TAM iron metabolism impacts on breast cancer biology

Iron is a microelement of vital importance for every metabolically active and dividing cell. Its relevance for invading pathogens and mutagenic properties implies the necessity of its tight regulation. Under physiological conditions, one of the key players in this process are macrophages, which orchestrate iron uptake, recycling, storage and redistribution on a systemic as well as local scale. Vigorously dividing malignant cells in general and breast cancer in particular exhibit an exacerbate demand for iron needed for extensive metabolism and sustained activation of pro-survival, pro-inflammatory and pro-metastatic signaling. Accordingly, the aberrant modulation of iron turnover proteins in human breast cancer tissue was demonstrated to correlate with tumor progression and metastatic dissemination. In spite of this evidence, the cellular and molecular mechanisms of iron homeostasis in breast carcinoma are only incompletely characterized. Breast neoplasms are highly infiltrated by a pro-angiogenic and immunosuppressive population of leukocytes called Tumor-Associated Macrophages (TAMs), whose abundance negatively influences patient`s prognosis. These cells, in analogy with macrophages in non-malignant tissue, may perform key functions in iron ingestion and provision to rapidly proliferating tumor epithelium. Yet, this hypothesis has never been experimentally followed in depth. In the proposed project we will explore iron capture and storage capabilities of TAMs and discern whether TAM-specific alterations of iron flux can impact on tumor growth and metastatic spreading as well as the differentiation properties of tumor-infiltrating macrophages. To achieve this we plan experiments with clinically relevant murine models of autochthonous mammary carcinogenesis, TAM depletions and macrophage specific knockouts of the exclusive iron export protein ferroportin and the crucial storage molecule ferritin. In addition, such animals will be treated with diverse iron donors. Finally, we intend to verify the findings of the animal experiments in the human setting by analyzing the expression of iron- metabolism genes in TAMs of primary breast cancer lesions and by correlating these data with clinical parameters and probability of recurrence and survival. The results are expected to substantially augment our knowledge on the mutual interaction of macrophages and malignant cells in terms of iron metabolism. Furthermore, they will help to clarify safety issues in the treatment of cancer-accompanying anemia by iron application and the potential usefulness of ferroportin modulation. The findings of the patient study will provide a valuable insight into prognostic relevance of local iron homeostasis and interplay between iron turnover and the presence of TAMs.

Iron is a crucial element for every living cell as a co-factor of numerous metabolic enzymes and the transporter of oxygen. However, surplus iron, if not bound by a storing protein ferritin (FT) or expelled from the cell by the sole known exporter ferroportin (FPN1), promotes generation of reactive oxygen species damaging cell components and causing toxicity. Hence, systemic and local iron flux needs to be tightly controlled. In normal tissues, a resident population of leukocytes, macrophages, perform this task by orchestrating iron uptake, storage and export. In the finalized project, we postulated that a special subset of macrophages infiltrating cancerous tissue, so called Tumor Associated Macrophages or TAMs, may serve similar functions and supply rapidly dividing and metabolizing malignant cells with the crucial element. To address this hypothesis, we used mice carrying macrophage-specific genetic deletions of FT and FPN1-coding genes, so called knockouts which were implanted mammary carcinoma cells to induce malignancy. In addition, animals were treated with a clinically applicable iron supplement used for treatment of anemia, also in tumor patients. Our results show that iron supplementation leads to a significant acceleration of tumor growth especially in mice with the deletion of macrophage ferritin. By more detailed investigation, we found that such ferritin-deficient leukocytes which could not store the element increase levels of the exporter FPN1 and export iron at a much faster pace than ferritin-proficient macrophages. This implicates that ferritin-knockout macrophages provide iron to tumor cells more efficiently, which may explain the faster cancer progression. However, we found as well that iron supplementation influences neither the division nor the death rate of malignant cells. Instead, iron was found particularly toxic to activated T cells directed against the tumor and iron negatively interfered with the outcomes of T cell-stimulating cancer therapies in our murine mammary cancer model. These observations may have implications for our view on safety and efficacy of iron supplements used for treatment of cancer-associated anemia. In particular, administration of iron to cancer individuals treated with T cell-stimulating drugs needs to be considered with caution.