To analyze whether mechanisms leading to Bim transcription are particularly at stake in HER2 overe pressing tumors, we went back to our investigation of published gene e pres sion profiles of breast cancer patients using a probe matching approach as described above. As shown in Table 1, we found a statistically significant enrichment, in HER2 overe pressing breast tumors compared to other breast kinase inhibitor Sorafenib tumors, in one BCL2L11 specific probe. Regarding pro apoptotic genes, a statistical enrichment in one BID specific probe and in one BIK specific probe was also found. In contrast, other breast tumors appeared statistically enriched for two PMAIP1 specific probes and for one BAD specific one. While this tends to suggest that pathways leading to Bim transcription might be more active in HER2 overe pressing breast cancers, this should nevertheless be taken cautiously.

Indeed, we did not confirm a statistical enrichment for Bim e pression in HER2 overe pressing cancers by our gene matching approach involving 5 cohorts, even though enrichment for BID and BIK and impoverish ment for BAD and NO A were confirmed. In an independent attempt to confirm that HER2 overe pressing tumors e press Bim, we prepared lysates from five tumors that had been diagnosed as HER2 overe pressing ones by immunohistochemistry and per formed western blot analysis. As shown in Figure 4, these lysates e pressed detectable levels of anti apoptotic Bcl 2, Bcl L and Mcl 1. They also e pressed detectable levels of Bim. Most importantly here, we picked these samples because they correspond to tumors that had received no treatment prior surgery.

The e pression of pro apoptotic Bim detected does not, therefore, result from stress induced by treatment, but is more likely to result from signals that are inherent to the biology of these tumors. c Myc contributes to Bim e pression and Mcl 1 dependence of BT474 cells We investigated which signaling pathways might contri bute to Bim e pression in BT474 cells. Fo o3a is a member of the Fo o class of the forkhead family of winged heli transcription factors, which was reported to directly induce the transcription of Bim, in particular in some breast cancer cells. However, a RNA inter ference approach that successfully down regulated Fo o3A e pression in BT474 cells had no discernible effect on constitutive Bim protein e pression, ruling out that Fo o3A activity is responsi ble for this constitutive e pression.

c Myc is a transcription factor that resembles tran scription factors of the basic heli loop heli leucine zipper family. It is a major regulator of cell proliferation but it is also capable of promoting apopto sis. In particular, it was Drug_discovery reported to induce Bim in cer tain settings. We used a RNA interference approach to specifically down regulate c Myc in BT474 cells and we found that it induced a significant decrease in the e pression of Bim in the resulting cells.