SPDEF is a mediator of tumorigenicity in SSEA1- tumor-initiating cells in high grade serous ovarian cancer

High Grade Serous Ovarian Adenocarcinoma (HGSOC) is a highly aggressive disease with poor prognosis and the leading cause of gynecological tumor-related deaths. The poor prognosis is related to the fact that already during early stages tumor cells start to spread into the peritoneum. This metastatic spread and the colonization of organs located within the peritoneum are the biggest problems with regard to therapy of HGSOC. Tumors often consist of a functionally heterogeneous population of cancer cells with distinct features. Subsets of tumor cell populations are able to promote tumor progression, metastatic spread and colonization, as well as outgrowth of tumor cells at distant organs. Therefore, the identification and targeting of so-called tumor-initiating cells is crucial. Knowledge about the intrinsic features of tumor-initiating cells and targeting them may ultimately lead to tumor regression and improved patient survival. However, no conclusive evidence about markers for a tumor-initiating population has been provided so far and even less is understood regarding the molecular mechanisms driving tumor-initiating cancer cell populations in HGSOC. My work shows that Stage Specific Embryonic Antigen 1 negative (SSEA1-) cells are enriched for tumor-initiating abilities in human HGSOC. Furthermore, SSEA1- cells can give rise to both SSEA1- and SSEA1+ cells whereas SSEA1+ cells only give rise to SSEA1+ cells demonstrating a hierarchical organization with SSEA1- cells being on top. Gene expression profiling demonstrated an enrichment of the transcription factor SAM-pointed ETS domaincontaining factor (SPDEF) in SSEA1- cells. Lentiviral knockdown of SPDEF impaired in vivo tumor growth and in vitro colony formation, whereas overexpression of SPDEF resulted in increased colony formation in vitro and tumor formation in vivo. Strikingly, also SSEA1+ cells acquired the capacity to initiate tumors in vivo and form colonies in vitro after SPDEF expression was re-introduced. I also show, that SPDEF negatively regulates the expression of the transcription factor Forkhead box protein A2 (FOXA2). FOXA2 overexpression resulted in decreased tumor-promoting capacity in an in vivo tumor formation assay. Based on these results, I propose that the transcriptional programs modulated by SPDEF, as well as those genes changed due to suppressed FOXA2 target gene transcription, lead to increased survival, clonogenicity and stemness of SSEA1- SPDEFhigh FOXA2low cells and may therefore promote ovarian cancer tumor initiation and metastatic spread. In summary, the data I generated indicate that SSEA1- cells represent a cellular subpopulation with increased tumor-initiating ability in HGSOC. These cells express higher levels of SPDEF, which exerts its tumorigenic potential by suppressing FOXA2 expression. Developing SPDEF inhibitors might be promising to target the SSEA1- tumor-initiating population and might ultimately lead to tumor regression and improved patient survival

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the ‘Phenylalanine, tyrosine and tryptophan biosynthesis’ pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin

Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC–MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones

Investigating the gut microbiome and metabolome following treatment with artificial sweeteners acesulfame potassium and saccharin in young adult Wistar rats

To elucidate if artificial sweeteners modify fecal bacterial composition and the fecal and plasma metabolomes, Wistar rats from both sexes were treated for 28 days with acesulfame potassium (40 and 120 mg/kg body weight) and saccharin (20 and 100 mg/kg body weight). Targeted MS-based metabolome profiling (plasma and feces) and fecal 16S gene sequencing were conducted. Both sweeteners exhibited only minor effects on the fecal metabolome and microbiota. Saccharin treatment significantly altered amino acids, lipids, energy metabolism and specifically, bile acids in the plasma metabolome. Additionally, sex-specific differences were observed for conjugated primary and secondary bile acids. Acesulfame potassium treated male rats showed larger alterations in glycine conjugated primary and secondary bile-acids than females. Other changes in the plasma metabolome were more profound for saccharin than acesulfame potassium, for both sexes. Changes in conjugated bile-acids in plasma, which are often associated with microbiome changes, and the absence of similarly large changes in microbiota suggest an adaptative change of the latter, rather than toxicity. Further studies with a high resolution 16S sequencing data and/or metagenomics approach, with particular emphasis on bile acids, will be required to explore the mechanisms driving this metabolic outcome of saccharin in Wistar rats

Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics

Gut Microbiota as Well as Metabolomes of Wistar Rats Recover within Two Weeks after Doripenem Antibiotic Treatment

An understanding of the changes in gut microbiome composition and its associated metabolic functions is important to assess the potential implications thereof on host health. Thus, to elucidate the connection between the gut microbiome and the fecal and plasma metabolomes, two poorly bioavailable carbapenem antibiotics (doripenem and meropenem), were administered in a 28-day oral study to male and female Wistar rats. Additionally, the recovery of the gut microbiome and metabolomes in doripenem-exposed rats were studied one and two weeks after antibiotic treatment (i.e., doripenem-recovery groups). The 16S bacterial community analysis revealed an altered microbial population in all antibiotic treatments and a recovery of bacterial diversity in the doripenem-recovery groups. A similar pattern was observed in the fecal metabolomes of treated animals. In the recovery group, particularly after one week, an over-compensation was observed in fecal metabolites, as they were significantly changed in the opposite direction compared to previously changed metabolites upon 28 days of antibiotic exposure. Key plasma metabolites known to be diagnostic of antibiotic-induced microbial shifts, including indole derivatives, hippuric acid, and bile acids were also affected by the two carbapenems. Moreover, a unique increase in the levels of indole-3-acetic acid in plasma following meropenem treatment was observed. As was observed for the fecal metabolome, an overcompensation of plasma metabolites was observed in the recovery group. The data from this study provides insights into the connectivity of the microbiome and fecal and plasma metabolomes and demonstrates restoration post-antibiotic treatment not only for the microbiome but also for the metabolomes. The importance of overcompensation reactions for health needs further studies

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients