Integrative Kinome Profiling Identifies mTORC1/2 Inhibition as Treatment Strategy in Ovarian Clear Cell Carcinoma.

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models.Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient-derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055.Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models.Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928-40. ©2018 AACR.

Quantitative proteomics analyses of activation states of human THP-1 macrophages.

Macrophages display large functional and phenotypical plasticity. They can adopt a broad range of activation states depending on their microenvironment. Various surface markers are used to characterize these differentially polarized macrophages. However, this is not informative for the functions of the macrophage. In order to have a better understanding of the functional changes of macrophages upon differential polarization, we studied differences in LPS- and IL4-stimulated macrophages. The THP-1 human monocytic cell line, was used as a model system. Cells were labeled, differentiated and stimulated with either LPS or IL-4 in a quantitative SILAC proteomics set-up. The resulting sets of proteins were functionally clustered. LPS-stimulated macrophages show increased secretion of proinflammatory peptides, leading to increased pressure on protein biosynthesis and processing. IL4-stimulated macrophages show upregulation of cell adhesion and extracellular matrix remodeling. Our approach provides an integrated view of polarization-induced functional changes and proves useful for studying functional differences between subsets of macrophages. Moreover, the identified polarization specific proteins may contribute to a better characterization of different activation states in situ and their role in various inflammatory processes.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects.

BACKGROUND: Adipose tissue is a primary site of obesity-induced inflammation, which is emerging as an important contributor to obesity-related diseases such as type 2 diabetes. Dietary fibre consumption appears to be protective. Short-chain fatty acids, e.g. propionic acid, are the principal products of the colonic fermentation of dietary fibre and may have beneficial effects on adipose tissue inflammation. MATERIALS AND METHODS: Human omental adipose tissue explants were obtained from overweight (mean BMI 28·8) gynaecological patients who underwent surgery. Explants were incubated for 24 h with propionic acid. Human THP-1 monocytic cells were differentiated to macrophages and incubated with LPS in the presence and absence of propionic acid. Cytokine and chemokine production were determined by multiplex-ELISA, and mRNA expression of metabolic and macrophages genes was determined by RT-PCR. RESULTS: Treatment of adipose tissue explants with propionic acid results in a significant down-regulation of several inflammatory cytokines and chemokines such as TNF-α and CCL5. In addition, expression of lipoprotein lipase and GLUT4, associated with lipogenesis and glucose uptake, respectively, increased. Similar effects on cytokine and chemokine production by macrophages were observed. CONCLUSION: We show that propionic acid, normally produced in the colon, may have a direct beneficial effect on visceral adipose tissue, reducing obesity-associated inflammation and increasing lipogenesis and glucose uptake. Effects on adipose tissue as a whole are at least partially explained by effects on macrophages but likely also adipocytes are involved. This suggests that, in vivo, propionic acid and dietary fibres may have potential in preventing obesity-related inflammation and associated diseases.

Human primary adipocytes exhibit immune cell function: adipocytes prime inflammation independent of macrophages.

BACKGROUND: Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT. METHODS AND RESULTS: Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-ß highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%. CONCLUSION: Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes.

Regulation of adipokine production in human adipose tissue by propionic acid.

BACKGROUND: Dietary fibre (DF) has been shown to be protective for the development of obesity, insulin resistance and type 2 diabetes. Short-chain fatty acids, produced by colonic fermentation of DF might mediate this beneficial effect. Adipose tissue plays a key role in the regulation of energy homeostasis, therefore, we investigated the influence of the short-chain fatty acid propionic acid (PA) on leptin, adiponectin and resistin production by human omental (OAT) and subcutaneous adipose tissue (SAT). As PA has been shown to be a ligand for G-protein coupled receptor (GPCR) 41 and 43, we investigated the role of GPCR's in PA signalling. MATERIALS AND METHODS: Human OAT and SAT explants were obtained from gynaecological patients who underwent surgery. Explants were incubated for 24 h with PA. Adipokine secretion and mRNA expression were determined using ELISA and RT-PCR respectively. RESULTS: We found that PA significantly stimulated leptin mRNA expression and secretion by OAT and SAT, whereas it had no effect on adiponectin. Furthermore, PA reduced resistin mRNA expression. Leptin induction, but not resistin reduction, was abolished by inhibition of Gi/o-coupled GPCR signalling. Moreover, GPCR41 and GPCR43 mRNA levels were considerably higher in SAT than in OAT. CONCLUSIONS: We demonstrate that PA stimulates expression of the anorexigenic hormone leptin and reduces the pro-inflammatory factor resistin in human adipose tissue depots. This suggests that PA is involved in regulation of human energy metabolism and inflammation and in this way may influence the development of obesity and type 2 diabetes.

Factors related to colonic fermentation of nondigestible carbohydrates of a previous evening meal increase tissue glucose uptake and moderate glucose-associated inflammation.

BACKGROUND: Evening meals that are rich in nondigestible carbohydrates have been shown to lower postprandial glucose concentrations after ingestion of high-glycemic-index breakfasts. This phenomenon is linked to colonic fermentation of nondigestible carbohydrates, but the underlying mechanism is not fully elucidated. OBJECTIVE: We examined the way in which glucose kinetics and related factors change after breakfast as a result of colonic fermentation. DESIGN: In a crossover design, 10 healthy men ingested as an evening meal white wheat bread (WB) or cooked barley kernels (BA) that were rich in nondigestible carbohydrates. In the morning after intake of 50 g (13)C-enriched glucose, the dual-isotope technique was applied to determine glucose kinetics. Plasma insulin, free fatty acid, interleukin-6, tumor necrosis factor-alpha, and short-chain fatty acid concentrations and breath-hydrogen excretion were measured. RESULTS: The plasma glucose response after the glucose drink was 29% lower after the BA evening meal (P = 0.019). The insulin response was the same, whereas mean (+/-SEM) tissue glucose uptake was 30% higher (20.2 +/- 1.9 compared with 15.5 +/- 1.8 mL/2 h; P = 0.016) after the BA evening meal, which indicated higher peripheral insulin sensitivity (P = 0.001). The 4-h mean postprandial interleukin-6 (19.7 +/- 5.1 compared with 5.1 +/- 0.7 pg/mL; P = 0.024) and tumor necrosis factor-alpha (7.8 +/- 2.1 compared with 5.3 +/- 1.6 pg/mL; P = 0.008) concentrations after the glucose drink were higher after the WB evening meal. Butyrate concentrations (P = 0.041) and hydrogen excretion (P = 0.005) were higher in the morning after the BA evening meal. CONCLUSION: In healthy subjects, factors related to colonic fermentation of nondigestible carbohydrates increase peripheral insulin sensitivity and moderate glucose-associated inflammation.