Introduction
Growth of the majority of breast cancers is stimulated by oestrogen and this oestrogen receptor (ER) signalling can be successfully blocked by anti-hormonal treatments, including aromatase inhibitors or the oestrogen receptor antagonists, tamoxifen or fulvestrant. Anti-hormone treatment is effective in a high proportion of initially responsive patients but subsequently a significant number acquire resistance with resulting poorer survival rates [
1]. Consequently, there is an urgent need for treatments for breast cancer that improve responses to prevent or delay endocrine resistance. In an attempt to overcome endocrine resistance, studies have focussed on developing novel agents that can reverse resistance by targeting growth factor signalling pathways. Endocrine resistant cells can be highly dependent on the use of activated growth factor signalling pathways including epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2) [
2]. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signalling network is also often prominent in endocrine resistant breast cancer, extending to tamoxifen resistant and oestrogen deprivation resistant MCF-7-derived cell lines [
3‐
5]. In patients with invasive breast cancer, increased activation of this pathway is associated with poor prognosis [
6] and, thus, mTOR has recently been recognised as an important drug target for breast cancer therapy [
7].
mTOR is a highly conserved serine/threonine protein kinase that belongs to the PI3K- related family and serves as a central regulator of cell metabolism, growth, proliferation and survival [
8]. Extensive knowledge about the function of this protein has come from the experimental use of the natural bacterial antibiotic rapamycin, which inhibits the activity of mTOR. mTOR consists of two separate multi-protein complexes, mTORC1 and mTORC2, that are both activated by growth factor stimulation. The mTORC1 complex is rapamycin sensitive; rapamycin binds the FK506-binding protein (FKBP-12) which binds to and causes allosteric inhibition of the signalling complex mTORC1 [
9] which contains mTOR, the regulatory associated protein (RAPTOR), mLST8, PRAS40 and DEPTOR proteins. mTORC1 positively regulates protein translation and synthesis via its main substrates, p70 ribosomal S6 kinase (p70S6K) and the eukaryotic initiation factor 4E binding protein-1 (4E-BP1). Upon phosphorylation, 4E-BP1 dissociates from the mRNA cap-binding protein eIF4E and allows it to interact with eIF4G to form a translation initiation complex [
10]. In the less well-defined rapamycin-insensitive mTORC2 complex [
11], mTOR is associated with the rapamycin insensitive companion (RICTOR), LST8, mSIN1, PROCTOR and DEPTOR and phosphorylation of 4E-BP1 on t37/46 is also considered rapamycin insensitive [
12]. The mTORC2 complex is involved in cytoskeletal organisation via paxillin, rho/rac and PKBα, but it also plays a key role in cell proliferation and survival via activation of serum and glucocorticoid protein kinase 1 (SGK1) and direct activation of Akt [
8]. However, the characterisation of mTORC1 and mTORC2 as rapamycin-sensitive and insensitive complexes may not always be entirely accurate, as chronic rapamycin treatment has also been reported to inhibit mTORC2 activity by blocking its assembly [
8,
13].
The mTOR inhibitor rapamycin (sirolimus) has been used clinically as an immunosuppressant drug in transplant medicine [
14]. However, it has recently been realised that the increased activity of the mTOR pathway caused by upstream changes in regulators, such as phosphatidylinositol-3 (PI3)-phosphatase (PTEN) and PI3K, also makes mTORC1 an attractive anti-cancer target [
15] and a number of rapamycin analogues (rapalogues) have been produced: RAD001 (everolimus, Afinitor®, Novartis), CCI-779 (temsirolimus, Wyeth) and AP23573 (MK-8669) (ridaforolimus ARIAD and Merck pharmaceuticals). The first clinical cancer trials in metastatic breast cancer with temsirolimus as a monotherapy resulted in only partial responses [
16,
17]. The unexpectedly modest outcomes, with patients acquiring resistance or exhibiting intrinsic resistance to these allosteric mTORC1 inhibitors, may be associated with a paradoxical increase in the activation of Akt and PI3K caused by inhibition of a negative feedback loop from S6 kinase to IRS-1 in response to mTORC1 inhibition by rapalogues [
18‐
21], with the subsequent enhanced Akt activation reportedly being associated with rapalogue monotherapy failure in some patients [
22‐
24].
However, while monotherapy studies with several signal transduction inhibitors, including rapalogues, have shown only modest success in advanced breast cancers, preclinical data indicate that a combination of anti-hormone and signal transduction inhibitors (STI's) can provide significantly greater inhibition than either agent alone [
21]. Although breast cancers may become resistant to first-line anti-hormone treatment they often retain an active ER and will still respond to an alternative endocrine agent as a second-line therapy, but longer-term success is more likely to be achieved by also targeting up-regulated growth factor pathways that can be independent from, or interactive with, ER signalling pathways. Pre-clinical data were supportive of the use of current mTOR antagonists alongside endocrine therapy in breast cancer which resulted in a number of clinical trials using such combination therapies [
25]. One of the earliest phase 3 trials combined letrozole and temsirolimus and was used on advanced breast cancer but the trial had to be terminated early due to failure to demonstrate any benefit [
19]. Later studies have been more successful with very promising results obtained recently in advanced endocrine resistant disease where RAD001 (everolimus) was used in combination with the steroidal aromatase inhibitor exemestane or with tamoxifen in phase 2/3 trials. These have shown significant improvement in progression-free survival from 4.1 months with exemestane alone to 10.6 months with a combination of exemestane and everolimus in the BOLERO 2 trial [
26] and an improved time to progression from 4.5 months with tamoxifen alone to 8.6 months with tamoxifen plus everolimus in the TAMRAD GINECO trial [
27]. These clinical findings indicated value for the allosteric mTOR inhibitors used alongside tamoxifen or aromatase inhibitors in advanced endocrine resistant tumours and there has been recent USA Food and Drug Administration approval for everolimus in combination with exemestane in ER+/HER2- metastatic breast cancer after non-steroidal aromatase inhibitor failure [
28].
Nevertheless, there remains a group of patients who are initially refractory to everolimus/anti-hormone therapy while others relapse at a later point during such treatment [
26,
27]. It is feasible that these patients may gain more benefit from treatment with alternative mTOR inhibitors that, unlike rapalogues, are not restricted to inhibition of only mTORC1 signalling. It is thus interesting that several types of new mTOR inhibitors are currently under development. The dual mTOR and PI3K inhibitors (SF1126-Semafore, NVP-BEZ235- Novartis, xL765-Exelis-Sanofi and GDC-0980- Roche-Genentech) simultaneously block both the PI3K and mTOR signalling and, therefore, have the theoretical advantage of totally shutting down the PI3K/Akt/mTOR network [
14]. These have the possible drawback of association with greater toxicity but in early small clinical trials are reported to induce stable disease or partial response (see [
14]). A new variety of mTOR inhibitors has also recently emerged which are ATP-competitive inhibitors that target the mTOR kinase domain and, thus, dually-inhibit activity of both mTORC1 and mTORC2 complexes. This approach should be an alternative way to mitigate the problem of Akt/PI3K activation by negative feedback seen with rapalogues. Preclinical data for two such agents, PP242 and PP30, suggest that along with the additional benefit of mTORC2 inhibition, these drugs can also be more effective than rapamycin at inhibiting mTORC1 activity [
14,
29,
30]. Several pan mTOR (mTORC1/2) dual kinase inhibitors (AZD8055 and its related compound AZD2014 (Astra Zeneca), as well as INK128 (Intellikine) and OSI-027 (OSI Pharmaceuticals)) are currently in phase I/II studies on solid tumours and breast cancer or lymphoma [
30‐
33]. However, the value of such dual mTORC1/2 inhibitory strategies remains unknown in the context of endocrine resistance in breast cancer.
Here, for the first time we show that in comparison with RAD001, an mTOR kinase inhibitor AZD8055 is significantly superior as a single agent, modulating both mTORC1 and mTORC2 signalling, cell growth and survival in tamoxifen (TamR) and oestrogen deprivation (MCF7-X) resistant cell lines that aim to model clinical relapse following first-line endocrine treatment. Furthermore, we demonstrate that in these endocrine resistant RAD001-resistant models, AZD8055 results in superior growth inhibition when used alongside fulvestrant and is additionally effective alongside anti-hormones during the earlier, endocrine responsive phase of this disease in vitro. Cumulatively, these data suggest considerable potential for mTOR kinase inhibitors that target both mTORC1 and 2 to subvert resistance during anti-hormonal management of breast cancer.
Methods
Cell culture
The parental ER + breast cancer cell lines were from American Type Culture Collection (ATCC) (Manassas, Virginia, USA) (T47D) or a gift from AstraZeneca (MCF-7) Alderly Park, Macclesfield, (Cheshire, UK). Experimental cells were grown in phenol red-free RPMI-1640 supplemented with 5% FCS (foetal calf serum), penicillin/streptomycin (10 U/ml and 10 μg/ml), fungizone (2.5 μg/ml) and 4 mM glutamine. All cell culture reagents and FCS were from Invitrogen Life Technologies (Fisher Scientific, Loughborough, UK). Cell lines were used within a window of 20 passages. The acquired ER + tamoxifen-resistant cell line, TamR, was derived from MCF-7 cells continuously exposed to 10
-7 M 4-hydroxytamoxifen (Sigma-Aldrich, Gillingham, Dorset, UK) until emergence (from six months) of a cell line resistant to the growth inhibitory properties of this anti-hormone as previously described [
34]. ER + acquired tamoxifen-resistant T47D-tamR cells were also available for this study, similarly derived by our group from T47D cells following continuous exposure to 10
-7 M 4-hydroxytamoxifen. Stable TamR cells were routinely maintained in phenol-red free RPMI-1640, 5% charcoal-stripped FCS (sFCS) and 10
-7 M 4-hydroxytamoxifen, with T47D-tamR cells also maintained in the presence of this anti-hormone. The ER + model used for acquired resistance to severe oestrogen deprivation was MCF7-X, derived from MCF-7 cells grown in phenol-red free RPMI containing 5% heat inactivated (65°C, 40 minutes) charcoal stripped FCS (X medium) as described previously [
3]. Two ER negative acquired fulvestrant (Faslodex)-resistant cell lines were also available for study generated from MCF-7 (FasR) and T47D (T47D-fasR) cells continuously exposed to fulvestrant (10
-7 M) for >2 years as previously described [
35].
Growth curves
Cells were seeded overnight at 40,000 cells/well (24-well plate) in their respective growth media. Cells were grown for seven days with 1 nM to 1,000 nM of the mTOR inhibitors AZD8055 or RAD001 (gifts from AstraZeneca) or appropriate vehicle control (dimethyl sulphoxide (DMSO)). Cell growth was evaluated by trypsin dispersion of cell monolayers and cell number was measured using a Coulter Counter. All TamR, MCF-7, MCF7-X, T47D-tamR and T47D-fasR experiments were performed at least in triplicate. In combination studies, fulvestrant was routinely used at 100 nM, a concentration shown previously to be growth inhibitory in TamR and MCF7-X models [
2,
3]. The growth impact of AZD8055 (0 to 100 nM) alongside oestrogen deprivation (using X cell medium as described above) or 10
-7 M 4-OH tamoxifen was also evaluated in MCF-7 cells.
Western blotting
Cell lines were grown to 70% confluence in their respective media and then treated with a concentration range (1 nM to 100 nM) of AZD8055 or RAD001 for 15 minutes to 24 hours. Monolayers were washed with PBS and lysed in ice-cold lysis buffer (50 mM TRIS, 5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% Triton-X100 pH 7.5) supplemented with protease and phosphatase inhibitors as previously described [
4]. Lysates were clarified by centrifugation (12,000 rpm, 15 minutes, 4°C) and the protein concentration of the supernatant was determined. A total of 20 μg protein was boiled for five minutes in SDS/dithiothreitol (DTT) sample buffer and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and after blocking for one hour in 5% skimmed milk (10 mM TRIS, 150 mM NaCl, 0.05% Tween 20 pH 7.6), they were incubated overnight with primary antibodies (1:1,000 dilution): mTOR (Cell Signalling Technology, Boston, Massachusetts, USA #2972); P
(ser2481) mTOR (Cell Signalling #2974); P
(ser 2448)mTOR (Cell Signalling #2971); Akt (Cell Signalling #9272); P
(ser 473) Akt (Cell Signalling #9271); p70S6k (Cell Signalling #9202); P
(thr389)-p70S6k (Cell Signalling #9205); S6 ribosomal protein (Cell Signalling #2217); P
(ser235/236)-S6ribosomal protein (Cell Signalling #2211); 4EBP-1 (Cell Signalling #9644); P
(thr37/46)4E-BP-1 (Cell Signalling #2855); P
(thr246) PRAS40 (Cell Signalling #2997); PRAS40 (Cell Signalling #2610); P-
(thr202/tyr402) erk42/44 (Cell Signalling #9101); p44/42 MAPK/erk1/2 (Cell Signalling #9102); ERα (Santa Cruz Biotechnology, Dallas, Texas, USA sc543); P
(ser167)ER (Upstate Merck Millipore, Billerica, Massachusetts, USA #07-481); and P
(ser118) ER (Santa Cruz 12915). Actin (Sigma # A5441, at 1:50,000) was used as a loading control. Blots were washed with TBS/Tween and bound antibodies were detected after one hour incubation with horseradish peroxidase (HRP)-labelled secondary antibodies (1:10,000). Bound proteins were visualised by enhanced chemiluminescence (Pierce, Thermoscientific, Rockford, USA). Where appropriate, signal quantification was performed by densitometry (AlphaEase system) and normalised relative to actin.
Polymerase Chain Reaction
Total RNA was extracted from monolayers of cells treated for 72 hours with 0 to 100 nM AZD8055 using TRIzol (Sigma) according to the manufacturer’s instructions. Reverse transcription was performed on 1 μg total RNA and PCR was performed as previously described [
4]. Oligonucleotide primers were synthesised by Invitrogen and co-amplification was performed with β-actin used as a loading control. Samples treated with oestradiol or fulvestrant were used as a positive control to show ER-regulated modulation of target genes in MCF7-X and TamR cells [
3,
36]. PCR products were quantified by densitometry (AlphaEase) and normalised relative to actin. The following primers were used in this study:
ß-Actin – GGA GCA ATG ATC TTG ATC T and CCT TCC TGG GCA TGG AGT CCT (202 bp)
Amphiregulin - TCC TCG GGA GCC GAC TAT GAC and GGA CTT TTC CCC ACA CCG (330 bp)
pS2 - CAT GGA GAA CAA GGT GAT CTG and CAG AAG CGT GTC TGA GGT GTC (336 bp)
cyclin D1-GCC TGT GAT GCT GGG CAC TTC ATC TG and TTT GGT TCG GCA GCT TGC TAG GTG AC (358 bp)
c-myc- TTG CAG CTG CTA GAC GCT G and CCA CAT ACA GTC CTG GAT GA (470 bp)
bcl2- CAC CTG TGG TCC ACC TGA C and AGC CAG GAG AAA TCA AAC AGA G (376 bp)
Immunocytochemistry
Subconfluent monolayers of TamR or MCF7-X cells were treated for one hour (phospho-ERα) or 72 hours (Ki67, ERα, pS2) in their respective growth media in the presence of AZD8055 (1 to 100 nM) on 3-aminopropyltriethoxysilane coated glass coverslips. Staining for the proliferation marker Ki67 (MIB1 antibody) was performed on cells fixed in 3.7% formaldehyde/0.15 M NaCl for ten minutes, five minutes in 100% ethanol with a final wash in PBS before assay. Staining for phosphorylated ERα (ser167 or ser118 sites), pS2 and total ERα was performed on cells optimally-fixed for fifteen minutes with 3.7% formaldehyde in PBS, five minutes PBS, five minutes methanol (-20°C), five minutes acetone (-20°C) and five minutes PBS or with 2% paraformaldehyde with 20 mM orthovanadate for twenty minutes followed by 2 x five minutes PBS washes (ERα 118). Coverslips were blocked with PBS/0.02%Tween for five minutes and incubated with 1:150 MIB1 (M7074 Dako Ltd, Ely, Cambridgeshire, UK) in PBS for one hour, or with 1:175 ERα (AB ER clone 6 F11 Vector Laboratories, Peterborough, UK) for 90 minutes; 1:100 ERα 167 (Upstate #07-481); 1:400 pS2 (Novocastra, Leica Biosystems, Newcastle-upon-Tyne, UK) for 90 minutes or 1:25 ERα 118 (Cell Signalling # 2515) overnight at room temperature. Unbound antibody was removed by washing with PBS. The EnVision (Dako) system was used for visualization (one to two hours at room temperature). Coverslips were washed in PBS and detected with diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide chromogen substrate (Dako) and counterstained with methyl green. Immunostaining was evaluated at 20× magnification using an Olympus BH-2 light microscope and representative photographs were taken. For Ki67, estimates of percentage of cells deemed positive versus negative/equivocally-stained were determined from five fields of view for each coverslip (20x magnification) from three independent experiments.
