Carol Mary Dutkowski

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

IntroductionUpregulation 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.MethodsIn 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.ResultsRAD001 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.ConclusionsThis 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.

Tamoxifen-induced epigenetic silencing of oestrogen-regulated genes in anti-hormone resistant breast cancer.

In the present study, we have taken the novel approach of using an in vitro 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.

Antihormone induced compensatory signalling in breast cancer: an adverse event in the development of endocrine resistance.

Abstract Using MCF7 breast cancer cells, it has been shown that antihormones promote expression/activity of oestrogen-repressed tyrosine kinases, notably EGFR, HER2 and Src. These inductive events confer responsiveness to targeted inhibitors (e.g., gefitinib, trastuzumab, saracatinib). We observed that these antihormone-induced phenomena are common to ER+HER2– and ER+HER2+ breast cancer models in vitro, where targeting of EGFR, HER2 or Src alongside antihormone improves antitumour response and delays/prevents endocrine resistance. Such targeted inhibitors also subvert acquired endocrine resistant cells which retain increased EGFR, HER2 and Src (e.g., TAMR and FASR models derived after 6–12 months of tamoxifen or Faslodex treatment). Thus, antihormone-induced tyrosine kinases comprise “compensatory signalling” crucial in limiting maximal initial antihormone response and subsequently driving acquired resistance in vitro. However, despite such convincing preclinical findings from our group and others, clinical trials examining equivalent antigrowth factor strategies have proved relatively disappointing. Our new studies deciphering underlying causes reveal that further antihormone-promoted events could be pivotal in vivo. Firstly, Faslodex induces HER3 and HER4 which sensitise ER+ cells to heregulin, a paracrine growth factor that overcomes endocrine response and diminishes antitumour effect of agents targeting EGFR, HER2 or Src alongside antihormone. Secondly, extended antihormone exposure (experienced by ER+ cells prior to adjuvant clinical relapse) can “reprogramme” the compensatory kinase profile in vitro, hindering candidate antigrowth factor targeting of endocrine resistance. Faslodex resistant cells maintained with this antihormone for 3 years in vitro lose EGFR/HER2 dependency, gaining alternative mitogenic/invasion kinases. Deciphering these previously unrecognised antihormone-induced events could provide superior treatments to control endocrine relapse in the clinic.

Abstract P3-05-19: A new cell panel to study oestrogen receptor loss in acquired endocrine resistant breast cancer

Background: Oestrogen receptor positive (ER+) breast cancer patients can acquire endocrine resistance and 10-20% tumours have lost ER at relapse. While growth factor pathway hyperactivity and ER promoter methylation contribute to de novo ER negativity, ER loss in acquired resistance is largely unexplored. We have recently developed 11 lines from MCF7, T47D, BT474 & MDAMB361 cells to model acquired resistance emerging with prolonged (3 year) endocrine treatment both in ER+/HER2- and ER+/HER2+ disease. Here we establish prevalence of acquired ER loss in the panel, determine any association with aggressiveness, and explore ER loss mechanisms in acquired endocrine resistance. Methods: Authenticated acquired resistant models derived from endocrine responsive lines cultured for 3 years with 10-7M tamoxifen (TamR), 10-7M fulvestrant (FasR) or oestrogen deprivation (5% charcoal-stripped foetal calf serum SFCSR) were profiled for ER & PR by PCR, immunocytochemistry and Western blotting (+/- 1-2wk antihormone withdrawal), for 2nd-line endocrine responsiveness and for migration using Boyden chamber assays vs. time-matched controls. Src kinase, EGFR, HER2, MAPK & AKT activity were examined and whether their respective inhibition using saracatinib or gefitinib (1μM), trastuzumab (100nM), U0126 or LY294002 (5μM) for 1wk restored ER. ESR1 promoter methylation was examined by bisulfite modification & MethyLight PCR. Results: Substantial ER mRNA & protein loss occurred in 7/11 long-term acquired endocrine resistant lines. This was irreversible by antihormone withdrawal and paralleled by complete PR loss and endocrine growth-insensitivity. While seen in all fulvestrant resistant lines, ER loss was less frequent with tamoxifen (in MCF7TamR & MDATamR) and only seen in oestrogen deprived resistant T47DSFCSR cells. Increased migration accompanied acquired ER loss in ER+/HER2- derived MCF7TamR, MCF7FasR & T47DSFCSR cells and was saracatinib-sensitive. Src and further growth factor pathway activity increased in several acquired resistant models, and ER loss associated with increased EGFR/HER2 in the MCF7- & MDA-derived cells and with increased MAPK activity in all lines. Weak ER recovery was seen in antioestrogen resistant models treated with saracatinib (MDATamR, MCF7FasR), gefitinib (MDATamR, BT474FasR, MCF7FasR/TamR) or trastuzumab (MCF7TamR). ESR1 DNA methylation was only prominent in MDATamR and MCF7TamR cells. No inhibitor restored ER in the T47D-derived cells, including T47DSFCSR which also lacked ESR1 methylation. Conclusions: Although ER loss is very prominent in this acquired resistant cell panel, it demonstrates there is capacity of prolonged antihormones, chiefly antioestrogens, to promote receptor loss independent of initial HER2 status. Acquired ER loss clinically would be expected to confer endocrine insensitivity and poorer prognosis given the panel findings. Where ER loss emerged with antioestrogens there was some mechanistic-overlap with de novo ER negativity, including ER promoter methylation for acquired tamoxifen resistance. Our future studies will use the panel to address if targeting these mechanisms can be optimized for ER recovery or if further mechanisms also drive ER loss in acquired resistance, notably for prolonged oestrogen deprivation. Citation Format: Julia M Gee, Mei Yee Meng, Richard A McClelland, Huw J Mottram, Susan R Kyme, Pauline Finlay, Lindy Goddard, Carol M Dutkowski, Denise Barrow, Walther Parson, Heidi Fiegl. A new cell panel to study oestrogen receptor loss in acquired endocrine resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-19.

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.