a strong upregula-tion of phosphorylated glycogen synthase kinase 3β (GSK3β)via activation of PI3K–AKTandMAPK feedback loops. Such

GSK3β inactivation leads to β-catenin accumulation thatentails drug resistance. The ability of cells to metastasize isreflected in their capacity to adhere to extracellular matrix andendothelium. Laminin cell adhesion assay demonstrated thatPRP-1 in the same concentrations that inhibit mTOR kinaseinhibited JJ012 chondrosarcoma cell adhesion. The neuropep-tide did not have any effect on the expression of total focaladhesion kinase and its phosphorylated form. Thus, it was notaccompanied by total HAT downregulation and total HDACupregulation. Combinatorial treatments of PRP-1 withMAPKand PI3K/AKT inhibitors most probably will lead to fullcytotoxicity overcoming drug resistance.

Keywords Chondrosarcoma . mTORC1 .Multidrugresistance . ECM–cell adhesion

Targeting mammalian target of rapamycin (mTOR) complex1 (mTORC1) downstream from phosphatidylinositol 3 ki-nase–Protein kinase B (PKB) (PI3K-AKT) pathway, whichregulates general protein translation and cancer bioenergeticlife span, represents one of the most attractive approaches totreating cancer. Nevertheless, the use of rapamycin and itsanalogs in the clinic has revealed that mTORC1 pathway isembedded in a network of signaling cross-talks and feedbacks,which might reduce its effectiveness in cancer. The negativefeedback loop of PI3K–AKT and PI3K–RAS–MAPK activa-tion is well documented [1]. Extracellular signal-regulatedkinase/mitogen-activated protein kinase (ERK/MAPK) acti-vation by rapamycin is stemming from an S6K-negative feed-back loop signaling to PI3K–rat sarcoma (RAS) pathways.Authors demonstrated that on mTORC1 inhibition, RAS–MAPK axis is activated. S6 kinase 1 (S6K1) usually servesas a brake for PI3K activation, which is not the case in thepresence of mTORC1 inhibitors [2]. The question whether

K. Galoian (*)Miller School of Medicine,University of Miami Health System,1600 NW 10th Avenue, Suite 8006 (R-2),Miami, FL 33136, USAe-mail: [email protected]

H. T. TempleMiller School of Medicine,University of Miami Health System,1400 NW 12 Avenue, UMH R 4036 East Building,Miami, FL 33136, USA

A. GaloyanInstitute of Biochemistry,NAS RA. 5/1, P. Sevag Str.,0014, Yerevan, Armenia

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mTORC1 inhibition is always accompanied by concomitantactivation of AKTand RAS–MAPKmediated by S6K–PI3K–RAS signaling feedback loop remains open [1–4]. Othersreported that mTORC1 inhibition mediated an increase ofRTK/IRS-1/PI3K activity towards RAS/MAPK, thereforepromoting both AKT activation and ERK phosphorylation,constituting a dual feedback mechanism [3]. In patient speci-mens, activation of ERK and the PTEN/PI3K/AKT/mTORpathway was associated with prostate cancer progression;moreover, the authors found that combined MEK/ERK andmTOR inhibition was effective in the adjuvant setting [4]. Inthis study, we explored the consequences of mTORC1 inhibi-tion with antiproliferative cytokine PRP-1 currently beinginvestigated in our laboratory [5–9]. Neuropeptide PRP-1(galarmin) [5, 6] produced by the brain neurosecretory cellsis a mTOR kinase (mTORC1) inhibitor [8, 9] with powerful80% antiproliferative effect in high-grade chondrosarcomaand other mesenchymal tumors [6–9]. The involvement ofthe negative feedback loops and its key players in PRP-1-mediated mTORC1 inhibition and the exploration of accom-panying cytoskeletal events triggering cytostatic effect anddrug resistance were the subjects of these investigations.

