Dynamics of the phosphoinositide 3-kinase p110δ interaction with p85α and membranes reveals aspects of regulation distinct from p110α

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.     

Genome-wide meta-analysis identifies regions on 7p21 (AHR) and 15q24 (CYP1A2) as determinants of habitual caffeine consumption

We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4 × 10(-19)), near AHR, and 15q24 (P = 5.2 × 10(-14)), between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production

The recombinant expression of human G protein-coupled receptors usually yields low production levels using commonly available cultivation protocols. Here, we describe the development of a high yield production protocol for the human neuropeptide Y receptor type 2 (Y2R), which provides the determination of expression levels in a time, media composition, and process parameter dependent manner. Protein was produced by Escherichia coli in a defined medium composition suitable for isotopic labeling required for investigations by nuclear magnetic resonance spectroscopy. The Y2 receptor was fused to a C-terminal 8x histidine tag by means of the pET vector system for easy one-step purification via affinity chromatography, yielding a purity of 95-99% for every condition tested, which was determined by SDS-PAGE and Western blot analysis. The Y2 receptor was expressed as inclusion body aggregates in complex media and minimal media, using different carbon sources. We investigated the influences of media composition, temperature, pH, and set specific growth rate on cell behavior, biomass wet weight specific and culture volume specific amounts of the target protein, which had been identified by inclusion body preparation, solubilization, followed by purification and spectrometric determination of the protein concentration. The developed process control strategy led to very high reproducibility of cell growth and protein concentrations with a maximum yield of 800 μg purified Y2 receptor per gram wet biomass when glycerol was used as carbon source in the mineral salt medium composition (at 38 °C, pH 7.0, and a set specific growth rate of 0.14 g/(gh)). The maximum biomass specific amount of purified Y2 receptor enabled the production of 35 mg Y2R per liter culture medium at an optical density (600 nm) of 25.     

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.     

Characterizing associations and SNP-environment interactions for GWAS-identified prostate cancer risk markers--results from BPC3

Genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. However, whether these associations can be consistently replicated, vary with disease aggressiveness (tumor stage and grade) and/or interact with non-genetic potential risk factors or other SNPs is unknown. We therefore genotyped 39 SNPs from regions identified by several prostate cancer GWAS in 10,501 prostate cancer cases and 10,831 controls from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). We replicated 36 out of 39 SNPs (P-values ranging from 0.01 to 10⁻²⁸). Two SNPs located near KLK3 associated with PSA levels showed differential association with Gleason grade (rs2735839, P = 0.0001 and rs266849, P = 0.0004; case-only test), where the alleles associated with decreasing PSA levels were inversely associated with low-grade (as defined by Gleason grade <8) tumors but positively associated with high-grade tumors. No other SNP showed differential associations according to disease stage or grade. We observed no effect modification by SNP for association with age at diagnosis, family history of prostate cancer, diabetes, BMI, height, smoking or alcohol intake. Moreover, we found no evidence of pair-wise SNP-SNP interactions. While these SNPs represent new independent risk factors for prostate cancer, we saw little evidence for effect modification by other SNPs or by the environmental factors examined.     

Comprehensive analysis of 5-aminolevulinic acid dehydrogenase (ALAD) variants and renal cell carcinoma risk among individuals exposed to lead

Background

Epidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD) gene affects lead toxicokinetics and may modify the adverse effects of lead.

Methods

The objective of this study was to evaluate single-nucleotide polymorphisms (SNPs) tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC). Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression.

Results

The adjusted risk associated with the ALAD variant rs8177796^CT/TT was increased (OR = 1.35, 95%CI = 1.05–1.73, p-value = 0.02) when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles (^GGOR = 2.68, 95%CI = 1.17–6.12, p = 0.01; ^GAOR = 1.79, 95%CI = 1.06–3.04 with an interaction approaching significance (p_int = 0.06).. No significant modification in RCC risk was observed for the functional variant rs1800435^(K68N). Haplotype analysis identified a region associated with risk supporting tagging SNP results.

Conclusion

A common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure.     

The antiangiogenic 16K prolactin impairs functional tumor neovascularization by inhibiting vessel maturation

Background

Angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology.

Methodology/Principal Findings

Here we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling.

Conclusions/Significance

Taken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.