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. 2013 Apr 2:13:174.
doi: 10.1186/1471-2407-13-174.

V体育平台登录 - The Wnt signalling pathway is upregulated in an in vitro model of acquired tamoxifen resistant breast cancer

Yan Ni Loh  1 , Ellen L Hedditch, Laura A Baker, Eve Jary, Robyn L Ward, Caroline E Ford
Affiliations

The Wnt signalling pathway is upregulated in an in vitro model of acquired tamoxifen resistant breast cancer

Yan Ni Loh et al. BMC Cancer. .

Abstract

Background: Acquired resistance to Tamoxifen remains a critical problem in breast cancer patient treatment, yet the underlying causes of resistance have not been fully elucidated VSports手机版. Abberations in the Wnt signalling pathway have been linked to many human cancers, including breast cancer, and appear to be associated with more metastatic and aggressive types of cancer. Here, our aim was to investigate if this key pathway was involved in acquired Tamoxifen resistance, and could be targeted therapeutically. .

Methods: An in vitro model of acquired Tamoxifen resistance (named TamR) was generated by growing the estrogen receptor alpha (ER) positive MCF7 breast cancer cell line in increasing concentrations of Tamoxifen (up to 5 uM). Alterations in the Wnt signalling pathway and epithelial to mesenchymal transition (EMT) in response to Tamoxifen and treatment with the Wnt inhibitor, IWP-2 were measured via quantitative RT-PCR (qPCR) and TOP/FOP Wnt reporter assays V体育安卓版. Resistance to Tamoxifen, and effects of IWP-2 treatment were determined by MTT proliferation assays. .

Results: TamR cells exhibited increased Wnt signalling as measured via the TOP/FOP Wnt luciferase reporter assays. Genes associated with both the β-catenin dependent (AXIN2, MYC, CSNK1A1) and independent arms (ROR2, JUN), as well as general Wnt secretion (PORCN) of the Wnt signalling pathway were upregulated in the TamR cells compared to the parental MCF7 cell line. Treatment of the TamR cell line with human recombinant Wnt3a (rWnt3a) further increased the resistance of both MCF7 and TamR cells to the anti-proliferative effects of Tamoxifen treatment. TamR cells demonstrated increased expression of EMT markers (VIM, TWIST1, SNAI2) and decreased CDH1, which may contribute to their resistance to Tamoxifen. Treatment with the Wnt inhibitor, IWP-2 inhibited cell proliferation and markers of EMT. V体育ios版.

Conclusions: These data support the role of the Wnt signalling pathway in acquired resistance to Tamoxifen. Further research into the mechanism by which activated Wnt signalling inhibits the effects of Tamoxifen should be undertaken. As a number of small molecules targeting the Wnt pathway are currently in pre-clinical development, combinatorial treatment with endocrine agents and Wnt pathway inhibitors may be a useful therapeutic option in the future for a subset of breast cancer patients VSports最新版本. .

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Figures

Figure 1
Figure 1
TamR cells are resistant to Tamoxifen. a: TamR cells were larger, flatter and exhibited a more mesenchymal phenotype than the parental cell line MCF7. 10X magnification. b: MTT proliferation assays were performed on parental MCF7 cells and TamR cells treated with increasing doses of Tamoxifen(0.0–15.0 μM) over 24 hours. 50% DMSO was used as a control as it is known to eliminate cells independent of ER status. TamR cells were successfully selected for acquired Tamoxifen resistance from its parental cell line MCF7 as they continued to proliferate in Tamoxifen concentrations, 5 μM, 7.5 μM, 10 μM, 11.5 μM and 13.5 μM, that eliminate MCF7 cells. Graph represents the average cell proliferation in percentage of 10 replicates with standard deviation represented by error bars. *** P<0.001. Abbreviation: Dimethyl sulfoxide (DMSO). c: mRNA expression of ER and HER2 was measured using qPCR. Graph represents the mRNA fold-regulation values of TamR cells relative to MCF7 cells, normalized against three housekeeping genes with standard deviation of triplicate experiments represented by error bars** P<0.01. Abbreviations: Estrogen receptor alpha (ER), Human Epidermal Growth Factor Receptor 2 (HER2).
Figure 2
Figure 2
TamR cells exhibit increased Wnt signalling. a: mRNA expression of Wnt-related genes was measured using qPCR. Graph represents the mRNA fold-regulation values of TamR cells relative to MCF7 cells, normalized against three housekeeping genes with standard deviation (s.d) of triplicate experiments represented by error bars. *P<0.05, ** P<0.01. b: mRNA expression changes of Wnt target genes were measured using RT Profiler PCR arrays and normalised to five housekeeping genes. Criteria for significant change include a statistically significant validation determined by the manufacturer and fold regulation of greater or less than 4. c: MCF7 and TamR cells were co-transfected with pRL-TK (Renilla) and either TOPflash or FOPflash expression plasmids. Cells were subsequently treated with 0.1 μg/ml recombinant Wnt3a (rWnt3a) for 24 hours prior to luciferase activities being measured using a Glomax 96 Microplate Luminometer. Average activity and s.ds were derived from octopulate transfected samples. Results represent the average of 3 experiments and bars represent the s.d of the mean. ***P<0.001. d: MTT proliferation assays were performed on parental MCF7 cells and TamR cells treated with 5 μM Tamoxifen and 0.1 μg/ml recombinant Wnt3a (rWnt3a) for 24 hours. 50% DMSO was used as a control. Treatment with rWnt3a and 5 μM Tamoxifen inhibited the proliferation of both the MCF7 and TamR cells compared to treatment with Tamoxifen alone. rWnt3a treatment in the TamR cells enhanced their resistance to Tamoxifen back to near basal levels. Graph represents the average cell proliferation in percentage of 10 replicates with s.d represented by error bars. *P<0.05, *** P<0.001. e: mRNA expression of EMT markers was measured using qPCR. Graph represents the mRNA fold-regulation values of TamR cells relative to MCF7 cells, normalized against three housekeeping genes with s.d of five independent experiments represented by error bars. **P<0.01, ***P<0.001*.
Figure 3
Figure 3
The Wnt inhibitor IWP-2 inhibits proliferation and EMT in TamR cells. a: TamR cells were pre-treated with 5 μM IWP-2 for 24 hours prior to co-transfection with pRL-TK (Renilla) and either TOPflash or FOPflash expression plasmids. Cells were subsequently treated with 5 μM IWP-2 for 24 hours prior to luciferase activities being measured using a Glomax 96 Microplate Luminometer. Average activity and standard deviations were derived from triplicate transfected samples. Results represent the average of 4 experiments and bars represent the standard deviation (s.d) of the mean. **P<0.01. b: MTT proliferation assays were performed on TamR cells treated with increasing doses of Tamoxifen(0.0–15.0 μM) over 24 hours, with or without the addition of 5 μM IWP-2. The graph represents the average cell proliferation of triplicate wells in three independent experiments, with standard deviation represented by error bars. ** P<0.01. c: mRNA expression of EMT markers was measured using qPCR in TamR cells treated for 48 hours with 5 μM IWP-2. Graph represents the mRNA fold-regulation values of control cells relative to IWP-2 treated cells, normalized against three housekeeping genes. This is a single experiment, and error bars represent the standard deviation of triplicate wells.
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