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Among them, activation of alternative cellular compensatory pathways could explain primary resistance or the emergence of rapid resistance. Open in a separate window. In vitro experiments were performed at least two times and at least in triplicate. Moreover, ER protein expression showed a marked increase with BYL treatment that was mitigated by the addition of fulvestrant. A representative number of biopsies at least two biological replicates per condition was processed to obtain RNA and submitted for gene expression analysis. Consistent with our PDX data, these clinical studies indicate that dual PI3K and ER blockade is effective even in patients who had progressed on previous anti-estrogen therapies.

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This bidirectional crosstalk seems to also occur in the breast where, in addition to our findings showing ER activation upon PI3K inhibition, inactivation of ER appears to be associated with activation of PI3K signaling 18

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Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER binding sites and increased occupancy by the ER of promoter regions of upregulated genes. Mice were sacrificed and tumors obtained on day 4, 2 hours after the last dose, and processed to obtain RNA for microarray gene expression analysis. These data suggest that blockade of estrogen function mitigates the effects mediated by PI3K inhibition. We also used RNAseq to evaluate gene expression changes in breast cancer patients who underwent BYLbased therapy to validate our in vitro findings on ER expression. This indeed proved to be the case, and we found a consistent increase in ER transcript and induction of a luminal signature typical of hormone-responsive breast cancers in cell lines, murine models, and patient samples upon suppression of the PI3K pathway.

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