Edwin C.A. Stigter

Interplay between metabolic identities in the intestinal crypt supports stem cell function

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism.

Mutations in the daf‐2 gene of the conserved Insulin/Insulin‐like Growth Factor (IGF‐1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf‐16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf‐2 mutants in comparison with N2 and daf‐16; daf‐2 double mutants. This revealed a remarkable longevity‐specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf‐2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf‐2 and daf‐16;daf‐2 double mutants confirmed a daf‐16‐dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction‐mediated longevity, which was independent of germline activity. RNA interference (RNAi)‐mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF‐1‐mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF‐1‐mediated extension of life.

Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3-isoprostane measurement

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3‐isoprostanes. F3‐isoprostanes are prostaglandin‐like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev‐1 and clk‐1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro‐oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress‐resistant and long‐lived daf‐2 insulin/IGF‐1 receptor mutant involved distinct daf‐16‐dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS.

Development and validation of a quantitative LC–tandem MS assay for hexadeca-4,7,10,13-tetraenoic acid in human and mouse plasma

Upon exposure to platinum analogs, mesenchymal stem cells were recently found to excrete minute amounts of specific lipid mediators that induce chemotherapy resistance. One of these lipids is hexadeca-4,7,10,13-tetraenoic acid (FA(16:4)n-3). Importantly, FA(16:4)n-3 is present in high concentrations in certain fish oils and algae and oral intake of these products also potently induced chemotherapy resistance. These findings suggested that certain foods could negatively affect clinical chemotherapy treatment. In order to allow further study of the relation between FA(16:4)n-3 and clinical chemotherapy resistance, a method for the detection and quantification of FA(16:4)n-3 in plasma is required. Therefore, a quantification method for FA(16:4)n-3 in human and mouse plasma was developed consisting of a liquid-liquid extraction, solid phase clean-up and LC-MS/MS (MRM) analysis. The method was fully validated over a period of three weeks according to the standard protocols and requirements. The linearity range of the method is 1-100 nmol/L (r(2)>0.99) using deuterated FA(16:3)n-3 as an internal standard. The limits of quantification and detection are 1.0 nmol/L and 0.8 nmol/L, respectively. Recoveries for spiked concentrations range from 103 to 108%. The intra-day and inter-day mean precision amounts to 98-106% and 100-108%, respectively. No significant loss of FA(16:4)n-3 is observed upon storage at -80 °C. The developed assay for the detection and quantification of FA(16:4)n-3 in human plasma is robust and reproducible. The validation parameters are within limits of acceptance.

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency

Background & Aims Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. Methods We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7. Results In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. Conclusions We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

A Single Complex Agpat2 Allele in a Patient With Partial Lipodystrophy

Genetic lipodystrophies are a group of rare syndromes associated with major metabolic complications – including severe insulin resistance, type 2 diabetes mellitus, and hypertriglyceridemia – which are classified according to the distribution of adipose tissue. Lipodystrophies can be present at birth or develop during life and can range from local to partial and general. With at least 18 different genes implicated so far, definite diagnosis can be challenging due to clinical and genetic heterogeneity. In an adult female patient with clinical and metabolic features of partial lipodystrophy we identified via whole genome sequencing (WGS) a single complex AGPAT2 allele [V67M;V167A], functionally equivalent to heterozygosity. AGPAT2 encodes for an acyltransferase implicated in the biosynthesis of triacylglycerol and glycerophospholipids. So far homozygous and compound heterozygous mutations in AGPAT2 have only been associated with generalized lipodystrophy. A SNP risk score analysis indicated that the index patient is not predisposed to lipodystrophy based on her genetic background. The partial phenotype in our patient is therefore more likely associated to the genetic variants in AGPAT2. To test whether the resulting double-mutant AGPAT2 protein is functional we analyzed its in vitro enzymatic activity via mass spectrometry. The resulting AGPAT2 double mutant is enzymatically inactive. Our data support the view that the current classification of lipodystrophies as strictly local, partial or generalized may have to be re-evaluated and viewed more as a continuum, both in terms of clinical presentation and underlying genetic causes. Better molecular understanding of lipodystrophies may lead to new therapies to treat adipose tissue dysfunction in common and rare diseases.

