Abstract
Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer.
Highlights
From the ‡Department of Basic Pharmaceutical Sciences, College of Pharmacy, §Research Centers in Minority Institutions (RCMI) Cancer Research Program, ¶Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125
Complement component 3 (C3), an ER responsive gene was significantly up-regulated by 250-fold, an observation that contradicts a recent study where C3 expression is often consistent with ER expression level [43]
It was noted that Bcl-2 levels decreased by over 340-fold in the letrozole resistant cell line, consistent with a previous report on the role of Bcl-2 in breast carcinoma [44]
Summary
From the ‡Department of Basic Pharmaceutical Sciences, College of Pharmacy, §Research Centers in Minority Institutions (RCMI) Cancer Research Program, ¶Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125. The aromatase activity in breast tumors can be elevated in situ by cytokines, cAMP, and cancerpromoting agents which stimulate protein kinase C activity (14 –17) These MCF-7 and AC1 cell lines are appropriate models for studying the balance between the androgenic and estrogenic effect in breast cancer as they express significant levels of AR and ER. In the mouse xenograft model established in Brodie’s laboratory, MCF-7 cells were stably transfected with the human aromatase gene and grown in ovariectomized female nude mice treated with letrozole for over 56 weeks [12, 22] Subsequent studies of these long-term letrozole treated (LTLTCa) cells isolated from these tumors confirmed the up-regulation of Her2/MAPK signaling cascade and the p160 coactivator, amplified in breast cancer 1 (AIB1), as an adaptive survival pathway [5, 12, 13, 23]. Identified protein markers were analyzed by bioinformatics tools to gain insight into global adaptive signaling modulations as a result of acquired resistance to letrozole
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