Materials and methods

Cell culture

Chondrosarcoma cells (JJ012) were cultured in monolayeruntil they were semiconfluent. Media consisted of Dulbecco'smodified Eagle's medium (DMEM)/MEM supplementedwith F12, 10% fetal bovine serum, 25 microgram/mlascorbic acid, 100 ng/ml insulin, 100 nM hydrocortisone,and 1% penicillin/streptomycin. Methelynedioxyamphet-amine (MDA) 231 (ER-) and luminal T47(ER+) cellswere cultured in DMEM media (with high glucose, L-glutamine and Na Pyruvate, 1% NEAA and 10% FBS,and 1% penicillin/streptomycin).

Adhesion experiments: ECM–cell adhesion

Cell culturing and the protocol were done according to themanufacturer's instructions (Cultrex Coat Laminin I 96 wellcell adhesion assay, Catalog #3491-096-K Cultrex). Thefinal fluorescence (485 nm excitation/520 nm emission)for each well read on the microplate reader allowed tocalculate the percent of cell adhesion to extracellular matrix(ECM) (laminin in this case). Measuring focal adhesionkinase (FAK) and phospho-FAK levels in treated and con-trolled PRP-1 cells, FAK protein levels in treated and con-trolled PRP-1 were estimated using FAK STAR ELISA KitCatalog #17-479 Millipore (upstate). Phospho-FAK was

detected with STAR Phospho-FAK (Tyr 397) ELISA KitCatalog #17-480 Millipore (upstate).

MDR assay

Multidrug resistance (MDR) cells expressing high levels ofPgp rapidly extrude nonfluorescent calcein AM from theplasma membrane reducing the accumulation of calcein.The degree of inhibition of Pgp activity can be quantitatedby measuring the increase in intracellular calcein fluores-cence. The protocol was done by following the VybrantMultidrug Resistance Assay Kit (V-13180) MolecularProbes instructions.

Human phospho-MAPK array

Proteome Profiler Array (human phospho-mitogen-activatedprotein kinase (MAPK) array kit) Catalog # ARY002B Rand D Systems allowed us to analyze the phosphorylationstatus of the major families of MAPKs as well as othersignal transduction regulators.

HDAC and HAT assays

The nuclear proteins were extracted using the application formonolayer adherent cells according to the instructions fromEpiQuik Nuclear Extraction Kit Catalog #OP-0022 Epigentek.Colorimetric measurement of histone acetyltransferase/histonedeacetylase (HAT/HDAC) activity and inhibition was possiblein utilizing EpiQuikHATActivity/InhibitionAssayKit Catalog#P-4003 (Epigentek) and EpiQuik HDAC Activity /InhibitionAssay Kit Catalog #P-4002 (Epigentek), correspondingly.

Results

PRP-1 inhibits ECM–cell adhesion in humanchondrosarcoma JJ012 cells and metastatic breast carcinomaMDA 231 (ER-) cell lines

Increased ECM–cell adhesion is extremely important fortumor survival [10]. The goal of these series of experimentswas to determine the PRP-1 role in ECM–cell adhesion inchondrosarcoma. Since we have reported previously thatPRP-1 is a novel mTORC1 inhibitor for the tumors ofmesenchymal and epithelial mesenchymal transition [8, 9]in Figs. 1 and 2, we demonstrated the strong similaritypattern of response to this neuropeptide by chondrosarcomaand breast carcinoma in adhesion-related reactions. CultrexCoat Laminin I 96 well cell adhesion assay was used toexamine the effect of PRP-1 on the process of ECM–celladhesion. Chondrosarcoma JJ012 (Fig. 1a) and breast car-cinoma MDA 231 (ER-) cell lines (Fig. 1b) were treated

