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Effect of cyclin E overexpression on resistance to trastuzumab and phosphorylation of SMAD3 in HER2+ breast cancer.

e12006 Background: Trastuzumab, an effective treatment for HER2+ breast cancer, can be limited by development of resistance. Cyclin E (CCNE) overexpression was implicated in trastuzumab resistance, though the association is not well understood. We sought to explore a potential mechanism focusing on altered phosphorylation of the TGFβ signaling protein, SMAD3, in CCNE overexpressing HER2+ breast cancer cells, leading to trastuzumab resistance. Methods: A transcriptional activity cell array (TRACER) was used to examine the relationship between specific transcription factors and trastuzumab resistance in BT474 HER2+ human breast cancer cells. Immunoblotting examined phosphorylation patterns of SMAD3 in trastuzumab sensitive and resistant conditions. Effects of blocking CCNE overexpression with a CDK2 inhibitor were measured. To test the relative influence of SMAD3 and SMAD3 linker region phosphorylation on cell proliferation, BT474R2 cell lines were created overexpressing SMAD3 (BT474R2-SMAD3), a mutant construct blocking CCNE-mediated SMAD3 linker region phosphorylation (BT474R2-5M), and a control (BT474R2-Blank). In vitro and in vivo studies measured response to trastuzumab. Results: Resistance to trastuzumab was confirmed for BT474R2 cells, showing a markedly increased resistance to treatment when compared to the parental line. Network analysis of TRACER data identified decreased SMAD3 activity was central to resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < 0.01). Noncanonical phosphorylation of SMAD3 was increased in CCNE-overexpressing cells. Blocking CCNE mediated noncanonical phosphorylation of SMAD3 with CDK2 inhibition resulted in decreased proliferation of BT474R2 cells. In vitro studies examining response to trastuzumab showed an increased sensitivity for BT474R2-5M cells. Findings were validated in NSG mice inoculated with BT474R2-5M cells that developed significantly lower tumor volumes (p < 0.001) when treated with trastuzumab. Conclusions: These results identified a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, which may be effectively treated with CDK2 inhibition to help enhance the efficacy of trastuzumab for patients with HER2+ breast cancer.