Dysregulated D-dopachrome tautomerase, a hypoxia-inducible factor-dependent gene, cooperates with macrophage migration inhibitory factor in renal tumorigenesis

Abstract

Clear cell renal cell carcinomas (ccRCCs) are characterized by biallelic loss of the von Hippel-Lindau tumor suppressor and subsequent constitutive activation of the hypoxia-inducible factors, whose transcriptional programs dictate major phenotypic attributes of kidney tumors. We recently described a role for the macrophage migration inhibitory factor (MIF) in ccRCC as an autocrine-signaling molecule with elevated expression in tumor tissues and in the circulation of patients that has potent tumor cell survival effects. MIF is a pleiotropic cytokine implicated in a variety of diseases and cancers and is the target of both small molecule and antibody-based therapies currently in clinical trials. Recent work by others has described D-dopachrome tautomerase (DDT) as a functional homologue of MIF with a similar genomic structure and expression patterns. Thus, we sought to determine a role for DDT in renal cancer. We find that DDT expression mirrors MIF expression in ccRCC tumor sections with high correlation and that, mechanistically, DDT is a novel hypoxia-inducible gene and direct target of HIF1α and HIF2α. Functionally, DDT and MIF demonstrate a significant overlap in controlling cell survival, tumor formation, and tumor and endothelial cell migration. However, DDT inhibition consistently displayed more severe effects on most phenotypes. Accordingly, although dual inhibition of DDT and MIF demonstrated additive effects in vitro, DDT plays a dominant role in tumor growth in vivo. Together, our findings identify DDT as a functionally redundant but more potent cytokine to MIF in cancer and suggest that current attempts to inhibit MIF signaling may fail because of DDT compensation.

Keywords: Cancer Biology; Clear Cell Renal Cell Carcinoma; DDT; Gene Regulation; Hypoxia; Hypoxia-inducible Factor (HIF); Kidney; MIF; VHL; von Hippel-Lindau.

FIGURE 1.

DDT and MIF expression are strongly correlated in ccRCC. A, representative images showing high, moderate, and low immunohistochemical staining for DDT and MIF in serial ccRCC tumor tissues. A normal kidney control is also shown on the array. Scale bar = 25 μm. B, quantitation of staining. DDT, like MIF, is expressed in the vast majority of ccRCC tissues. Mod, moderate; Neg., negative. C, correlation between DDT staining and MIF staining on the ccRCC tissue microarray shown by Pearson correlation coefficient (R² = 0.3686, p = 0.0001. D and E, significant correlation of DDT and MIF gene expression in two ccRCC studies in Oncomine. The axes display log2-transformed, median-centered expression values.

FIGURE 2.

DDT is a hypoxia-inducible HIF target gene. A, qRT-PCR of DDT and MIF expression in the presence or absence of VHL in RCC4 cells. B, Western blot analyses of HIF1α or HIF2α and DDT in the presence or absence of VHL in RCC4 cells (left panel) and 786-O cells (right panel). C, qRT-PCR of RCC4VHL cells under 21% oxygen or hypoxia (0.5% O₂) for 24 h. D, Western blot analysis of HIF1α and DDT proteins in 293T cells under 21% oxygen or hypoxia (2% O₂ and 0.5% O₂) for 24 h. E, Western blot analysis of HIF1α or HIF2α after knockdown by shRNAs in RCC4 cells. F, qRT-PCR of GLUT4, VEGF, and DDT in RCC4VHL after HIF1α or HIF2α knockdown.

FIGURE 3.

HIF1α and HIF2α bind to the DDT promoter. A, diagram of the proximal promoter region of DDT, identified putative HREs, and ChIP primers. Shown is a ChIP analysis of RCC4VHL cells exposed to 21% of 0.5% oxygen for 24 h and amplified with primers to an upstream region (B), HREs1 and 2 (C), and HRE3 (D) following pull-down with IgG, HIF1α, or HIF2α antibodies.

FIGURE 4.

