In contrast, in MCF7 X cells there was evidence from sytox green assays that while an anti proliferative effect occurred this was without any significant cell death with AZD8055 when used as a single agent. TamR cells have increased migratory ability compared to parental MCF 7 cells. Al though numbers of migrated TAMR cells were very modest, Trichostatin A following 24 hours treatment with AZD8055 TamR migration was shown to be reduced by 40% showing that dual mTORC1 2 blockade has the cap acity to impact on both resistant tumour cell growth and aggressiveness. Investigation of any cross talk between ER and mTOR signalling targeted by AZD8055 in TamR and MCF7 X cells Both TamR and MCF7 X cells were derived from oestrogen dependent MCF 7 breast cancer cells that have acquired tamoxifen or oestrogen deprivation resist ance, respectively, but still grow in an ER dependent manner.

In the TamR cell line, it is already known that there is prominent cross talk between erk1 2 and breast cancer cell growth amphiregulin, c myc and cyclinD1. mRNA expression of these ER regu lated genes Inhibitors,Modulators,Libraries was measured after Inhibitors,Modulators,Libraries 72 hours treatment with a concentration range of AZD8055 but failed to show any significant altered gene transcription in TamR or MCF7 X cells. ICC in TamR and MCF7 X cells confirmed that 72 hours treatment with 1 to 100 nM AZD8055 caused a concentration dependent reduction in cell number but did not alter expression of pS2 or ER protein in TamR or MCF7 X cells.

While only examining a limited panel of ER regulated genes, these data do suggest that the mTOR Inhibitors,Modulators,Libraries inhibitor impact was independent of changes in ER regulated transcriptional events and, hence, that mTOR and ER signalling are unlikely to be closely inter active in these acquired resistant models. The capacity for ER independent impact of AZD8055 was further supported by the demonstration that 25 nM AZD8055 phosphorylation of the ER s118 site in the ER AF 1 domain. In MCF7 X cells, MAPK and PI3K Akt Inhibitors,Modulators,Libraries also have the capacity to cross talk with ER at pERser118 and pERser167, respectively. Activation at such ER sites by cross talk can contribute to driving transcription of oestrogen ER regulated genes, notably amphiregulin which plays a part in the growth of TamR cells and also pS2 expression in MCF 7X cells. The pos sible contribution of cross talk between mTOR signal ling and ER in these models of tamoxifen or oestrogen deprivation resistance was thus investigated using AZD8055 in the current study.

Western blotting of MCF7 X and TamR confirmed prominent basal ER phosphorylation levels at ser118 and 167 in the latter model. Both models treated for one hour with Inhibitors,Modulators,Libraries AZD8055 showed, in conjunction with downregula tion of mTOR activity at s2448 and blog of sinaling pathways s2481, a concentra tion dependent inhibition of ER phosphorylation at s167. Total ER and phosphorylation of ER at s118 were not significantly affected by AZD8055.