Crosstalk between ERα and Receptor Tyrosine Kinase Signalling and Implications for the Development of Anti-Endocrine Resistance
"> Figure 1
<p>Assessment of tamoxifen (<b>upper panels</b>) and fulvestrant (<b>lower panels</b>) sensitivity in breast cancer cell lines. An MTT assay was used to measure sensitivity to tamoxifen and a clonogenic assay was used for assessment of fulvestrant sensitivity (<span class="html-italic">n</span> ≥ 4). Dose–response curves were fitted using the Prism (version 6) program. IC<sub>50</sub> values indicating the levels of drug resistance are shown by bar graphs using a paired <span class="html-italic">t</span>-test: *** <span class="html-italic">p</span> < 0.001; **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 2
<p>Levels of HER expression in breast cancer cell lines with acquired resistance to anti-oestrogen therapy. Representative Western blot data obtained from whole-cell lysates (<b>A</b>) separated by SDS-PAGE and immunoblotted onto PVDF membranes before immunodetection (<span class="html-italic">n</span> = 4). The head and neck cancer HN5 cell line HER1 + ve and breast cancer SKBR3 cell line HER2 + ve were used as positive controls. (<b>B</b>) Expression levels of parental cells (set at 1.0) were compared with the resistant variant using the following paired <span class="html-italic">t</span>-test levels of statistical significance for densitometric scans: * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.001; *** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 3
<p>Sensitivity of breast cancer cell lines with acquired resistance to anti-oestrogen therapy to tyrosine kinase inhibitors (TKIs), assessed by MTT assay (<span class="html-italic">n</span> ≥ 4), (<b>A</b>) MCF-7 and (<b>B</b>) T47D cell lines. Dose–response curves were fitted using the Prism (version 6) program. Data show a broad collateral sensitivity of anti-endocrine-resistant cells to lapatinib, gefitinib, and afatinib. See <a href="#sec2dot3-cancers-10-00209" class="html-sec">Section 2.3</a> for statistical analyses.</p> "> Figure 4
<p>HER1 and HER2 expression with and without tyrosine kinase inhibitor (TKI) treatment in parental MCF-7 and MCF-7-TR (tamoxifen-resistant) and T47D and T47D-FR (fulvestrant-resistant) breast cancer cell lines using confocal fluorescence microscopy. Cells were treated with 5 µM lapatinib and incubated for 48 h. TO-PRO-3 (blue) was used to label nuclei, and HER1 (<b>A</b>) and HER2 (<b>B</b>) antibody reactions were conjugated to Alexa-Fluor 488 secondary antibody. Images are representative of at least three separate experiments. Settings for the microscope were maintained as consistently as possible from one experiment to another.</p> "> Figure 5
<p>(<b>A</b>) Levels of p27<sup>kip1</sup> expression in drug-sensitive and -resistant MCF-7 and T47D breast cancer cell lines in the absence or presence of the 5 µM lapatinib and anti-oestrogen therapy, with densitometry levels shown (<b>B</b>). (<b>C</b>) Constitutive expression of Skp2 in breast cancer cell lines. Representative Western blot data obtained from whole-cell lysates separated by SDS-PAGE, immunoblotted onto PVDF membranes before immunodetection (<span class="html-italic">n</span> = 3). Expression levels were set as 1.0 (control cells) using densitometric analysis and are then shown in response to the various drug treatments relative to each control: * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.001.</p> "> Figure 6
