No abstract

Epithelial-to-mesenchymal transition (EMT) is fundamental to both normal tissue development and cancer progression. We hypothesized that EMT plasticity defines a range of metabolic phenotypes and that individual breast epithelial metabolic phenotypes are likely to fall within this phenotypic landscape. To determine EMT metabolic phenotypes, the metabolism of EMT was described within genome-scale metabolic models (GSMMs) using either transcriptomic or proteomic data from the breast epithelial EMT cell culture model D492. The ability of the different data types to describe breast epithelial metabolism was assessed using constraint-based modeling which was subsequently verified using 13C isotope tracer analysis. The application of proteomic data to GSMMs provided relatively higher accuracy in flux predictions compared to the transcriptomic data. Furthermore, the proteomic GSMMs predicted altered cholesterol metabolism and increased dependency on argininosuccinate lyase (ASL) following EMT which were confirmed in vitro using drug assays and siRNA knockdown experiments. The successful verification of the proteomic GSMMs afforded iBreast2886, a breast GSMM that encompasses the metabolic plasticity of EMT as defined by the D492 EMT cell culture model. Analysis of breast tumor proteomic data using iBreast2886 identified vulnerabilities within arginine metabolism that allowed prognostic discrimination of breast cancer patients on a subtype-specific level. Taken together, we demonstrate that the metabolic reconstruction iBreast2886 formalizes the metabolism of breast epithelial cell development and can be utilized as a tool for the functional interpretation of high throughput clinical data.

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here, we used 13 C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro. We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors.Implications: EMT in breast cells causes an increased demand for glutamine for fatty acid biosynthesis, altering its contribution to glutathione biosynthesis which sensitizes the cells to mTOR inhibitors.

Purpose To evaluate the outcome of arthroscopic treatment for iliopsoas impingement after total hip arthroplasty (THA) 2 years after surgery using patient reported outcomes (PROM). Methods In this study 12 patients (13 hips) were included from a local hip arthroscopy registry. Patients completed web-based PROMs preoperatively and at a minimum of 2 years postoperatively. The PROMs included the International Hip Outcome Tool short version (iHOT-12), the Copenhagen Hip and Groin Outcome Score (HAGOS), the European Quality of Life-5 Dimensions Questionnaire (EQ-5D), the Hip Sports Activity Scale (HSAS) for physical activity level, the Visual Analog Scale (VAS) for overall hip function and a single question regarding overall satisfaction with the surgery. Results The mean age was 64.4 years (±15.1SD), mean body mass index (BMI) was 26.6 (±4.3SD), mean follow-up time was 49.8 months (±25SD). Comparing PROMs preoperatively with 2-year follow up showed an improvement for many of the PROMs used. The PROMs scores were iHOT-12 (24.9 vs 34.5, p = 0.13), HAGOS subscales (symptoms 38.2 vs 54.5, p = 0.05; pain 36 vs 53, p = 0.04; sport 14.1 vs 35.1, p = 0.03; daily activity 31 vs 47.5, p = 0.04; physical activity 21.8 vs 24, p = 0.76; quality of life 24 vs 35, p = 0.03), EQ-VAS (57.9 vs 58, p = 0.08), EQ-5D (0.34 vs 0.13, p = 0.07) and VAS for overall hip function (43.1 vs 46.2, p = 0.14). In total, 10 out of the 12 patients (83%) were satisfied with the intervention. Conclusion Patients undergoing surgery for iliopsoas impingement after previous THA showed improved self-reported hip function where most patients were satisfied with treatment.

Lanthipeptides are ribosomally-synthesized natural products from bacteria featuring stable thioether-crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class-IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence-based structural models identified the N-terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α-helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class-IV and class-III lanthipeptide synthetases.

Lanthipeptides are ribosomally‐synthesized natural products from bacteria featuring stable thioether‐crosslinks and various bioactivities. Herein, we report on a new clade of tricyclic class‐IV lanthipeptides with curvocidin from Thermomonospora curvata as its first representative. We obtained crystal structures of the corresponding lanthipeptide synthetase CuvL that showed a circular arrangement of its kinase, lyase and cyclase domains, forming a central reaction chamber for the iterative substrate processing involving nine catalytic steps. The combination of experimental data and artificial intelligence‐based structural models identified the N‐terminal subdomain of the kinase domain as the primary site of substrate recruitment. The ribosomal precursor peptide of curvocidin employs an amphipathic α‐helix in its leader region as an anchor to CuvL, while its substrate core shuttles within the central reaction chamber. Our study thus reveals general principles of domain organization and substrate recruitment of class‐IV and class‐III lanthipeptide synthetases.

Background: Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention.Methods: We utilize 13C carbon analogs of glucose and glutamine to examine difference in internal flux patterns of metabolites in central carbon and lipid metabolism following EMT in breast epithelial cell lines. Furthermore, an isotopomer spectral analysis, 13C-metabolic flux analysis and weighted correlation network analysis are utilized for investigation of the alterations in metabolic functionality following EMT in breast.Results: There are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of TCA-cycle flux characterized by increased mitochondrial IDH2 -mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Our network approach identified specific subtype of cancer drugs that are significantly associated with GSH abundance and we confirmed this in vitro.Conclusions: EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to PI3K/Akt/mTOR inhibitors.

<p>UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with A) 1-13C-glutamine and B) 5-13C-glutamine for 6 hours. Results are shown as mean + standard deviation (n = 3).</p>