Iron is an essential element for virtually all organisms. On the one hand, it facilitates cell proliferation and growth. On the other hand, iron may be detrimental due to its redox abilities, thereby contributing to free radical formation, which in turn may provoke oxidative stress and DNA damage. Iron also plays a crucial role in tumor progression and metastasis due to its major function in tumor cell survival and reprogramming of the tumor microenvironment. Therefore, pathways of iron acquisition, export, and storage are often perturbed in cancers, suggesting that targeting iron metabolic pathways might represent opportunities towards innovative approaches in cancer treatment. Recent evidence points to a crucial role of tumor-associated macrophages (TAMs) as a source of iron within the tumor microenvironment, implying that specifically targeting the TAM iron pool might add to the efficacy of tumor therapy. Here, we provide a brief summary of tumor cell iron metabolism and updated molecular mechanisms that regulate cellular and systemic iron homeostasis with regard to the development of cancer. Since iron adds to shaping major hallmarks of cancer, we emphasize innovative therapeutic strategies to address the iron pool of tumor cells or cells of the tumor microenvironment for the treatment of cancer.

Tumour cell‐secreted factors skew infiltrating immune cells towards a tumour‐supporting phenotype, expressing pro‐tumourigenic mediators. However, the influence of lipocalin‐2 (Lcn2) on the metastatic cascade in the tumour micro‐environment is still not clearly defined. Here, we explored the role of stroma‐derived, especially macrophage‐released, Lcn2 in breast cancer progression. Knockdown studies and neutralizing antibody approaches showed that Lcn2 contributes to the early events of metastasis in vitro. The release of Lcn2 from macrophages induced an epithelial–mesenchymal transition programme in MCF‐7 breast cancer cells and enhanced local migration as well as invasion into the extracellular matrix, using a three‐dimensioanl (3D) spheroid model. Moreover, a global Lcn2 deficiency attenuated breast cancer metastasis in both the MMTV–PyMT breast cancer model and a xenograft model inoculating MCF‐7 cells pretreated with supernatants from wild‐type and Lcn2‐knockdown macrophages. To dissect the role of stroma‐derived Lcn2, we employed an orthotopic mammary tumour mouse model. Implantation of wild‐type PyMT tumour cells into Lcn2‐deficient mice left primary mammary tumour formation unaltered, but specifically reduced tumour cell dissemination into the lung. We conclude that stroma‐secreted Lcn2 promotes metastasis in vitro and in vivo, thereby contributing to tumour progression. Our study highlights the tumourigenic potential of stroma‐released Lcn2 and suggests Lcn2 as a putative therapeutic target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

While the importance of iron for tumor development is widely appreciated, the exact sources of tumor-supporting iron largely remain elusive. The possibility that iron might be provided by stromal cells in the tumor microenvironment was not taken into account so far. In the present study, we show that tumor-associated macrophages (TAM) acquire an iron-release phenotype upon their interaction with tumor cells, thereby increasing the availability of iron in the tumor microenvironment. Mechanistically, TAM expressed elevated levels of the high-affinity iron-binding protein lipocalin-2 (LCN-2), which appeared to be critical for the export of iron from TAM, and in turn enhanced tumor cell proliferation. Moreover, in PyMT-mouse tumors as well as in primary human breast tumors LCN-2 was predominantly expressed in the tumor stroma as compared to tumor cells. LCN-2 expression in the stroma further correlated with enhanced tumor proliferation in vivo. Our data suggest a dominant role of TAM in the tumor iron-management and identify LCN-2 as a critical iron transporter in this context. Targeting the LCN-2 iron export mechanism selectively in stromal cells might open for future iron-targeted tumor therapeutic approaches.

A growing body of evidence suggests that macrophage polarization dictates the expression of iron-regulated genes. Polarization towards iron sequestration depletes the microenvironment, whereby extracellular pathogen growth is limited and inflammation is fostered. In contrast, iron release contributes to cell proliferation, which is important for tissue regeneration. Moreover, macrophages constitute a major component of the infiltrates in most solid tumors. Considering the pivotal role of macrophages for iron homeostasis and their presence in association with poor clinical prognosis in tumors, we approached the possibility to target macrophages with intracellular iron chelators. Analyzing the expression of iron-regulated genes at mRNA and protein level in primary human macrophages, we found that the iron-release phenotype is a characteristic of polarized macrophages that, in turn, stimulate tumor cell growth and progression. The application of the intracellular iron chelator (TC3-S₎₂ shifted the macrophage phenotype from iron release towards sequestration, as determined by the iron-gene profile and atomic absorption spectroscopy (AAS). Moreover, whereas the addition of macrophage supernatants to tumor cells induced tumor growth and metastatic behavior, the supernatant of chelator-treated macrophages reversed this effect. Iron chelators demonstrated potent anti-neoplastic properties in a number of cancers, both in cell culture and in clinical trials. Our results suggest that iron chelation could affect not only cancer cells but also the tumor microenvironment by altering the iron-release phenotype of tumor-associated macrophages (TAMs). The study of iron chelators in conjunction with the effect of TAMs on tumor growth could lead to an improved understanding of the role of iron in cancer biology and to novel therapeutic avenues for iron chelation approaches.

Background: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. Methods: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. Results: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. Conclusions: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.

While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.