<p>Oestrogen receptor-α (ERα) re-activation following treatment of anti-oestrogen-resistant breast cancer cell lines with lapatinib. (<b>A</b>,<b>B</b>) Representative Western blots of ERα and activated pERα at time points up to 72 h for MCF-7 and T47D cell lines, respectively. Representative Western blot data obtained from whole-cell lysates (upper panel) separated by SDS-PAGE, immunoblotted onto PVDF membranes before immunodetection (<span class="html-italic">n</span> = 3). (<b>C</b>,<b>D</b>) Levels of fold change in ESR mRNA as measured by qPCR and corrected to GAPDH as the housekeeping gene, using the ΔΔC<sub>t</sub> equation. Expression in MCF-7 (<b>C</b>) and T47D (<b>D</b>) parental cell line control (untreated) values were normalised to 1.0. Statistical analysis was performed using a two-tailed <span class="html-italic">t</span>-test: * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 7
<p>Flow cytometry data using annexin V (conjugated to fluoroscein isothiocyanate—FITC) (FL1) and propidium iodide (PI) (FL3) showing the effect of afatinib in combination with anti-oestrogens (48 h treatment) in MCF-7 parent and MCF-7-TR (tamoxifen-resistant) cell lines (<b>A</b>), and in T47D parent and T47D-FR (fulvestrant-resistant) cell lines (<b>B</b>) using the lowest dose levels of drugs (2.5 µM afatinib, 5.0 µM tamoxifen, or 0.5 µM fulvestrant). The lower left-hand quadrant of each representative data point cytogram represents the live cell population (FITC- and PI-negative). The percentages displayed indicate the live cell component in the given examples; (<b>C</b>) shows the combination index (CI) values obtained for the various drug treatments at three different levels (afatinib: 2.5 µM, 5.0 µM, and 10.0 µM; tamoxifen: 5 µM, 10.0 µM, and 20.0 µM; fulvestrant: 0.5 µM, 1.0 µM, and 2.0 µM). Comparisons between parent and resistant cell line CI values were made by two-way ANOVA with Bonferroni correction: * <span class="html-italic">p</span> < 0.001; ** <span class="html-italic">p</span> < 0.0001. (<b>D</b>) The various phases of the cell cycle according to drug treatment; data shown are representative of repeat experiments (<span class="html-italic">n</span> = 4). See <a href="#sec2dot8-cancers-10-00209" class="html-sec">Section 2.8</a> for statistical analysis.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Levels of Anti-Endocrine Resistance in Breast Cancer Cell Lines
2.2. Development of Anti-Endocrine-Resistant Breast Cancer Cells Gives Rise to Changes in Expression of HER Receptors
2.3. Anti-Endocrine-Resistant Cells Show Collateral Sensitivity to TKI Therapies
2.4. HER1 and HER2 Expression in Endocrine-Resistant Cell Lines Following TKI Treatment (Confocal Microscopy)
2.5. TKI Therapy Amplifies p27kip1 in the Presence of Anti-Endocrine Therapies as a Mechanism of Overcoming Drug Resistance
2.6. The Impact of TKI Treatment on ERα Expression in Drug-Resistant Cells
2.7. The Co-Administration of TKI and Anti-Oestrogens Enhances Cell Death and Overcomes Anti-Endocrine Resistance
2.8. Treatment of Anti-Endocrine-Resistant Cell Lines with Anti-Oestrogens and TKIs Produced Increases in the Sub-G1 Component of the Cell Cycle
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Cell Culture
4.3. Cytotoxicity Testing
4.4. Western Blot Analysis
4.5. Real-Time Quantitative PCR Assay
4.6. Optical and Fluorescence Confocal Microscopy (Confocol Laser Scanning Microscopy)
4.7. Annexin V with Propidium Iodide Methodology for Apoptosis with Flow Cytometry
4.8. Cell-Cycle Analysis (Flow Cytometry)
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Montaser, R.Z.; Coley, H.M. Crosstalk between ERα and Receptor Tyrosine Kinase Signalling and Implications for the Development of Anti-Endocrine Resistance. Cancers 2018, 10, 209. https://doi.org/10.3390/cancers10060209
Montaser RZ, Coley HM. Crosstalk between ERα and Receptor Tyrosine Kinase Signalling and Implications for the Development of Anti-Endocrine Resistance. Cancers. 2018; 10(6):209. https://doi.org/10.3390/cancers10060209
Chicago/Turabian StyleMontaser, Rugaia Z., and Helen M. Coley. 2018. "Crosstalk between ERα and Receptor Tyrosine Kinase Signalling and Implications for the Development of Anti-Endocrine Resistance" Cancers 10, no. 6: 209. https://doi.org/10.3390/cancers10060209