Abstract P4-03-05: AP-1 as a potential mediator of resistance to the cyclin-dependent kinase (CDK) 4/6-inhibitor palbociclib in ER-positive endocrine-resistant breast cancer
Authors: De Angelis C , Nardone A , Cataldo ML , Veeraraghavan J , Fu X , Giuliano M , Malorni L , Jeselsohn R , Osborne KC , Schiff R 
Published: February 2018

Abstract
Background: The CDK4/6-inhibitor palbociclib (Palbo) in combination with endocrine therapy (ET) substantially improves progression-free survival compared to ET alone. However, almost all initial responders eventually develop resistance and relapse. Delineating the early adaptive signaling and the mechanisms underlying resistance to CDK4/6 inhibition is therefore crucial to identify new biomarkers and therapeutic targets to enhance the efficacy of Palbo and improve patient outcome.
Materials and Methods: MCF7 parental (P) cells and derivative lines made resistant (R) to tamoxifen (TamR), estrogen deprivation (EDR), or fulvestrant (FulR) were used. The MCF7P line and its endocrine-R (EndoR) derivatives were exposed to increasing concentrations of Palbo to generate acquired Palbo-R (PDR) models. The proteomic/signaling profiles of P and EndoR cells upon short-term Palbo treatment and as PDR develops were determined using reverse-phase protein arrays (RPPA). Transcriptional activity of the activator protein-1 (AP-1) transcription factor (TF) was measured by luciferase reporter assay. Global AP-1 blockade was achieved using a pINDUCER system to express doxycycline (Dox)-inducible dominant-negative (DN) c-Jun that lacks the transcriptional activation domain. Cell growth and colony formation were assessed using methylene blue staining and clonogenic assays, respectively. Levels of phosphorylated (p)-RB and CDK2 were assessed by Western Blot.
Results: In P and all EndoR cell models, Palbo inhibited cell growth and colony formation in a dose-dependent manner, though the inhibitory effect was greater in the EndoR cells compared to P cells [IC50 value of P cells &gt;3 times that of EndoR lines (p&lt;0.001); clonogenic % inhibition at 100nM = 54 in P and &gt;85 in EndoR lines (p&lt;0.001)]. Across the P and all EndoR models, short-term Palbo treatment resulted in increased levels of several membrane and intracellular signaling molecules, TFs, and enzymes. Among these, the AP-1 TF components c-Jun and p-c-Jun showed the highest increase across all models, with the utmost change observed in the TamR model (Fold-change = 4.4, 4.0 for total and p-c-Jun, respectively). Since we also observed that acquired resistance to Palbo in the TamR model was associated with higher AP-1 transcriptional activity and increased total and p-c-Fos levels, we assessed the efficacy of combining Palbo with AP-1 blockade in the TamR model. In two independent TamR clones ectopically expressing inducible DN-c-Jun, AP-1 blockade (+Dox) in combination with Palbo was highly effective in inhibiting cell growth and reducing p-RB and CDK2 levels compared to single agent treatments. In addition, in both the TamR/DN-c-Jun-expressing clones, the combination of Palbo, AP-1 blockade, and fulvestrant resulted in cell death and a significantly greater cell growth inhibition compared to any dual or mono treatments.
Conclusion: Our results suggest activation of AP-1 as a potential mechanism of resistance to Palbo in ER+ EndoR models. Transcriptomic profiling of the Palbo-sensitive and R cells, currently underway, will provide an in-depth understanding of the role of AP-1 as well as other key targets and associated molecular mechanisms in Palbo resistance.
Citation Format: De Angelis C, Nardone A, Cataldo ML, Veeraraghavan J, Fu X, Giuliano M, Malorni L, Jeselsohn R, Osborne KC, Schiff R. AP-1 as a potential mediator of resistance to the cyclin-dependent kinase (CDK) 4/6-inhibitor palbociclib in ER-positive endocrine-resistant breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-05.