Introduction: Studies suggest that iron metabolism is associated with tumor progression, but a mechanism and the relative importance of different iron handling proteins still remain to be elucidated. We investigated the effects of the frequently polymorphic high iron gene (HFE), on tumor growth. This study is significant because of the ability of HFE mutant protein to alter iron handling and because our previous studies have shown the presence of this mutation is associated with altered cancer phenotype in cells.
Hypothesis: Mutations in the HFE protein modify tumor progression and metastatic potential.
Methods: C57BL/6 mice possessing either H67D/H67D (H63D in humans) or WT/WT HFE genotype, aged 18 months, were injected subcutaneously with 4x106 B16F10 cells. After 2 weeks, mice were sacrificed and the tumors were weighed and prepared for analysis of RNA, protein, and histology. At the same time, plasma was collected by cardiac puncture. Animal methods were approved by our IACUC, protocol 04-166. RT-PCR analysis of tumors was performed using the Mouse Cancer Pathwayfinder Array (SABiosciences). In parallel, bone marrow derived macrophages were cultured to determine how B16F10 cell cultures react to conditioned media from macrophages of each genotype. ELISAs were performed for ferroportin and ferritin. Secreted cytokine and chemokine content of macrophage media was assessed with an ELISA array (Qiagen).
Results: H67D mice had significantly smaller tumors (t-test, P = 0.0166) after two weeks. The two-week time frame was selected because rapid tumor growth affected animal survival at longer incubations. Exploratory qRT-PCR analysis of tumors revealed that the H67D host may promote epithelial phenotype and suppress angiogenesis and growth. In culture, macrophages derived from H67D mice express higher levels of MCP1 (p = 0.0169). Conditioned media from the H67D macrophages was associated with significantly less B16F10 cell growth compared to WT (p < 0.0001 MTT, p = 0.0008 BrdU). In macrophage cell lysates, H67D is associated with higher levels of Ferroportin (p = 0.0297), and WT macrophages showed higher iron levels in response to FAC (p = 0.0295).
Conclusions: Tumors grown in H67D mice are significantly lower in weight than control mice. Macrophages from H67D mice neither load iron nor support tumor cell proliferation as well as WT controls. Our data reveal the importance of HFE genotype on macrophage function and tumor growth. The H63D HFE allele is seen in approximately 15% of the human population so its impact on cancer studies is likely significant.
Citation Format: Cody L. Weston, Ahmed Alkhateeb, William Hund, James R. Connor. HFE genotype affects tumor progression and macrophage behavior in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2014-24