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Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

ABSTRACT: While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger RNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F, confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here, we show that the eIF4F translation pathway was hyperactive in tamoxifen-resistant (TamR) MCF-7L breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the eIF4E-eIF4G interaction also decreased the proliferation and anchorage-dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and, potentially, other endocrine therapies.

SUBMITTER: Fagan DH

PROVIDER: S-EPMC5518927 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

Fagan Dedra H DH Fettig Lynsey M LM Avdulov Svetlana S Beckwith Heather H Peterson Mark S MS Ho Yen-Yi YY Wang Fan F Polunovsky Vitaly A VA Yee Douglas D

Hormones & cancer 20170602 4

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger ...[more]

PMID: 28577281

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Project description: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.

Project description:BackgroundChemoresistance is a major factor involved in a poor response and reduced overall survival in patients with advanced breast cancer. Although extensive studies have been carried out to understand the mechanisms of chemoresistance, many questions remain unanswered.MethodsIn this research, we used two isogenic MCF-7 breast cancer cell lines selected for resistance to doxorubicin (MCF-7DOX) or docetaxel (MCF-7TXT) and the wild type parental cell line (MCF-7CC) to study mechanisms underlying acquired resistance to taxanes in MCF-7TXT cells. Cytotoxicity assay, immunoblotting, indirect immunofluorescence and live imaging were used to study the drug resistance, the expression levels of drug transporters and various tubulin isoforms, apoptosis, microtubule formation, and microtubule dynamics.ResultsMCF-7TXT cells were cross resistant to paclitaxel, but not to doxorubicin. MCF-7DOX cells were not cross-resistant to taxanes. We also showed that multiple mechanisms are involved in the resistance to taxanes in MCF-7TXT cells. Firstly, MCF-7TXT cells express higher level of ABCB1. Secondly, the microtubule dynamics of MCF-7TXT cells are weak and insensitive to the docetaxel treatment, which may partially explain why docetaxel is less effective in inducing M-phase arrest and apoptosis in MCF-7TXT cells in comparison with MCF-7CC cells. Moreover, MCF-7TXT cells express relatively higher levels of β2- and β4-tubulin and relatively lower levels of β3-tubulin than both MCF-7CC and MCF-7DOX cells. The subcellular localization of various β-tubulin isoforms in MCF-7TXT cells is also different from that in MCF-7CC and MCF-7DOX cells.ConclusionMultiple mechanisms are involved in the resistance to taxanes in MCF-7TXT cells. The high expression level of ABCB1, the specific composition and localization of β-tubulin isoforms, the weak microtubule dynamics and its insensitivity to docetaxel may all contribute to the acquired resistance of MCF-7TXT cells to taxanes.

Project description:BackgroundTamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance.MethodsMTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth.ResultsMTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression.ConclusionsOur results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.

Dataset Information

Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

Publications

Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation.

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