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Introduction

Breast cancer is clinically divided into four categories, based on the following receptor profiles: Luminal A [estrogen receptor (ER)+, progesterone receptor (PgR)+, human epidermal growth factor receptor 2 (HER2)−], luminal B (ER+, PgR+, HER2+), basal like (ER−, PgR−, HER2−) and HER2-like (ER−, PgR−, HER2+). Luminal A/B tumors correspond to hormone-responsive breast cancer phenotypes (1) and anti-hormonal therapy, including anti-estrogens and aromatase inhibitors, represents the gold standard of treatment for this type of cancer (2). However, resistance to these agents may ultimately occur (3). Resistance to standard chemotherapy and to anti-hormone therapy is the main cause of treatment failure in patients with solid tumors.

Chemoresistance has a multifactorial origin. Molecular changes in different signaling pathways, including apoptosis and cell cycle regulation, modifications of the cellular phenotype, metabolic alterations of chemotherapeutics and dysregulation of inhibitors of apoptosis proteins (IAPs) are all events which may be involved in chemoresistance and in the increased malignancy of several types of tumor (4). As the steroid hormone signaling pathway activates phosphoinositide 3-kinase (PI3k)/protein kinase B (Akt) and that mammalian target of rapamycin (mTOR) is a downstream mediator of Akt, certain drugs acting as mTOR inhibitors have been developed and tested in order to overcome resistance to anti-hormonal therapy (5).

Sirolimus (SRL) was the first mTOR inhibitor molecule to be developed. It was obtained from the bacterium Streptomyces hygroscopicus and has been approved for renal transplantation (6). Everolimus (RAD001) is derived from SRL and contains a 2-hydroxy-ethyl chain that makes the drug more hydrophilic than SRL, increasing its oral bioavailability by ~10–16% (7). The mechanism underlying the action of SRL and RAD001 is the inhibition of mTOR complex 1 and the regulation of factors involved in several cellular functions, including protein synthesis, regulation of angiogenesis, lipid biosynthesis, mitochondrial biogenesis and function, cell cycle and autophagy (8). RAD001 has received approval from the US Food and Drug Administration for the treatment of hormone receptor-positive advanced breast cancer in combination with exemestane in post-menopausal patients with non-steroidal aromatase inhibitor-refractory disease (9). Unfortunately, conflicting data have been reported concerning the responsiveness of breast cancer cells to mTOR inhibitors. For example, a previous study has demonstrated that not all hormone-sensitive mammary tumor cell lines have good reactivity to RAD001 (10).

Among the multiple mechanisms involved in chemoresistance is escape from apoptosis, which is often determined by the increased expression of IAPs. Our group previously demonstrated that the upregulation of one of these IAPs, survivin, is involved in the resistance of human breast cancer cells to taxanes and also to K858, an inhibitor of kinesins (11,12). Given that survivin and estrogens are involved in the PI3k/Akt/mTOR transduction pathway, it is possible to hypothesize that survivin may be involved in the establishment of RAD001 resistance in certain hormone-responsive breast cancers. For this purpose, the effects of RAD001 in two human breast cancer cell lines, BT474 (luminal B) and MCF7 (luminal A) were analyzed, and the former was demonstrated to be responsive while the latter was resistant to RAD001. Following this, the potential involvement of survivin in the establishment of this resistance to RAD001 was examined.

Materials and methods

Cell culture and treatments

Two human breast cancer cell lines were utilized in the present study: MCF7 (Luminal A; ER+/PgR+/HER2−) and BT474 (Luminal B; ER+/PgR+/HER2+), both obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were grown at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine and 50 U/ml penicillin-streptomycin (all Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). RAD001 (a gift from Novartis International AG, Basel, Switzerland) was solubilized in DMSO to form a 100 mM stock solution that was utilized at final concentration of 100 nM. YM155 (Selleck Chemicals LLC, Houston, TX, USA) was solubilized in DMSO to form a 10 mM stock solution and used at a final concentration of 2.5 nM. Cells used as negative controls were treated with equivalent quantities of DMSO rather than RAD001, but were otherwise treated identically.

