Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro

Abstract

Introduction: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells.

Methods: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR.

Results: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER.

Conclusions: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.

Figure 1

Comparison of RAD001 and AZD8055 effect on cell growth and mTORC1/2 signalling pathways in TamR and MCF7-X cells. TamR (black squares) and MCF7-X cells (white squares) were grown for seven days in the presence of RAD001 (0 to 1,000 nM) (A) or AZD8055 (B) (0 to 100 nM) and cell number determined by Coulter Counting. The effect of AZD8055 on seven days growth of tamoxifen-resistant T47D-tamR cells similarly determined is shown in (C). Results expressed as% control are the mean (+/- SEM) of three to five independent experiments. *P <0.05 versus appropriate cell line control (0), **P <0.01 versus appropriate cell line control (0), ***P <0.001 versus appropriate cell line control (0). Western blot of 70% confluent TamR and MCF7-X cells treated for one hour with RAD001 or AZD8055 (0 to 100 nM) (D). Blots were probed with phospho- and total antibodies for mTORC1 (rapamycin sensitive) and mTORC2 (rapamycin insensitive) signalling pathways. Blot shown is representative of at least two independent experiments.

Figure 2

Time course of AZD8055 effect on mTORC1 and TORC2 signalling pathways in TamR and MCF7-X cells. Western blots of 70% confluent TamR (A) and MCF7-X (B) cells treated for 15 minutes to 24 hours with AZD8055 (0 to 100 nM). Blots were probed with phospho- and total antibodies for mTORC1 and two signalling pathways. In both cell lines mTORC1 and mTORC2 signalling pathways were rapidly inhibited by AZD8055. Blots shown are representative of at least two independent experiments. Western blots of 70% confluent TamR and MCF7-X cells treated for 15 minutes to 48 hours with AZD8055 (0 to 100 nM) probed with antibodies for MAPK activity (p-erk1/2) showing no effect (C). Blots shown are from representative experiments.

Figure 3

Effect of AZD8055 on proliferation and viability in TamR and MCF7-X cells. Immunocytochemical evaluation of MIB1 proliferation marker (Ki67) in TamR (A) and MCF7-X cells (B) treated for three days with AZD8055 (0 to 100 nM). Multiple fields of view (x20) were assessed for% cells expressing no/equivocal MIB1 staining. Results are from three independent experiments. * P <0.05 versus untreated control (ANOVA with post-hoc test). Sytox green impermeable nuclear stain was used to measure live cell count in TamR and MCF7-X cells in a viability cell assay before and after three days treatment with AZD8066 (C). After three days in the presence of 25 nM AZD8055, TamR live cell number fell below the pre-treatment count indicating some cell death with this agent. Live cell count (total minus dead cells) was a mean of eight replicates in three independent experiments. *P <0.05, ***P <0.001 for three days treatment with AZD8055 versus appropriate day 0 control ANOVA with post-hoc test. ANOVA, analysis of variance.

Figure 4

Effect of AZD8055 on migration in TamR cells. Twenty-four hours migration was measured over an 8 μm pore membrane coated with fibronectin in the presence or absence of 25 nM AZD8055. Results shown are from a representative experiment (n = 2).

Figure 5

One hour treatment with AZD8055 affects pER_ser167 protein expression in TamR and MCF7-X cells. Western blot of 70% confluent TamR and MCF7-X cells treated for one hour with AZD8055 (25 to 100 nM). Images show blots probed for pER (s118 and s167), total ER, mTOR phosphorylation on s2481 and s2448 to demonstrate mTOR inhibition and actin to indicate equal loading. The accompanying bar charts show densitometric analysis of western blot data normalised to actin. pER_ser167 but not pER_ser118 protein was inhibited in MCF7-X and TamR cells. Blots are representative of two independent experiments (A). Accompanying immunocytochemical images confirm the effect of AZD8055 on pER_ser167 but not pER_ser118 protein expression in TamR and MCF7-X cells. Immunocytochemistry was performed on TamR and MCF7-X cells fixed after treatment +/- AZD8055 (100 nM) for one hour and cells were stained with antibodies for pER_ser118 and pER_ser167(B). At least five fields of view were examined for staining and representative images (x20) shown. Results are shown from at least three independent experiments.

Figure 6

AZD8055 does not significantly affect ER regulated gene expression in MCF7-X and TamR cells. RNA was extracted from MCF7-X (A) and TamR (B) after 72 hours treatment with AZD8055 (0 to 100 nM) and gene expression analysed by semi-quantitative PCR. The image shows an ethidium bromide stained 2% agarose gel with the upper band showing cyclin D1, pS2 c-myc, bcl2 (only expressed in MCF7-X) and amphiregulin (only expressed in TamR) and the β-actin PCR product (lower band). The bar charts show actin-normalised values for the ER regulated genes which are not significantly affected by AZD8055. Data are representative of two independent experiments. Accompanying immunocytochemical images confirm that 72 hours treatment with a concentration range of AZD8055 (0 to 100 nM) does not significantly affect pS2 or total ER protein expression in MCF7-X or TamR cells (C). Images shown (x20) are representative of three independent experiments.

Figure 7

AZD8055 effect on growth of ER- acquired fulvestrant-resistant MCF7 (FasR) and T47D cells (T47D-fasR). FasR (A) and T47D-fasR (B) cells were grown for seven days in the presence of AZD8055 (0 to 100 nM) and cell number determined by Coulter counting. Results expressed as% control. **P <0.01, ***P <0. 001.

Figure 8

Greater inhibition of TamR (A) and MCF7-X (B) cell growth by the pure anti-oestrogen fulvestrant is apparent when the drug is used in combination with AZD8055. TamR cells were grown for seven days in the absence of tamoxifen. TamR and MCF7-X cells were then grown for seven days in the presence of fulvestrant (10^-7 M) and AZD8055 (0 to 25 nM). Cells were counted and the results expressed as a percentage of the untreated control. Results are means of four independent experiments. Statistical analyses used ANOVA with a post hoc test to compare doses of AZD8055 plus fulvestrant versus AZD8055 alone and AZD8055 plus fulvestrant versus the fulvestrant alone control (0). *P <0.05, **P <0.01, ***P <0. 001. ANOVA, analysis of variance.

Figure 9

Greater inhibition of MCF-7 cell growth is apparent when AZD8055 is used in combination with tamoxifen or oestrogen deprivation. Seven days growth of oestrogen dependent MCF-7 cells was compared in standard control medium (black squares), in the presence of 10^-7 M tamoxifen (grey square) or oestrogen-deprived medium (X cell medium) (white squares) with a concentration range of AZD8055 (0 to 100 nM). Triplicate wells of cells were counted by Coulter Counter and the results expressed as a percentage of the untreated control cells. Combination with 10 nM AZD8055 significantly increased growth inhibition in the presence of tamoxifen or oestrogen-deprived medium by 66% and 56%, respectively, versus anti-hormone alone. *P <0.05, **P <0.01, ***P <0.001 versus appropriate anti-hormone control in the absence of AZD8055 (0). Results shown are means of three independent experiments. Statistical analysis was performed with ANOVA and post hoc test. ANOVA, analysis of variance.