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.

Post-ischemic inactivation of HIF prolyl hydroxylases in endothelium promotes maladaptive kidney repair by inducing glycolysis.

Ischemic acute kidney injury (AKI) is common in hospitalized patients and increases the risk for chronic kidney disease (CKD). Impaired endothelial cell (EC) functions are thought to contribute in AKI to CKD transition, but the underlying mechanisms remain unclear. Here, we identify a critical role for endothelial oxygen sensing prolyl hydroxylase domain (PHD) enzymes 1-3 in regulating post-ischemic kidney repair. In renal endothelium, we observed compartment-specific differences in the expression of the three PHD isoforms in both mice and humans. We found that post-ischemic concurrent inactivation of endothelial PHD1, PHD2, and PHD3 but not PHD2 alone promoted maladaptive kidney repair characterized by exacerbated tissue injury, fibrosis, and inflammation. Single-cell RNA-seq analysis of the post-ischemic endothelial PHD1, PHD2 and PHD3 deficient (PHDTiEC) kidney revealed an endothelial glycolytic transcriptional signature, also observed in human kidneys with severe AKI. This metabolic program was coupled to upregulation of the SLC16A3 gene encoding the lactate exporter monocarboxylate transporter 4 (MCT4). Strikingly, treatment with the MCT4 inhibitor syrosingopine restored adaptive kidney repair in PHDTiEC mice. Mechanistically, MCT4 inhibition suppressed pro-inflammatory EC activation reducing monocyte-endothelial cell interaction. Our findings suggest avenues for halting AKI to CKD transition based on selectively targeting the endothelial hypoxia-driven glycolysis/MCT4 axis.

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.

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.

Hypoxic preconditioning protects against ischemic kidney injury through the IDO1/kynurenine pathway.

Prolonged cellular hypoxia leads to energetic failure and death. However, sublethal hypoxia can trigger an adaptive response called hypoxic preconditioning. While prolyl-hydroxylase (PHD) enzymes and hypoxia-inducible factors (HIFs) have been identified as key elements of oxygen-sensing machinery, the mechanisms by which hypoxic preconditioning protects against insults remain unclear. Here, we perform serum metabolomic profiling to assess alterations induced by two potent cytoprotective approaches, hypoxic preconditioning and pharmacologic PHD inhibition. We discover that both approaches increase serum kynurenine levels and enhance kynurenine biotransformation, leading to preservation of NAD+ in the post-ischemic kidney. Furthermore, we show that indoleamine 2,3-dioxygenase 1 (Ido1) deficiency abolishes the systemic increase of kynurenine and the subsequent renoprotection generated by hypoxic preconditioning and PHD inhibition. Importantly, exogenous administration of kynurenine restores the hypoxic preconditioning in the context of Ido1 deficiency. Collectively, our findings demonstrate a critical role of the IDO1-kynurenine axis in mediating hypoxic preconditioning.

Natural products as a means of overcoming cisplatin chemoresistance in bladder cancer.

Cisplatin remains an integral part of the treatment for muscle invasive bladder cancer. A large number of patients do not respond to cisplatin-based chemotherapy and efficacious salvage regimens are limited. Immunotherapy has offered a second line of treatment; however, only approximately 20% of patients respond, and molecular subtyping of tumors indicates there may be significant overlap in those patients that respond to cisplatin and those patients that respond to immunotherapy. As such, restoring sensitivity to cisplatin remains a major hurdle to improving patient care. One potential source of compounds for enhancing cisplatin is naturally derived bioactive products such as phytochemicals, flavonoids and others. These compounds can activate a diverse array of different pathways, many of which can directly promote or inhibit cisplatin sensitivity. The purpose of this review is to understand current drug development in the area of natural products and to assess how these compounds may enhance cisplatin treatment in bladder cancer patients.

Inhibition of Endothelial PHD2 Suppresses Post-Ischemic Kidney Inflammation through Hypoxia-Inducible Factor-1.