Cytotoxicity assay

To determine cytotoxicity, a sulforhodamine B colorimetric assay was performed. Cells (1.5×104) were plated on a 96-well plate, grown for 24 h and then treated with 100 nM RAD001 for 24, 48 and 72 h at 37°C. Cells were then fixed with 50 % trichloroacetic acid for 1 h at 4°C and stained for 30 min at room temperature with 0.4% sulforhodamine B in 1% acetic acid. Excess dye was removed by washing four times with 1% acetic acid. Protein-bound dye was dissolved in 10 mM TRIS (pH 10), and optical density was determined at 510 nm using a microplate reader.

Western blotting

Cells (2×106) were treated with 100 nM RAD001 for 48 h at 37°C, and then with and without 2.5 nM YM155 1 h prior to RAD001 treatment. Control cells were treated with equivalent quantities of DMSO. Cells then were lysed by incubating in lysis buffer (1% Triton, 0.1 % sodium dodecyl sulfate-SDS, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 2 mM EDTA) plus a protease inhibitor cocktail tablet (Roche Applied Science, Penzburg, Germany) for 30 min at 4°C. Lysates were then centrifuged at 16,000 × g for 15 min at 4°C and the supernatant was collected. The protein concentration was evaluated using the Bio-Rad Protein Concentration assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein lysate samples (50–100 µg) were separated by molecular weight on 10, 12 or 14% SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk and then incubated with primary antibodies overnight at 4°C, washed in Tris-buffered saline with 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated anti-mouse IgG (cat. no. A4416) or anti-rabbit IgG (cat. no. A0545) (both 1:5,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. The filters were then developed using enhanced chemiluminescence (Super Signal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Kodak X-Omat films (Kodak, Rochester, NY, USA). The primary antibodies used were as follows: Rabbit anti-survivin (1:1,000; cat. no. NB500-201; Novus Biologicals, Littleton, CO, USA), mouse anti-poly (ADP-ribose) polymerase 1 (PARP1; 1:500; cat. no. SC-8007; Santa Cruz Biotechnology, Dallas, TX, USA), which is able to detect both cleaved and uncleaved PARP1, mouse anti-caspase-8 (1:500; cat. no. 9746; Cell Signaling Technology, Danvers, MA, USA) which is able to detect both cleaved and uncleaved caspase-8, mouse anti-caspase-9 (1:500; cat. no. 9508; Cell Signaling Technology), mouse anti-B cell lymphoma 2 (Bcl2; 1:250; cat. no. Sc7382; BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-BCL2 associated X, apoptosis regulator (Bax; 1:250; cat. no. Sc493; BD Biosciences), rabbit anti-LC3 (1:500; cat. no. ABC232; Sigma-Aldrich, St. Louis, MO, USA), which is capable of recognizing the doublet of LC3 composed of the two single bands of LC3I and LC3II; rabbit anti-beclin-1 (1:500; cat. no. 62557; Abcam, Cambridge, UK), mouse anti-β-actin (1:750; cat. no. A5060; Sigma Aldrich, St. Louis, MO, USA).

Experiments were performed in triplicate and each band from the blots was quantified using ImageJ v.1.48 software (National Institutes of Health, Bethesda, MD, USA) and the mean value was calculated and expressed as densitometric units (DU).

