Contribution of three-dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer

Abstract

Hepcidin is a peptide hormone that negatively regulates iron efflux and plays an important role in controlling the growth of breast tumors. In patients with breast cancer, the combined expression of hepcidin and its membrane target, ferroportin, predict disease outcome. However, mechanisms that control hepcidin expression in breast cancer cells remain largely unknown. Here we use three-dimensional breast cancer spheroids derived from cell lines and breast cancer patients to probe mechanisms of hepcidin regulation in breast cancer. We observe that the extent of hepcidin induction and pathways of its regulation are markedly changed in breast cancer cells grown in three dimensions. In monolayer culture, BMPs, particularly BMP6, regulate hepcidin transcription. When breast cancer cells are grown as spheroids, there is a >10 fold induction in hepcidin transcripts. Microarray analysis combined with knockdown experiments reveal that GDF-15 is the primary mediator of this change. The increase in hepcidin as breast cells develop a three-dimensional architecture increases intracellular iron, as indicated by an increase in the iron storage protein ferritin. Immunohistochemical staining of human breast tumors confirms that both GDF-15 and hepcidin are expressed in breast cancer specimens. Further, levels of GDF-15 are significantly correlated with levels of hepcidin at both the mRNA and protein level in patient samples, consistent with a role for GDF-15 in control of hepcidin in human breast tumors. Inclusion of tumor-associated fibroblasts in breast cancer spheroids further induces hepcidin. This induction is mediated by fibroblast-dependent secretion of IL-6. Breast cancer cells grown as spheroids are uniquely receptive to IL-6-dependent induction of hepcidin by tumor-associated fibroblasts, since IL-6 does not induce hepcidin in cells grown as monolayers. Collectively, our results suggest a new paradigm for tumor-mediated control of iron through the control of hepcidin by tumor architecture and the breast tumor microenvironment.

Growth/Differentiation Factor-15 (GDF-15): From Biomarker to Novel Targetable Immune Checkpoint

“…In breast cancer, where GDF-15 has been linked to metastasis and to resistance toward trastuzumab, the following signaling pathways were suggested for GDF-15: Signaling via Smad 1/5/8 leading to up-regulation of hepcidin (145); induction of IGF1R-FoxM1 signaling leading to activation of Snail and Slug, epithelial-to-mesenchymal transition and MMP2/9-mediated cellular invasion (109); triggering of HER2-independent, TGFRand Src-dependent phosphorylation of the HER2-Akt-Erk1/2 axis (146,147); activation of p38 MAPK (148) and of JNK mitogen-activated protein kinases (MAPKs) (149) resulting in enhanced invasion of HER2-positive cancer cells. In contrast, another study found inhibition of invasion and of metastasis via activation of the Yes associated protein (YAP) leading to (YAP)-dependent transcriptional repression (150).…”

Altered Iron Metabolism and Impact in Cancer Biology, Metastasis, and Immunology

“…High levels of hepcidin are often observed in tumors to maintain an iron-utilization phenotype within tumor cells. CAFs can induce hepcidin in tumor cells through interleukin 6 (IL-6) secretion and stimulation of signal transducer and activator of transcription 3 (STAT3) signaling (170), illustrating one of the mechanisms where iron metabolism underpins tumor-stroma crosstalk.…”

New highlight in breast cancer development: the key role of hepcidin and iron metabolism

“…In line with these considerations, Blanchette-Farra and collaborators recently proposed a new paradigm for tumor-mediated control of iron via hepcidin by tumor architecture and the breast tumor microenvironment (5). Authors used 3D cell culture method by investigating breast cancer spheroids derived from both MCF-7 and MCF-10a cell lines and primary cell culture from breast cancer patients to study the possible role of cancer-associated fibroblasts in the regulation of breast tumor pathways by iron and hepcidin (5). Hepcidin is a peptide hormone that binds to ferroportin and elicits its degradation, playing a not redundant role in breast disease by increasing iron cytoplasmic concentration (6).…”

“…Thus, the identification of molecular mechanisms involved in breast cancer occurrence and development represents one of the most important goals of the biomedical research. In line with these considerations, Blanchette-Farra and collaborators recently proposed a new paradigm for tumor-mediated control of iron via hepcidin by tumor architecture and the breast tumor microenvironment (5). Authors used 3D cell culture method by investigating breast cancer spheroids derived from both MCF-7 and MCF-10a cell lines and primary cell culture from breast cancer patients to study the possible role of cancer-associated fibroblasts in the regulation of breast tumor pathways by iron and hepcidin (5).…”