Role of MIF1/MIF2/CD74 interactions in bladder cancer.

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.

Aging induces changes in cancer formation and microbial content in a murine model of bladder cancer.

Bladder cancer (BCa) incidence is tightly linked to aging. Older patients with BCa present with higher grade tumors and have worse outcomes on Bacillus-Calmette-Guerin (BCG) immunotherapy. Aging is also known to result in changes in the gut microbiome over mammalian lifespan, with recent studies linking alterations in the gut microbiome to changes in tumor immunity. There is limited information on the microbiome in BCa models though, despite known links to aging and immunotherapy. The purpose of this study was to evaluate how aging impacts tumor formation, inflammation, and the microbiome of mice given the model BCa carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We hypothesized old animals would have larger, more inflamed tumors and a shift in their fecal microbiome compared to their younger counterparts. Young (~8-week-old) or old (~78-week-old) C57Bl/6J animals were administered 0.05% BBN in drinking water for 16 weeks and then euthanized or allowed to progress for an additional 4 weeks. After 16 weeks of BBN, old mice had higher bladder to body weight ratio than young mice, and also muscle invasive tumors, which were not seen in their young counterparts. Old animals also had increased innate immune recruitment, but CD4+/CD8+ T cell recruitment did not appear different. BBN dramatically altered the microbiome in both sets of animals as measured by ß-diversity, including changes in multiple genera of bacteria. These data suggest old mice have a differential response to BBN-induced BCa. Given the median age of patients with BCa, understanding how the aged phenotype interacts with BCa is imperative.

Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts.

The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.

Pyruvate Dehydrogenase Kinase 4 Deficiency Increases Tumorigenesis in a Murine Model of Bladder Cancer.

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.

Tumor M2-PK: A novel urine marker of bladder cancer.

PURPOSE: Bladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment. METHODS: HTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values. RESULTS: The three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects. CONCLUSIONS: Our studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

The Role of Pyruvate Dehydrogenase Kinase-4 (PDK4) in Bladder Cancer and Chemoresistance.

Advanced bladder cancer remains a major source of mortality, with poor treatment options. Cisplatin-based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1-PDK4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high-grade compared with low-grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize bladder cancer cells to cisplatin. Upregulation of PDK4 in malignant bladder cancer cell lines as compared with benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0-G1 phase arrest in bladder cancer cells. Similarly, siRNA knockdown of PDK4 inhibited bladder cancer cell proliferation. Cotreatment of bladder cancer cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro Although daily treatment with 200 mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared with either DCA or cisplatin alone. This was attributed to substantial intratumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs. Mol Cancer Ther; 17(9); 2004-12. ©2018 AACR.