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with different concentrations (1 μg/ml and 10 μg/ml) of thecytokine. Next, we examined the changes in total FAK andphospho-FAK protein levels to determine whether the de-crease in ECM–cell adhesion is mediated by the changes intotal FAK or its phosphorylated form (Fig. 2). No statisticalsignificant changes were observed both in total or phospho-FAK levels after the treatment with PRP-1, indicating thatobvious decrease in percent of ECM–cell adhesion (Fig. 1)did not involve any changes in total or phospho-FAK. Since,like many mTORC1 inhibitors, PRP-1 exerted cytostaticeffect [6–9], it is important to understand how PRP-1impacts multidrug resistance in mesenchymal cell lines.MDR resistance phenotype was characterized by the develop-ment of tumor resistance to anticancer drugs, was associatedwith overexpression of transmembrane transporter ATP-dependent 170 kDA permeability glycoprotein (Pgp), andwas encoded by MDR1 gene. Neither PRP-1 nor calciumchannel blocker verapamil did inhibit Pgp activity, (data notshown); however, cyclosporin inhibited Pgp activity in bothcell lines at IC50 of 6 μg/ml (Fig. 3). Thus, it was clear that

PRP-1 did not inhibit Pgp-mediated drug resistance, yet thecytokine caused ECM–cell adhesion to decrease (Fig. 1).Weproceeded with the investigation of phosphorylation status ofmajor MAPK families as well as other signal transductionregulators in PRP-1-treated versus untreated samples usingProteome Profiler Array in JJ012 human chondrosarcoma cellline (Fig. 4). Phospho-MAPK array in human chondrosar-coma cell line treated with cytokine (10 μg/ml) demonstratedalmost 16- and 8.6-fold upregulation of phosphorylation, thusinactivating GSK3αβ and GSK3β, correspondingly. Suchinactivation eventually leads to β-catenin accumulation thatentails drug resistance. Data indicated strong MAPK pathwayactivation as well.Very similar results were obtained withbreast carcinoma (data not shown). The upregulations ofdifferent MAPKs, especially p38 (50-fold) and GSK3βphosphorylation along with p53 downregulation, made itclear that PRP-1 as mTORC1 inhibitor mediated 4-fold.(AKT)3 upregulation triggered PI3K–AKT and PI3K–RAS–MAPK negative feedback loops. Next series ofexperiments demonstrated that after treatment with thisneuropeptide in chondrosarcoma cells, HDAC was upre-gulated (twofold), whereas HAT was downregulated (two-fold) (Figs. 5 and 6). PRP-1 did not display the propertiesof HDAC inhibitor in that model.

Fig. 1 Dose–response effect of PRP-1 on human JJ012 chondrosar-coma (a) and MDA 231 (ER-) breast carcinoma (b) cell lines inCultrex Coat Laminin 96 well cell adhesion assay. Data are presentedas mean±SD percent cell adhesion, obtained in three independentexperiments. ECM –cell adhesion was inhibited by 62 % of 10 μg/ml PRP-1 in both cell lines

Fig. 2 Total focal adhesion kinase (FAK) levels (a) and phospho-FAKlevels in human JJ012 breast MDA 231 (ER-) carcinoma cell lines (b)treated with different concentrations of PRP-1

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Discussion

Drug resistance and inactivation of GSK3

According to the literature data, the chemotherapeutic resis-tance was attributed also to PI3K/AKT pathway activation[11, 12] and not only to MDR, which is characterized by thedevelopment of tumor resistance to anticancer drugs and isassociated with overexpression of transmembrane transporter.AKT phosphorylation that entails inhibition of a highly con-served glycogen synthase kinase 3 (GSK3). So, by inactivat-ing GSK3, AKT may enhance the functions of some of thesetranscription factors. Our experiments with phospho-MAPKarray in human chondrosarcoma cell line treated with PRP-1(10 μg/ml) demonstrated strong upregulation of phosphoryla-tion, thus inactivating GSK3αβ and GSK3β (16- and 8.6-

