Bode, AM

Abstract Background: The primary mechanism by which tamoxifen (Tam) and its metabolites exert their biologic effects is through estrogen receptor (ER) binding and inhibition of ER signaling. We and others demonstrated that endoxifen (Endx) has greater antitumor activity in vitro and in vivo compared to Tam and the first-in-human Endx phase I study demonstrated its antitumor activity in patients with prior progression on Tam (Goetz SABC 2015). PKCs are a family of serine/threonine-specific protein kinases that regulate signaling pathways involved in cell proliferation and tumorigenic transformation. Our prior protein docking studies suggested endoxifen may be a substrate for PKCs. Here we report the effects of Tam and Endx on PKCβ1 binding, kinase activity, as well as interactions between PKCβ1 and ERα. Methods: Surface Plasmon Resonance (SPR, Biacore T200, GE Healthcare) was used to evaluate binding of Tam, N-desmethyl Tam (NDMT), 4-HT, and Endx to PKCβ1 and PKCβ2. The effects of Tam and Endx on PKCβ1 kinase activity were determined. Proliferation and colony formation in MCF7 parental and PKCβ1 overexpressing cells were evaluated. siRNA silencing was used to knockdown PKCβ1 expression in the following cells: MCF7 aromatase expressing cells that were either sensitive (MCF7/AC1) or resistant to letrozole (MCF7/AC1 L-resistant); T47D; and MDA-MB-361. Coimmunoprecipitation assay and DUOlink in situ proximity ligation were used to investigate the interaction between PKCβ1 and ERα. Results: Endx more potently inhibited PKCβ1 kinase activity compared to Tam ( IC50 350 nM vs 47.8 μM ) with KDs for PKCβ1 binding as follows: Endx (100 nM ), Tam ( 2 μM ), 4-HT ( 2 μM ) and NDMT (> 7 μM ). None of the SERMs exhibited PKCβ2 binding. In the MCF7/AC1 and MCF7/AC1 L-resistant cells, PKCβ1 knockdown resulted in ERα degradation and potently inhibited cell proliferation. These results were confirmed in T47D and MDA-MB-361 cells. Notably, PKCβ1 knockdown in MCF7/AC1 cells resulted in significantly greater E2 induced proliferation comparing siRNA knockdown vs. control. To further explore these effects, we evaluated the effects of PKCβ1 overexpression in MCF7 cells and demonstrated that PKCβ1 overexpression reduced cell proliferation and colony formation compared to parental MCF-7 cells without affecting ERα protein stability. Coimmunoprecipitation assays in transient transfected MCF-7 cells with exogenous PKCβ1 as well as PKCβ1 expressing MDA-MB-231 cells transiently or stably transfected with ERα demonstrated PKCβ1 and ERα interaction, with confirmation by Duolink assay that this interaction occurs in the cytoplasm. Conclusions: Our findings demonstrated that endoxifen binds and inhibits PKCβ1 at relevant concentrations achieved in the endoxifen clinical trial studies. PKCβ1 interacts with cytoplasmic ERα and PKCβ1 knockdown inhibits cell proliferation and enhances ERα turnover. However, in PKCβ1 overexpressing cells, PKCβ1 may exhibit tumor suppressive effects. These data suggest a complex interaction between PKCβ1 and ERα and that endoxifen's effects on PKCβ1 may alter drug response of endocrine therapy. Further studies are ongoing to characterize the role of PKCβ1 and its role in ER biology and response to endoxifen. Citation Format: Guo C, Kuffel MJ, Kudgus RA, Huang Z, Bode AM, Cheng J, Suman VJ, Reid JM, Bruinsma ES, Subramaniam M, Ames MM, Hawse JR, Goetz MP. Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-03.

Abstract Background: Endoxifen (Endx) is being developed as a drug for patients with estrogen receptor (ER)+ breast cancer (BC). We previously demonstrated that Endx was superior to TAM in both a letrozole (L) sensitive and resistant MCF7/AC1 model. Tamoxifen (TAM) and its most commonly studied metabolite, 4-hydroxy TAM (4HT), are known to activate critical ER co-regulators such as SRC3, which are oncogenic, and contribute to acquired and de novo endocrine resistance. While both Endx and 4HT are more potent inhibitors of genomic ERα functions compared to TAM, the effects of Endx on non-genomic signaling have not been characterized and its efficacy as an anti-cancer agent compared to RAD001 in AI resistant BC is unknown. Methods: We evaluated the 1 hour in vitro effects of TAM, 4HT, Endx, and L, in terms of activation of multiple signaling cascades using an MCF7/AC1 L-resistant cells and long term estrogen deprived (LTED) cells (MDA 361 and ZR75). Protein Docking studies (shape screening, pharmacophore modeling and ligand-protein docking) for oncogenic kinases, and their associated IC50's were determined for TAM and Endx. The in vivo activity of Endx (50 mg/kg, oral) was determined in MCF/AC1 L-resistant xenograft tumors and compared with L control (10 ug/day, sc), Exemestane (E) (250 ug/d, sc) and E+RAD001 (2.5 mg/kg/d, oral). Results: In MCF7/AC1 L-resistant cells, 1 hr treatment with either TAM (100 nM or 5 uM) or 4HT (100 nM or 5 uM) phosphorylated SRC3 (AIB1) and resulted in downstream activation (phosphorylation) of IGF1R, PDK1, AKT, and p70S6K. In contrast, Endx (100 nM and 5 uM) did not phosphorylate SRC3 nor any downstream kinases but resulted in a dose dependent decrease in AKT phosphorylation. Similar findings were observed in LTED cells MDA 361 and ZR75. Docking studies identified a significant decrease in the binding energy (>2 kcal/mol) of Endx compared to TAM for PKC and PI3K and subsequent kinase profiling demonstrated that Endx more potently inhibits PKCβ1 (IC50 357nM) and PI3KΔ (2.5uM) compared to TAM (IC50 4.8μM and 6.2μM respectively). Therefore, we evaluated the in vivo anti-tumor activity of Endx with E and E+RAD001 in MCF7 L-resistant xenograft tumors. After 9 weeks, the% change in tumor volume relative to baseline was significantly lower for both Endx (median -34%, p = 0.0002) and E+RAD001 (median +1.5%, p = 0.0002) as compared to L controls (median +301%). In contrast, E alone was non-significantly lower (median +172%, (p = 0.09). No significant differences were observed comparing Endx and E+RAD001 (p = 0.56). Conclusions: Endx inhibits PKCβ1 and PI3KΔ at concentrations that are pharmacologically relevant, and unlike TAM and 4HT, does not activate SRC3 nor induce oncogenic signaling pathways in AI resistant cells. Importantly, Endx elicits substantial anti-tumor effects in AI resistant models that are similar to the current standard of care of E+RAD001. These data suggest that Endx may be superior to TAM for the treatment of ER+ BC and may result in clinical benefit for patients with AI resistant disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-08.