Volume 2, Issue 2, Pages 270-282 (August 2012) Luminal Iron Levels Govern Intestinal Tumorigenesis after Apc Loss In Vivo  Sorina Radulescu, Matthew J. Brookes, Pedro Salgueiro, Rachel A. Ridgway, Ewan McGhee, Kurt Anderson, Samuel J. Ford, Daniel H. Stones, Tariq H. Iqbal, Chris Tselepis, Owen J. Sansom  Cell Reports  Volume 2, Issue 2, Pages 270-282 (August 2012) DOI: 10.1016/j.celrep.2012.07.003 Copyright © 2012 The Authors Terms and Conditions

Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 1 Effect of Modulating Wnt Signaling on Expression of Iron Transport Proteins (A) Culturing RKO cells with iron (IL) for 24 hr repressed TfR1 and DMT1, and increased ferritin protein levels. LiCl treatment increased the level of TfR1, DMT1, and c-Myc expression relative to control. Costimulation with LiCl and IL (IL+LiCl) further increased c-MYC, TfR1, and DMT1 levels and suppressed ferritin expression. p < 0.0001 c-MYC, DMT1, p = 0.0071 TfR1 in IL+LiCl versus IL alone; p = 0.003 c-MYC, p = 0.0017 TfR1, p = 0.0007 DMT1 in IL+LiCl versus LiCl alone, Student’s t test; ∗ denotes statistical significance compared with control; † denotes statistical significance compared with LiCl alone. β-actin was used for protein normalization. Data are presented as mean ± SEM. (B) To assess cellular IL levels, a ferrozine assay was performed in RKO cells in control or in the presence of IL or LiCl, or both (IL+LiCl) for 24 hr. Wnt activation further increases IL content (p < 0.0001 IL+LiCl versus LiCl alone, p = 0.0006 IL+LiCl versus IL alone, Student’s t test; ∗ denotes statistical significance compared with control; † denotes statistical significance compared with LiCl alone). Data are presented as mean ± SEM. (C) Culturing SW480 cells for 24 hr with IL resulted in upregulation of c-MYC, TfR1, and DMT1, and repression of ferritin. Reconstitution of WT human Apc and culture with IL (IL+APC) significantly reduced c-MYC, TfR1 and DMT1 and ferritin expression (p = 0.002 TfR1, p = 0.003 DMT1, p < 0.0001 ferritin IL+APC versus IL, Student’s t test; ∗ denotes statistical significance compared with control). Data are presented as mean ± SEM. (D) SW480 cells reconstituted with WT human APC resulted in significantly decreased cellular IL content (ferrozine assay; control+APC versus control, p = 0.0053; control+APC versus IL +APC, p < 0.0048, Student’s t test; ∗ denotes statistical significance compared with control; † denotes statistical significance compared with IL alone). Data are presented as mean ± SEM. (E) To assess the association of c-MYC with TfR1, DMT1, and ferritin in vivo, mRNA expression levels were assessed in eight murine polyps from individual ApcMin/+ mice, 20 human colonic polyps, and 10 human colorectal carcinomas. Expression levels were assessed relative to matched normal control mucosa. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 2 Effect of c-Myc Overexpression on Cellular Iron Metabolism RKO cells were transiently transfected with either pCDNA3.1/Zeo-MYCER or empty vector, and the chimeric protein was then activated by the addition of 4-OHT. (A) Representative western blot demonstrating the expression of the chimeric protein. (B) c-MYC overexpression resulted in increased TfR1 and DMT1 mRNA expression (p = 0.017 TfR1, p = 0.0005 DMT1, Student’s t test). Data are presented as mean ± SEM. (C) Representative western blot indicating expression levels of TfR1, DMT1, and ferritin in control and c-MYC overexpressing cells. B-actin was employed for protein normalization. (D) c-MYC overexpression resulted in increased TfR1 and DMT1, and decreased ferritin protein expression (p = 0.0025 TfR1, p = 0.046 DMT1, p < 0.0001 ferritin, Student’s t test). Data are presented as mean ± SEM. (E) To assess the influence of activated c-MYC ER on cellular iron (IL) levels, cells were cultured in the presence and absence of 4-OHT and IL. Activated c-MYC ER and IL (c-Myc ER+4-OHT+IL) enhanced cellular IL loading compared with cells with no IL (c-Myc ER+4-OHT) and in nonactivated c-MYC ER cells challenged with IL (c-Myc ER+IL; p < 0.0001 c-Myc ER+4-OHT+IL versus c-Myc ER+4-OHT, p = 0.0016 c-Myc ER+4-OHT+IL versus c-Myc ER+IL, Student’s t test). Data are presented as mean ± SEM; ∗ denotes statistical significance compared with control; † denotes statistical significance compared with c-Myc ER+IL. