FOXC1 is a critical mediator of EGFR function in human basal-like breast cancer

Abstract

Background: Human basal-like breast cancer (BLBC) has a poor prognosis and is often identified by expression of the epidermal growth factor receptor (EGFR). BLBC remains a major clinical challenge because its pathogenesis is not well understood, thus hindering efforts to develop targeted therapies. Recent data implicate the forkhead box C1 (FOXC1) transcription factor as an important prognostic biomarker and functional regulator of BLBC, but its regulatory mechanism and impact on BLBC tumorigenesis remain unclear.

Methods: The association between FOXC1 and EGFR expression in human breast cancer was examined by immunohistochemistry in formalin-fixed tissues and analysis of the TCGA database. The regulation of FOXC1 by EGFR activation was investigated in MDA-MB-468 cells using immunoblotting, qRT-PCR, and luciferase activity assays. This EGFR effect on FOXC1 expression was confirmed using the MDA-MB-468 xenograft model.

Results: Both FOXC1 mRNA and protein levels significantly correlated with EGFR expression in human breast tumors. EGFR activation induced FOXC1 transcription through the ERK and Akt pathways in BLBC. EGFR inhibition in vivo reduced FOXC1 expression in xenograft tumors. We also found that FOXC1 knockdown impaired the effects of EGF on BLBC cell proliferation, migration, and invasion.

Conclusions: Our findings uncover a novel EGFR-FOXC1 signaling axis critical for BLBC cell functions, supporting the notion that intervention in the FOXC1 pathway may provide potential modalities for BLBC treatment.

Figure 1. FOXC1 expression significantly correlates with EGFR expression in human BLBC tumors

(A) Distribution of EGFR and FOXC1 by immunohistochemical staining scores in 34 human triple-negative samples (P=0.0356). Association was examined using the Fisher's Exact Test. (B) Representative images for FOXC1 and EGFR IHC in a human breast tumor. Magnification: × 400. (C) Correlation between EGFR and FOXC1 mRNA expression in invasive breast cancer samples from the TCGA database (R=0.5151, P<0.0001).

Figure 2. EGFR activation up-regulates FOXC1 expression in BLBC cells

(A) MDA-MB-468 and BT-20 EGFR-positive human breast cancer cells and MCF-10A human mammary epithelial cells were serum-starved overnight and treated with EGF, TGFα, or HB-EGF (50 ng/ml) for 24 h. FOXC1 level was analyzed by immunoblotting. Actin was used as a loading control. (B) Serum-starved MDA-MB-468 cells were treated with increasing concentrations of EGF for 24 h (top) or with EGF (100 ng/ml) for the indicated time periods (bottom), followed by immunoblotting analysis. (C) MDA-MB-468 cells were serum starved overnight. Then cells were treated with EGF for 24 h in the presence or absence of the EGFR inhibitor AG1478 (1 μM), Lapatinib (1 μM) or Erlotinib (3 μM). FOXC1 protein levels were measured by immunoblotting. (D) MDA-MB-468 cells (left) and MCF-10A cells (right) were serum-starved overnight and treated with EGF for the indicated time periods. FOXC1 mRNA levels were analyzed by qRT-PCR. *, P<0.05; ***, P<0.0001; **, P<0.001. (E) MDA-MB-231 cells were transfected with pBABE-EGFR construct for 48 h. FOXC1 mRNA levels were detected by qRT-PCR. *, P<0.05. (F) MDA-MB-468 cells were serum-starved overnight. Then cells were treated with EGF for 6 h after DRB pretreated for 1 h. FOXC1 mRNA levels were detected by qRT-PCR. *, P<0.05. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Data represent mean ± SD from 3 independent experiments.

Figure 3. EGFR inhibitor Gefitinib abrogates the growth of xenograft mammary tumor in nude mice

BALB/c nu/nu nude mice were subcutaneously inoculated with MDA-MB-468 cells. When tumors reached ~150 mm³, these mice were randomized into 2 groups (n=10) for Iressa treatment. (A) Comparisons of mice and tumors of between the two groups are shown. (B) The tumor growth curves are plotted. Error bars represent 95% confidence intervals, P<0.05. (C) The levels of p-EGFR and FOXC1 in the tumor tissues were detected by immunoblotting with the indicated antibodies.

Figure 4. EGFR activation up-regulates FOXC1 expression through ERK and PI3K/Akt pathways

(A) MDA-MB-468 cells were-serum starved overnight. Cells were treated with EGF for 24 h in the presence or absence of 5 μM U0126 (MEK inhibitor), 20 μM LY294002 (PI3K inhibitor), or 10 μM SP600125 (JNKII inhibitor) after preincubation with these inhibitors for 30 minutes. p-ERK (T202/Y204), ERK, p-Akt (S473), Akt, p-JNK (T183/Y185), JNK, and FOXC1 levels were analyzed by immunoblotting. (B) MDA-MB-468 cells were transiently transfected with the FOXC1-luc and then pretreated with the inhibitors for 30 minutes followed by EGF treatment for 24 h in the presence or absence of these inhibitors, followed by luciferase assays. **, P<0.001; ***, P<0.0001. (C) MDA-MB-468 cells were transiently transfected with the FOXC1 promoter-luc and then pretreated with 5 μM U0126, 1 μM AI-IV (Akt inhibitor) or their combination for 30 minutes followed by EGF treatment for 24 h in the presence or absence of these inhibitors, followed by luciferase assays. *, P<0.05; **, P<0.001. (D) MDA-MB-468 cells were serum-starved overnight, then pretreated with 5 μM U0126, 1 μM AI-IV or their combination for 30 minutes followed by EGF treatment for 6 h in the presence or absence of these inhibitors. FOXC1 mRNA levels were detected by qRT-PCR. **, P<0.001; ***, P<0.0001. Data represent mean ± SD of 3 independent experiments.

Figure 5. FOXC1 is essential for EGF-mediated cell proliferation, migration, and invasion

(A) MDA-MB-157 or BT-20 cells were transiently transfected with control or FOXC1 siRNA for 24 h, and then treated with EGF for another 24 h after starvation overnight. FOXC1 knockdown was confirmed by immunoblotting. (B) Control or FOXC1-knockdown cells were stimulated with EGF and cell proliferation was evaluated by CellTiter-Glo assays at the indicated time points. Data represent 3 independent experiments. (C) MDA-MB-468 cells were transfected with control or FOXC1 siRNA for 24 h and then treated with EGF for 24 h. FOXC1 expression was assessed using immunoblotting. (D) Migration and invasion of control and FOXC1-knockdown cells were analyzed using transwell chamber assays. Migrating or invasive cells were counted in 3 randomly selected fields. Data represent mean ± SD. **, P<0.001.