SNAI2 upregulation is associated with an aggressive phenotype in fulvestrant-resistant breast cancer cells and is an indicator of poor response to endocrine therapy in estrogen receptor-positive metastatic breast cancer

Abstract

Background: Endocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance.

Methods: In the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting.

Results: SNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004).

Conclusions: Our results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.

Keywords: Endocrine resistance; Epithelial-mesenchymal transition; Estrogen receptor-positive breast cancer; Fulvestrant; SNAI2.

Fig. 1

Upregulation of SNAI2 in endocrine-resistant cells compared to parental endocrine-sensitive cell lines. Evaluation of the gene expression of epithelial-mesenchymal transition (EMT) transcription regulators in MCF-7-based fulvestrant-resistant (FulvR) vs. fulvestrant-sensitive (a) and tamoxifen-resistant (TamR) vs. tamoxifen-sensitive (b) cells by gene array. Verification of altered expression of SNAI2 in four fulvestrant-resistant (c) and four tamoxifen-resistant cell lines (d), by reverse transcription (RT)-quantitative (q)PCR (RT-qPCR). Gene expression was normalized using PUM1 and depicted as the relative expression in endocrine-resistant vs. endocrine-sensitive cells: *p < 0.05. Data are shown with error bars representing mean ± standard deviation. SNAI2 protein expression alteration in the same fulvestrant-resistant (e) and tamoxifen-resistant cell lines (f), as determined by western blotting. β-actin was used as loading control. A representative of three independent experiments is shown

Fig. 2

Fulvestrant-resistant cells exhibit a more motile mesenchymal phenotype compared to parental fulvestrant-sensitive cells. The motility of MCF-7-based fulvestrant-sensitive and fulvestrant-resistant cells was evaluated by a transwell assay. a Representative micrographs (purple-stained cells, × 20 magnification) and b column diagram analysis of the percentage of cells that migrated through the membrane: *p < 0.05. Data are shown with error bars representing mean ± standard deviation. c Protein expression levels of E-cadherin, vimentin and estrogen receptor (ER)α by western blotting. β-actin was used as loading control. A representative of three independent experiments is shown

Fig. 3

Inhibition of SNAI2 induces epithelial characteristics and impairs growth of fulvestrant-resistant breast cancer cell lines. a MCF-7-based fulvestrant-resistant and parental sensitive cells were transfected with small interfering RNA (siRNA) against SNAI2 leading to a reduction at the messenger RNA (mRNA) level, as evaluated by reverse transcription (RT)-quantitative (q)PCR (RT-qPCR). SNAI2 knockdown resulted in a significant reduction in the migration ability of both fulvestrant-resistant and fuvestrant-sensitive cells, as depicted in b representative micrographs (purple-stained cells, × 20 magnification) and c by column diagram analysis of the percentage of cells that migrated following SNAI2 knockdown. d E-cadherin mRNA expression after SNAI2 silencing determined by RT-qPCR. Gene expression was normalized using PUM1. e Effect of SNAI2 knockdown on growth of fulvestrant-resistant and parental sensitive cells, as measured by crystal violet-based colorimetric assay. f Cell growth of fulvestrant-resistant and parental sensitive cells following treatment with the selective p53-SNAIL binding inhibitor (GN25), fulvestrant, the two drugs in combination or vehicle (control), as measured by crystal violet-based colorimetric assay: *p < 0.05. Data are shown with error bars representing mean ± standard deviation. g Immunocytochemical analysis of SOX2 protein in formalin-fixed paraffin-embedded fulvestrant-resistant and fulvestrant-sensitive cells (× 20 magnification). h Protein expression levels of SOX2 following SNAI2 knockdown determined by western blotting. β-actin was used as loading control. A representative of three independent experiments is shown

Fig. 4

SNAI2 expression correlates with progression-free survival (PFS) in patients with estrogen receptor (ER)+ metastatic breast cancer treated with endocrine therapy. Kaplan-Meier plots evaluating PFS according to expression of SNAI2 in ER+ metastatic lesions from a an initial and b a second cohort of patients with breast cancer treated with endocrine therapy in the advanced setting. c Survival analysis of PFS according to SNAI2 levels in fulvestrant-treated patients from cohorts 1 and 2. A two-sided p value (*p < 0.05) was calculated using log-rank testing. Representative micrographs of breast cancer metastasis sections showing low SNAI2 expression (d and e) or high SNAI2 expression (f and g) (× 20 magnification)