Abstract 903: Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells

Abstract

Abstract ErbB2/Her2 overexpression is detected in approximately 30% of human breast cancers and is associated with poor prognosis. Lapatinib, a small molecule dual inhibitor targeting ErbB2 and EGFR, is FDA-approved for the treatment of ErbB2-positive (ErbB2+) advanced or metastatic breast cancer. However, lapatinib resistance (LR) is emerging as a critical issue in clinical oncology. To understand the molecular mechanisms of LR, we first developed a lapatinib-resistant cell line from ErbB2-overexpressing BT474 breast cancer cells by prolonged exposure of parental BT474 cells to gradually increasing concentrations of lapatinib (up to 8 µM). We performed microarray analyses on parental and LR cells to identify novel factors/pathways that contribute to LR. Our microarray data indicated that the gene expression profile of the BT474/LR cells is significantly different from the parental BT474 cells. We found that DARPP32/PPP1R1B is one of the most significantly upregulated genes in the BT474/LR cells. To investigate the role of DARPP32 in LR development, we examined the effect of DARPP32 knockdown via lentiviral shRNA on BT474/LR cell proliferation and apoptosis. Results from MTT, clonogenic, cell cycle, and cleaved caspase-3 and PARP analyses indicated that DARPP32 knockdown significantly sensitized BT474/LR cells to lapatinib. We also found that DARPP32 knockdown renders MDA-MB-361 breast cancer cells, which express high endogenous levels of DARPP32, more sensitive to lapatinib. These data demonstrate that DARPP32 overexpression contributes to LR. To understand the mechanism of DARPP32 overexpression-mediated LR, we focused on the regulation of transcription factor cAMP response element-binding protein (CREB) in this process. As such, DARPP32 overexpression was generally associated with CREB upregulation. We also found that CREB-mediated transcription was increased in BT474/LR cells, and DARPP32 knockdown in BT474/LR and MDA-MB-361 cells significantly downregulated CREB protein levels and CREB-mediated transcription, as indicated by luciferase reporter assays. CREB overexpression rendered BT474 cells resistant to lapatinib and reversed DARPP32 knockdown-induced sensitization in BT474/LR cells. In contrast, CREB knockdown sensitized the cells to lapatinib. Further analysis with MG132 blockage suggests that DARPP32 may regulate CREB protein levels through the proteasomal degradation pathway. Taken together, we have identified DARPP32 overexpression as a critical mediator of acquired lapatinib resistance. The underlying mechanisms involve the deregulation of CREB protein and activity levels. These novel findings advance our understanding of the molecular mechanisms of LR and provide fundamental support for testing the DARPP32-CREB axis in clinical settings for the management of LR breast cancers, which is of significant translational value. Citation Format: Zhikun Ma, Erin W. Howard, Amanda B. Parris, Xiaohe Yang. Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 903.