Proteomic Signatures of Acquired Letrozole Resistance in Breast Cancer: Suppressed Estrogen Signaling and Increased Cell Motility and Invasiveness

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.Aromatase inhibitors (AIs)¹ have increasingly been used to treat ER positive breast cancer in postmenopausal women as adjuvant or neoadjuvant therapy (1, 2). AIs block estrogen production by inhibiting the aromatization reaction that converts androgens to estrogens, resulting in the remission of estrogen dependent breast tumors. However, acquired resistance occurs in the majority of patients after several years of AI treatment, and this may ultimately lead to eventual relapse of the disease (3, 4). Therefore it is necessary to better understand the resistance mechanisms to abrogate or delay the onset of AI resistance. Numerous studies have used in vitro and in vivo models to dissect the adaptive signaling events in the development of AI resistance in breast cancer (5, 6). The in vitro long term estrogen deprivation (LTED) model has been proposed to represent AI resistance in breast cancer that also showed cross-resistance to other hormonal treatment (7). However, the LTEDaro lines did not cluster with the AI-resistant lines (8). Alternatively, in vivo models using cells derived from mouse xenografts have been widely used to study the mechanism of AI resistance and potential therapeutic strategies (3, 9–11). Notably, ER expression levels were up-regulated in the LTED model (7) but down-regulated in the letrozole resistant breast cancer cells (12, 13).In in vitro experiments, it has been found that although MCF-7 and T47D (both ER+ breast cancer cell lines) express detectable levels of aromatase, the enzymatic activity is rather low for experimental studies of the action of aromatase inhibitors and subsequent resistance to AIs. It is recognized that the aromatase activity in MCF-7aro cells is significantly higher than that in typical breast cancer and surrounding adipose stromal cells. However, the aromatase activity in breast tumors can be elevated in situ by cytokines, cAMP, and cancer-promoting agents which stimulate protein kinase C activity (14–17). Thus 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. Clinically, it has been observed that ∼66% of breast carcinomas contain aromatase (the rate-limiting enzyme responsible for estrogen biosynthesis), and the estrogen synthesized in situ has a role in stimulating the tumor (18, 19). In breast, ovarian, uterine, and prostate cancer, activation of PI.3/II leads to the overexpression of aromatase within the tissues themselves, and this has been associated with an increase in tumor growth (20, 21).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 (LTLT-Ca) 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). Inhibition of MAPK led to reduced cell proliferation and restoration of ER expression, implicating the occurrence of crosstalk between ER and growth factor receptor signaling. Moreover, inhibition of Her2 restored the sensitivity of LTLT-Ca cells to letrozole (10, 23). In the genome-wide analysis of several hormone resistant cell lines, elevated DNA replication, recombination, repair function, cell cycle control, and the pyrimidine metabolism pathway were also found as an enhanced survival mechanism in the resistant cells (6, 7). Given the complex signaling network involved in AI refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of AI resistance by a global proteomics-based systems biology approach may reveal previously unconsidered molecular changes that could be used as therapeutic targets. Therefore, the objective of this study is to characterize global proteomic alterations occurring in the letrozole resistant cell line, originating from the Brodie laboratory (12).Here we have performed a quantitative proteomic analysis of the whole cell lysis samples of LTLT-Ca against the letrozole sensitive control cell line, AC-1, using a gel-free approach. To achieve relative quantitation of proteins we used a tandem mass tag labeling technique where each sample is triply labeled for analytical precision. Fractionation and nanoflow reverse phase HPLC were employed in combination with a high-resolution tandem mass spectrometer for peptide separation and identification. Identified protein markers were then analyzed by bioinformatics tools to gain insight into global adaptive signaling modulations as a result of acquired resistance to letrozole.