Mass spectrometry imaging identifies metabolic patterns associated with malignant potential in pheochromocytoma and paraganglioma.

OBJECTIVE: Within the past decade, important genetic drivers of pheochromocytoma and paraganglioma (PPGLs) development have been identified. The pathophysiological mechanism that translates these alterations into functional autonomy and potentially malignant behavior has not been elucidated in detail. Here we used MALDI-mass spectrometry imaging (MALDI-MSI) of formalin-fixed paraffin-embedded tissue specimens to comprehensively characterize the metabolic profiles of PPGLs. DESIGN AND METHODS: MALDI-MSI was conducted in 344 PPGLs and results correlated with genetic and phenotypic information. We experimentally silenced genetic drivers by siRNA in PC12 cells to confirm their metabolic impact in vitro. RESULTS: Tissue abundance of kynurenine pathway metabolites such as xanthurenic acid was significantly lower (P = 2.35E-09) in the pseudohypoxia pathway cluster 1 compared to PPGLs of the kinase-driven PPGLs cluster 2. Lower abundance of xanthurenic acid was associated with shorter metastasis-free survival (log-rank tests P = 7.96E-06) and identified as a risk factor for metastasis independent of the genetic status (hazard ratio, 32.6, P = 0.002). Knockdown of Sdhb and Vhl in an in vitro model demonstrated that inositol metabolism and sialic acids were similarly modulated as in tumors of the respective cluster. CONCLUSIONS: The present study has identified distinct tissue metabolomic profiles of PPGLs in relation to tumor genotypes. In addition, we revealed significantly altered metabolites in the kynurenine pathway in metastatic PPGLs, which can aid in the prediction of its malignant potential. However, further validation studies will be required to confirm our findings.

Impact of Extrinsic and Intrinsic Hypoxia on Catecholamine Biosynthesis in Absence or Presence of Hif2α in Pheochromocytoma Cells.

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).

Direct Pathway Cloning (DiPaC) to unlock natural product biosynthetic potential.

Specialized metabolites from bacteria are an important source of inspiration for drug development. The genes required for the biosynthesis of such metabolites in bacteria are usually organized in so-called biosynthetic gene clusters (BGCs). Using modern bioinformatic tools, the wealth of genomic data can be scanned for such BGCs and the expected products can often structurally be predicted in silico. This facilitates the directed discovery of putatively novel bacterial metabolites. However, the production of these molecules often requires genetic manipulation of the BGC for activation or the expression of the pathway in a heterologous host. The latter necessitates the transplantation of the BGC into a suitable expression system. To achieve this goal, powerful cloning strategies based on in vivo homologous recombination have recently been developed. This includes LCHR and LLHR in E. coli as well as TAR cloning in yeast. Here, we present Direct Pathway Cloning (DiPaC) as an efficient complementary BGC capturing strategy that relies on long-amplicon PCR and in vitro DNA assembly. This straightforward approach facilitates full pathway assembly, BGC refactoring and direct transfer into any vector backbone in vitro. The broad applicability and efficiency of DiPaC is demonstrated by the discovery of a new phenazine from Serratia fonticola, the first heterologous production of anabaenopeptins from Nostoc punctiforme and the transfer of the native erythromycin BGC from Saccharopolyspora erythraea into Streptomyces. Due to its simplicity, we envisage DiPaC to become an essential method for BGC cloning and metabolic pathways construction with significant applications in metabolic engineering, synthetic biology and biotechnology.

Biocatalytic Total Synthesis of Ikarugamycin.

Nature provides an inexhaustible diversity of small organic molecules with beautiful molecular architectures that have strong and selective inhibitory activities. However, this tremendous biomedical potential often remains inaccessible, as the structural complexity of natural products can render their synthetic preparation extremely challenging. This problem is addressable by harnessing the biocatalytic procedures evolved by nature. In this work, we present an enzymatic total synthesis of ikarugamycin. The use of an iterative PKS/NRPS machinery and two reductases has allowed the construction of 15 carbon-carbon and 2 carbon-nitrogen bonds in a biocatalytic one-pot reaction. By scaling-up this method we demonstrate the applicability of biocatalytic approaches for the ex vivo synthesis of complex natural products.

Promiscuous hydroxylases for the functionalization of polycyclic tetramate macrolactams--conversion of ikarugamycin to butremycin.

Polycyclic tetramate macrolactams (PTMs) are a structurally, biomedically and biosynthetically intriguing class of bacterial metabolites. By combining parts of the machineries of different PTM biosynthetic pathways, we demonstrate for the first time the substrate promiscuity of a class of PTM tailoring enzymes, thereby facilitating the (bio)synthesis of butremycin.

A systematic study on the influence of the main ingredients of an ivy leaves dry extract on the ß2-adrenergic responsiveness of human airway smooth muscle cells.

The bronchospasmolytic and secretolytic effects of ivy leaves dry extracts can be explained by an increased ß2-adrenergic responsiveness of the bronchi. Recently, it was shown that α-hederin inhibits the internalization of ß2-adrenergic receptors (ß2AR) under stimulating conditions. α-Hederin pretreated alveolar type II cells and human airway smooth muscle cells revealed an increased ß2AR binding and an elevated intracellular cAMP level, respectively. In order to identify whether additional compounds also mediate an increased ß2-adrenergic responsiveness, we examined the ingredients of an ivy leaves dry extract (EA 575) protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, 3,4-, 3,5- and 4,5-dicaffeoylquinic acid, hederacoside B, and ß-hederin. Within all the tested substances, only ß-hederin inhibited the internalization of GFP-tagged ß2AR in stably transfected HEK293 cells. Using fluorescence correlation spectroscopy ß-hederin (1 µM, 24 h) pretreated HASM cells showed a statistically significant increase in the ß2AR binding from 33.0 ± 8.9% to 44.1 ± 11.5% which was distributed with 36.0 ± 9.5% for τbound1 and 8.1 ± 2.6% for τbound2, respectively (n = 8, p < 0.05). The increased binding was selectively found for the receptor-ligand complex with unrestricted lateral mobility (τbound1 of 0.9 ± 0.1 ms, D1 = 9.1 ± 0.2 µm(2)/s, n = 8), whereas the binding of ß2AR with hindered lateral mobility (τbound2 of 64.2 ± 47.6 ms, D2 = 0.15 ± 0.02 µm(2)/s, n = 8) was not affected. Compared to control cells, a statistically significant increase of 17.5 ± 6.4% (n = 4, p < 0.05) and 24.2 ± 5.8% (n = 4, p < 0.001) in the cAMP formation was found for ß-hederin pretreated HASM cells after stimulation with 10 µM of terbutaline and simultaneous stimulation with 10 µM terbutaline and 10 µM forskolin, respectively. Within this systematic study focusing on the influence of the ingredients of an ivy leaves dry extract on HASM cells it was possible to identify ß-hederin as further component presumably responsible for the ß2-mimetic effects.