Iron Uptake via DMT1 Integrates Cell Cycle with JAK-STAT3 Signaling to Promote Colorectal Tumorigenesis

Abstract

Dietary iron intake and systemic iron balance are implicated in colorectal cancer (CRC) development, but the means by which iron contributes to CRC are unclear. Gene expression and functional studies demonstrated that the cellular iron importer, divalent metal transporter 1 (DMT1), is highly expressed in CRC through hypoxia-inducible factor 2α-dependent transcription. Colon-specific Dmt1 disruption resulted in a tumor-selective inhibitory effect of proliferation in mouse colon tumor models. Proteomic and genomic analyses identified an iron-regulated signaling axis mediated by cyclin-dependent kinase 1 (CDK1), JAK1, and STAT3 in CRC progression. A pharmacological inhibitor of DMT1 antagonized the ability of iron to promote tumor growth in a CRC mouse model and a patient-derived CRC enteroid orthotopic model. Our studies implicate a growth-promoting signaling network instigated by elevated intracellular iron levels in tumorigenesis, offering molecular insights into how a key dietary component may contribute to CRC.

Figure 1. DMT1 is overexpressed in CRC

(A) Studies of iron regulatory genes determined by RNA-Seq in the TCGA database and (B) gene expression analysis of DMT1 by qPCR in 8 pairs of primary human CRCs and adjacent normal tissues collected at University of Michigan (UM). (C) Quantification of DMT1 staining and (D) representative fluorescent microscopy in CRCs and adjacent normal tissues (left), and confocal images demonstrating apical colocalization of DMT1 and E-Cadherin (E-cad) co-staining (right). *p<0.05, **p<0.01 and ***p< 0.001 and error bars represent SEM.

Figure 2. Colon-selective disruption of DMT1 decreases sporadic and colitis-associated colon tumors

(A) Gene expression of Dmt1 in CRCs and their adjacent normal tissues, (B) tumor number and burden, (C) gross image and H&E staining, and (D) representative images and quantification of Ki67 staining of CRCs from CDX2 Dmt1^F/F/Apc^F/+ mice and their littermate controls at 3 months old. (E) Gene expression of Dmt1 in CRCs and their adjacent normal tissues, (F) tumor number and burden, (G) quantification of Ki67 staining of colon tumors from CDX2^ERT2 Dmt1^F/F/Apc^F/+, CDX2^ERT2 Dmt1^F/+/Apc^F/+ mice and their littermate controls. *p<0.05, **p<0.01 and ***p< 0.001 and error bars represent SEM. See also Figure S1 and S2.

Figure 3. HIF-2α is essential in the transcriptional regulation of DMT1

Dmt1 expression in CRCs and adjacent normal tissues from Vil Hif-2α^F/F/Apc^min/+ mice (A), CDX2 Hif-2α^F/F/Apc^F/+ mice (B) and their littermate controls. (C) Tumor number, (D) gross image, H&E and enhanced Perls’ staining of CRCs, from CDX2 Hif-2α^F/F/Apc^F/+, CDX2 Hif-2α^F/+/Apc^F/+ mice and their littermate controls at 3 months old. (E–H) DMT1 promoter luciferase assay in HCT116 cells. (I) High throughput screening for genes critical for DMT1 activation. *p<0.05, **p<0.01 and ***p< 0.001 and error bars represent SEM.

Figure 4. Inflammatory responses are increased in iron-driven colon tumorigenesis

(A) Enrichment plot and heatmap derived by gene set enrichment analysis (GSEA) for genes in regulation of immune response and inflammatory response. (B) Gene expression of inflammatory genes in colon tumors (T) and adjacent normal tissues (N) from CDX2; Dmt1^F/F/Apc^F/+ mice and their littermate controls. *p<0.05, **p<0.01and ***p< 0.001 error bars represent SEM. See also Figure S3.

