Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses Ivanova et al. Ivanova et al. Proteome Science 2011, 9:60 http://www.proteomesci.com/content/9/1/60 (27 September 2011) Ivanova et al. Proteome Science 2011, 9:60 http://www.proteomesci.com/content/9/1/60 RESEARCH Open Access Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses MM Ivanova1, SM Abner1, WM Pierce Jr2 and CM Klinge1* Abstract Background: A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor b (ERb); however, the role of ERb and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERb in human lung adenocarcinoma cells that respond proliferatively to estradiol (E2) are distinct from those in non-E2-responsive cells. Methods: FLAG affinity purification of FLAG-ERb-interacting proteins was used to isolate ERb-interacting proteins in whole cell extracts from E2 proliferative H1793 and non-E2-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation. Results: LC-MS/MS identified 27 non-redundant ERb-interacting proteins. ERb-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERb-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERb and EGFR interact in a gender-dependent manner and in response to E2 or EGF. BRCA1 interacted with ERb in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue. Conclusion: Our results identify specific differences in ERb-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses. Background A recent epidemiological study reported reduced risk of lung cancer mortality in breast cancer patients using antiestrogens, suggesting further study is needed to examine the potential of antiestrogens to reduce lung cancer risk [1]. The role of estrogens in lung cancer initiation and disease progression remains unclear; however, estrogens are known to induce differentiation and * Correspondence: 1 Department of Biochemistry & Molecular Biology, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY. 40292 USA Full list of author information is available at the end of the article maturation of normal lung tissue [2,3]. Some epidemiologic data indicate that women have a higher risk of lung adenocarcinoma, a type of non-small cell lung cancer (NSCLC), compared to men [4,5]. A positive correlation between post-menopausal estrogen replacement therapy, smoking, and lung adenocarcinoma was reported in one study [6]. The mechanisms underlying the apparent role of gender and estrogens in NSCLC is not yet understood [7]. Local estrogen production may play a role since NSCLC carcinomas had higher estradiol (E2) concentrations compared to the corresponding non-neoplastic lung tissues from the same patient, © 2011 Ivanova et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ivanova et al. Proteome Science 2011, 9:60 http://www.proteomesci.com/content/9/1/60 regardless of gender [8]. E 2 concentrations correlated with aromatase (CYP19A1) mRNA, but not with estrogen receptor a or b (ERa or ERb) staining [8]. E2 concentration was positively associated with tumor size and Ki-67 staining in ERb-positive NSCLC tumors from male patients but not postmenopausal female patients [8]. Likewise, cytosolic ERb was a prognostic indicator of reduced survival in male, but not female NSCLC tumors [9]. Aromatase and ERb expression were correlated, reflecting a more differentiated and less invasive phenotype [10]. Estrogens may contribute to lung tumorigenesis through mechanisms involving genomic, membraneinitiated, and mitochondrial ER-regulated activities. ERs bind directly to estrogen response elements (EREs) or interact with other DNA-bound transcription factors, e. g., AP-1, Sp1, and NF-B, via a “tethering mechanism” [11,12]. These interactions recruit coregulators and either activate or suppress gene transcription in a ligand- and gene- specific manner (reviewed in [13]). A second mechanism by which estrogens regulate cell function is by a membrane-initiated, ‘pre-genomic’ or ‘nongenomic’ signaling pathway involving activation of intracellular protein kinases, e.g., PI3K, MAPK, JNK, within minutes of treatment. These rapid signaling events are mediated through plasma membrane-associated ERa and/or GPR30/GPER [14] and involve crosstalk with other plasma membrane receptors, e.g., EGFR and IGF-R [12,15-17]. ERb is in mitochondria of NSCLC cells [18-21]. ERb interacts with proapoptotic Bad in a ligand-independent manner protecting NSCLC cells from apoptosis-inducing agents, e.g., cisplatin [20]. These data indicate that downregulating ERb may be beneficial in NSCLC. Both ERa and ERb are expressed in normal lung tissue and in lung adenocarcinomas [18,21-25]. ERb is the predominant ER subtype in adult human lung and ERb expression is higher in lung adenocarcinoma than in normal lung tissue [26-28]. Interestingly, men with ERb-positive tumors had a significant reduction in mortality compared with those with ERb-negative tumors; whereas women with ERb-positive tumors exhibited increased mortality [29]. Studies from our lab showed that E 2 did not stimulate estrogenic responses, including proliferation, in normal lung bronchial epithelial cells [18], but stimulated proliferation of lung adenocarcinoma cell lines from females, but not males, through genomic ER regulation [22]. E2 had no effect on the intracellular distribution of ERb and showed no gender difference [18]. Since the biochemical function of ERb in lung adenocarcinoma is unknown, the identification of ERb interacting proteins is essential to dissect ERb’s role in the lung cancer progression. Page 2 of 14 Since ERb’s discovery in 1996 [30], 47 proteins have been report (...truncated)