Abstract: PP06-2

Different Mechanisms Utilized By Mutant Estrogen Receptor Alpha Proteins to Activate Transcription in a Ligand-Independent Manner

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Abstract


Approximately 70% of breast cancers are estrogen receptor + (ER)-positive and are driven by estrogen. Targeting ER+ with endocrine therapies, such as tamoxifen and aromatase inhibitors, provides effective adjuvant treatment for this patient subset with reductions in recurrence approaching 50%. Still, many tumors acquire resistance, and in ~20% of these cases, gain-of-function mutations in the ER+ gene (ESR1) are implicated. view more

Approximately 70% of breast cancers are estrogen receptor + (ER)-positive and are driven by estrogen. Targeting ER+ with endocrine therapies, such as tamoxifen and aromatase inhibitors, provides effective adjuvant treatment for this patient subset with reductions in recurrence approaching 50%. Still, many tumors acquire resistance, and in ~20% of these cases, gain-of-function mutations in the ER+ gene (ESR1) are implicated. Amino acid substitutions in the receptor's ligand binding domain (LBD) result in mutant receptors displaying ligand-independent activity, enhanced binding to some steroid receptor coactivators (SRCs), and resistance to subsequent endocrine therapy (e.g., anti-estrogen fulvestrant). In addition, rarer translocations of ESR1 to other genes create fusion proteins that lack the LBD and cannot be targeted with anti-estrogens. Therefore, new approaches are critically needed in treating ER+-positive metastatic breast cancer in patients expressing these different ER+ mutants. Our studies focus on the most prominent of the ER+ LBD mutants, Y537S and D538G, and the ESR1-YAP1 fusion protein. While efforts to develop more effective anti-estrogens (e.g. new oral Selective ER Downregulators, SERDs) may be useful, additional therapeutic strategies designed to concomitantly target coactivators (CoAs) offer a unique approach to enhance treatment (or prevent development) of recurrent disease. To accomplish targeting of the proper CoA, the set of CoAs that bind mutant ER+ proteins must first be determined. Here, we have setup a mass spectrometric, unbiased proteomic approach, based on our prior published "estrogen response element (ERE) DNA pulldown" system with wild-type (WT) ER+ (1), to profile the entire CoA "complexome" for each mutant ER+ protein. Mass spectrometric data of CoA recruitment to each mutant was compared to the WT receptor to identify potential new therapeutic targets. Interestingly, we observed specific CoAs (or complexes) indeed display robust enhanced binding to a mutant ER+ protein over the WT receptor (e.g., SRCs, p300, MLL4 complex with Y537S; SRC-1, SRC-3, p300, and SAGA complex with D538G; 26S proteasome with ESR1-YAP1 fusion protein). Based on these findings, we tested the effect of inhibiting select CoA candidates on their ability to activate transcription of an ERE-driven luciferase reporter mediated by the different ER+ mutant receptors. We found that a new "pan-SRC" small molecule inhibitor (SMI) called SI-1 reduced Y537S and D538G activities, the proteasome inhibitor MG-132 reduced ESR1-YAP1 activity, and siRNA targeting MLL4 reduced Y537S activity. These data suggest that SMIs targeting these distinct CoAs may be promising new therapeutics, as single agents or in combination with new oral SERDs, to inhibit growth of breast cancer cells expressing these ER+ mutant proteins.

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