BACKGROUND: Prolyl-4-hydroxylase domain-containing proteins 1-3 (PHD1 to PHD3) regulate the activity of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2, transcription factors that are key regulators of hypoxic vascular responses. We previously reported that deficiency of endothelial HIF-2 exacerbated renal ischemia-reperfusion injury, whereas inactivation of endothelial PHD2, the main oxygen sensor, provided renoprotection. Nevertheless, the molecular mechanisms by which endothelial PHD2 dictates AKI outcomes remain undefined. METHODS: To investigate the function of the endothelial PHD2/HIF axis in ischemic AKI, we examined the effects of endothelial-specific ablation of PHD2 in a mouse model of renal ischemia-reperfusion injury. We also interrogated the contribution of each HIF isoform by concurrent endothelial deletion of both PHD2 and HIF-1 or both PHD2 and HIF-2. RESULTS: Endothelial deletion of Phd2 preserved kidney function and limited transition to CKD. Mechanistically, we found that endothelial Phd2 ablation protected against renal ischemia-reperfusion injury by suppressing the expression of proinflammatory genes and recruitment of inflammatory cells in a manner that was dependent on HIF-1 but not HIF-2. Persistence of renoprotective responses after acute inducible endothelial-specific loss of Phd2 in adult mice ruled out a requirement for PHD2 signaling in hematopoietic cells. Although Phd2 inhibition was not sufficient to induce detectable HIF activity in the kidney endothelium, in vitro experiments implicated a humoral factor in the anti-inflammatory effects generated by endothelial PHD2/HIF-1 signaling. CONCLUSIONS: Our findings suggest that activation of endothelial HIF-1 signaling through PHD2 inhibition may offer a novel therapeutic approach against ischemic AKI.

Metabolic Flexibility in Cancer: Targeting the Pyruvate Dehydrogenase Kinase:Pyruvate Dehydrogenase Axis.

Cancer cells use alterations of normal metabolic processes to sustain proliferation indefinitely. Transcriptional and posttranscriptional control of the pyruvate dehydrogenase kinase (PDK) family is one way in which cancer cells alter normal pyruvate metabolism to fuel proliferation. PDKs can phosphorylate and inactivate the pyruvate dehydrogenase complex (PDHC), which blocks oxidative metabolism of pyruvate by the mitochondria. This process is thought to enhance cancer cell growth by promoting anabolic pathways. Inhibition of PDKs induces cell death through increased PDH activity and subsequent increases in ROS production. The use of PDK inhibitors has seen widespread success as a potential therapeutic in laboratory models of multiple cancers; however, gaps still exist in our understanding of the biology of PDK regulation and function, especially in the context of individual PDKs. Efforts are currently underway to generate PDK-specific inhibitors and delineate the roles of individual PDK isozymes in specific cancers. The goal of this review is to understand the regulation of the PDK isozyme family, their role in cancer proliferation, and how to target this pathway therapeutically to specifically and effectively reduce cancer growth.

The Endothelial Prolyl-4-Hydroxylase Domain 2/Hypoxia-Inducible Factor 2 Axis Regulates Pulmonary Artery Pressure in Mice.

Hypoxia-inducible factors 1 and 2 (HIF-1 and -2) control oxygen supply to tissues by regulating erythropoiesis, angiogenesis and vascular homeostasis. HIFs are regulated in response to oxygen availability by prolyl-4-hydroxylase domain (PHD) proteins, with PHD2 being the main oxygen sensor that controls HIF activity under normoxia. In this study, we used a genetic approach to investigate the endothelial PHD2/HIF axis in the regulation of vascular function. We found that inactivation of Phd2 in endothelial cells specifically resulted in severe pulmonary hypertension (∼118% increase in right ventricular systolic pressure) but not polycythemia and was associated with abnormal muscularization of peripheral pulmonary arteries and right ventricular hypertrophy. Concurrent inactivation of either Hif1a or Hif2a in endothelial cell-specific Phd2 mutants demonstrated that the development of pulmonary hypertension was dependent on HIF-2α but not HIF-1α. Furthermore, endothelial HIF-2α was required for the development of increased pulmonary artery pressures in a model of pulmonary hypertension induced by chronic hypoxia. We propose that these HIF-2-dependent effects are partially due to increased expression of vasoconstrictor molecule endothelin 1 and a concomitant decrease in vasodilatory apelin receptor signaling. Taken together, our data identify endothelial HIF-2 as a key transcription factor in the pathogenesis of pulmonary hypertension.

