Macrophage migration inhibitory factor (MIF1) is a pleiotropic cytokine involved in inflammation and cancer. Genetic knockout of Mif1 in the validated N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine (BBN) model of bladder cancer (BCa) resulted in stage arrest at non‐muscle‐invasive disease in prior studies. Small‐molecule inhibition of MIF1 reduced cancer‐associated outcomes, but it did not fully recapitulate genetic models. D‐dopachrome tautomerase (gene symbol DDT), commonly referred to as MIF2, is a functional homolog of MIF1, and both MIF1 and MIF2 can bind the cell surface receptor CD74 on multiple cell types to initiate a signaling cascade. It has been proposed that this interaction mediates part of the protumorigenic effects of MIF1 and MIF2 and may explain the discordance in prior studies. We hypothesized that MIF2 functions redundantly with MIF1 in BCa development and progression. The Cancer Genome Atlas (TCGA) analysis indicated MIF and DDT expression were increased in BCa patients compared to control. 4‐Iodopyridine (4‐IPP), a combined MIF1/MIF2 inhibitor, was more efficacious than ISO‐1, a MIF1‐only inhibitor, in preventing cellular proliferation in BCa cell lines. To evaluate these findings in vivo, wild‐type (WT) and Mif1−/− animals were exposed to 0.05% BBN in drinking water for 16 weeks to initiate tumorigenesis and then evaluated over the subsequent 4 weeks for tumor formation and progression in the presence or absence of 4‐IPP. 4‐IPP reduced bladder weights in WT animals and bladder weights/tumor stage in Mif1−/− animals. To determine whether MIF1/MIF2 functioned through CD74 in BCa, WT or Cd74−/− animals were used in the same BBN model. Although these animals were partially protected against BBN‐induced BCa, 4‐IPP did not enhance this effect. In conclusion, our data suggest that MIF2 mechanistically functions in a similar protumorigenic manner to MIF1, and this is at least partially through CD74. Dual inhibition of MIF homologs is more efficacious at reducing tumor burden in this model of BCa. © 2022 The Pathological Society of Great Britain and Ireland.
Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial isozyme in the PDK family (PDK1-4) partially responsible for phosphorylation of pyruvate dehydrogenase (PDH). Phosphorylation of PDH is thought to result in a pro-proliferative shift in metabolism that sustains growth of cancer cells. Previous data from our lab indicate the pan-PDK inhibitor dichloroacetate (DCA) or acute genetic knockdown of PDK4 blocks proliferation of bladder cancer (BCa) cells. The goal of this study was to determine the role of PDK4 in an in vivo BCa model, with the hypothesis that genetic depletion of PDK4 would impair formation of BCa. PDK4−/− or WT animals were exposed to N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 16 weeks, and tumors were allowed to develop for up to 7 additional weeks. PDK4−/− mice had significantly larger tumors at later time points. When animals were treated with cisplatin, PDK4−/− animals still had larger tumors than WT mice. PDK4 expression was assessed in human tissue and in mice. WT mice lost expression of PDK4 as tumors became muscle-invasive. Similar results were observed in human samples, wherein tumors had less expression of PDK4 than benign tissue. In summary, PDK4 has a complex, multifunctional role in BCa and may represent an underrecognized tumor suppressor.
Macrophage migration inhibitory factor (MIF1) is a pro-tumorigenic cytokine overexpressed in multiple cancers. Our previous data in a carcinogen (BBN) induced murine bladder cancer (BCa) model indicates genetic depletion of MIF1 induces stage arrest. Pharmacological inhibition of MIF1 with the inhibitor CPSI-1306 reduces tumor stage and bladder weights. These data indicate MIF1 is a therapeutic target in BCa. However, a second protein with significant homology to MIF1 was recently identified and termed MIF2. Both MIF1 and MIF2 bind and activate their receptor CD74 yielding significant functional homology and thus, targeting both proteins may be more efficacious. We hypothesize MIF2 recapitulates the pro-tumorigenic effects of MIF1 in BCa in a CD74 dependent manner. We assessed MIF1 and MIF2 by ELISA in serum from human patients with BCa and MIF1/2 expression were evaluated in TCGA. WT mice (C57Bl/6 background), MIF-/-, and CD74-/- mice were treated with 0.05% BBN for 16 weeks to induce BCa, and then switched to water. Tumors were allowed to develop until 20 weeks post initiation of BBN, a time frame consistent with muscle invasive tumor development. Some animals were given either vehicle, or a dual MIF1/MIF2 inhibitor 4-IPP (80mg/kg i.p. daily) for 4 weeks after cessation of BBN. We assessed bladder weights as a surrogate for tumor weight, tumor stage and immunohistochemistry for proliferation, angiogenesis and inflammation. In vitro cell proliferation was measured using the hexosaminidase assay and tautomerase activity was measured using two separate enzymatic assays for MIF tautomerase activity. MIF1 and MIF2 mRNA levels were elevated in TCGA. Analysis of serum from BCa patients indicated increased MIF1 and MIF2 in BCa patients, although this didn't reach statistical significance. 4-IPP treated C57Bl/6 animals had reductions in bladder weight, although tumor staging was similar between groups. 4-IPP treatment had no effect on bladder weights or tumor stage in CD74-/- mice supporting a role for CD74 mediated MIF signaling. To more fully assess the role of MIF2 directly, MIF1-/- mice were treated with 4-IPP to solely block MIF2 4-IPP treatment lead to major reductions in tumor stage, tumor incidence and bladder weight indicating inhibition of MIF2 may be more effective when MIF1 is chronically absent. Inflammation and proliferation trended lower, but did not reach statistical significance. MIF1 depletion did not affect MIF2 mRNA levels in bladder, heart or liver. In vitro treatment with 4-IPP substantially reduced proliferation of two high grade BCa cell lines (HTB-5, HTB-9) at concentrations consistent with doses found to inhibit recombinant MIF activity. These data indicate MIF1/2 enhance BCa tumorigenesis through activation of CD74. Combined MIF1/MIF2 may improve therapeutic targeting of MIF, especially during chronic MIF1 inhibition. Citation Format: Ganeshkumar Rajendran, Benjamin L. Woolbright, Erika Abbott, Austin Martin, Katie Dennis, John Arthur Taylor. MIF-2 in bladder cancer: potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5150.
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