Glutamine catabolism supports amino acid biosynthesis and suppresses the integrated stress response to promote photoreceptor survival.

Photoreceptor loss results in vision loss in many blinding diseases, and metabolic dysfunction underlies photoreceptor degeneration. So, exploiting photoreceptor metabolism is an attractive strategy to prevent vision loss. Yet, the metabolic pathways that maintain photoreceptor health remain largely unknown. Here, we investigated the dependence of photoreceptors on Gln catabolism. Gln is converted to glutamate via glutaminase (GLS), so mice lacking GLS in rod photoreceptors were generated to inhibit Gln catabolism. Loss of GLS produced rapid rod photoreceptor degeneration. In vivo metabolomic methodologies and metabolic supplementation identified Gln catabolism as critical for glutamate and aspartate biosynthesis. Concordant with this amino acid deprivation, the integrated stress response (ISR) was activated with protein synthesis attenuation, and inhibiting the ISR delayed photoreceptor loss. Furthermore, supplementing asparagine, which is synthesized from aspartate, delayed photoreceptor degeneration. Hence, Gln catabolism is integral to photoreceptor health, and these data reveal a novel metabolic axis in these metabolically-demanding neurons.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Metabolic Alterations Caused by Simultaneous Loss of HK2 and PKM2 Leads to Photoreceptor Dysfunction and Degeneration.

HK2 and PKM2 are two main regulators of aerobic glycolysis. Photoreceptors (PRs) use aerobic glycolysis to produce the biomass necessary for the daily renewal of their outer segments. Previous work has shown that HK2 and PKM2 are important for the normal function and long-term survival of PRs but are dispensable for PR maturation, and their individual loss has opposing effects on PR survival during acute nutrient deprivation. We generated double conditional (dcKO) mice lacking HK2 and PKM2 expression in rod PRs. Western blotting, immunofluorescence, optical coherence tomography, and electroretinography were used to characterize the phenotype of dcKO animals. Targeted and stable isotope tracing metabolomics, qRT-PCR, and retinal oxygen consumption were performed. We show that dcKO animals displayed early shortening of PR inner/outer segments, followed by loss of PRs with aging, much more rapidly than either knockout alone without functional loss as measured by ERG. Significant alterations to central glucose metabolism were observed without any apparent changes to mitochondrial function, prior to PR degeneration. Finally, PR survival following experimental retinal detachment was unchanged in dcKO animals as compared to wild-type animals. These data suggest that HK2 and PKM2 have differing roles in promoting PR neuroprotection and identifying them has important implications for developing therapeutic options for combating PR loss during retinal disease.

Flow cytometric method for the detection and quantification of retinal cell death and oxidative stress.

Retinal cell death is the major cause of vision loss in many forms of blinding retinal disease. A plethora of research is focused on understanding the mechanisms of retinal cell death to identify potential neuroprotective strategies that prevent vision loss in these diseases. Traditionally, histological techniques have been used to determine the type and extent of cell death in the retina. These techniques, such as TUNEL labeling and immunohistochemistry, are laborious and time consuming, resulting in low throughput and variable results depending on the experimenter. To increase throughput and reduce variability, we developed several flow cytometry-based assays to detect and quantify retinal cell death. The methods and accompanying data presented demonstrate that flow cytometry can readily detect both retinal cell death and oxidative stress and importantly, the efficacy of neuroprotective agents. These methods will be of interest to investigators looking to increase throughput and efficiency without compromising sensitivity as the methods herein reduce analysis time from several months to less than a week. As such, the flow cytometry methods presented have the potential to expedite research efforts focused on developing novel strategies for retinal cell neuroprotection.

Development of Novel Small-Molecule Activators of Pyruvate Kinase Muscle Isozyme 2, PKM2, to Reduce Photoreceptor Apoptosis.

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2's efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

Rod photoreceptor-specific deletion of cytosolic aspartate aminotransferase, GOT1, causes retinal degeneration.