Reverse transcription polymerase chain reaction (RT-PCR) assay

A total of 2×106 cells were treated with 100 nM RAD001 for 48 h at 37°C and then total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Moloney murine leukemia virus reverse transcriptase (New England BioLabs, Inc., Ipswich, MA, USA) was used to reverse-transcribe 1 µg total RNA into cDNA at 42°C. cDNA (5 µg) was then subjected to PCR in a buffer (New England BioLabs, Inc.) containing 25 pmol upstream and downstream primers and 1.25 U Platinum Taq polymerase and dNTP (both EuroClone, Pero, Italy). The number of amplified products, expressed in arbitrary optical density units, were normalized to GADPH expression, which was used as the housekeeping gene. The amplification reaction was performed in a Piko-Thermal Cycler (Finnzymes Instruments; Thermo Fisher Scientific, Inc.). The resulting PCR products were separated on 2% agarose gel and visualized using Gel-Red (Biotium, Inc., Fremont, CA, USA). The sequences of the human gene-specific primers and the sizes of the amplified products were as follows: GAPDH forward, 5′-AGATGTTCCAATATGATTCC-3′ and reverse, 5′-TGGACTCCACGACGTACTCAG-3′; 161 bp (Sigma-Aldrich, St. Louis, MO, USA); survivin forward, 5′-CAGATTTGAATCGCGGGACCC-3′ and reverse, 5′-CCAAGTCTGGCTCGTTCTCAG-3′; 206 bp (Primm, Milano, Italy). The PCR program was as follows: 94°C for 5 min, followed by 30 cycles at 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and a final step of 72°C for 5 min.

Cell apoptosis assay

Cells (2×106) were treated with 100 nM RAD001 or DMSO for 48 h at 37°C. Detached and adherent cells were harvested using trypsin-EDTA and washed with cold PBS. The cells were double stained with APC-Annexin V-allophycocyanie (cat. no. 550474, BD Biosciences, Franklin Lakes, NJ, USA) and 7-amino-actinomycin (7-AAD) (cat. no. 559925; BD Biosciences) in a calcium binding buffer (cat. no. 556454; BD Biosciences) according to the manufacturer's protocol and analyzed using the fluorescence activated cell sorting (FACS) cytofluorimeter FACSCalibur (BD Biosciences) and the CellQuest Pro software version 5.1 (BD Biosciences).

Statistical analysis and graphic programs

All results were analyzed using one-way analysis of variance and the significance was evaluated using the Tukey honest significant difference post hoc test. All figures were created using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA), all graphs were produced and statistical analyses conducted using Graph Pad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

The effect of RAD001 on cell viability was assessed, and the concentration of 100 nM RAD001 was selected based on the results of a previous study performed on a large panel of breast cancer cell lines (10). MCF7 and BT474 cells were treated for 24, 48 and 72 h with 100 nM RAD001. No significant effect was evident on cell viability in either cell line following 24 h of treatment, while a significant decrease in cell viability was evidenced following treatment for 48 h in BT474 cells, but not in MCF7 cells. The described effects on cell viability were similar when cells were treated for 72 h, without any significant variation compared with 48 h treatment (Fig. 1A). Based on these results, BT474 cells were indicated as sensitive and MCF7 cells indicated as resistant to RAD001. According to these data, RAD001 was used for the following experiments at the concentration of 100 nM for 48 h.

Figure 1. Responsiveness of breast cancer cell lines to RAD001 (A) Viability of BT474 and MCF7 cells following RAD001 treatment, expressed as the mean ± standard deviation. (B) Annexin V-allophycocyanin and 7-AAD expression as evaluated by FACS. Quadrant location for the representative images: Lower left indicates living cells, lower and upper right indicate apoptotic cells and the upper left indicates necrotic cells. (C) Each bar represents the FACS-reported value of apoptotic (black) and necrotic (grey) cells, expressed as mean ± standard deviation of three different experiments. (D) Western blotting analysis for PARP1, caspase-8, Bax, Bcl2 and β-actin. The densitometry quantitation is indicated for Bax/Bcl2 ratio. RAD001, everolimus; 7-AAD, 7-amino-actinomycin; FACS, fluorescence activated cell sorting; PARP1, poly(ADP-ribose) polymerase 1; Bax, BCL2 associated X, apoptosis regulator; Bcl2, B cell lymphoma 2.

