Dataset Information

Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

ABSTRACT: Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen-receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small-GTPase signaling is implicated in many types of cancer. In this study, we employed a targeted, quantitative proteomic approach that relies on stable-isotope labeling by amino acids in cell culture (SILAC), gel fractionation, and scheduled multiple-reaction-monitoring (MRM) analysis, to assess the differential expression of small GTPases in MCF-7 and the paired tamoxifen-resistant breast cancer cells. The method displayed superior sensitivity and reproducibility over the shotgun-proteomic approach, and it facilitated the quantification of 96 small GTPases. Among them, 13 and 10 proteins were significantly down- and up-regulated (with >1.5-fold change), respectively, in the tamoxifen-resistant line relative to in the parental line. In particular, we observed a significant down-regulation of RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic analysis and downstream validation experiments, supported a role for RAB31 in tamoxifen resistance in ER-positive breast-cancer cells. Together, our results demonstrated that the targeted proteomic method constituted a powerful approach for revealing the role of small GTPases in therapeutic resistance.

SUBMITTER: Huang M

PROVIDER: S-EPMC6456261 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

Huang Ming M Wang Yinsheng Y

Analytical chemistry 20181130 24

Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen-receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small-GTPase signaling is implicated in many types of cancer. In this study, we employed a targeted, quantitative proteomic approach that relies on stable-isotope labeling by amino acids in cell culture (SILAC), gel fractionation, and scheduled mu ...[more]

PMID: 30431262

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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.

Dataset Information

Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

Publications

Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells Revealed by Targeted, Quantitative Proteomic Analysis.

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