CXCR4 activation maintains a stem cell population in tamoxifen-resistant breast cancer cells through AhR signalling

Abstract

Background: Tamoxifen is commonly used for breast cancer therapy. However, tamoxifen resistance is an important clinical problem. Continuous treatment with conventional therapy may contribute to cancer progression in recurring cancers through the accumulation of drug-resistant cancer progenitors.

Methods: To investigate signalling mechanisms important for the maintenance and viability of drug-resistant cancer progenitors, we used microarray analysis, PCR array for genes involved in cancer drug resistance and metabolism, flow cytometry, soft agar colony formation assay, in vivo tumourigenicity assay and immunohistochemical analysis using tamoxifen-sensitive and tamoxifen-resistant breast cancer MCF7 cells.

Results: Downregulation of CXCR4 signalling by small molecule antagonist AMD3100 specifically inhibits growth of progenitor cell population in MCF7(TAM-R) cells both in vitro and in vivo. Microarray analysis revealed aryl hydrocarbon receptor (AhR) signalling as one of the top networks that is differentially regulated in MCF7(TAM-R) and MCF7 xenograft tumours treated with AMD3100. Further, small molecule antagonists of AhR signalling specifically inhibit the progenitor population in MCF7(TAM-R) cells and growth of MCF7(TAM-R) xenografts in vivo.

Conclusion: The chemokine receptor CXCR4 maintains a cancer progenitor population in tamoxifen-resistant MCF7 cells through AhR signalling and could be a putative target for the treatment of tamoxifen-resistant breast cancers.

Figure 1

High expression level of ABCG2 in tamoxifen-resistant MCF7 cells line is associated with an increase in the cancer progenitor population. (A) MCF7(TAM-R) cells showed an increased level of ABCG2 expression as analysed by RT–PCR and western blot analysis. (B) Side population analysis and (C) Aldefluor assay showed a significant enrichment of the progenitor population within MCF7(TAM-R) cells compared with the tamoxifen-sensitive MCF7 cells (*P value<0.05).

Figure 2

MCF7(TAM-R) cells are more sensitive to modulation of CXCR4 signalling than MCF7 cells and tamoxifen-resistant progenitor cell population, and could be specifically inhibited by CXCR4 antagonist AMD3100. (A) AKT inhibitor IV (IC₅₀=1.94 × 10⁻⁷) and CXCR4 antagonist AMD3100 (IC₅₀=2.55 × 10⁻⁷) are potent inhibitors of SP in tamoxifen-resistant cell. MCF7(TAM-R) cells were cultured in tamoxifen-free growth medium in the presence of inhibitors at indicated concentration for 5 days. The culture medium was replenished daily. (B) The CXCR4 knockdown in MCF7(TAM-R) cells showed a decrease in the SP and growth inhibition compared with scrambled shRNA-transduced control MCF7(TAM-R) cells. (C) MCF7(TAM-R) cells are more sensitive to modulation of CXCR4 signalling than MCF7 cells. The cells were cultured in tamoxifen-free growth medium in the presence of inhibitors at indicated concentration for 5 days. The culture medium was replenished daily. (D) The CXCR4 antagonist AMD3100 specifically inhibits SP in the tamoxifen-resistant MCF7(TAM-R) cells in a dose-dependent manner. The cells were cultured in tamoxifen-free growth medium in the presence of inhibitors at indicated concentration for 3 days. The culture medium was replenished daily.

Figure 3

Inhibition of CXCR4 reduces tamoxifen-resistant tumour growth in vivo and alters AhR signalling. (A) MCF7(TAM-R) xenograft tumours treated with AMD3100 showed more than a twofold decrease in the growth rate compared with a control group. (B) The tamoxifen-sensitive xenograft tumours did not show a significant growth inhibition in response to AMD3100 treatment. (C) Haematoxylin and eosin staining of the xenografts showed regressive MCF7(TAM-R) tumours in AMD3100-treated animals. Two histological sections of two different tumours were analysed for each treatment condition. Representative images are showed. (D) Histological analysis of MCF7(TAM-R) xenograft tumours treated with AMD3100 revealed a decrease in ABCG2 expression as compared with untreated MCF7(TAM-R) xenografts and tumours formed by MCF7 cells. (E) Gene expression analysis revealed the aryl hydrocarbon receptor signalling in top ten networks that are deregulated in MCF7(TAM-R) tumours as compared with MCF7 xenograft tumours. TP53, FOS, PERG, SMARCA4 and HSP90AA1 genes that are involved in AhR signalling network are similarly downregulated in MCF7(TAM-R) tumours treated with CXCR4 inhibitor AMD3100 and tamoxifen and upregulated in MCF7 tamoxifen-sensitive tumours under the same treatment conditions. (F) Microarray results were validated by RT–PCR analysis.

Figure 4

AhR signalling differentially regulates MCF7(TAM-R) and MCF7 cells. (A) Structure of AhR antagonists SR1 and SR2. (B) Small molecule antagonists of AhR signalling SR1, SR2 and CH-223191could specifically inhibit ALDH-positive MCF7(TAM-R) cells and induce ALDH-positive population in MCF7 cells, (C). The cells were cultured in tamoxifen-free growth medium in the presence of SR1, SR2 and CH-223191at concentration 1 μℳ, 3 μℳ and 3 μℳ, respectively for 5 days. The culture medium was replenished daily. (D) AhR agonist methylchloroanthrene specifically increases ALDH population in MCF7(TAM-R) cells. The cells were cultured in tamoxifen-free growth medium in the presence of methylchloroanthrene at concentration 5 μℳ for 5 days. The culture medium was replenished daily. (E) AhR antagonists SR1 and SR2 specifically increase AhR-depedent expression of LY6E gene in tamoxifen-sensitive MCF7 cells. (F) Preincubation of MCF7(TAM-R) cells with small molecule antagonists of AhR before subcutaneous injection into nude mice significantly delayed tumour growth compared with MCF7 (TAM-R) xenografts treated with DMSO or MCF7 xenografts treated with AhR antagonist. The cells were cultured in tamoxifen-free growth medium in the presence of SR2 at concentration 3 μℳ or DMSO for 5 days. The culture medium was replenished daily.

Figure 5

Mechanism of CXCR4-dependent maintenance of prostate cancer progenitors in tamoxifen-resistant MCF7 cells. CXCL4 signalling induces the activation of AhR-dependent gene transcription contributing to expanded progenitor population and tumour growth. Targeting CXCR4 signalling with small molecule inhibitors may be beneficial in eliminating prostate cancer stem-like cells in tamoxifen-resistant tumours.