Abstract 840: High levels of hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)) in fish oil induce resistance to chemotherapy in vivo

Environment-mediated resistance to chemotherapy is emerging as a cause of treatment failure. We recently discovered that two fatty acids, 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), induced resistance to a broad spectrum of chemotherapeutics in mice. These fatty acids were produced by mesenchymal stem cells upon platinum stimulation, and were therefore called platinum-induced fatty acids (PIFAs; Roodhart et al, Cancer Cell 2011). Fish oil, a complex, non-standardized mixture of fatty acids, was also shown to contain high levels of these PIFAs. Fish oil supplements are commonly used by cancer patients due to their perceived positive health effects. First, to determine the percentage of cancer patients taking fish oil supplements, a questionnaire was handed out at the UMC Utrecht medical oncology department. In an interim analysis, 12% of responders (11/90) were found to use fish oil supplements, the majority of whom continued supplementation during chemotherapy (82%). Second, we analyzed 5 commercially available fish oils for 16:4(n-3) content by ultra-high pressure liquid chromatography coupled to Orbitrap mass spectrometry. Even though 16:4(n-3) values differed, all tested fish oils contained significant amounts of the resistance-inducing PIFA. Concentrations ranged between 0.1 - 6.5 μM 16:4(n-3). We previously determined that as little as 25 nM purified 16:4(n-3) was sufficient to induce chemoresistance in mice. Here, we show that fish oils abundant in 16:4(n-3) interfered with chemotherapy actions in BALB/c mice bearing subcutaneous C26 tumors. Fish oil alone, administered by oral gavage, did not alter tumor volume compared to untreated mice. However, a single oral administration of each of three different fish oils together with intraperitoneal cisplatin therapy induced potent chemoresistance (tumor volume on day 4 after fish oil and chemotherapy was 222%, 223% and 246% compared to tumor volume after cisplatin alone). These results were confirmed in LLC-bearing C57Bl/6 mice. Finally, the fish oil containing the highest 16:4(n-3) level (6.5 μM) was tested for dose dependency by oral administration of 100, 10, 1 and 0.1 αl. As little as 1 αl of this fish oil was sufficient to induce resistance to cisplatin. On day 4, tumor volumes were 118±44 mm3 for vehicle-treated mice, 41±16 mm3 for cisplatin-treated and 94±63 mm3 for mice treated with cisplatin + 1 αl of fish oil (p 0.02). Concluding, commercially available fish oils contained variable levels of resistance-inducing PIFA 16:4(n-3). In clinically relevant doses, fish oil induced resistance to chemotherapy in mice. This implicates that fish oil use can interfere with chemotherapy outcome. 12% of cancer patients use fish oil, urging the need for clinical studies to determine the effects of fish oil intake on PIFA concentrations in human plasma, and on response to chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 840. doi:1538-7445.AM2012-840

Abstract 705: Interfering with mesenchymal stem cell-induced chemoresistance via COX-1 and thromboxane synthase inhibition

