Jordan Nicola J, Dutkowski Carol M, Barrow Denise, Mottram Huw J, Hutcheson Iain R, Nicholson Robert I, Guichard Sylvie M, Gee Julia M W
Breast Cancer Res. 2014 Jan 23;16(1):R12. doi: 10.1186/bcr3604.
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.
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.
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.
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.
内分泌抵抗性乳腺癌（BC）中PI3K/Akt/mTOR信号通路的上调已确定mTOR是与抗激素药物联合以控制耐药性的一个有吸引力的靶点。RAD001（依维莫司/Afinitor®），一种变构mTOR抑制剂，在这种情况下已被证明具有价值；然而，一些患者在治疗期间固有难治或复发，需要替代策略。在此，我们通过与RAD001在雌激素受体阳性（ER+）内分泌抵抗性BC细胞中进行比较，评估新型双mTORC1/2 mTOR激酶抑制剂（以AZD8055为例）的潜力。
他莫昔芬耐药（TamR）或雌激素剥夺耐药（MCF7-X）的体外模型单独用RAD001或AZD8055处理，或与抗激素药物氟维司群联合处理。终点指标包括生长、细胞增殖（Ki67）、活力和迁移，通过蛋白质印迹法监测PI3K/AKT/mTOR信号通路的影响。通过免疫细胞化学和逆转录-聚合酶链反应研究潜在的ER相互作用。
RAD001对MCF7来源的TamR和MCF7-X细胞的生长抑制作用较差（IC50≥1μM），能快速抑制mTORC1，但不抑制mTORC2/AKT信号通路。相比之下，AZD8055能快速抑制mTORC1和mTORC2/AKT活性，是TamR（IC50 18 nM）和MCF7-X（IC50 24 nM）以及另一个T47D来源的他莫昔芬耐药模型T47D-tamR（IC50 19 nM）的高效（P<0.001）生长抑制剂。AZD8055显著（P<0.05）抑制耐药细胞增殖，增加细胞死亡并减少迁移。此外，AZD8055加氟维司群对TamR或MCF7-X细胞进行联合处理，与单独使用任何一种药物相比，能更好地控制耐药细胞生长（P<0.05）。与他莫昔芬（P<0.01）或雌激素剥夺（P<0.05）联合使用AZD8055也能有效抑制内分泌反应性MCF-7细胞。尽管AZD8055抑制了TamR和MCF7-X中雌激素受体（ER）的ser167磷酸化，但对ER的ser118活性或几个ER调控基因的表达没有影响，这表明mTOR激酶抑制剂的作用在很大程度上不依赖于ER。两个获得性氟维司群耐药且缺乏ER的模型的生长抑制（IC50分别为18和20 nM）进一步证明了AZD8055具有不依赖ER的活性。
这是首份证明双mTORC1/2 mTOR激酶抑制剂有潜力控制获得性内分泌抵抗性BC的报告，即使在依维莫司无效的情况下也是如此。此类抑制剂在作为内分泌抵抗性疾病的二线治疗与抗激素治疗联合使用时，以及在早期内分泌反应阶段与抗激素药物联合使用以阻碍耐药性发展时，可能会显示出特别的益处。
