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Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells

Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/5356219 Hiscox, S. et al. Dual targeting of Src and ERprevents acquired antihormone resistance inbreast cancer cells. Breast Cancer Res. Treat.115, 57-67  ARTICLE   in  BREAST CANCER RESEARCH AND TREATMENT · JUNE 2008 Impact Factor: 3.94 · DOI: 10.1007/s10549-008-0058-6 · Source: PubMed CITATIONS 44 READS 28 8 AUTHORS , INCLUDING:Steve HiscoxCardiff University 104   PUBLICATIONS   3,184   CITATIONS   SEE PROFILE Kathryn Mary TaylorCardiff University 45   PUBLICATIONS   1,255   CITATIONS   SEE PROFILE Tim P GreenCSL Limited 31   PUBLICATIONS   1,272   CITATIONS   SEE PROFILE Available from: Kathryn Mary TaylorRetrieved on: 14 January 2016  PRECLINICAL STUDY Dual targeting of Src and ER prevents acquired antihormoneresistance in breast cancer cells S. Hiscox   N. J. Jordan   C. Smith   M. James   L. Morgan   K. M. Taylor   T. P. Green   R. I. Nicholson Received: 5 February 2008/Accepted: 7 May 2008/Published online: 21 May 2008   Springer Science+Business Media, LLC. 2008 Abstract  Acquired resistance to endocrine therapiespresents a major obstacle to the successful treatment of breast cancer patients. Previously, we have shown thatacquisition of resistance to tamoxifen in breast cancer cellsis accompanied by an elevation in Src kinase activity whichpromotes an aggressive, invasive phenotype in vitro. Here,we have explored the potential therapeutic effects of com-bining Src inhibition with anti-oestrogen treatment on thedevelopment of endocrine insensitivity in breast cancercells. Treatment of MCF7 and T47D cells with tamoxifenalone resulted in an initial growth inhibitory phase followedby the eventual development of tamoxifen resistance toge-ther with an elevation of Src kinase activity, which wascentral to their increased invasive capacity. Chronic expo-sure of both cell types to the Src inhibitor, AZD0530, as amonotherapy resulted in outgrowth of AZD0530-resistantcells, in which Src kinase activity remained suppressed asdid their in vitro invasive nature. Treatment of both MCF7and T47D cells with AZD0530 in combination withtamoxifen resulted in a reduction of Src activity togetherwith inhibition of focal adhesion kinase phosphorylationand a complete abrogation of their in vitro invasivebehaviour. Furthermore, combination therapy significantlysuppressed expression of cyclinD1 and c-myc and pre-vented cell proliferation and the subsequent emergence of aresistant phenotype, with total cell loss occurring by 12weeks. These data demonstrate that pharmacological tar-geting of Src kinase, in conjunction with antihormonetherapies, effectively prevents antihormone resistance inbreast cancer cells in vitro and suggests a potential noveltherapeutic benefit of Src kinase inhibitors clinically. Keywords  Src    ER    Tamoxifen-resistance   Breast cancer Introduction Steroid hormones are central in directing the growth anddevelopment of oestrogen receptor (ER) positive breastcancers; as such, endocrine therapies which seek to perturbthe steroid hormone environment of the tumour cells canpromote extensive remissions in established disease andprovide significant benefits in terms of patient survival[1, 2]. However, despite the undoubted improvements brought by endocrine therapies, in practice their benefitsare limited by the capacity of a significant proportion of breast cancer patients that show an initial positive responseto ultimately suffer a recurrence due to acquired anti-oestrogen resistance [3–6]. In these patients, relapse on such therapies clinically presents as local and/or regionalrecurrences, frequently with distant metastases [7] and theoutlook for these patients is poor. Recent in vitro studieshave demonstrated that chronic exposure of breast cancercells to tamoxifen ultimately results in the acquisition of tamoxifen resistance [8, 9] which is accompanied by the gain of aggressive, invasive characteristics [10, 11]. Overexpression of the non-receptor tyrosine kinase, Src,and/or an increase in its kinase activity has been associatedwith the development and progression of a number of human malignancies including breast cancer [12, 13] where S.Hiscox( & )    N.J.Jordan    C.Smith    M.James    L.Morgan   K. M. Taylor    R. I. NicholsonTenovus Centre for Cancer Research, Welsh School of Pharmacy, Redwood Building, King Edward VII Avenue,Cardiff, Wales CF10 3XF, UK e-mail: hiscoxse1@cf.ac.uk T. P. GreenAstraZeneca, Alderley Park, Macclesfield, UK   1 3 Breast Cancer Res Treat (2009) 115:57–67DOI 10.1007/s10549-008-0058-6  it has been implicated in metastatic progression [14].Interestingly, it has recently been reported that expressionof activated Src is associated with a reduced recurrence-free survival in ductal carcinoma in situ [15]. Steroid-induced cellular responses involve activation of Src and,although the molecular pathways involved in theseresponses have yet to be fully defined, can involve growthfactor receptor signalling. For example, targeting of the C-terminal region of the ER to the membrane of ER-negativebreast cancer cells results in oestrogen-stimulated, c-Src-dependent responses including activation of the epidermalgrowth factor receptor (EGFR) [16]. Furthermore, oestro-gen stimulation results in the recruitment and activation of Src leading to activation of Shc, MEK/MAPK, and PI3K/ PKB [17]. Significantly, we have recently identified thatSrc kinase activity is significantly elevated in tamoxifen-resistant breast cancer cells, where it promotes an aggres-sive, invasive phenotype [11]. A role for Src kinase inacquired endocrine resistance has also been suggested byothers, where it may contribute to cellular growth viaregulation of Cas-mediated EGFR signalling [18] orthrough interplay with focal adhesion kinase (FAK) [19].Given the putative role of Src in acquired tamoxifenresistance, we hypothesised that targeting of Src alongsidethe ER would present a novel strategy to suppress thedevelopment of an aggressive endocrine-resistant pheno-type. In this report, we demonstrate that combinedtargeting of the ER and Src kinase using tamoxifen and thenovel Src inhibitor, AZD0530 [20] in two separate, ER-positive breast cancer cell lines, greatly enhances thegrowth inhibitory effects seen with either agent alone,preventing the development of acquired tamoxifenresistance. Materials and methods Cell cultureTamoxifen-responsive MCF7 and T47D breast cancer cellswere routinely cultured in home medium (RPMI mediumsupplemented with 5% foetal calf serum (FCS), 10 IU/mlpenicillin, 10  l g/ml streptomycin, 2.5  l g/ml fungizone and200 mM glutamine). Subsequent treatment of these cellswas performed in experimental medium (phenol-red freeRPMI containing 5% charcoal-stripped, steroid-depletedFCS with antibiotics and glutamine as above) supplementedwith 10 - 7 M tamoxifen (4-hydroxy tamoxifen), AZD0530(0–1  l M) or both agents in combination as indicated. In allcases the tissue culture medium ( ± tamoxifen/AZD0530)was replaced every 3 days unless otherwise stated. Stock solutions of AZD0530 and tamoxifen were dissolved inDMSO and ethanol respectively prior to diluting in tissueculture medium. Corresponding DMSO/ethanol controlshad no effect on the parameters analysed. Treatment of cellswith AZD0530 alone produced similar results irrespectiveof whether cells were cultured in home medium or experi-mental medium.Cell growth assaysCells were grown for 10 days in the presence of tamoxifen(10 - 7 M), AZD0530 (0–1  l M) or a combination of bothagents. Changes in cell growth were then evaluated bymeans of trypsin dispersion and subsequent counting usinga Coulter Counter. To determine the effects of chronicexposure to these agents on the growth of both cell lines,log-phase cell cultures were either grown in home mediumwith no treatments or exposed to tamoxifen  ±  AZD0530(0–1  l M) in experimental medium with routine passagingonce cell confluency had reached  * 80%. Each passagewas subsequently recorded and plotted against time inculture to allow the comparison of the ability of thesetreatments to maintain their growth suppressive effects.Measurement of apoptosisCells were harvested at various times during long-termculture and apoptotic events analysed using a VybrantApoptosis Assay (Molecular Probes, Eugene, OR, USA) asfollows: harvested cells were washed and resuspended to1  9  10 6 cells/ml in PBS prior to the addition of FITC-labelled YO-PRO-1 (final concentration of 100 nM) andTRITC-labelled propidium iodide (final concentration of 1.5 nM) for 30 min at 4  C. Cells were immediately read ona FACScan flow cytometer. Negative controls consisted of cells alone without antibody. The mean fluorescence of aminimum of 1  9  10 4 cells was determined for each sampleand the experiments were repeated 4 times, each time induplicate. Data represents mean of three separate experi-ments, each performed in duplicate.RT-PCRTotal RNA was extracted from MCF7 and T47D cellsusing Trizol reagent (Life Technologies) and converted tocDNA using random hexamers and Super-Script II reversetranscriptase (Invitrogen). cDNA was amplified using PCRprimer pairs (MWG Biotech) specific for c-myc (Fwd:5 0 -ccaagctcgtctcagagaag-3 0 , Rev:5 0 -cagcaggatagtccttccga-3 0 )and cyclin-D1 (Fwd:5 0 -ggatgctggaggtctgcgag-3 0 , Rev:5 0 -gagaggaagcgtgtgaggcg-3 0 ). All PCR reactions were per-formed as multiplex reactions with PCR primers specificfor  b -actin (Fwd:5 0 -ccttcctgggcatggagtcct-3 0 , Rev:5 0 -ggagcaatgatcttgatctt-3 0 ) as an internal control. PCR was per-formed in a semi-quantitative manner using 25 cycles so 58 Breast Cancer Res Treat (2009) 115:57–67  1 3  that products were in the linear range of amplification. PCRproducts were separated on a 1.0% agarose gel, stainedwith ethidium bromide and visualized and photographedunder ultraviolet light.Measurement of cellular proliferationKi67 antigen was assessed in both cell types followingtreatment with tamoxifen  ±  AZD0530 using the MIB-1anti-Ki67 antibody (Coulter Electronics, Luton, UnitedKingdom). Briefly, cells were grown and treated in petridishes containing coverslips as indicated then washed andfixed in formal saline. Primary antibody (MIB-1) wasapplied to the coverslips after blocking with PBS/Tweenand incubation performed for 60 min. After washing inPBS, the secondary antibody (Mouse Envision, DAKO UK Ltd., Ely, Cambridgeshire) was applied to the coverslipsfor 75 min. After further PBS washing, the chromogen(‘‘SigmaFast’’ DAB, Sigma, Poole, UK) was added to thecells for 10 min after which the coverslips were rinsed indistilled water. Samples were counterstained with 20%haematoxylin for 3 min and mounted for examination bylight microscopy. Control coverslips (no primary antibody)were checked for non-specific binding before assessingstaining intensity in the test samples. The percentage of Ki67-positive cells was estimated after counting at least1,000 tumour cells.Cell cycle analysisCells were cultured in the presence or absence of tamoxi-fen, AZD0530 or the combination for up to 8 weeks. Foranalysis of cell cycle, the cells were harvested by tryp-sinisation and resuspended at 10 - 7 cells/ml before usingthe Cycletest Plus DNA reagent kit (Becton Dickinson)following the manufacturers instructions to produce uni-form suspensions of single nuclei with DNA stained andsuitable for reading on a FACScan Flow Cytometer (Bec-ton Dickinson).Cell migration and invasion assaysCells were exposed to tamoxifen  ±  AZD0530 for the timesindicated in the figures prior to seeding onto fibronectin-coated polycarbonate, microporous membranes (8  l m poresize) at 5  9  10 4 cells/membrane and allowed to migrate tothe underside of the membrane for a period of 24 h.Migratory cells were fixed, stained with 0.5% crystal violetand counted. To determine the cells’ invasive capacity,cells were seeded onto Matrigel-coated, porous membranesas described previously [10]. After 72 h culture, invadedcells were fixed, stained with DAPI and counted. Cellmigration/invasion was quantified as the mean number of cells observed in each of eight random fields of view persample, in duplicate with each experiment performed aminimum of three times.Western blottingCells were harvested at 2 week intervals during theirexposure to tamoxifen  ±  AZD0530 and lysates resolvedby SDS-PAGE using 8% gels. Proteins were immobilisedon nitrocellulose membranes and subsequently probed withphospho-specific antibodies that recognised the activeforms of Src (Y419) and FAK (Y861, both from Biosource,Nivelles, Belgium). Tyrosine 419 in Src is a major posi-tive-regulatory site, the phosphorylation of which increasesSrc activity and is widely used as a indicator of changes inSrc activity both in vitro and in vivo (for example, see [21].Although other tyrosine residues on FAK are phosphory-lated in a Src-dependent manner, phosphorylation of FAK at Y861 was investigated as this is well known to regulatecancer cell migration and to be increased in metastaticbreast cancer cells [22, 23] and thus may be important in regulating the effects of Src kinase in cancer cells. Repeatimmunoprobings were performed using pan antibodies todetermine total levels of these proteins and with  b -actin tocorrect for loading. Western blots were then scanned toprovide data for quantitation, with normalization againstactin first. Each experiment was performed at least threetimes with representative gels shown. Statistical analysis Data was analysed using one-way ANOVA with post-hocanalysis in order to examine whether inclusion of AZD0530 (0.1 and 1  l M) significantly affected theobservations obtained with tamoxifen alone. Significancewas assumed at  P \ 0.05. Results AZD0530 and tamoxifen together showed improvedgrowth inhibitory effects compared with either agentaloneTreatment of both MCF7 and T47D cells with AZD0530resulted in a dose-dependent inhibition of Src kinaseactivity, as shown by a decrease in phosphorylation of Src atY419 (Fig. 1a) whilst having no effect on the level of Srcprotein in these cells. Despite both cell type having similarlevels of Src kinase, T47D cells exhibited a small but non-significant