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. 2012 Nov 8;31(45):4798-802.
doi: 10.1038/onc.2011.635. Epub 2012 Jan 16.

FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling

J Wang  1 , P S Ray, M-S Sim, X Z Zhou, K P Lu, A V Lee, X Lin, S P Bagaria, A E Giuliano, X Cui
Affiliations

FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling

J Wang et al. Oncogene. .

Abstract

Human basal-like breast cancer (BLBC) is an enigmatic and aggressive malignancy with a poor prognosis. There is an urgent need to identify therapeutic targets for BLBC, because current treatment modalities are limited and not effective. The forkhead box transcription factor FOXC1 has recently been identified as a critical functional biomarker for BLBC. However, how it orchestrates BLBC cells was not clear. Here we show that FOXC1 activates the transcription factor nuclear factor-κB (NF-κB) in BLBC cells by increasing p65/RelA protein stability. High NF-κB activity has been associated with estrogen receptor-negative breast cancer, particularly BLBC. The effect of FOXC1 on p65/RelA protein stability is mediated by increased expression of Pin1, a peptidyl-prolyl isomerase. FOXC1 requires NF-κB for its regulation of cell proliferation, migration and invasion. Notably, FOXC1 overexpression renders breast cancer cells more susceptible to pharmacological inhibition of NF-κB. These results suggest that BLBC cells may rely on FOXC1-driven NF-κB signaling. Interventions of this pathway may provide modalities for the treatment of BLBC.

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Conflict of interest statement

Conflict of Interest

Dr. Wang, Dr. Ray, Dr. Bagaria, and Dr. Cui are named inventors on patent applications regarding the role of FOXC1 in cancer.

Figures

Figure 1
Figure 1. FOXC1 induces NF-κB activity in breast cancer cells
(A) Significant canonical signaling pathways in basal-like (red), HER2 (green) and luminal (blue) breast cancers from the Richardson et al. dataset were identified using Ingenuity Pathway Analysis and ranked by the impact factor (see Supplementary Information for detailed methods). (B) Immunoblotting of NF-κB components in MDA-MB-231 cells overexpressing FOXC1 or the vector. (C) Immunoblotting of p65 in control or FOXC1 shRNA-expressing BT-549 breast cancer cells. (D) Nuclear proteins were isolated from vector- or FOXC1-overexpressing MDA-MB-231 cells, followed by immunoblotting of p65 and a nuclear marker Lamin A/C. (E) Nuclear localization of p65 protein was visualized by fluorescence microscopy (green, p65; blue, nuclear DNA staining by DAPI). (F) MDA-MB-231 cells were transiently transfected with NF-κB-luc or the vector pGL4-luc, FOXC1, and IκBα-SR. NF-κB activity was assessed by luciferase assays. Datarepresent mean ± SD (n = 3).
Figure 2
Figure 2. FOXC1 increases p65 protein stability by upregulating Pin1 in breast cancer cells
(A) Immunoblotting of Pin1 in MDA-MB-231 cells overexpressing FOXC1 or the vector. (B) Real-time RT-PCR analysis of Pin1 mRNA in the same cells. The Pin primers are 5′-TGGGTGCCTTCAGCAGAGGTCAG-3′ and 5′-CCGGAATCCGTGAACACGGGC-3′ (see Supplementary Information for detailed methods). (C) A 2.3 kb Pin1 promoter-luciferase reporter construct was co-transfected into MDA-MB-231 cells with FOXC1 or the vector, followed by luciferase assays. (D) Lysates from FOXC1- or vector-overexpressing MDA-MB-231 cells were immunoprecipitated with an anti-p65 antibody, followed by immunoblotting of IκBα, Pin1, and SOCS-1. (E) Same cells were transfected with a ubiquitin construct, treated with 10 μM MG-132, and subjected to immunoprecipitation with an anti-p65 antibody, followed by immunoblotting of ubiquitin. (F) Same cells were treated with cycloheximide (10 μg/ml) and harvested at the indicated time points, followed by immunoblotting and densitometry of protein bands. Band intensities were normalized to that of actin, then normalized to the t = 0 controls.
Figure 3
Figure 3. NF-κB mediates the effects of FOXC1 on cell proliferation, migration, and invasion
(A) FOXC1- or vector-overexpressing MDA-MB-231 cells were treated with the NF-κB inhibitor BMS-345541 (2 μM), followed by MTT assays (left), transwell migration assays (middle), and transwell invasion assays (right). Data represent mean ± SD (n = 3). (B) Wild-type (wt) and IKKα/IKKβ-null MEFs were transfected with FOXC1 or the vector, followed by cell proliferation MTT assays at the indicated time points (left). Deficiency of IKK expression in knockout MEFs is shown by immunoblotting (right). (C) Wild-type (wt) and IKKα/IKKβ-null MEFs were transfected with FOXC1 or the vector, followed by cell cycle analysis using flow cytometry (left). *, P < 0.05. CyclinD1 expression was assessed by immunoblotting (right).
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