Discussion Resistance to endocrine therapy in breast cancer remains a major problem in the clinic. The mechanism behind this resistance is complex and it is still unclear whether tamoxifen kinase assay resistance is based on 1 decreased transcrip tion inhibition and consequent proliferation inhibition, 2 decreased proliferation inhibition via non classical genomic or non genomic actions of the ER, or 3 ER independent mechanisms. Here we studied the role of EGFR signalling in this process, using estrogen responsive MCF7 cells that have increased expression of wild type EGFR. We showed that EGF driven signalling in these cells is sufficient to maintain ER independent cell proliferation. We generated a MCF7 cell line with ectopic expression of EGFR, which allowed the unbiased analysis of the inter action of EGFR and ER signalling.

In contrast, in many studies on the mechanism of tamoxifen Inhibitors,Modulators,Libraries resistance, MCF7 cells are used that already have an increased expression or constitutive activation of EGFR and/or Inhibitors,Modulators,Libraries downstream MAPK or Akt activation due to long term culture in the presence of tamoxifen. This prohibits the investigation of the intrinsic effect of EGFR signalling on the antagonistic activity of tamoxifen in cells that, in the absence of EGF, respond similarly as the parental MCF7 cells. With respect to ER expression, this was similar in our MCF7 EGFR and parent Inhibitors,Modulators,Libraries MCF7 cells, and resembles tamoxifen resistant ER positive human tumours that express ER at normal levels. Therefore, our MCF7 EGFR cell line represents an important tool to study the mechanisms of tamoxifen resistance in a more clinically relevant model.

Ectopic expression of human EGFR in MCF7 cells induced cell proliferation upon stimulation with EGF, which was ER independent, since ER knock down did not affect EGF induced proliferation. In agreement with this, EGF induced proliferation was not blocked by tamoxifen or fulvestrant. Inhibitors,Modulators,Libraries Therefore, increased EGFR expression in ER positive breast cancers may be a sole important determinant for prediction of anti estrogen resistance. Although our data are consistent with litera ture data showing tamoxifen, and also fulvestrant resistance upon increased EGFR expression in breast cancer cells, typically these studies involved human breast cancer cell lines that were long term cultured in the pres ence of these antagonists. It cannot be excluded that additional changes in other cellular Inhibitors,Modulators,Libraries signalling path ways parallel or downstream of the EGFR may be mutated in these models as well. It is relevant to note that while EGF induced cell proliferation in MCF7 EGFR cells find more information was ER independent and tamoxifen insensitive, the majority of E2 induced transcriptional changes in MCF7 EGFR cells remained sensitive to tamoxifen after EGF stimulation.