Figure 5. Iron is essential for STAT3 activation in colon tumorigenesis

(A) TCGA reverse phase protein array (RPPA) analysis based on iron transporter expression in tumor tissues (high iron transporters, n=85; low iron transporters, n=531). (B) Western blotting analysis of in colon tumors (T) and adjacent normal tissues (N) from CDX2 Dmt1^F/F/Apc^F/+ mice and littermate controls. (C) Immunofluorescence staining and quantification for p-STAT3 in CRCs from CDX2^ERT2 Dmt1^F/F/Apc^F/+ mice (n=3). (D) STAT3 activity reporter assay in cells transfected with empty vector (EV), JAK1, JAK2 or JAK3 and treated with deferoxamine (DFO) 100 μM or control for 24 hours. Western blot analysis in HCT116 or SW480 cells treated (E) with DFO (100 μM) and IL6 (10 ng/mL) for 24 hours, or (F) with ferrous sulfate (FS, 100 μM) and JAK1/2 inhibitor (JAKi, 3 μM Ruxolitinib) for 24 hours. (G) Western blot analysis in HCT116 or HEK293T stable cells overexpressing DMT1 or parental controls treated with JAKi (3 μM) for 24 hours. (H) Western blot analysis in HCT116 or SW480 cells treated with FS (0 or 100 μM) and STAT3 inhibitor (STAT3i, 0 or 100 μM S3I-201) for 24 hours. (I) Representative gross image of CRCs, (J) tumor number, size, burden and number grouped by size from CDX2^ERT2 Apc^F/+ mice maintained on regular iron diet (35 mg/kg iron, 35 Fe) or high iron diet (1000 mg/kg iron, 1000 Fe). (K) STAT3 expression level in stable adenoma enteroids with STAT3 knockdown (shSTAT3) or scramble shRNA (shScr). (L) Representative bright-field images and quantification of enteroid size and Ki67 staining of adenoma enteroids grown in iron-reduced RPMI media or RPMI media supplemented with FS 100 μM for 7 days. *p<0.05, **p<0.01 and ***p<0.001. ##p<0.01 compared with FS treated shScr stable adenoma enteroids. Errors bars represent SEM. Western blot data was quantitated and the values above the blots represent p-STAT3/STAT3. See also Figure S4 and S5.

Figure 6. Iron-dependent CDK1 kinase activity is critical for JAK-STAT3 activation

(A) Schematic diagram for identification of Fe²⁺ binding proteins with LC-MS/MS based proteomics analysis. (B) Immunoblot analysis of proteins pulled down (PD) by Fe²⁺ or empty beads in HCT116 cells transfected with HA-tagged CDK1 or empty vector (EV). (C) Immunoblot analysis of CDK1 protein PD by Fe²⁺ or empty beads from mouse colon tumors lysates. (D) Kinase assay assessing histone H1 (H1) phosphorylation by purified recombinant (r) CDK1 in the presence or absence of FS 10μM. (E) STAT3 activity reporter assay in HCT116 cells transfected with EV or CDK1 and treated with IL6 10 ng/mL, FS 100 μM, DFO 100 μM or vehicle control for 24 hours. (F) STAT3 activity reporter assay in HCT116 cells transfected with 50 nM siRNA for CDK1 (siCDK1) or scrambled control (siScr) and treated with vehicle control or IL6 10 ng/mL. Western blot analysis in HCT116 or SW480 transfected with siCDK1 for 24 hours and then treated with (G) FS or (H) IL6 for 24 hours. Western blot analysis in HCT116 or SW480 cells treated with (I) FS and CDK1/2 inhibitor (CDKi, 0 or 10 nM Dinaciclib), or (J) with IL6 and CDKi for 24 hours. STAT3 activity reporter assay in HCT116 cells transfected with (K) EV, CDK1, JAK1 or JAK2, (L) siCDK1, siScr and/or JAK1, or (M) EV, CDK1, JAK1 and/or DFO for 24 hours. (N) Phospho-transfer assay assessing JAK1 phosphorylation by rCDK1 in the presence of FS (0 or 10 μM). (O) Representative bright-field images and quantification of enteroid size and Ki67 staining of adenoma enteroids grown in iron-reduced RPMI media or RPMI media supplemented with FS 100 μM after treatment with CDKi (0 or 10 nM) for 7 days. *p<0.05, **p<0.01 and ***p<0.001. ##p<0.01 compared with iron-reduced conditions. Error bars represent SEM. Western blot data was quantitated and the values above the blots represent p-STAT3/STAT3. See also Figure S6, Table S2 and S3.

Figure 7. Pharmacological inhibition of DMT1 decreases growth of colon tumors

(A) Tumor number and burden from CDX2^ERT2 Apc^F/+ mice orally administered with 50 mg/kg DMT1i, or vehicle. (B) Representative bright field images, and quantification of size and Ki67 staining of adenoma enteroids grown in iron-reduced RPMI media or RPMI media supplemented with FS 100 μM after treatment with DMT1i (0 or 3 μM) for 7 days. (C) Intrarectal implantation of adenoma enteroid in NOD/SCID mice visualized by endoscopy examination. Dotted line represents the established adenoma xenograft. (D) Immunofluorescent staining of a mouse colon tumor and adenoma xenograft from human specific DMT1 antibody (hDMT1). (E) Immunofluorescent staining and quantification of co-localization of hDMT1 and PCNA for established tumors from human adenoma enteroids grown in NOD/SCID mice for 2 weeks and then mice orally administered with 50 mg/kg DMT1i, or vehicle for 2 weeks. (F) Proposed mechanism for iron-driven colon tumorigenesis. Fe²⁺ is transported by DMT1 into intestinal epithelial cells and binds with CDK1, which increases CDK1 kinase activity to phosphorylate JAK1 kinase and activates STAT3 signaling pathway for tumor growth. *p<0.05 and **p<0.01 and error bars represent SEM. See also Figure S7.