Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

Abstract

Introduction: Acquired 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).

Methods: We 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.

Results: Quantitative 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.

Conclusions: Our 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.

Figure 1

Treatment of MCF-7 cells with 4-OH Tam resulted in a resistant phenotype. A. Survival ratio vs time (zero to six months). MCF-7 cells cultured with 10^-7 M 4-OH tamoxifen for zero to six months were plated in six-well plates at a density of 50,000 per well in 5% FBS DMEM medium. The cells were counted after treatment with 10^-7 M 4-OH tamoxifen for five days; the final concentration of ethanol was 0.1%. B. Survival ratio vs 4-hydroxytamoxifen concentration. MCF-7 cells cultured for six months with 10^-7 M 4-OH tamoxifen (MCF-7-TamR) and 0.1% ethanol (MCF-7-control) were seeded in 96-well plates at a density of 3,000 each in 5% FBS phenol-red free DMEM medium. The cells were then treated with 10^-7 to 10^-5 M 4-OH tamoxifen for five days.

Figure 2

MCF-7-TamR cells are resistant to tamoxifen in clonogenic assays. A. Effect of tamoxifen on colony formation of MCF-7-TamR and MCF-7-control. Number of colonies formed in resistant and sensitive cell lines was plotted as a function of treatment (DMSO and tamoxifen). B. Representative colony images of the resistant and sensitive cells.

Figure 3

Quantitative RT-PCR confirms that mRNA levels are consistent with protein expressions for the selected genes.

Figure 4

Western blot analysis confirms protein changes in MCF-7-TamR cells initially identified in quantitative proteomics. A) A representative Western blot of control (con) and tamoxifen resistant (MCF-7-TamR) cells showing the different expression levels of the indicated proteins. B) Four independent samples from each cell line were used to quantify the levels of each protein. The housekeeping protein Rho-GDI was used as an internal loading control. For comparative purposes, the control samples were set to 100%.

Figure 5

MCF-7-TamR cells exhibit significantly down-regulated ER mediated signaling pathways. A) MCF-7-control and MCF-7-TamR cells were grown in 5% phenol free DMEM for 48 hours prior to 24 hours treatment with 1 nM 17β-estradiol (E₂) or vehicle control (DMSO). Q PCR was performed for ER regulated genes ER, PgR, and SDF-1. Normalization was to MCF-7-control cells treated with vehicle; B) E₂ induced ER regulated gene expression for tamoxifen resistant cells (normalization to MCF-7-TAMR treated with DMSO). qPCR was performed for genes ER, SDF-1, and PgR. Results reflect average fold change in cycle number for mRNA levels +/-. Cycle number was normalized to β-actin. * Significantly different from DMSO control, P < 0.05.

Figure 6

Regulation of actin cytoskeleton in the tamoxifen resistant cell line, MCF-7-TamR. The pathway illustrates changes to proteins expression, TamR cell line vs. control, involved in regulating the actin cytoskeleton. All red proteins are significantly up-regulated and all blue proteins are significantly down-regulated. The ratio value for each significantly regulated protein is indicated to the right. Solid lines with no arrow indicate physical interaction. Solid lines with an arrow or bar at the end indicate direct activation or inhibition, respectively. Dotted lines indicate indirect or unknown method of interaction. The orange lines highlight the Rho activation pathways and the green lines highlight integrin mediated focal adhesions.

Figure 7

Acquired resistance to tamoxifen in MCF-7 cells leads to enhanced migration capacity. 2.5 × 10⁴ MCF-7 or MCF-7-TamR cells were seeded on the upper chamber of a transwell system where the lower wells contained media with 5% serum. After 24 h, cells were fixed and stained with crystal violet and the number of migrated cells counted. Bars represent the percent of migrated MCF-TamR cells per 100 × field of view ± SEM. ***, P < 0.001.

Figure 8

S100P up-regulation in MCF-7 cells confers tamoxifen resistance and enhanced migration. A) Western blot of S100P expression in MCF-7, MCF-7-TamR, MCF-7-S100P. B) Survival ratio of MCF-7, MCF-7-TamR, MCF-7-S100P after treatment of 4-hydroxytamoxifen for five days. C) Migration assay for MCF-7, MCF-7-TamR, MCF-7-S100P.

Figure 9

Kaplan-Meier survival plots demonstrate the prognostic relevance of S100P overexpression on patient survival. A) Overexpression of S100P is predictive of lower relapse free survival (P = 1.76e-6) for all patients; B) overexpression of S100P is correlated with decreased distant metastasis free survival (P = 0.029); C) Untreated breast cancer patients had lower relapse free survival if their tumors overexpressed S100P (P = 0.017); D) Weak correlation between the metastasis free survival and overexpression of S100P (P = 0.18); E) Higher level of S100P expression is predictive of poor relapse free survival for ER positive breast cancer patients (P = 0.00037); F) S100 overexpression is not associated with survival of ER negative breast cancer patients; G) S100 overexpression is not statistically significant for prognosis of ER positive endocrine treated patients; H) Overexpression of S100P is not predictive of metastasis-free survival of ER positive endocrine treated patients.