DDT knockdown impairs growth and survival of RCC4 cells in vitro. A, Western blot analysis of control (shGFP) or DDT knockdown (shDDT-1 and shDDT-2) in RCC4 cell lysates probed with DDT, phospho-ERK (phos-ERK), total ERK, p27, and β-actin antibodies. B, cell proliferation assay of control or DDT knockdown RCC4 cells. *, p < 0.00015 for both shDDTs versus shGFP at all points measured. C, colony formation assay of control or DDT knockdown RCC4 cells. *, p = 0.0002; **, p = 0.0008.

FIGURE 5.

DDT knockdown impairs growth and survival of 786-O cells both in vitro and in vivo. A, qRT-PCR of DDT expression following knockdown with shDDT in 786-O cells. exp., expression. B, colony formation assay of control (shGFP) or DDT knockdown (shDDT) in 786-O cells. *, p = 0.015. C, xenograft tumor assay of 786-O cells with DDT knockdown (n = 10) and control (n = 10). *, p < 0.033 for all data points beyond day 16.

FIGURE 6.

DDT and MIF additively regulate ccRCC growth and survival. A, Western blot analysis of control (shGFP/shGFP), DDT knockdown (shGFP/shDDT), MIF knockdown (shMIF/shGFP), and dual DDT/MIF knockdown (shDDT/shMIF) RCC4 cell lysates probed with MIF, DDT, phospho-ERK (p-ERK), total ERK (t-ERK), phospho-Akt (p-Akt), total Akt (t-Akt), p27, and β-actin antibodies. B, colony formation assay of RCC4 cells with DDT, MIF, or dual knockdown. *, p = 0.002; **, p = 0.005; ***, p = 0.001, ****, p = 0.0007, *****, p = 0.00001, ******, p = 0.003. C, qRT-PCR of 786-O cells with DDT, MIF, or dual knockdown. D, colony formation assay of 786-O cells with DDT, MIF, or dual knockdown. *, p = 0.001; **, p = 0.004; ***, p = 0.0007, ****, p = 0.006, *****, p = 0.0008, ******, p = 0.006. E, in vivo bioluminescence imaging of orthotopically implanted, luciferase-expressing 786-O cells with DDT, MIF, or dual knockdown. Differences between shGFP/shGFP (n = 5) and shMIF/shGFP (n = 6) are statistically significant at day 14 (p = 0.04). shGFP/shDDT and shMIF/shDDT differences from shGFP/shGFP are statistically significant at all points after day 10 (p < 0.025).

FIGURE 7.

DDT and MIF knockdown tumors display increased p27 staining and decreased vascular density in xenograft orthotopic 786-O tumors. shGFP, shDDT, shMIF, and shDDT/MIF tumors were sectioned and stained for MIF, DDT, p27, and CD31. Decreases in CD31-positive vessels (arrows) averaged from 10 high power fields are significant: shGFP versus shDDT, p = 0.0017; shGFP versus shMIF, p = 0.0003; shGFP versus shDDT/MIF, p = 5.3 × 10⁻⁹; shDDT versus shDDT/MIF, p = 0.003; and shMIF versus shDDT/MIF, p = 0.027. Representative sections are shown. Scale bar = 50 μm.

FIGURE 8.

DDT and MIF regulate cell migration. A, wound healing assay of control (shGFP), shDDT, shMIF, and shDDT/MIF RCC4 cells. B, quantitation of the percentage of unrepaired wound in A. *, p = 0.0334; **, p = 0.0069; ***, p = 0.0063; ****, p = 0.0343. C, mean velocity (micrometers/second) of GFP-, DDT-, MIF-, and DDT/MIF-depleted cells in time-lapse video microscopy. *, p = 6.92 × 10⁻⁷; **, p = 5.34 × 10⁻⁷; ***, p = 1.57 × 10⁻¹⁰; ****, p = 2.52 × 10⁻³; *****, p = 1.52 × 10⁻³. D, wound healing assay of HUVECs in media with and without rMIF (100 ng/ml). E, quantitation of the percentage of unrepaired wound in D (*, p = 0.0053) and the percentage of HUVEC-unrepaired wound in the presence of conditioned media from control, shDDT, shMIF, and shDDT/MIF RCC4 cells (**, p = 0.0059; ***, p = 0.0176; ****, p = 0.0190).