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.