In order to detect whether the decrease of cell viability determined by RAD001 was due to necrosis or to apoptosis, untreated cells and cells treated with 100 nM RAD001 for 48 h were double-stained with APC-conjugated Annexin V and with 7-AAD, and then analyzed by FACS. There was no significant difference in MCF7 cells following RAD001 treatment, and the cells remained practically negative for Annexin V and 7-AAD, confirming the resistance to RAD001. BT474 cells responded to RAD001 with a significant increase of Annexin V staining and a lower increase of 7-AAD, indicative of a higher rate of apoptosis (a 10-fold increase compared with the control) as compared with the rate of necrosis (a 5-fold increase compared to the control) (Fig. 1B and C).

The apoptosis induced by 48 h treatment with 100 nM RAD001 was further investigated by evaluating the expression of PARP, which is a key mediator involved in DNA repair and apoptosis that is activated in stress conditions by caspase-dependent cleavage in the late phase of apoptosis: Treatment with RAD001 resulted in PARP cleavage in BT474 cells but not in MCF7 cells, as evidenced by an uncleaved PARP1 band of 116 kDa present in every sample and a cleaved PARP1 band of 89 kDa appearing only in BT474-treated cells (Fig. 1D). The cleavage of caspase-8 and caspase-9 was also analyzed, and RAD001 treatment was revealed not to induce any cleavage of caspase-9 in either cell lines (data not shown). However, RAD001 treatment induced the cleavage of caspase-8 exclusively in BT474 cells, visualized as a band migrating at 43 kDa, while only the uncleaved band of caspase-9 of 57 kDa was visible in MCF7 cells (Fig. 1D). These data confirmed MCF7 resistance to RAD001-induced apoptosis, and demonstrated that the activation of apoptosis in BT474 cells followed the extrinsic pathway. The state of Bax, which induces apoptosis, and Bcl2, which inhibits apoptosis, was then investigated. These proteins are members of the Bcl2 family, which controls mitochondria permeability and cytochrome C release, and an increase of Bax/Bcl2 ratio is often indicative of apoptosis. The 21 kDa Bax band increased and 26 kDa Bcl-2 band decreased in BT474 cells treated with 100 nM RAD001 for 48 h, but not in MCF7 cells, resulting in an increase of the Bax/Bcl2 ratio in treated BT474 cells, with 0.94 densitometric units (DU) in the control compared with 2.20 DU in the treated cells, while it was not modified in MCF7 (Fig. 1D). These data confirmed the sensitivity of BT474 cells to RAD001 and the induction of apoptosis, and at the same time confirmed the lack of responsiveness of MCF7 cells to RAD001.

In order to investigate the mechanisms underlying the resistance of MCF7 to RAD001, the effect of 48 h 100 nM RAD001 treatment on survivin was analyzed. The 16 kDa survivin band and survivin mRNA were downregulated in BT474 cells, while survivin protein and mRNA were upregulated in MCF7 cells (Fig. 2A). Notably, RAD001 affected survivin in opposite ways in the two cell lines, which may justify the sensitivity of BT474 cells and, at the same time, the resistance of MCF7 cells. Thus, it was speculated that forced negative control of the upregulation of survivin in MCF7 may restore the sensitivity of these cells to RAD001. YM155, an inhibitor of survivin, was selected, however, since YM155 has also been described as an inducer of apoptosis (13–15), we different concentrations of YM155 were tested on MCF7 cells in order to identify the concentration capable of inhibiting survivin expression without inducing immediate apoptosis. The concentration of 2.5 nM was selected for YM155 and used for the following experiments. Consequently, MCF7 cells were treated with 2.5 nM YM155 1 h prior to treatment with 100 nM RAD001 for 48 h, and upregulation of survivin, which was visible in the cells treated with RAD001 only, was reversed by treatment with YM155 (Fig. 2B). At the same time, treatment of MCF7 cells with RAD001 and YM155 induced apoptosis, as supported by the presence of PARP1 and caspase-8 cleavage (Fig. 2B). Treatment with YM155 alone was also performed as control and a slight downregulation of survivin was observed, but PARP1 and caspase-8 cleavage were not induced (Fig. 2B). Treatment of MCF7 cells with RAD001 and YM155 did not induce cleavage of caspase-9 (data not shown), confirming that only the extrinsic pathway and not the intrinsic pathway was activated, as demonstrated in Fig. 1D.

