Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

Sustained plasma hepcidin suppression and iron elevation by Anticalin-derived hepcidin antagonist in cynomolgus monkey.

BACKGROUND AND PURPOSE: Anaemia of chronic disease (ACD) has been linked to iron-restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin-derived, small protein-based anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. EXPERIMENTAL APPROACH: Anticalin- and hepcidin-specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS-080-PEG30 administration in vivo. KEY RESULTS: PRS-080 had a hepcidin-binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS-080-PEG30), a t1/2 of 43 h in cynomolgus monkeys. Dose-dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS-080-PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment-free interval, all iron parameters returned to pre-dose values. CONCLUSIONS AND IMPLICATIONS: In conclusion, we developed a dose-dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron-restricted erythropoiesis that can address the root cause of ACD. PRS-080-PEG30 is currently in early clinical development.

Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform.

Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40) isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25(+40) as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.

Strong association between a single gene and fertilization efficiency of males and fecundity of their mates in the bulb mite.

Although variation in male fertilization efficiency has been shown to have a genetic basis in several species, the genes responsible for the effect are generally unknown. Here, we show a strong association between the fertilization success of males and their phosphogluconate dehydrogenase (Pgdh) genotype in the bulb mite Rhizoglyphus robini. Males homozygous for the slow (S) allele fathered a significantly greater proportion of offspring when competing with males homozygous for the fast (F) allele. There was no evidence that female fecundity was influenced by their Pgdh genotype. The fecundity of FF females did not differ significantly from the fecundity of SS females but female fecundity was significantly influenced by the genotype of their mate. Females paired with SS males laid significantly fewer eggs than females paired with FF males. Altogether these data show a trade-off, with the male SS genotype associated with their higher fertilization efficiency but at the cost of a negative impact on the fecundity of females mating with them.

Enzyme polymorphisms in Rhizoglyphus robini and R. echinopus and their application in paternity analysis.

Cellulose acetate electrophoresis (CAE) was used to investigate enzyme polymorphism in two congeneric species of Acaridae, Rhizoglyphus robini and R. echinopus. Using homogenates of individual mites, 27 enzymes were examined in two buffers. Five enzymes showed interspecific polymorphism, 15 exhibited intraspecific variation in R. robini and 12 in R. echinopus. Polymorphic PGDH was used to analyse paternity in progeny of R. robini females that had mated with two males. The second male sired on average 67% of progeny. Enzyme polymorphism can be used to study poorly known aspects of acarid mites biology, such as their mating systems and population structure under natural conditions and in stored foods.