The parental MCF7 and MCF7 EGFR cells showed a small increase in MAPK1/3 activation after E2 stimulation which seems consistent with the results of Migliaccio et al. who observed a 2 3 fold MAPK1/3 activation in MCF7 cells several minutes after estradiol exposure by measuring radiolabelled phosphate incorporation in a MAPK substrate. However, the increase MAPK1/3 activa tion by EGF in our MCF7 sellekchem and MCF7 EGFR cells is much bigger than the activation by estradiol. In tamoxifen resistant breast tumour cells an agonistic effect was observed by tamoxifen both at the level of ER mediated transcription and cell proliferation. It has been suggested that these effects of tamoxifen depend on the phosphorylation of ER by MAPK1/3.

However, not all groups find agonistic effects of tamoxifen on transcription and/or proliferation after increased MAPK activation and ER serine 118 phosphorylation. Similarly, Inhibitors,Modulators,Libraries in our MCF7 EGFR model also no agonistic effects of tamoxifen were observed on proliferation and transcription. This is not surprising as the proliferation of MCF7 EGFR cells after EGF stimulation is already high, and any possible additional agonistic effects of tamoxifen Inhibitors,Modulators,Libraries may therefore not become manifest. However, it may also be the result of other cell types being used in the previous studies compared to our present cell lines. The lack of an agonistic effect of tamoxifen on transcription after EGFR activation actually suggests that no agonistic effects of tamoxifen are induced in our Inhibitors,Modulators,Libraries MCF7 EGFR cells by enhanced EGFR signalling.

EGFR activation in MCF7 EGFR cells caused strong downstream activation of both the MAPK and Akt signalling cascades. Using specific inhibitors we demon strated that the Inhibitors,Modulators,Libraries MEK/MAPK pathway is not dominant in EGFR driven proliferation. Recently, using insertion mutagenesis in an estrogen dependent breast carcinoma cell line, a panel of 7 candidate breast cancer anti estrogen resistant genes were identified that directly underlie estrogen independence leading to tamoxifen resistance, including both EGFR, AKT1, and AKT2. Importantly, the mRNA levels of these latter candidates in breast cancer material were significantly correlated with progression or metastasis free survival. These data support our findings about the importance of the PI3K/Akt in the EGFR signalling leading to estrogen independent proliferation and tamoxifen insensitivity.

Inhibitors,Modulators,Libraries The remaining question is which of the downstream targets of AKT are ultimately responsible for EGF induced prolifer ation in MCF7 EGFR cells. One of the candidates may be c Jun NH2 terminal kinase, which can regulate activator protein 1 transcription of e. g. cyclin D1 and other proliferation and survival genes. This kinase inhibitor Dorsomorphin hypothesis is strengthened e. g. by the data of Johnston et al, showing increased JNK activity and AP 1 DNA binding in tumours of resistant patients.