Photoreceptor cell death is the cause of vision loss in many forms of retinal disease. Metabolic dysfunction within the outer retina has been shown to be an underlying factor contributing to photoreceptor loss. Therefore, a comprehensive understanding of the metabolic pathways essential to photoreceptor health and function is key to identifying novel neuroprotective strategies. Glutamic-oxaloacetic transaminase 1 (Got1) encodes for a cytosolic aspartate aminotransferase that reversibly catalyzes the transfer of an amino group between glutamate and aspartate and is an important aspect of the malate-aspartate shuttle (MAS), which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Previous work has demonstrated that the activity of this enzyme is highest in photoreceptor inner segments. Furthermore, ex vivo studies have demonstrated that the retina relies on aspartate aminotransferase for amino acid metabolism. Importantly, aspartate aminotransferase has been suggested to be an early biomarker of retinal degeneration in retinitis pigmentosa and a possible target for neuroprotection. In the present study, we characterized the effect of Got1 deletion on photoreceptor metabolism, function, and survival in vivo by using a rod photoreceptor-specific, Got1 knockout mouse model. Loss of the GOT1 enzyme from rod photoreceptors resulted in age-related photoreceptor degeneration with an accumulation of retinal aspartate and NADH and alterations in the expression of genes involved in the MAS, the tricarboxylic acid (TCA) cycle, and redox balance. Hence, GOT1 is critical to in vivo photoreceptor metabolism, function, and survival.

Identification of TP53RK-Binding Protein (TPRKB) Dependency in TP53-Deficient Cancers.

Tumor protein 53 (TP53; p53) is the most frequently altered gene in human cancer. Identification of vulnerabilities imposed by TP53 alterations may enable effective therapeutic approaches. Through analyzing short hairpin RNA (shRNA) screening data, we identified TP53RK-Binding Protein (TPRKB), a poorly characterized member of the tRNA-modifying EKC/KEOPS complex, as the most significant vulnerability in TP53-mutated cancer cell lines. In vitro and in vivo, across multiple benign-immortalized and cancer cell lines, we confirmed that TPRKB knockdown in TP53-deficient cells significantly inhibited proliferation, with minimal effect in TP53 wild-type cells. TP53 reintroduction into TP53-null cells resulted in loss of TPRKB sensitivity, confirming the importance of TP53 status in this context. In addition, cell lines with mutant TP53 or amplified MDM2 (E3-ubiquitin ligase for TP53) also showed high sensitivity to TPRKB knockdown, consistent with TPRKB dependence in a wide array of TP53-altered cancers. Depletion of other EKC/KEOPS complex members exhibited TP53-independent effects, supporting complex-independent functions of TPRKB. Finally, we found that TP53 indirectly mediates TPRKB degradation, which was rescued by coexpression of PRPK, an interacting member of the EKC/KEOPS complex, or proteasome inhibition. Together, these results identify a unique and specific requirement of TPRKB in a variety of TP53-deficient cancers. IMPLICATIONS: Cancer cells with genomic alterations in TP53 are dependent on TPRKB.

miR-34a Regulates Expression of the Stathmin-1 Oncoprotein and Prostate Cancer Progression.

In aggressive prostate cancers, the oncoprotein STMN1 (also known as stathmin 1 and oncoprotein 18) is often overexpressed. STMN1 is involved in various cellular processes, including cell proliferation, motility, and tumor metastasis. Here, it was found that the expression of STMN1 RNA and protein is elevated in metastatic prostate cancers. Knockdown of STMN1 resulted in reduced proliferation and invasion of cells and tumor growth and metastasis in vivo Furthermore, miR-34a downregulated STMN1 by directly binding to its 3'-UTR. Overexpression of miR-34a in prostate cancer cells reduced proliferation and colony formation, suggesting that it is a tumor suppressor. The transcriptional corepressor C-terminal binding protein 1 (CtBP1) negatively regulated expression of miR-34a. Furthermore, gene expression profiling of STMN1-modulated prostate cancer cells revealed molecular alterations, including elevated expression of growth differentiation factor 15 (GDF15), which is involved in cancer progression and potentially in STMN1-mediated oncogenesis. Thus, in prostate cancer, CtBP1-regulated miR-34a modulates STMN1 expression and is involved in cancer progression through the CtBP1\miR-34a\STMN1\GDF15 axis.Implications: The CtBP1\miR-34a\STMN1\GDF15 axis is a potential therapeutic target for treatment of aggressive prostate cancer. Mol Cancer Res; 16(7); 1125-37. ©2017 AACR.