fold, correspondingly) via negative feedback loops of PI3K–AKT and PI3K–RAS–MAPK activation. Data indicated si-multaneous strong MAPK pathway activation of JNK1/2;ERK1/2; p38α, β, σ, γ; and MKK3/6. Results proved thatp38 MAPK was most strongly activated (50-fold) in JJ012chondrosarcoma cells on the treatment with PRP-1, whileAKT was activated 4-fold in comparison with the control.P38 MAPK is known also to inactivate GSK3β by directphosphorylation at its C terminus. P38 MAPK-mediatedphosphorylation of GSK3β independent fromAKTwas docu-mented before, primarily in the brain and thymocytes thatprovide potential mechanism for p38 MAPK-mediated sur-vival in those tissues [13] since inactivated GSK3β is notcapable to phosphorylate β-catenin for its ubiquitination andsubsequent degradation that will lead to β-catenin accumula-tion. Phospho-MAPK arrays demonstrated upregulation ofdownstream survival signaling effectors—Hsp27, RSK1,

Fig. 3 Vybrant multidrug resistance assay in human JJ012 chondro-sarcoma and MDA 231 (ER-) breast carcinoma cell lines with com-petitive Pgp drug inhibitor cyclosporin (three independentexperiments)

Fig. 4 Human phospho-protein profiler MAPK array (R&D SystemsInc.) in human chondrosarcoma JJ012 cell line treated with 10 μg/mlPRP-1. The arrays are semi-quantitative pixel density measurement ofthe X-ray film looking at fold differences between control (not treated)and (PRP-1-treated) samples. Data are presented as mean±SD foldpixel density increase obtained in three independent experiments(Figs. 5 and 6). PRP-1 did not display the properties of HDACinhibitor in that model

Fig. 5 Histone deacetylase (HDAC) activity in human JJ012 chon-drosarcoma cell line treated with PRP-1 (a) and histone deacetylase(HDAC) activity in human primary low-intermediate chondrosarcomacell line (b) treated with PRP-1. EpiQuik HDACT Activity /InhibitionColorimetric Assay Kit (Epigentek) was used

Fig. 6 Histone acetyltransferase (HAT ) activity in human JJ012human chondrosarcoma cell line treated with PRP-1 was measuredcolorimetrically using EpiQuik HAT Activity/Inhibition Assay Kit(Epigentek). Data are presented as mean±SD fold inhibition of HATlevels in PRP-1-treated samples versus control samples obtained inthree independent experiments. PRP-1 (15 μg/ml and 10 μg/ml)caused almost twofold inhibition of HAT in the experimental sample

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and CREB—when treated with PRP-1 [14–16]. It becomesevident that fourfold activation of AKT is evidently enough totriggerMAPK cascade feedback loop, and strong activation ofp38 MAPK is enough to inactivate GSK3β independentlyfromAKT, which in its turn contributes to its phosphorylation.Simultaneous complete downregulation of phospho p53 wasobserved in our AKT3 activation-linked experiments, knownto increase nuclear MDM2, that ubiquitinate and impair p53[17].

HDAC/HAT activity and GSK3β

The study addressed also the involvement of HDACs andHAT in the PI3K–AKT feedback loop. It was known fromthe literature that HDACIs are capable to induce AKT de-phosphorylation by disrupting HDAC-protein phosphatase 1(PP1) complexes [18]. This disruption results in the increasedassociation of PP1 with AKT, resulting in the dephosphoryla-tion and consequent inactivation of the kinase. The overex-pression of specific HDACS occurs in several cancer types, andthe growth of cell lines derived from these tumors is sup-pressed by small interfering RNA (siRNA)-mediated knock-down of the overexpressed HDAC [19]. Both in JJ012 humanchondrosarcoma cell and in primary intermediate chondrosar-coma cells was total HDAC inhibition not observed in ourexperiments after the treatment in comparison with the con-trol, on the contrary. PRP-1 is not an HDAC inhibitor inchondrosarcoma and that is why AKT is not getting dephos-phorylated after the PI3K–AKT feedback loops are triggeredas a consequence of mTORC1 inhibition. It was reported byothers that global HAT is upregulated when GSK3β is inacti-vated [20]. However, total HATwas downregulatedwith PRP-1 in our experiments with chondrosarcoma JJ012 and MDA231 (ER-) breast carcinoma cell lines. One can speculate that,perhaps, concomitant activation of AKT feedback loop isprevented, if the mTORC1 inhibitor is also involved in HDACinhibition [20–26].

