Abstract P4-03-16: Cyclin E affects Smad3 pathway in trastuzumab resistant HER2+ breast cancer

Abstract

Abstract Introduction and Objective: HER2 amplification is found in approximately 20% of patients with breast cancer and is associated with poor prognosis. Trastuzumab, a humanized anit-HER2 antibody, has been shown to improve patient outcomes, but clinical efficacy for this treatment is limited by the high rate of drug resistance. Cyclin E overexpression and cyclin-dependent kinase 2 (CDK2) activation correlate with trastuzumab resistance, and the underlying mechanisms of this correlation are an area of active study. In this work, we examined the contribution of cyclin E/CDK2-mediated Smad3 noncanonical phosphorylation to trastuzumab resistance in HER2+ breast cancer. Methods: Trastuzumab resistant BT474R2 cells were developed by culturing BT474 cells in trastuzumab-supplemented media for 18 months. These cells were then inoculated into immunodeficient mice and treated with trastuzumab twice weekly. BT474 and BT474R2 cells were used to study cell proliferation, phosphorylation in the Smad3 linker region and transcription factor activities with a living cell array. Results: Inhibition of cyclin E activity with CDK2 inhibitor therapy (CDK2i, 600 nM) significantly decreased BT474R2 cell proliferation. CDK2i treatment led to decreased phosphorylation at T179 (p<0.01), S204 (p<0.01) and S213 (p<0.05) in the Smad linker region. Further, CDK2i increased expression of p15 (p<0.05) and decreased expression of c-myc (p<0.05) in comparison with trastuzumab treatment. Transduction of BT474R2 cells with a 5M Smad3 construct containing inhibitory mutations in 5 CDK2 phosphorylation sites resulted in decreased cell proliferation. In a transfected cell array using BT474 and BT474R2 cells, activities of cell proliferation and metastasis-associated transcription factors were significantly different between the trastuzumab sensitive and resistant cells. Conclusions: Taken together, overexpression of cyclin E leads to Smad3 noncanonical phosphorylation and results in trastuzumab resistance in HER2+ breast cancer. CDK2i treatment may be a promising therapeutic strategy for patients with trastuzumab resistance. Citation Format: Decker JT, Wan L, Shea LD, Jeruss JS. Cyclin E affects Smad3 pathway in trastuzumab resistant HER2+ 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 P4-03-16.