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

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

Increased Ret signalling and impact of vandetanib in acquired tamoxifen resistant breast cancer

Deregulation of the tyrosine kinase Ret and its coreceptors (GFRα) has been implicated in neoplasia, and Ret is of interest as a therapeutic target in endocrine-treated breast cancer. This study evaluated in vitro impact of vandetanib, a tyrosine kinase inhibitor able to target Ret in addition to EGFR and VEGFR2, in ER+ breast cancer cells that have acquired resistance to tamoxifen treatment. Tamoxifen resistant TAMR and endocrine responsive MCF7 cells were grown in vitro for 7 days +/- vandetanib (0.5-5μM) +/- exogenous growth factors (10-50ng/ml), and also in continuous culture with vandetanib (1μM), to monitor growth impact and emergence of vandetanib resistance. For Western blotting or immunoprecipitation, log phase cells were transferred for 24hr to serum-free medium and pre-treated for 1hr +/- vandetanib followed by Ret ligands GDNF or artemin for 5mins. Immunohistochemistry for Ret activity was performed on an ER+ tamoxifen-treated clinical breast cancer TMA sample series using Y1062 Ret phospho-antibody with HScore staining assessment. Growth of TAMR cells was substantially inhibited by vandetanib (p<0.001, IC50 0.6μM) with complete cell loss by 17weeks, contrasting rapid emergence of resistance in endocrine responsive MCF7 cells. TAMR cells were more sensitive to vandetanib vs. gefitinib (p<0.001; 1μM each agent), indicating mitogenic signalling in addition to EGFR contributed to TAMR growth. TAMR cells had elevated basal Ret expression, activity and interaction with elevated GFRα3 coreceptor expression; mature VEGFR2 was not detected in TAMR cells. Exogenous GFRα3 ligands artemin or GDNF modestly stimulated TAMR cell growth and hyperactivated Ret, downstream kinases (including MAPK, AKT) and ER Ser167, confirming functional GFRα/Ret signalling and its cross-talk with ER. Increased phospho-Ret immunostaining was also associated with shortened DFI (p=0.036) and survival (p=0.011) in tamoxifen-treated clinical ER+ breast cancers. Vandetanib (0.5-1μM) depleted GFRα3/Ret activity and decreased phospho-EGFR in TAMR cells under basal and Ret ligand-stimulated conditions, inhibited MAPK, p70S6K and S6RP phosphorylation, and partially-reduced levels of phospho-AKT and phospho-ER. However, vandetanib failed to consistently impact on HER2, 3 or 4 activity; moreover, hyperactivation of all erbB receptors by exogenous heregulin B1 (10ng/ml) recovered AKT, MAPK, p70S6K and ER AF-1 phosphorylation in the presence of vandetanib and was able to overcome the basal growth-inhibitory impact of this agent in TAMR cells. These findings demonstrate a central importance for increased Ret signalling and its cross-talk with ER in tamoxifen resistant breast cancer that can be targeted in vitro by vandetanib. Further studies are required to determine optimal combination treatments with vandetanib to circumvent potential intrinsic resistance in clinical breast cancers that exhibit heregulin B1 enrichment

Multiple responses to EGF receptor activation and their abrogation by a specific EGF receptor tyrosine kinase inhibitor

BACKGROUND: Epidermal growth factor receptor (EGF-R) autophosphorylation is essential for its intracellular mitogenic signaling via the MAPK pathway and for interaction in other cellular processes. Inhibition of this activity in tumor cells that predominantly utilise EGF-R therefore offers an alternative approach to therapy. METHODS: The ability of a specific inhibitor of EGF-R tyrosine kinase, ZM 252868, (TKI) to alter various parameters related to growth in DU145 and PC3 cell lines was investigated, by immunocytochemistry, Northern blotting, Western blotting and invasion assays. RESULTS: In DU145 cultures, the total cell population and number of cells in cell cycle decreased in the presence of TKI whilst the apoptotic rate was significantly increased. Reduction in autophosphorylation of the EGF-R, membrane expression of EGF-R, activation of the MAPK, p38, and JNK enzymes and the invasive capacity of DU145 cells was observed in the TKI treated cells. Under the same conditions, PC3 cell growth and EGF-R expression and MAPK activation were not affected. The use of inhibitors of intracellular signaling indicated that the DU145 cells, in contrast to PC3 cells, predominantly utilize EGF-R activation of the MAPK signaling pathway for growth. CONCLUSIONS: In prostatic cancer patients, in whom androgen resistance has developed and whose tumors have upregulated EGF-R for growth, specific TKI's may offer an important therapy option

Tamoxifen-Induced Epigenetic Silencing of Oestrogen-Regulated Genes in Anti-Hormone Resistant Breast Cancer

<div><p>In the present study, we have taken the novel approach of using an <em>in vitro</em> model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.</p> </div

5-Aza/E2 inhibits TAM-Wd cell proliferation.

<p>A. TAM-Wd and MCF-7 cell concentration response to E2 challenge (1×10⁻¹² M to 1×10⁻⁷ M) ±5-Aza (1×10⁻⁶ M). Cell counts were taken on day 7 of culture. Data shown represent E2-treated cell counts as a percentage of non-E2 treated control cells. Cell counts are significantly lower in 5-Aza treated TAM-Wd cells, co-treated with E2 at a concentration of 1×10⁻¹⁰ M and greater (*p<0.017). B. TAM-Wd cell concentration response to E2 challenge (1×10⁻¹² M to 1×10⁻⁷ M) +5-Aza (1×10⁻⁶ M) ±TAM (1×10⁻⁷ M). Cell counts were taken on day 7. Data shown represent E2-treated cell counts as a percentage of non-E2 treated control cells. The co-addition of TAM to 5-Aza treated TAM-Wd cells significantly changes the effect of E2 from a concentration of 1×10⁻⁹ M and greater (*p<0.026). C. Anchorage-dependent proliferation assay of TAM-Wd cells treated with E2 (1×10⁻⁹ M) ± TAM (1×10⁻⁷ M) in the presence of 5-Aza (1×10⁻⁶ M) for 14 days. The data shown represent actual cell number/well recorded over 3 independent experiments. By day 14, there are significantly more cells in the 5-Aza and 5-Aza/E2/TAM treated cells compared to TAM-Wd +5-Aza/E2 treated cells (*p<0.001). D. Cell cycle analysis using flow cytometric analysis of propidium iodide-stained TAM-Wd cells. Data represent percentage of cell in each phase relative to the total population.</p

Tamoxifen resistant cells retain ER expression and TAM-resistance following withdrawal from the drug.