Sytox green viability assay
A Sytox green viability assay was modified from Jones and Singer [
37]. Cells were seeded into 96-cell plates (5,000 cells/well) and left to adhere overnight (37°C, 5% CO
2). After 24 hours (day 0), Sytox green, a cell impermeant green-fluorescent nucleic acid stain (Invitrogen), was added to each control well (n = 8) (final concentration 1:25,000) and after one hour the number of cells with a compromised plasma membrane (that is, late apoptotic and necrotic cells) that had taken up the sytox green was counted (number/well) using a fluorescent microscope. Cell membranes were permeabilised overnight with 0.25% saponin (Sigma) in the presence of sytox green, and then total cell number/well was counted. The remaining experimental cells were cultured for 72 hours in the presence of AZD8055 (0 to 100 nM), then the sytox green assay was performed to give a count for dead and total cell numbers as described above. Data were subsequently analysed by comparing live cell counts on day 0 with live cell counts on day 3. Cell death was considered to have occurred when the viable cell number fell below the day 0 pre-treatment control number. Each treatment was assessed from eight replicates and three independent experiments.
Migration assay
Pore inserts (8 μm, Costar #3422) were incubated with 300 μl sterile fibronectin (Sigma) in PBS (10 μg/ml) for two hours at 37°C. Excess fibronectin was removed from the bottom of the insert by washing in PBS. Inserts were air dried for 15 minutes, then 650 μl cell culture medium +/- 25 nM AZD8055 was placed in the well and the insert suspended in it. A total of 40,000 TamR cells in normal growth medium were added to the insert and the plate was incubated for 24 hours at 37°C. Cells on the insert were fixed with 3.7% formaldehyde (15 minutes). Cells on the inside of the insert were removed with a cotton swab and cells that had migrated to the lower side of the insert membrane were stained with 0.5% crystal violet for 30 minutes. Excess stain was removed by repeated washing with distilled water. Migrated cells were counted under an inverted light microscope over five fields of view (x20). Each experiment included triplicate wells and the experiment was repeated twice.
Statistics
Statistical analyses were carried out using a two sided t-test and ANOVA with post hoc test. P <0.05 was considered significant.
Competing interests
JMWG, RIN and IRH are in receipt of research funding from AstraZeneca. SMG is an employee of AstraZeneca. NJJ and the work included in this study was predominantly research funded by AstraZeneca. The remaining authors have no conflict of interest.
Authors’ contributions
As Principal Investigator, JMWG conceived the study, participated in its design and execution and helped to draft and provide critical revision of the manuscript. RIH, RIN and SMG participated in the design of the study and provided further critical revision of the manuscript. NJJ drafted the manuscript, designed the experiments and carried out cell culture, Western blotting, ICC, proliferation, viability assays and PCR and interpreted the data. CMD, HJM and DB designed and carried out growth studies, analysed and interpreted this data. HJM also carried out migration assays. All authors read and approved the final manuscript.