de showed the presence of a sizable num ber of potential c Myc binding web-sites. To identify if c Myc binds to the Bim promoter, we analyzed its recruitment by chromatin immunoprecipita tion assays in BT474 cells. Benefits presented in Figure 7B show that c Myc is recruited for the initiation transcription site of BCL2L11 gene. Of note, we found this to become associated with all the binding of histone three acetylation and that of RNA polymerase II, which is indicative of gene transcription. Interestingly, we also noticed the recruitment from the E2F1 transcription aspect on this gene. Following mTORC1 inhibition by RAD001 remedy, as expected in the reduce of c Myc expression beneath these con ditions, an inhibition of c Myc binding to the Bim promoter was observed. This correlated using a loss of your transcription indicators.
In contrast, E2F1 binding was not impacted following RAD001 treatment suggesting that RAD001 mediated inhibition of Bim expression is E2F1 independent. Altogether, these information indicate that mTORC1 pro motes Bim expression by stabilizing c Myc on BCL2L11 promoter in the HER2 overexpressing inhibitor OTX015 breast cancer cell lines BT474. Discussion We used, in this study, BT474 cells that overexpress HER2 neu, and in which signaling downstream of this member in the EGF receptor family members is extremely active. Our benefits establish that, despite the potent and many survival signals which are related with HER2 activity, these cells depend on the expression of a single anti apop totic protein for their survival, because the down regulation of Mcl 1 is adequate to induce substantial rates of sponta neous apoptosis in these cells.
Mcl 1 appears to become cru cial even for the subpopulation of BT474 which have functions of cancer initiating cells, as its depletion signifi cantly reduces the number of mammospheres from this source these cells can form. Since the co depletion of pro apoptotic Bim mitigates the effects of Mcl 1 knock down on mammosphere formation, these effects probably result in the induction of cell death in sphere forming cells. We can not formally rule out, how ever, that Mcl 1 contributes to the biology of cancer initiating cells by mechanisms apart from regulation of cell survival stricto sensu. This aspect is at the moment getting investigated in our laboratory.
Given the part played by Mcl 1 in maintaining the survival of HER2 expressing cells, and in keeping a substantial pool of cancer initating cells amongst them, pathways that bring about the expression in the anti apopto tic protein Mcl 1 are expected to contribute for the pathogenesis of HER2 amplified mammary tumors. Con versely, pharmacological manipulations of these path ways may perhaps be of therapeutic benefit. Our investigation of published expression data hint on a selective enrichment for Mcl 1 trancripts in HER2 amplified mammary tumors compared to other mammary tumors.