Figure 2. Regulation of apoptosis by survivin expression. (A) Western blot (first and second rows) and reverse-transcription polymerase chain reaction (third and fourth rows) analysis of BT474 and MCF7 cells for survivin, β-actin and GADPH. (B) Western blot analysis of survivin, PARP1, caspase-8 and β-actin. (C) Viability of MCF7 cells treated with RAD001 and YM155, expressed as the mean ± standard deviation. PARP1, poly(ADP-ribose) polymerase 1.

MCF7 viability was investigated using sulforhodamine B staining following 24, 48 and 72 h of treatment with 100 nM RAD001 and 2.5 nM YM155, and a significant decrease of cell viability was observed when MCF7 cells were treated for 48 and 72 h with RAD001 together with YM155 (Fig. 2C). This effect was not evident following treatment for 24 h, and there was no significant effect on cell viability when the cells were treated alone with RAD001 or with YM155 (Fig. 2C). Taken together, these results demonstrated that YM155 was able to reverse the upregulation of survivin induced by RAD001, and at the same time was able to restore sensitivity to RAD001 with the induction of apoptosis in MCF7 cells.

The potential involvement of autophagy was then investigated, since RAD001 and mTOR inhibitors are described as autophagy activators (8,16), and also because the induction of apoptosis required a longer duration of RAD001 treatment. Autophagy and the autophagosome are controlled by specific interactions of several protein complexes: A protein complex including beclin-1 is required for phagophore nucleation, while another complex including the cytosolic and the lipidated form of LC3 protein (LC3I/LC3II) is necessary for elongation and closure of the autophagosome (17). Therefore, the effect of RAD001 on the expression of LC3I, LC3II and beclin-1 was evaluated. There was an increase of 14 kDa LC3II and of 52 kDa beclin-1 in BT474 cells treated with RAD001 for 4 h, but LC3II and beclin-1 expression decreased so that it was similar to the untreated condition following treatment with 100 nM RAD001 for 8 and 12 h (Fig. 3). A high ratio between the LC3II 16 kDa band and LC3I 14 kDa band is indicative of autophagy, and an increase of this ratio was observed following treatment for 4 h (Fig. 3), confirming that RAD001 induced autophagy in BT474 cells, but only as an early event. At the same time, RAD001 induced autophagy in MCF7 cells, as evidenced by an increase of LC3II and beclin-1 expression following 4 and 8 h of treatment with 100 nM RAD001, while the beclin-1 and LC3II/LC3I ratio were slightly reduced following treatment for 12 h (Fig. 3). The addition of YM155 to RAD001 treatment in MCF7 failed to modify the described effects on LC3 and beclin-1. Longer treatment of BT474 and MCF7 cells with 100 nM RAD001 also failed to modify the expression of LC3I–II and beclin-1 (data not shown). These results confirmed that autophagy was induced by RAD001 as an early event, and that it stopped following longer treatment durations.

Figure 3. Induction of autophagy following short RAD001 treatment times, as assessed by western blotting. Densitometry quantitation of bands are indicated for LC3I, LC3II and for the LC3II/LC3I ratio; the densitometry value of LC3I and LC3II was normalized to the β-actin value. LC3, microtubule associated LC3 (LC3-I–II).

