Background: Iron overload is known to cause cancer. Iron depletion treatment has been reported to have an anti-cancer effect. However, it is unclear whether iron depletion treatment is also effective in cancer stem cells (CSCs) or not. Recently, new CSCs model, miPS-LLCcm, was epigenetically established from murine induced pluripotent stem cells (miPS cells) in Okayama university (Chen at el. PLOS ONE 2012). In this model, Green Fluorescent Protein (GFP) was designed under Nanog promoter lesion. Therefore, GFP positive cells indicated the undifferentiated cancer progenitor cells. The tumor tissue derived from miPS-LLCcm cells involved glandular structure, angiogenesis, and cytokeratin positive lesion in immunostaining. We hypothesized that CSCs depended on iron in proliferation and differentiation. Thus, we also hypothesized iron depletion treatment might have a novel therapeutic effect.

Methods: We used miPS-LLCcm cells as CSCs, colon26 and 4T1 cells as differentiated cancer cells. Iron depleted condition was simulated by iron free medium with 1% fetal bovine serum. Transferrin (Holo) and iron chelators (Deferoxamine and Deferasirox) additional examinations were done to reveal the dependency of iron. Cell viability was examined by XTT assay 48 hours after administration of transferrin and iron chelators. Western blot analysis was done to examine the effect of iron depletion on iron related markers and stemness markers including TfR-1, DMT-1, Nanog, SOX2, c-Myc, Oct3/4, Klf-4, E-cadherin. Subcutaneous tumor model of miPS-LLCcm cells was used in vivo. Deferoxamine (30mg/kg/day) and deferasirox (30mg/kg/day) were administrated directly into the tumor. Tumors were collected for immunostaining.

Results: Transferrin strongly promoted cell proliferation of miPS-LLCcm in iron depleted condition. However, Transferrin did not promote proliferation of colon26 and 4T1 in the condition. Iron depletion by iron chelators suppressed miPS-LLCcm proliferation and the expression of stemness markers including Nanog, SOX2, c-Myc, Oct3/4, Klf-4, and E-cadherin in vitro study. Deferasirox more strongly suppressed the expression of stemness markers than deferoxamine. Iron depletion by iron chelators suppressed subcutaneous tumor growth. The average tumor volume of the control group was 1270.8 ± 411.2 mm3 while that of deferoxamine treatment group was 502.5 ± 207.5 mm3 (p<0.05) and deferasirox treatment group was 360.2 ± 148.8 mm3 (p<0.01) on day 19. The expression of stemness markers was also more strongly suppressed in the deferasirox treatment group than deferoxamine treatment group.

Conclusions: Iron is a key element of the proliferation and stemness in CSCs model. Iron control therapy including iron chelators is a novel therapeutic target of CSCs.

Citation Format: Toshiaki Ohara, Takayuki Ninomiya, Kazuhiro Noma, Hajime Kashima, Yuki Katsura, Takuya Kato, Yasuko Tomono, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Iron control is a novel therapeutic target of cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2510.