Crosstalk between ERα and Receptor Tyrosine Kinase Signalling and Implications for the Development of Anti-Endocrine Resistance

Abstract

Although anti-endocrine therapies have significantly advanced the treatment of breast cancer, they pose the problem of acquired drug resistance. The oestrogen receptor (ER)-expressing breast cancer cell lines MCF-7 and T47D alongside their in vitro derived resistant counterparts MCF-7-TR (tamoxifen-resistant) and T47D-FR (fulvestrant-resistant) showed dual resistance to fulvestrant and tamoxifen in the presence of upregulated HER1 and HER2 growth factor receptors. Our study demonstrated that tamoxifen resistance and fulvestrant resistance are associated with collateral sensitivity to the tyrosine kinase inhibitors (TKIs) lapatinib (p < 0.0001) and afatinib (p < 0.0001). Further, we found that over time, the TKIs reactivated ER&alpha; protein and/or mRNA in tamoxifen- and fulvestrant-resistant cells. Combinations of anti-endocrine agents with afatinib gave rise to significantly enhanced levels of apoptosis in both T47D-FR and MCF-7-TR in a synergistic manner versus additive effects of agents used singly. This was associated with p27^kip1 induction for anti-endocrine-resistant cells versus parental cells. Our data supports the use of combination treatment utilising dual HER1/2 inhibitors in breast cancer patients showing resistance to multiple anti-endocrine agents.

Keywords: breast cancer; drug resistance; receptor tyrosine kinases.

Figure 1

Assessment of tamoxifen (upper panels) and fulvestrant (lower panels) 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 (n ≥ 4). Dose–response curves were fitted using the Prism (version 6) program. IC₅₀ values indicating the levels of drug resistance are shown by bar graphs using a paired t-test: *** p < 0.001; **** p < 0.0001.

Figure 2

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 (A) separated by SDS-PAGE and immunoblotted onto PVDF membranes before immunodetection (n = 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) Expression levels of parental cells (set at 1.0) were compared with the resistant variant using the following paired t-test levels of statistical significance for densitometric scans: * p < 0.05; ** p < 0.001; *** p < 0.0001.

Figure 3

Sensitivity of breast cancer cell lines with acquired resistance to anti-oestrogen therapy to tyrosine kinase inhibitors (TKIs), assessed by MTT assay (n ≥ 4), (A) MCF-7 and (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 Section 2.3 for statistical analyses.

Figure 4

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 (A) and HER2 (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.

Figure 5

(A) Levels of p27^kip1 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). (C) 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 (n = 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: * p < 0.05; ** p < 0.001.

Figure 6

Oestrogen receptor-α (ERα) re-activation following treatment of anti-oestrogen-resistant breast cancer cell lines with lapatinib. (A,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 (n = 3). (C,D) Levels of fold change in ESR mRNA as measured by qPCR and corrected to GAPDH as the housekeeping gene, using the ΔΔC_t equation. Expression in MCF-7 (C) and T47D (D) parental cell line control (untreated) values were normalised to 1.0. Statistical analysis was performed using a two-tailed t-test: * p < 0.05; ** p < 0.01.

Figure 7

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 (A), and in T47D parent and T47D-FR (fulvestrant-resistant) cell lines (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; (C) 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: * p < 0.001; ** p < 0.0001. (D) The various phases of the cell cycle according to drug treatment; data shown are representative of repeat experiments (n = 4). See Section 2.8 for statistical analysis.