Expression and Role of PAICS, a De Novo Purine Biosynthetic Gene in Prostate Cancer.

BACKGROUND: Our goal was to investigate de novo purine biosynthetic gene PAICS expression and evaluate its role in prostate cancer progression. METHODS: Next-generation sequencing, qRTPCR and immunoblot analysis revealed an elevated expression of a de novo purine biosynthetic gene, Phosphoribosylaminoimidazole Carboxylase, Phosphoribosylaminoimidazole Succinocarboxamide Synthetase (PAICS) in a progressive manner in prostate cancer. Functional analyses were performed using prostate cancer cell lines- DU145, PC3, LnCaP, and VCaP. The oncogenic properties of PAICS were studied both by transient and stable knockdown strategies, in vivo chicken chorioallantoic membrane (CAM) and murine xenograft models. Effect of BET bromodomain inhibitor JQ1 on the expression level of PAICS was also studied. RESULTS: Molecular staging of prostate cancer is important factor in effective diagnosis, prognosis and therapy. In this study, we identified a de novo purine biosynthetic gene; PAICS is overexpressed in PCa and its expression correlated with disease aggressiveness. Through several in vitro and in vivo functional studies, we show that PAICS is necessary for proliferation and invasion in prostate cancer cells. We identified JQ1, a BET bromodomain inhibitor previously implicated in regulating MYC expression and demonstrated role in prostate cancer, abrogates PAICS expression in several prostate cancer cells. Furthermore, we observe loss of MYC occupancy on PAICS promoter in presence of JQ1. CONCLUSIONS: Here, we report that evaluation of PAICS in prostate cancer progression and its role in prostate cancer cell proliferation and invasion and suggest it as a valid therapeutic target. We suggest JQ1, a BET-domain inhibitor, as possible therapeutic option in targeting PAICS in prostate cancer. Prostate 77:10-21, 2017. © 2016 Wiley Periodicals, Inc.

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

The miR-124-prolyl hydroxylase P4HA1-MMP1 axis plays a critical role in prostate cancer progression.

Collagen prolyl hydroxylases (C-P4HAs) are a family of enzymes involved in collagen biogenesis. One of the isoforms of P4HA, Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), catalyzes the formation of 4-hydroxyproline that is essential for the proper three-dimensional folding of newly synthesized procollagen chains. Here, we show the overexpression of P4HA1 in aggressive prostate cancer. Immunohistochemical analysis using tissue microarray demonstrated that P4HA1 expression was correlated with prostate cancer progression. Using in vitro studies, we showed that P4HA1 plays a critical role in prostate cancer cell growth and tumor progression. Expression profiling studies using P4HA1 modulated prostate cells suggested regulation of Matrix metalloproteases 1. The invasive properties of P4HA1 overexpressing cells were reversed by blocking MMP1. Our studies indicate P4HA1 copy number gain in a subset of metastatic prostate tumors and its expression is also regulated by microRNA-124. MiR-124 in turn is negatively regulated by transcriptional repressors EZH2 and CtBP1, both of which are overexpressed in aggressive prostate cancer. Chick chorioallantoic membrane (CAM) assay and mice xenograft investigations show that P4HA1 is required for tumor growth and metastasis in vivo. Our observations suggest that P4HA1 plays a critical role in prostate cancer progression and could serve as a viable therapeutic target.