Discussion

RAD001 is an inhibitor of m-TOR derived from SRL (7) that is utilized for the treatment of hormone receptor-positive advanced breast cancer in combination with exemestane (9). Unfortunately, a previous study has demonstrated that not all hormone-sensitive mammary tumors respond well to RAD001 in vitro (10). Consequently, two cell lines that responded differently to RAD001 were selected: One sensitive (BT474) and the other resistant (MCF7). Our group has previously demonstrated that the upregulation of survivin, a specific IAP component, contributes to establishing resistance to taxanes and kinesin inhibitors in breast cancer cells (11,12). Survivin is a downstream target of the PI3k/Akt/mTOR pathway that is involved in drug resistance (18). Therefore, the present study aimed to investigate whether survivin was involved in the described resistance of hormone-sensitive mammary tumors to RAD001. RAD001 was demonstrated to modify survivin expression in opposite ways in the two cell lines, characterized by downregulation in the sensitive cells and upregulation in the resistant cells. These data suggest a potential association between increased survivin expression and resistance to RAD001. In order to further investigate this association, YM155, an inhibitor of the promoter of survivin, was used to inhibit survivin expression. When upregulation of survivin was blocked with YM155, the sensitiveness to RAD001 was restored in MCF7 cells, as demonstrated by the increased apoptosis rate induced when MCF7 cells were treated with YM155 in combination with RAD001. These data strengthen the hypothesis that survivin is a mediator of drug resistance. This is supported by data suggesting that the increase of this protein in cancer tissues is an unfavorable prognostic marker and is associated with increased risk of recurrence (19). The overexpression of survivin has been proposed as a predictive factor in determining the response to chemotherapy (20).

Autophagy has been described as an alternative cell death pathway that is induced by rapamycin inhibitors when apoptosis is defective (21). Notably, short durations of RAD001 treatment were able to induce autophagy in the two cell lines, while 48 h of treatment were required for the induction of apoptosis. The dysregulation of autophagy serves a dual effect in cancer development, since its chronic inhibition promotes cancer but, at the same time, increased induction of autophagy is a mechanism of tumor cell survival in advanced conditions of acidosis and hypoxia (22). In particular, increased autophagy has been correlated with the development of resistance to chemotherapy in breast cancer (22,23). Autophagy is a multistep process that generates double-membrane vesicles called autophagosomes (24) which engulf the cytosolic form of LC3 (LC3-I) which is then conjugated to phosphatidylethanolamine to form LC3-II (18), so LC3-I is localized in the cytosol while LC3-II is present in the autophagosomes. The ratio between LC3-II and LC3-I, which measures the rate of autophagy, was significantly increased following treatment with RAD001 for 4 h. In parallel, beclin-1, which is also involved in the autophagy of breast cancer cells (25), demonstrated increased expression following treatment with RAD001 for 4 h. Therefore, a short period of RAD001 treatment induced autophagy in these cell lines, and autophagy was progressively depressed from 8–48 h, and at 48 h RAD001 activates apoptosis. These data suggest that autophagy and apoptosis are two sequential and independent effects induced by RAD001 in breast cancer cells. However, the inhibition of survivin induced by YM155 failed to modify the rate of autophagy induced by RAD001, which may be because the RAD001-induced control of survivin expression lasts longer than RAD001-induced autophagy. Consequently, it is possible to state that survivin controls apoptosis but does not interfere with the autophagy induced by RAD001.

In conclusion, survivin may be a potential target for the inhibition of the survival and proliferation of cancer cells, although its activation may involve several collateral pathways, switched on in case of interruption of the principal route of transduction. This is in accordance with different clinical trials conducted with YM155 in combination with other anti-cancer agents, as described in a previous review (26). A phase II study in HER2− metastatic breast cancer performed with YM155 plus docetaxel reported no significant differences compared with docetaxel alone (27). However, the authors have observed that, unless the pre-clinical data provided a good rationale for utilizing YM155, their study presented various limitations, including the lack of pharmacokinetic interaction analysis between the two drugs. At the same time, a phase II study on aggressive B-cell lymphoma treated with YM155 plus rituximab reported encouraging antitumor activity and a durable response (28).

Taken together with the results of previous studies, the results of the present study suggested that YM155 may be a valuable treatment for cancer. Furthermore, these data justify further research into drugs effective at targeting the survivin pathway, since the ability to negatively regulate survivin on multiple fronts may help to control the evasion of cancer cells from therapy-induced apoptosis.

References