Abstract
IntroductionAcquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam).MethodsWe cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions.ResultsQuantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility.ConclusionsOur data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.
Highlights
Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood
While numerous earlier studies in other laboratories have demonstrated that tamoxifen resistant breast cancer cell lines were generated by long term exposure of MCF-7 cells to 10-6 to 10-7M 4-OH Tam over a period of 6 to 12 months, adaptive signatures of the resulting resistant phenotypes may vary with different experimental conditions employed
Cell culture MCF-7 cell line was purchased from ATCC (ATCC #HTB-22, Manassas, VA, USA), and routinely cultured in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 5% FBS, 4 mM glutamine, 1 mM sodium pyruvate, 100 IU/ mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin
Summary
Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. The current study employed a quantitative proteomic strategy to capture global changes in protein expression in a tamoxifen resistant cell line derived from the wild type MCF-7 parental cells. EGFR expression was reported to be 10-fold higher in one tamoxifen-resistant model [14] but not in other models [15,16] It has been shown [13] that use of dextran coated charcoal-stripped (DCC) serum in tamoxifen treatment may introduce, in addition to adaptive changes of the cells in response to tamoxifen, effects of long term estrogen deprivation (LTED), complicating the interpretation of molecular signals of resistance development for tamoxifen. We used a phenol-red free DMEM medium containing 5% FBS so that the background estrogen level is in a range that is unlikely to induce adaptive changes due to estrogen deprivation and to minimize the agonistic action of tamoxifen in ER(+) breast cancer cells
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