TLR9-dependent IL-23/IL-17 is required for the generation of Stachybotrys chartarum-induced hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease that develops after repeated exposure to inhaled particulate Ag. Stachybotrys chartarum is a dimorphic fungus that has been implicated in a number of respiratory illnesses, including HP. In this study, we have developed a murine model of S. chartarum-induced HP that reproduces pathology observed in human HP, and we have hypothesized that TLR9-mediated IL-23 and IL-17 responses are required for the generation of granulomatous inflammation induced by inhaled S. chartarum. Mice that undergo i.p. sensitization and intratracheal challenge with 10(6) S. chartarum spores developed granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of T cells. S. chartarum sensitization and challenge resulted in robust pulmonary expression of IL-17 and IL-23. S. chartarum-mediated granulomatous inflammation required intact IL-23 or IL-17 responses and required TLR9, because TLR9(-/-) mice displayed reduced IL-17 and IL-23 expression in whole lung associated with decreased accumulation of IL-17 expressing CD4(+) and γδ T cells. Compared with S. chartarum-sensitized dendritic cells (DC) isolated from WT mice, DCs isolated from TLR9(-/-) mice had a reduced ability to produce IL-23 in responses to S. chartarum. Moreover, shRNA knockdown of IL-23 in DCs abolished IL-17 production from splenocytes in response to Ag challenge. Finally, the intratracheal reconstitution of IL-23 in TLR9(-/-) mice recapitulated the immunopathology observed in WT mice. In conclusion, our studies suggest that TLR9 is critical for the development of Th17-mediated granulomatous inflammation in the lung in response to S. chartarum.

Molecular cross-regulation between PPAR-γ and other signaling pathways: implications for lung cancer therapy.

Peroxisome proliferator-activated receptors (PPAR)-γ belongs to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. It is a mediator of adipocyte differentiation, regulates lipid metabolism and macrophage function. The ligands of PPAR-γ have long been in the clinic for the treatment of type II diabetes and have a very low toxicity profile. Activation of PPAR-γ was shown to modulate various hallmarks of cancer through its pleiotropic affects on multiple different cell types in the tumor microenvironment. An overwhelming number of preclinical-studies demonstrate the efficacy of PPAR-γ ligands in the control of tumor progression through their affects on various cellular processes, including cell proliferation, apoptosis, angiogenesis, inflammation and metastasis. A variety of signaling pathways have been implicated as potential mechanisms of action. This review will focus on the molecular basis of these mechanisms; primarily PPAR-γ cross-regulation with other signaling pathways and its relevance to lung cancer therapy will be discussed.

Cloning and characterization of zebrafish (Danio rerio) cyclin-dependent kinase 5.

Cyclin-dependent kinase 5 (cdk5) is a ubiquitous protein activated by neuron-specific activators, p35 and p39. Cdk5 regulates neuronal migration, differentiation, axonogenesis, synaptic transmission and apoptosis. However, its role in primary neurogenesis remains unexplored. Here, we have cloned and characterized the zebrafish cdk5 ortholog. Zebrafish cdk5 is 96% identical to its human counterpart. In situ hybridization analyses demonstrated that zebrafish cdk5 transcripts are ubiquitously expressed as early as the blastula stage. At 11.5h of development, cdk5 transcripts were present in the neural plate at the domains where primary neurons begin to be specified. RT-PCR analyses showed equal levels of cdk5 transcripts up to 72 h of development. SiRNA-mediated cdk5 knockdown resulted in a reduction in primary sensory neurons of the trigeminal ganglia of the peripheral nervous system, suggesting that cdk5 plays a crucial role in the development of the peripheral nervous system.

Comparative functional analysis of rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters suggest differential regulations.

We have carried out a comparative functional analysis of the rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-beta1 is low as compared to TGF-beta5 promoter in XTC cells but comparable in A6 cells, while TGF-beta5 promoter shows nearly negligible activity as compared to TGF-beta5 promoter in all the tested mammalian cell lines. Moreover, TGF-beta5 promoter is found to be repressed in XTC cells on treatment with TGF-beta5 protein. Thus, the regulation of TGF-beta1 and TGF-beta5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-beta1 and TGF-beta5 proteins, TGF-beta1 and TGF-beta5 genes have distinct functions in their respective species.