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

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 beta (ER beta); however, the role of ER beta and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ER beta in human lung adenocarcinoma cells that respond proliferatively to estradiol (E-2) are distinct from those in non-E-2-responsive cells. Methods: FLAG affinity purification of FLAG-ER beta-interacting proteins was used to isolate ER beta-interacting proteins in whole cell extracts from E-2 proliferative H1793 and non-E-2-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 ER beta-interacting proteins. ER beta-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ER beta-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 ER beta and EGFR interact in a gender-dependent manner and in response to E-2 or EGF. BRCA1 interacted with ER beta in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue. Conclusion: Our results identify specific differences in ER beta-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.