Background: Environment-mediated resistance to chemotherapy is emerging as a cause of treatment failure. We showed that mesenchymal stem cells (MSCs) induce resistance to a broad spectrum of chemotherapies. Upon platinum stimulation, two unique polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT(n-6)) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)) are produced by MSCs, which, via a paracrine mechanism, induce chemoresistance in tumors. Aim: Here, we aim to prevent MSC-induced chemoresistance by blocking PIFA production. Method: Various mouse models were established in which recruitment of MSCs to tumors was mimicked by iv administration of MSCs. Furthermore, cultured MSCs were treated with various drugs in order to block their chemo-protective capacity. Conditioned media were subsequently analyzed in vivo. Results: Conditioned medium of platinum-stimulated, cultured MSCs induced chemoresistance in tumor-bearing mice. However, the capacity to secrete the chemo-protective PIFAs was only retained by mesenchymal cells with multi-lineage differentiation potential: MSCs and mouse embryonic fibroblasts (MEFs). More differentiated progeny from the mesenchymal lineage including 3T3 Fibroblasts, pre-adipocytes (3T3-L1), differentiated adipocytes, pre-osteoblasts (MC3T3) and differentiated osteoblasts were not capable of PIFA production upon platinum stimulation. Furthermore, this cytoprotective response was specific for multipotent cells from the mesenchymal lineage, since administration of hematopoietic stem cells did not influence the tumor response to chemotherapy. One of the identified PIFAs, KHT(n-6), is known to be a by-product of thromboxane A2 synthesis, which is a downstream product of the cyclooxygenase (COX)-1 and thromboxane synthase (TxS) pathway. Interestingly, conditioned media from MSCs pre-treated with SC-560, a highly selective COX-1 inhibitor, indomethacin, a relatively selective COX-1 inhibitor or one of two specific TxS inhibitors (ozagrel and furegrelate) did not induce chemoresistance in vivo. Of note, selective COX-2 inhibition in MSC by celecoxib did not affect their capacity to induce chemoresistance, suggestive of a specific COX-1/TxS-dependent PIFA production. When mice were treated with either indomethacin or ozagrel as single agents no anti-tumor effect was observed. However, combining these drugs with cisplatin in vivo had an additive anti-tumor effect compared to cisplatin alone. Conclusion: We show a novel mechanism of chemoresistance mediated by PIFA released from early, multipotent cells of the mesenchymal lineage. The production of these PIFAs is dependent on COX-1 and TxS, and blocking these enzymes enhanced the antitumor effects of cisplatin in vivo, making these enzymes drugable targets to prevent PIFA-induced chemoresistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2011-705

Abstract 4739: Mesenchymal stem cells induce broad spectrum resistance to chemotherapy through the release of platinum-induced fatty acids

Background The development of resistance to chemotherapy is a major obstacle for lasting effective treatment of cancer. It is becoming increasingly clear that the tumor micro-environment plays a key role in the development of drug resistance. Mesenchymal stem cells (MSCs), key players in tissue repair and wound healing, are recruited in large numbers to the stroma of developing tumors where they are known to support tumor growth and enhance metastasis formation. Aim To determine whether MSCs confer resistance to chemotherapy, we established different murine tumor models in which we resembled the mobilization and recruitment of MSCs from the bone marrow to tumor. Results Administration of 50,000 MSCs intravenously (iv), or as little as 1,000 MSCs subcutaneously (sc) together with chemotherapy almost completely abolished the anti-tumor effect of the chemotherapy in a dose-dependent matter. Notably, this was only observed with the platinum-based chemotherapeutics, namely cisplatin, oxaliplatin and carboplatin. To show that the MSCs induce resistance in a paracrine fashion, we incubated cultured MSCs with platinum-based chemotherapy and injected the conditioned medium (CM+) from these cells sc concomitantly with the chemotherapy. Strikingly, the CM+ induced an almost complete resistance to multiple types of chemotherapy, including non-platinum based chemotherapeutics like 5-FU and irinotecan. The CM from untreated MSCs (CM-), did not induce resistance. Through a metabolomics approach, we identified two unique platinum-induced polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT(n-6)) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)) in the CM+ responsible for the induction of resistance by the MSCs. We tested the purified PIFAs in our mouse models and confirmed that in minute quantities (2pmol) these PIFAs induce resistance to a broad spectrum of chemotherapy. In contrast, other related poly-unsaturated fatty acids had no effect. Conclusion In summary, our study identifies a novel paracrine mechanism of resistance via activation of MSCs by platinum-based chemotherapy with the subsequent release of two specific resistance-inducing PIFAs. Our findings suggest that interventions directed against either MSC or the identified PIFAs may be explored to enhance the efficacy of chemotherapy and prevent the development of resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4739. doi:10.1158/1538-7445.AM2011-4739