Lactoferrin-endothelin-1 axis contributes to the development and invasiveness of triple-negative breast cancer phenotypes

Abstract

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathways responsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown. In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-α, PR, and HER-2 in a proteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- or HER-2-targeted therapies. Furthermore, we found that lactoferrin increases migration and invasiveness of both non-TNBC and TNBC cell lines. We discovered that lactoferrin directly stimulates the transcription of endothelin-1 (ET-1), a secreted proinvasive polypeptide that acts through a specific receptor, ET(A)R, leading to secretion of the bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor-antagonist blocked lactoferrin-dependent motility and invasiveness of breast cancer cells. The physiologic significance of this newly discovered Lf-ET-1 axis in the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue lactoferrin and ET-1 levels in patients with TNBC compared with those in ER(+) cases. These findings describe the first physiologically relevant polypeptide as a functional determinant in downregulating all three therapeutic receptors in breast cancer, which uses another secreted ET-1 system to confer invasiveness. Results presented in this article provide proof-of-principle evidence in support of the therapeutic effectiveness of ET-1 receptor antagonist to completely block the lactoferrin-induced motility and invasiveness of the TNBC as well as non-TNBC cells, and thus, open a remarkable opportunity to treat TNBC by targeting the Lf-ET-1 axis using an approved developmental drug.

Figure 1. Lactoferrin downregulates ERα, PR, and HER-2 in human breast cancer cells

(A) Effect of a single dose of Lf on the levels of indicated proteins in ZR-75 cells. Cells were treated with 20 μg/ml of Lf for indicated times and subjected to Western blot analysis (B) Effect of multiple and increasing doses of Lf on expression levels of proteins in ZR-75 cells. (C) Analysis of ERα, PR and HER-2 levels in ZR-75 cells by confocal microscopy. Cells were treated with 20μg/ml of Lf for 12 hrs, fixed and then stained with protein-specific antibodies. Alexa-488 (ERα and HER-2), Alexa-555 (PR) and DAPI (nucleus). Bar = 5μm.

Figure 2. Lactoferrin downregulates ERα, PR, and HER-2 at a transcriptional level

(A) Northern blot analysis of ERα, PR and HER-2 mRNAs in MCF-7 cells treated with or without 100 μg/ml of Lf for indicated times. (B) MCF-7 cells were treated with or without Lf for indicated time points or with different concentrations of Lf in the presence or absence of proteasomal inhibitor, MG-132. Vinculin was used as a loading control. (C) MDA-MB-231 stable clones expressing ERα or HER-2 were generated as described in the materials and methods. Vinculin was used as a loading control. (D) Effect of Lf on the levels of ERα and HER-2 receptors in MDA-MB-231 pooled clones stably expressing ERα or HER-2.

Figure 3. Lf promotes breast cancer cell migration and invasiveness and compromises anti-invasive activity of tamoxifen and Herceptin

(A) Effect of Lf on cell migration in tamoxifen-treated MCF-7 cells. On the right is the quantification of the average difference in wound closure between 0 and 24 hrs. Left micrographs show representative images of migrated cells after 24 hrs of treatment. (B) Effect of Lf on cell migration in Herceptin-treated MCF-7 cells. On the right is the quantification of the average difference in wound closure between 0 and 24 hrs. Left micrographs show representative images of migrated cells after 24 hrs of treatment. (C) Effect of Lf on cell invasion in tamoxifen-treated MCF-7 cells. E2 was added to the upper wells to enhance tamoxifen effect. (D) Effect of Lf on cell invasion in Herceptin-treated MCF-7 cells. Heregulin was added to the upper wells to enhance Herceptin effect. (E) Effect of Lf on the invasiveness of MDA-MB-231 and MDA-MB-468 cells. Left micrographs are representative images of invaded cells Right is the quantification of the average number of invaded cells. Error bars indicate standard deviation. Scale bars = 0.5mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure 4. Endothelin-1 is a transcriptional target of Lf

(A & B) Analysis of differentially regulated genes after Lf treatment in MDA-MB-231 and MDA-MB-468 cells by Affymetrix Human Exon 1.0 ST microarrays using Gene-Spring GX 10.0.2. (C) Effect of Lf on ET-1 mRNA levels in breast cancer cells. Actin was used as a loading control. (D) Effect of Lf in the presence or absence of cycloheximide on the induction of ET-1 mRNA in MDA-MB-231 cells.

Figure 5. Lf stimulates Endothelin-1 transcription

(A) Effect of Lf on ET-1 promoter activity in breast cancer cells. Cells were treated with 100 μg/ml of Lf for 36 hrs. (B) EMSA analysis of Lf binding to all three Lf-consensus DNA sequences in the ET-1 promoter using the nuclear extracts from MCF-7 cells treated with or without Lf for 6 hrs. (C) Effect of Lf on the ET-1 promoter with wild-type or mutant Lf-binding sequences in MDA-MB-468 cells. (D) EMSA analysis of Lf binding to oligonucleotides encompassing the wild-type Lf-consensus DNA sequence 3 or its three mutant versions using the nuclear extracts from MCF-7 cells treated with or without Lf for 6 hrs. (E) EMSA analysis of Lf-stimulated nuclear extracts from MCF-7 cells in the presence of antibody to Lf. Antibody to rabbit-IgG was used as a negative control (F) Effects of holo- or apo-Lf on the ET-1 promoter luc activity in MDA-MB-468 cells. Error bars indicate standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure 6. Endothelin-1 receptor antagonist blocks lactoferrin-dependent invasiveness

(A) Concentration of ET-1 in the conditioned media from breast cancer cells treated with or without 100 μg/ml of Lf for 24 hrs as measured by ELISA. (B) Effect of ET-1-receptor inhibitor BQ123 on cell migration of MCF-7 cells treated with or without Lf or ET-1, using Boyden chambers. (C) Effect of ET-1-receptor inhibitor BQ123 on cell invasion of MCF-7 cells treated with or without Lf or ET-1 using Matrigel^™ invasion chambers. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure 7. Lf-ET-1 axis promotes TNBC phenotypes

(A) Quantitative PCR analysis of Lf mRNA levels in tumor samples from TNBC and non-TNBC breast cancer patients. (B) Q-PCR analysis of ET-1 mRNAs in TNBC or non-TNBC breast tumors. mRNA experiments were repeated three times, with each point in triplicate. (C) Concentration of Lf and ET-1 in plasma samples from TNBC or non-TNBC patients by ELISA. (D) IHC evaluation of Lf and ET-1 expression in breast tumors from the same patients used in panel B. Table depicts TNBC and non-TNBC tissue samples with scoring of Lf and ET-1 staining intensity: +++ (strong), ++ (medium). (E) Working model for Lf-mediated downregulation of receptors and cell migration/invasion in triple negative breast cancer cells.