The mission of the Stem Cell Toxicology Group is to characterize responses to toxicants to elucidate mechanisms and identify the role of stem cells (SCs) in disease manifestation. The Group provides expertise in areas of SC biology and toxicology, and works on National Toxicology Program (NTP) Laboratory mission-related projects that involve SCs. While SCs/cancer SCs (CSCs) and potential mechanisms of action in inorganic carcinogenesis has been the major focus, research efforts are extending beyond cancer to include those diseases and conditions associated with exposure to NTP-relevant chemicals as well as effects of these chemicals at various life stages. Arsenic (As) and cadmium (Cd) are inorganic carcinogens that are major human health hazards and defining mechanisms is key to defining risk. We use mature differentiated and SC cell models of human target-relevant tissues of these carcinogens. Millions of people worldwide are exposed to unhealthy levels of these inorganics making elucidation of mechanisms all the more important. Our in vivo models combined with our in vitro work with mature differentiated and SC cell lines, show As targets SCs for transformation apparently due to a hyper-resistance and hyper-adaptability of SCs to As. These characteristics lead to CSC over-abundance in the tumors and in transformed human cell lines. We continue to discover and define the mechanisms involved in this SC targeting and transformation. Unlike As, Cd initially selectively kills 90% of SCs during exposure to a non-toxic, but transforming, level for the heterogeneous parental lines. The remaining SCs rapidly re-emerge and undergo transformation. We are determining if Cd has transformed these SCs and observing these SCs and the mature cell line for selection of hyper-resistant SCs. We are also defining the metabolic profiles during transformation of several As and Cd transformed SC/CSC models. Neither As nor Cd appears to be directly mutagenic, suggesting possible epigenetic mechanisms. Epigenetic modifications have been shown to play key roles in cancers induced As and Cd. The KRAS oncogene is highly up-regulated during As and Cd induced malignant transformation. We found no evidence that this KRAS increase is due to DNA damage or methylation processes, suggesting epigenetic factors are involved. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at a post-transcriptional level. We find As exposure dysregulates miRNA expression that appears to control RAS activation during As malignant transformation of mature and SCs. Knockdown of RAS or restoration of RAS-associated miRNAs partially mitigates this phenotype, suggesting miRNA-regulated RAS expression is a putative driver in As transformation. Depleting KRAS in Cd-transformed cells also reverses cancer phenotype, although this did not appear to be miRNA-regulated. These studies help determine roles of miRNAs and underlying epigenetic mechanisms involved in As and Cd carcinogenesis, and suggest possible miRNA biomarkers of inorganic transformation. We are working with the Biomolecular Screening Branch (NTP) using next-gen sequencing methods to further examine the KRAS upregulation in As transformation. In these studies, the major driver of cell transformation is an increased level of KRAS. We performed a genome-wide evaluation of DNA methylation and gene expression, but observed genomic changes appear to be secondary to elevated KRAS. Data show that KRAS expression appears unaffected by any changes in proximal methylation clusters at this genomic locus. Preliminary findings implicate the activation of endogenous retroviruses in As-transformed cells that may have incorporated KRAS. The potential of embryonic SCs (ESCs) and induced pluripotent SCs (iPSCs) in disease modeling, drug testing, drug development, and regenerative therapies have made SC biology one of the most active areas of research over the last decade. We are using ESCs and iPSCs to examine effects of environmental toxicants on embryogenesis, developmental toxicology and reproductive toxicology. With the Epigenetics and Stem Cell Biology Lab (DIR) we are developing assays to screen environmental toxicants/chemicals on human ESCs and human iPSCs to help discover and better predict developmental toxicants and teratogens. Ongoing studies are using a high-throughput transcriptomics (HTT) platform to examine chemical-induced gene expression alterations in hESCs and iPSCs, and between iPSCs derived from different patients. These studies will help determine the effects of chemicals on embryonic development, germ-layer differentiation, and/or teratogenicity. Polycyclic aromatic compounds (PACs) are environmental contaminants with extensive toxicities. With the Toxicology Branch (NTP), we are characterizing the toxicity of a broad range of PAHs and PAH mixtures. Initial studies (24 PACs, 5 cell lines) of structurally diverse PACs showed that, in general, activity levels of PACs corresponded across in vitro and in vivo testing platforms. These results offer promise for the development of an in vitro battery for predicting in vivo activity. This project has been expanded to include approximately 100 PAHs and PAH mixtures. Additionally, to cover more biological space the number of cell lines used is also being increased to 13 and will include non-tumorigenic and cancer cell lines. This expanded project is well underway and initial screens (i.e. cytotoxicity) will be completed in the near future. Crumb rubber (CR) is a major component of synthetic turfs. There is evidence of potential carcinogenic risk from playing on these turfs. CR is composed of ground tires and can contain volatile, semi-volatile, and non-volatile organic compounds, metals, and particulate matter. Major exposure pathways are dermal, inhalation and ingestion. With the Predictive Toxciology and Screening Group (NTPL) and the Toxicology Branch (NTP) we are investigating the toxicity of several crumb rubber samples. We find these samples are cytotoxic to human lung and skin cells at relatively low doses and short exposure times. We will continue these studies on additional cell lines (intestinal, hematopoietic) and will determine mechanisms of action, components and metabolites released from the samples, and use HTT approaches to examine possible altered gene expression caused by CR. With collaborators at Harvard and in Japan we are examining a unique Japanese cohort that was acutely exposed to high levels of As during infancy in order evaluate the association between this developmental exposure and the resultant differential gene expression and signaling pathway alterations that have persisted into adulthood. The overall purpose was to help identify the genes and pathways most affected by this early-life exposure as a possible method to help identify potential early indicators of disease. As-poisoned subjects showed DNA methylation and signaling pathway alterations in blood cells that suggested adverse effects on immune regulation and function, and respiratory and infectious disease vulnerability. Exosomes are extracellular vesicles that contain biomolecular cargo that can exert effects on recipient cells. We have shown that As-transformed epithelial cells can recruit nearby SCs into a CSC phenotype. We believe exosomes released by As-transformed cells play a key role in regulating the tumor microenvironment and are isolating and characterizing exosomes to determine the potential role of select cargo molecules in the SC recruitment process. We have found that As-transformed cells secret exosomes carrying cancer-favoring biomolecules (KRAS, specific miRNAs) which appear to recruit and transform nearby SCs. Knockdown of many of these factors decreases the ability of the transformed cells to recruit the SCs into a CSC phenotype.
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