Regulation of mitochondrial function and cellular energy metabolism by protein kinase C-λ/ι: a novel mode of balancing pluripotency.

Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis are key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self-renewing versus differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization, and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι-hypoxia-inducible factor 1α-PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts.

Inhibition of protein kinase C signaling maintains rat embryonic stem cell pluripotency.

Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.

EED and KDM6B coordinate the first mammalian cell lineage commitment to ensure embryo implantation.

The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages.

Epigenetic regulation of DNA methyltransferases: DNMT1 and DNMT3B in gliomas.

The role of epigenetics and significance of aberrant gene regulation in etiology of cancer is a well-established phenomenon. The hallmark of cancer epigenetics is aberrant DNA methylation consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes (TSGs) by DNA methyltransferases (DNMTs). In mammals, DNA methylation is catalyzed by DNMTs encoded by DNMT1, DNMT3A, and DNMT3B. Interestingly, little is known about variation in the methylation status of epigenetic regulators themselves in gliomas. Here, we report significant overexpression of DNMT1 and DNMT3B. A study of the methylation status and histone modifications at the promoter region of DNA methyltransferase I (DNMT1) gene revealed an unmethylated DNA promoter, similar to that detected in normal brain tissues. However, a differential histone code with distinct euchromatin marks--AcH3, AcH4, and H3k4me2--was specifically detected in tumors, unlike in normal brain tissues, which were found predominantly enriched with heterochromatin marks such as H3K9me2 and H3K27me3. In contrast, a differential methylation pattern of DNMT3B gene promoter occurred in glioma tumors, wherein it was found hypomethylated. Transcriptional silencing by CpG island methylation is a prevalent mechanism for inactivation of TSGs. Inhibiting DNMTs by 5-azacytidine (DNMT inhibitor) treatment led to significant inhibition of expression of DNMT1 and DNMT3B and enhanced expression of TSGs such as PTEN and p21 analyzed in this study. Our studies have identified effects of increased presence of DNMTs on inhibition of tumor suppressors that are epigenetically silenced in gliomas, thereby leading to aberrant regulation of cell cycle progression and failure to maintain genomic stability.

Spontaneous transformation of human adult nontumorigenic stem cells to cancer stem cells is driven by genomic instability in a human model of glioblastoma.

The presence of a CD133+/nestin+ population in brain tumors suggests that a normal neural stem cell may be the cell of origin for gliomas. We have identified human CD133-positive NSCs from adult glioma tissue and established them as long-term in vitro cultures human neuroglial culture (HNGC)-1. Replicative senescence in HNGC-1 led to a high level of genomic instability and emergence of a spontaneously immortalized clone that developed into cell line HNGC-2 with features of cancer stem cells (CSCs), which include the ability for self-renewal and the capacity to form CD133-positive neurospheres and develop intracranial tumors. The data from our study specify an important role of genomic instability in initiation of transformed state as well as its progression into highly tumorigenic CSCs. The activated forms of Notch and Hes isoforms were expressed in both non-neoplastic neural stem cells and brain tumor stem cells derived from it. Importantly, a significant overexpression of these molecules was found in the brain tumor stem cells. These findings suggest that this model comprised of HNGC-1 and HNGC-2 cells would be a useful system for studying pathways involved in self-renewal of stem cells and their transformation to cancer stem cells. Disclosure of potential conflicts of interest is found at the end of this article.