ECM–cell adhesion and drug resistance

During metastasis, tumor cells disperse via the vasculatureand the lymphatic systems, undergoing intravasation andextravasation before finally invading and growing at sec-ondary sites. To undertake successfully the stages requiredfor dissemination, tumor cells must be able to adhere to, andsubsequently detach from the components of the ECM andbasement membrane such as collagens, fibronectin, andlaminin, and ultimately the vascular endothelium. Adhesionmolecules, expressed by both the tumor and endothelium,mediate these adhesive interactions, and more specificallyintegrins are thought to be particularly important in cancermetastasis, forming a major group of adhesion moleculescoordinating ECM and cell–cell interactions. It was

demonstrated here that invasive uveal melanoma cells ad-here in higher numbers to ECM substrates and cells of thevasculature than noninvasive cells [27]. The importance ofcell–matrix interactions in promoting cell survival is nowwell established [28]. The morphology of the invading frontis influenced by the changes in the adhesiveness parameters,and the invasiveness of the tumor is related to adhesion.Cell–cell adhesion has less influence on invasion comparedwith cell–medium adhesion [29]. Focal adhesions serve asnodes to transmit environmental signals initiated by solublegrowth factors, ECM, and surrounding mechanical forces toregulate cell growth and migration. Within tumors, cancercells respond to changes in the expression and organizationof matrix proteins such as fibronectin and collagen. Tyrosineprotein kinases play a key role in regulating signals mediatedby integrin receptors. A central regulator of integrin sig-naling is the FAK, an ubiquitously expressed nonreceptortyrosine protein kinase [10]. It was suggested that integrin-mediated activation of MAP kinase is independent of FAK andindicates the probable existence of at least two distinct integrinsignaling pathways in fibroblasts [30]. It is not entirely clearwhether physical cellular adhesion and spreading are requiredfor integrin-mediated signal transduction [31]. Bozzo et al.[32] demonstrated that soluble ECM components applied tosuspension cells can activate β1 integrin signaling and sup-press apoptosis without cell adhesion and spreading. Theseresults showed that matrix proteins can promote cell survivalin neuronal cells via integrin receptors. This effect does notrequire cell adhesion and subsequent changes in cell shape as itcan be mediated by soluble integrin ligands in suspended cellsand involves a signaling pathway different from that triggeredby growth factors. Cell adhesion to ECM was reported toconfer resistance against cell-damaging agents, that is, drugsand radiation in tumor and normal cells in vitro [33], and FAKphosphorylation contributes to drug resistance [34]. In thisstudy, we present the evidence that it is not always the casethat drug resistance can be mediated by the abovementionednegative feedback loops leading to GSK3β phosphorylation,thus its inactivation, and be adhesion independent.ECM–cell adhesion can be downregulated, instead ofanticipating upregulation without the changes in totaland phospho-FAK. Summarizing all mentioned above,we can conclude that PRP-1-mediated mTORC1 inhibi-tion in chondrosarcoma and breast carcinoma cell lineled to the adhesion of independent drug resistance viaPI3K–AKT and PI3K–RAS–MAPK feedback loops andthat ECM–cell adhesion-independent drug resistance wasmediated by GSK3β inactivation. Combinatorial treat-ments of mTORC1 inhibitor immunomodulator PRP-1with MAPK and PI3K/AKT inhibitors most probablywill lead to full cytotoxicity. We assume that negativefeedback loops in general can be prevented if mTORC1inhibitor is involved in HDAC inhibition.

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Acknowledgment This study was supported by the Research Accountof the University of Miami, Miller School of Medicine Tissue Bank.

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