Abstract
Abstract Background: Dysregulation of the cyclin D-CDK4/6-Rb axis occurs in a substantial proportion of ER-positive (ER+) breast cancers and has been linked with endocrine resistance. Adding the CDK4/6 inhibitor palbociclib to endocrine treatment has led to a substantial improvement of the outcome of patients with ER+ metastatic breast cancer. However, with the increasing clinical use, acquired resistance to palbociclib is merging as a new major clinical challenge. Methods: The ER+ cell lines T47D and MCF7 have been shown to be highly sensitive to treatment with palbociclib. Using long-term co-culture with increasing doses of Palbociclib, we generated MCF7 and T47D cell line clones with acquired resistance to Palbociclib. Three distinct resistant clones were selected for each cell line showing an IC50 shift from sensitive to resistant of approximately 300nM to 3uM for MCF7 and 400nM to 3.5uM for T47D, respectively. Resistant cell lines were characterized using RNA sequencing and mass spectrometry-based phosphoproteomics. Effects on selected target proteins (eg pAKT, pS6, pRB, RB or Cyclin D1) were confirmed using Western Blots. To modify resistance to palbociclib, a targeted in vitro drug-screen was performed using a range of inhibitors of the PI3K/AKT/mTOR and MEK pathways. Results: Western blot analysis of resistant cell lines demonstrated sustained down-regulation of Rb and phospho-Rb in response to palbociclib, which was reversible after discontinuation of palbociclib. Mass spectrometry identified >6,000 peptides across parental and resistant cells corresponding to 4,757 phospho-peptides and 5,337 phosphorylation sites. Pathway analysis suggested increased activity in the P3IK/AKT/mTOR pathway in resistant clones (including Akt1, p90S6K and mTOR), as well as changes in p53 and apoptotic regulation (e.g. phosphorylation of BAD). In addition, resistant clones showed multiple phosphorylation changes in the Rho/Rac pathway, suggesting changes in cytoskeletal organisation and a more invasive phenotype. Targeted drug screening showed a variable pattern across resistant clones with increased sensitivity to co-treatment of palbociclib with AKT inhibitors, PI3K alpha/delta inhibitors and/or MEK inhibitors in selected resistant clones, whereas pan-PI3K or PI3K beta/delta inhibitors showed limited efficacy in the selected clones. Conclusions: Phosphoproteomic analysis of palbociclib-resistant ER+ breast cancer cell lines demonstrated up-regulation of PI3K/AKT/mTOR and anti-apoptotic pathways. Resistant cell lines were sensitive to inhibition of PI3K/AKT/mTOR and/or MEK pathways with distinct patterns of activity across resistant clones suggesting that co-treatment of CDK4/6 inhibitors and PI3K/AKT and/or MEK inhibitors warrants further investigation as potential new therapeutic strategies in palbociclib resistance. Citation Format: Lenihan C, Bouchekioua-Bouzaghou K, Shia A, Wilkes E, Casado-Izquierdo1 P, Cutillas P, Schmid P. Characterization of resistance to the selective CDK4/6 inhibitor palbociclib in ER positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-02.
Published Version
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