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 3 Iron Modifies Intestinal Tumorigenesis in the ApcMIN/+ Mouse (A–C) Tumor number, size, and burden comparison between CD- and IDD-fed mice at 80 days of age (p < 0.0001 number, burden, p = 0.0006 size, Mann-Whitney). (D) Tumor number comparison on section (first 10 cm of intestine) between CD- and CID-fed mice at 60 days of age (p = 0.002, Mann-Whitney). (E) Representative H&E pictures of intestines from CD- and CID-fed mice. Panels highlight numerous small adenomas in the CID-fed mice. Scale bar: 200 μm. (F) Perls’ Prussian blue staining of adenomas from control and CID-fed mice showing iron deposits within the CID tumors. Scale bar: 10 μm. See also Figures S1 and S2. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 4 High Iron Drives Intestinal Tumorigenesis and Lgr5 Expression (A) Kaplan-Meier survival plot for Lgr5creERT2+ Apcfl/fl mice on IDD, CD, or CID (p = 0.001, logrank, IDD versus CD, and CID versus CD). (B) Percentage of crypts with lesions (in intestines from Lgr5creERT2+ Apcfl/fl mice on CD or CID at day 15 post-induction. A lesion was defined as a clump of >10 cells with nuclear β-catenin accumulation. Each individual crypt (from a strip of 10 cm of intestine) was assessed for the presence or absence of a lesion (p = 0.04, Mann-Whitney). (C) The average number of cells displaying nuclear β-catenin per lesion (as a measure of size) in intestines from Lgr5creERT2+ Apcfl/fl mice on control or CID at day 15 post-induction (p = 0.04, Mann-Whitney). (D) Representative H&E pictures of intestines from Lgr5creERT2+ Apcfl/fl CD- and CID-fed mice at day 15 post-induction. Red lines highlight the size of the Apc-deficient crypts. Scale bar: 10 μm. (E) In vivo GFP fluorescence imaging of whole-mount intestines from Lgr5creERT2+ Apcfl/fl mice on CD or CID (end of tumorigenesis). Tumors express GFP. (F) Measurement of Lgr5 cell volume, via the GFP label, in vivo in normal crypts using a multiphoton laser microscope. MIN CID: Lgr5creERT2+ ApcMIN/+ on high iron for 2 weeks. WT CID: Lgr5creERT2+ Apc+/+ littermate on high iron for 2 weeks. WT control: Lgr5creERT2+ Apc+/+ on CD (age matched). A minimum of 60 crypts were measured per mouse (MIN CID versus WT CID, p < 0.0001, Mann-Whitney). Error bars represent standard deviation (SD). See also Figures S3 and S4. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 5 Iron Levels Modulate Proliferation and Apoptosis Immediately after Apc Loss In Vivo (A) Mitotic index as percentage of cells in mitosis per crypt in Apc-deficient enterocytes from AHCre+ Apc fl/fl mice on CD, IDD, or CID at day 4 post-induction (p = 0.015, CID versus CD, and CID versus IDD, Mann-Whitney). (B) Apoptotic index as percentage of cells in apoptosis per crypt in Apc-deficient enterocytes from AHCre+ Apc fl/fl mice on CD, IDD, or CID at day 4 post-induction (p = 0.015, IDD versus CD, and IDD versus CID, Mann-Whitney). (C) Representative H&E pictures of intestines from AHCre+ Apcfl/fl mice on CD, IDD, or CID at day 4 post-induction. Arrows indicate apoptotic bodies. Circles indicate mitotic figures. Scale bar: 10 μm. (D) Representative H&E pictures of adenomas from Lgr5creERT2+ Apcfl/fl mice on CD, IDD, or CID at endpoint tumorigenesis. Arrows indicate apoptotic bodies. Circles indicate mitotic figures. Scale bar: 10 μm. (E) Mitotic index as percentage of cells in mitosis per adenoma in Lgr5creERT2+ Apcfl/fl mice on CD, IDD, or CID at endpoint tumorigenesis (p = 0.006, CID versus CD, and CID versus IDD, Mann-Whitney). (F) Apoptotic index as percentage of cells in apoptosis per adenoma in Lgr5creERT2+ Apcfl/fl mice on CD, IDD, or CID at endpoint tumorigenesis (p = 0.006, IDD versus CD, and IDD versus CID, Mann-Whitney). See also Figures S5 and S6. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 6 High Iron Levels Alter Stem Cell Gene Expression following Apc Loss In Vivo A population of cells highly enriched in intestinal stem cells was obtained by LCM of the bottom half of the intestinal crypt in WT and Apc-deficient mice (day 4 post-induction). A second population of cells was isolated away from the stem cell zone but still within the recombined area of the crypt and villus, representing the non-stem-cell zone. qRT-PCR analysis of the expression of a panel of stem cell and Wnt target genes was performed in these stem-cell-enriched and non-stem-cell-zone populations. (A) mRNA levels of stem cell genes Olfm4, Lgr5, Troy, and Rgmb (left panel), as well as Wnt target genes