Abstract
Abstract Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., anastrozole and letrozole). However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated letrozole-resistant breast cancer cells undergo adaptive changes resulting in constitutive activation of the MAPKinase signaling pathway and increased expression of HER2 and IGFR with decreased expression of aromatase and ERα. Given the complex signaling network involving letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of AI resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. The objective of this study was to characterize for the fist time global proteomic alterations occurring in the LTLT-Ca letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca cells vs. AC1 cells (letrozole-sensitive) was performed to identify significant protein expression changes. Protein extracts were digested with trypsin and the resulting peptides were labeled with tandem mass tags. Fractionation and nanoflow HPLC-MS/MS was used for peptide separation, identification and quantification. Proteomic data were analyzed by bioinformatics tools to gain insight into global adaptive signaling modulations as a result of acquired resistance to letrozole. A total of 1743 proteins were identified and quantified that were present in both cell lines, of which 411 were significantly upregulated and 452 were significantly downregulated (p<0.05, fold change > 1.20). Bioinformatics analysis of these altered proteins revealed that acquired letrozole resistance is associated with a hormone-independent, more motile phenotype. In particular, overexpression of multiple proteins was implicated in the enhanced migration and invasion in the resistant cell line, including ROCK, RAC1, S100 family proteins and the actin bundling protein, fascin. Subsequent migration and invasion assays demonstrated more than a 25% increase in the motility of the LTLT-Ca cells. Treatment with pharmacologic inhibitors of fascin, ROCK, and RAC1 partially abrogated both invasion and migration of the letrozole-resistant cells. Moreover, targeted gene expression arrays confirmed a 28-fold and 6-fold increase in the expression of EGFR and HER2, respectively, while ERα and pS2 were dramatically suppressed by 28-fold and 1100-fold, respectively. Taken together, our study for the first time revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence and enhanced cell motility and the potential values of several altered proteins as novel therapeutic targets for treatment of acquired letrozole resistance in postmenopausal breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-415. doi:1538-7445.AM2012-LB-415
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