decrease in sensitivity to the Src inhibitor.However, at the highest concentration of AZD0530 (1  l M), Breast Cancer Res Treat (2009) 115:57–67 59  1 3  a corresponding inhibition of MAPK activity was alsoobserved in both cell types. Treatment of cells withAZD0530 also resulted in a dose-dependent decrease ingrowth over a period of 10 days (Fig. 1b). A modest sup-pression of cell growth was seen following culture withtamoxifen alone at this time point (Fig. 1c) with T47D cellsappearing more resistant to its growth inhibitory effects, ashas been previously reported [24]. Importantly, whenAZD0530 (1  l M) and tamoxifen were combined, maximalinhibition of cell growth over this time period was observed(Fig. 1c). Analysis of the growth data by isobologramsuggested that the combined effect of the two drugs wasadditive rather than synergistic (data not shown).AZD0530 and tamoxifen prevent the emergence of tamoxifen resistanceSince prolonged exposure of breast cancer cell lines to anti-oestrogens ultimately results in acquired resistance [8, 9], a process in which activated Src has been implicated [11, 18, 19], we wished to determine whether inhibition of Srcactivity alongside tamoxifen treatment would delay orprevent the development of resistance. Chronic exposure of both MCF7 and T47D cells to tamoxifen resulted in theemergence of a resistant phenotype after an initial period of growth suppression, as demonstrated by the ability of thesecells to regain their ability to grow in the presence of theantihormone (Fig. 2). Interestingly, treatment withAZD0530 as a monotherapy also ultimately resulted incells which were able to grow in the presence of thisinhibitor, suggestive of further acquired drug resistance.Significantly, our data revealed that cells exposed to thecombination of tamoxifen and AZD0530 did not grow butrather, a gradual loss of cells in culture was observed, withno viable cells visible by approximately 13 weeks (Fig. 2).Addition of AZD0530 to tamoxifen does not enhanceapoptosisWe next investigated whether tamoxifen and AZD0530combined increased cellular apoptosis. FACS analysis of cells cultured in the presence of tamoxifen  ±  AZD0530revealed that combining these agents did not significantlyincrease the extent of apoptosis seen with either compoundas a single agent. (Table 1 shows data from 1 and 8 weeks’continuous culture in the presence of these agents).AZD0530 and tamoxifen in combination significantlysuppress expression of the Ki67 antigenSince an induction of apoptosis did not appear to play asignificant role in AZD0530-induced prevention of acquired tamoxifen resistance, we next evaluated the pro-liferative capacity of MCF7 and T47D cells cultured in thepresence of tamoxifen with and without AZD0530. Anal-ysis of the Ki67 expression, a marker of cellularproliferation, revealed that the combination of AZD0530and tamoxifen significantly inhibited cellular proliferationin both cell lines, an effect observable as early as 10 dayspost treatment (Fig. 3a—MCF7 cells, 3b—T47D cells). A p-Srct-Srcp-Srct-Src    M   C   F   7   T   4   7   D [AZD0530] (µM) 0 0.1 1 B AZD0530 concentration (µM) 0204060801001200 0.1 0.5 1MCF7T47D ††††    M  e  a  n   %   g  r  o  w   t   h   i  n   h   i   b   i   t   i  o  n    M   C   F   7   T   4   7   D p-MAPKt-MAPKp-MAPKt-MAPK  0204060801001200 [AZD0530] (uM)   p  -   M   A   P   K   (   %   o   f  c  o  n   t  r  o   l   ) MCF7T47D 020406080100120 [AZD0530] (uM)   p  -   S  r  c   (   %   o   f  c  o  n   t  r  o   l   ) MCF7T47D ***** * β -actin 0.1 10 0.1 1 MCF7    M  e  a  n   %   g  r  o  w   t   h   i  n   h   i   b   i   t   i  o  n Tamoxifen concentration *** C 0204060801001200 10-9M 10-8M 10-7M 10-6M 10-5M0 µM AZD05300.1µM AZD05301µM AZD0530 T47D **** Tamoxifen concentration 0204060801001200 10-9M 10-8M 10-7M 10-6M 10-5M0 µM AZD05300.1µM AZD05301µM AZD0530    M  e  a  n   %   g  r  o  w   t   h   i  n   h   i   b   i   t   i  o  n   MCF7T47DMCF7T47D Fig. 1  Western blottinganalysis demonstrated thatAZD0530 inhibited Src kinasephosphorylation at Y419 in bothMCF7 and T47D cells in a dose-dependent manner whilstMAPK activity was inhibited atonly 1  l M ( a ). MCF7 and T47Dcell growth in response toincreasing concentrations of AZD0530 ( b ) ortamoxifen  ±  AZD0530 ( c ) over10 days was quantified using aCoulter counter. Whereas bothAZD0530 and tamoxifen aloneinhibited cellular growth in adose-dependent manner,AZD0530, when combined withtamoxifen, dramatically reducedcellular growth over this timepoint.   P \ 0.02 vs. control;* P \ 0.02 vs. tamoxifen aloneat this data point. Data aremeans of at least threeindependent experiments60 Breast Cancer Res Treat (2009) 115:57–67  1 3
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