Cyclin D2, c-Myc, and Axin2 (right panel), are all increased following Apc loss in mice on the CID compared with WT mice on the same diet (p < 0.01). (B) mRNA levels of stem cell genes Olfm4, Lgr5, Troy, and Rgmb are increased in the stem cell compartment of Apc-deficient mice on the CID compared with CD (left panel, p < 0.01). There is no difference in the expression of these stem cell genes in the non-stem cell compartment between diets. (C) mRNA levels of Wnt target genes Cyclin D2, c-Myc, and Axin2 are increased in both the stem cell compartment (left panel) and the non-stem cell compartment (right panel) of Apc-deficient mice on the CID compared with CD (p < 0.01). See also Figure S7. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure 7 The Luminal Iron Pool Is Responsible for Intestinal Tumor Formation In Vivo (A–C) Tumor number, size, and burden comparison between CD, IDD, and IDD+IDex treatment (IDD+IDex) ApcMIN/+ mice at 80 days of age. Systemic iron overload via IDex treatment does not affect the tumor protection conferred by the lack of dietary iron. (D) Tumor burden comparison between ApcMIN/+ mice with systemic iron overload (IDex), systemic iron chelation (DFO), or CD alone (CD). (E) Overview representing the therapeutic opportunity offered by dietary iron modulation. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S1 The IDD Does Not Affect the Weight Gain of APC MIN/+ Mice, Related to Figures 3 and 7 (A and B) Average weight gain in APC MIN/+ mice on CD, IDD, and CID in the first 2 weeks post-weaning. (C) Weights of APC MIN/+ mice during the first 3 weeks on diets. Note that mice on CID were taken after 2 weeks on the diet. Error bars represent standard deviation. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S2 Iron Deposits in APCMIN/+ Intestine on High and Low Iron Diets, Related to Figure 3 Perls’ Prussian blue stain is shown for intestinal sections taken from mice on the CID at day 60, and from mice on the IDD and the IDD in conjunction with IDex treatment (IDD+IDex) at day 80. Iron deposits are visible in the villus and crypt of the CID mice only. Scale bar: 10 μm. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S3 Iron Deposits and Weight Monitoring in Lgr5creERT2+ APCfl/fl Mice, Related to Figure 4 (A) Perls’ Prussian blue stain is shown for intestinal sections from Lgr5creERT2+ APCfl/fl mice on the CD or CID taken at day 15 post-induction. Iron deposits are visible as blue punctate staining. Scale bar: 10 μm (left) and 5 μm (right). (B) Weights of Lgr5creERT2+ APCfl/fl mice on IDD and CD during a survival experiment. None of the mice developed signs of illness during this period. Error bars represent standard deviation. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S4 Summed Lgr5-GFP Intensity Projections, Related to Figure 4 Intestines from APCMIN/+ (left two panels) and WT mice (right two panels) on the CID and carrying the Lgr5creERT2 construct. (A and B) Projected Lgr5-GPF intensity across intestinal crypts taken from multiphoton microscopy stacks. (C and D) 3D heat map reconstruction of Lgr5-GFP surfaces according to volume of GFP fluorescence. Blue end is low, red end is high. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S5 Effect of IDD and CID on WT Intestinal Crypts, Related to Figure 5 (A–C) Mitotic and apoptotic indices and average crypt size for WT mice on the IDD (3 weeks treatment). The difference between the mitotic and apoptotic indices is significant (p < 0.03) but there is no difference in crypt size. (D and E) Mitotic and apoptotic indices in crypts from CID-treated mice (2 weeks on diet). (F) Weights of WT and APC-deficient mice during the 3 weeks on IDD (the length of the experiment). Error bars represent standard deviation. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S6 Blood Counts from WT and APC-Deficient Mice, Related to Figure 5 (A–D) Blood counts from WT and APC-deficient mice placed on diet as adults (6 weeks of age) after 3 weeks on diet. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions

Figure S7 mRNA levels of TfR1, DMT1, and Ferritin In Vivo following APC Loss, Related to Figure 6 qRT-PCR showing the levels of TfR1, DMT1, and ferritin in crypts from APC-deficient mice (day 4 post-induction) on CID relative to WT mice on the same high-iron diet. Cell Reports 2012 2, 270-282DOI: (10.1016/j.celrep.2012.07.003) Copyright © 2012 The Authors Terms and Conditions