<p>A. RT-qPCR evaluation of the basal total ER mRNA expression in MCF-7, TAM-R in the presence of TAM) and TAM-Wd cells. Data are shown as arbitrary units after normalisation to actin (n = 3) (*p<0.001). Lysates were western blotted for total ER protein and β-actin (loading control). Data are representative of 3 independent experiments. B. MCF-7, TAM-R and TAM-Wd cell concentration response to TAM (1×10⁻¹⁰M to 1×10⁻⁶ M). Cell number was assessed after 7 days. The data shown represent the percentage cell number relative to non-treated control cells (*p<0.001). The bar graph depicts flow cytometry data was used to determine the percentage of cells in S-phase following TAM treatment (1×10⁻⁷ M). A significant reduction in S-phase was observed only in the MCF-7 cells (*p<0.001). C. Cell proliferation inhibition in response to fulvestrant (1×10⁻⁷ M). Cell counts were taken on days 4, 6, 8 and 11. The data shown represent the cell number relative to time-matched non-treated control cells. Cell counts for all cell-lines treated with fulvestrant were significantly reduced compared to vehicle treated controls by day 11 of culture (*p<0.001).</p

pS2 and PgR are silenced by DNA-methylation in tamoxifen-resistant cells.

<p>RT-qPCR evaluation of pS2 (A) and PgR (C) mRNA expression in MCF-7, TAM-R and TAM-Wd cells ± E2 (1×10⁻⁹ M) for 48 hrs. Data was normalised to actin (n = 3). Gene expression is significantly induced following E2 challenge in the MCF-7 cells (*p<0.001). The right panel of the figure shows parallel ICC for pS2 (A) and PgR (B) protein (10× magnification) (representative of 3 independent experiments). Clonal bisulphite sequencing analysis was performed across pS2 (B) and PgR (D) gene promoter regions in MCF7, TAM-R and TAM-Wd. PS2 (B) and PgR (D) promoter region maps are shown on the left panel. The PCR amplicons (441 bp for pS2 and 566 bp for PgR) interrogated a total number of 12 CpG sites pS2 gene (B) and 53 CpG sites for PgR (D) located downstream the TSS. Bisulphite maps determined by direct sequencing of individual clones show the density of methylated CpG site (black circle) and unmethylated CpG site (white circle) at individual CpG sites. The line plots on the right panel show the percentage of methylation of each CpG site interrogated in each cell line for pS2 (B) and PgR (D) genes. The lines on the gene promoter maps represent CpG sites and the arrow the Transcriptional Start Site (TSS). The dashed red box denotes the genomic region with the major changes in DNA methylation that was further analyzed in the line plot.</p

Genes associated with an anti-proliferative function are silenced by DNA methylation in tamoxifen-resistant cells.

<p>A. MeDIP coupled PCR to determine the methylation status of RASAL1, DUSP7, ATP2B4 and GDF15 in MCF-7, TAM-R and TAM-Wd cells, relative the expression of PEG3 (an imprinted gene). Data is expressed as 2^∧−ΔΔct (n = 3). GAPDH is included as a negative control to demonstrate the successful enrichment of methylated material. The data demonstrate a greater enrichment of methylated DNA in resistant vs tamoxifen-sensitive MCF-7 cells for all four genes (*p<0.05). B. RT-qPCR evaluation of GDF15 expression in MCF-7, TAM-R and TAM-Wd cells +E2 (1×10⁻⁹ M), +5-Aza (1×10⁻⁶ M), 5-Aza + E2 or 5-Aza + E2+ TAM (1×10⁻⁷ M) for 48 hrs. Data shown are normalised to GAPDH and presented relative to the expression calculated for vehicle-treated cells. In the absence of 5-Aza, GDF15 expression is only significantly increased by E2 challenge in MCF-7 cells (*p<0.001). The lower panel shows a western blot of mature, processed GDF15 protein (18 KDa) and β-actin (loading control) (representative of 3 independent experiments). C. Clonal bisulphite sequencing analysis for the GDF15 promoter region in TAM-Wd cells ±5-Aza (1×10⁻⁶ M) for 48 hrs. Colours and symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040466#pone-0040466-g003" target="_blank">Figure 3</a>.</p

Recombinant GDF15 is anti-proliferative and induces apoptosis in the TAM-Wd cells.

<p>A. Dose response to recombinant GDF-15 protein (1, 3, 10 ng/ml) in MCF-7, TAM-R and TAM-Wd cells. Cell number was assessed after 48 hrs post-treatment. The data shown represent the cell number relative to non-treated control cells. The dose-dependent anti-proliferative effect was statistically significant from 1 ng/ml across all cell-lines (*p<0.001). B. Both the addition of recombinant GDF15 and 5-Aza/E2 caused an increase in apoptotic cells as determined by flow cytometric analysis of M30 bound, TAM-Wd cells. Increases in apoptosis were significant for both treatments (*p<0.001), although 5-Aza/E2 induced a greater response.</p