Statistical considerations and database limitations in NMR-based metabolic profiling studies.

INTRODUCTION: Interpretation and analysis of NMR-based metabolic profiling studies is limited by substantially incomplete commercial and academic databases. Statistical significance tests, including p-values, VIP scores, AUC values and FC values, can be largely inconsistent. Data normalization prior to statistical analysis can cause erroneous outcomes. OBJECTIVES: The objectives were (1) to quantitatively assess consistency among p-values, VIP scores, AUC values and FC values in representative NMR-based metabolic profiling datasets, (2) to assess how data normalization can impact statistical significance outcomes, (3) to determine resonance peak assignment completion potential using commonly used databases and (4) to analyze intersection and uniqueness of metabolite space in these databases. METHODS: P-values, VIP scores, AUC values and FC values, and their dependence on data normalization, were determined in orthotopic mouse model of pancreatic cancer and two human pancreatic cancer cell lines. Completeness of resonance assignments were evaluated using Chenomx, the human metabolite database (HMDB) and the COLMAR database. The intersection and uniqueness of the databases was quantified. RESULTS: P-values and AUC values were strongly correlated compared to VIP or FC values. Distributions of statistically significant bins depended strongly on whether or not datasets were normalized. 40-45% of peaks had either no or ambiguous database matches. 9-22% of metabolites were unique to each database. CONCLUSIONS: Lack of consistency in statistical analyses of metabolomics data can lead to misleading or inconsistent interpretation. Data normalization can have large effects on statistical analysis and should be justified. About 40% of peak assignments remain ambiguous or impossible with current databases. 1D and 2D databases should be made consistent to maximize metabolite assignment confidence and validation.

NMR-based urine metabolic profiling and immunohistochemistry analysis of nephron changes in a mouse model of hypoxia-induced acute kidney injury.

Acute kidney injury can be caused by multiple factors, including sepsis, respiratory failure, heart failure, trauma, or nephrotoxic medications, among others. Here, a mouse model was used to investigate potential urinary metabolic biomarkers of hypoxia-induced AKI. Urine metabolic profiles of 48 Swiss Webster mice were assessed using nuclear magnetic resonance spectroscopy (NMR) for 7 days following 72 h exposure to a hypoxic 6.5% oxygen environment. Histological analyses indicated a lack of gross nephron structural changes in the aftermath of hypoxia. Immunohistochemical (IHC) analyses, however, indicated elevated expression of protein injury biomarkers in distal and proximal tubules but not glomeruli. Kidney injury molecule-1 levels peaked in distal tubules at 72 h and were still increasing in proximal tubules at 7 days posthypoxia, whereas cystatin C levels were elevated at 24 h but decreased thereafter, and were elevated and still increasing in proximal tubules at 7 days posthypoxia. Neutrophil gelatinase-associated lipocalin levels were modestly elevated from 24 h to 7 days posthypoxia. NMR-based metabolic profiling revealed that urine metabolites involved in energy metabolism and associated biosynthetic pathways were initially decreased at 24 h posthypoxia, consistent with metabolic suppression as a mechanism for cell survival, but were significantly elevated at 48 and 72 h posthypoxia, indicating a burst in organism metabolism associated with reactivation of cellular energetics during recovery after cessation of hypoxia and return to a normoxic environment. The IHC results indicated that kidney injury persists long after plasma and urine biomarkers of hypoxia return to normal values.

NMR spectroscopy and electron microscopy identification of metabolic and ultrastructural changes to the kidney following ischemia-reperfusion injury.

Cellular, molecular, and ultrastructural nephron changes associated with ischemia-reperfusion injury-induced acute kidney injury (IRI-AKI) are not completely understood. Here, a multidisciplinary study was used to identify nephron changes in a mouse model of IRI-AKI. Histological analyses indicated distended Bowman's glomerular spaces and proximal and distal tubules. Increased filtrate volume in nephrons was caused by reduced water reabsorption by severely damaged proximal tubule brush borders and blocked flow of filtrate into collecting tubules by mucoprotein casts in distal tubules. Immunohistochemistry revealed protein AKI biomarkers in proximal tubules and glomeruli but not in distal tubules. Nuclear magnetic resonance spectroscopy revealed several metabolites that increased such as valine, alanine, and lactate. Other metabolites such as trigonelline, succinate, 2-oxoisocaproate, and 1- methyl-nicotinamide decreased or were absent in urine following IRI due to altered kidney function or metabolism. Urinary glucose increased due to reduced reabsorption by damaged proximal tubule brush borders. Scanning electron microscopy revealed flattening of podocytes and pedicals surrounding glomerular capillaries, and transmission electron microscopy (TEM) revealed effacement of podocyte pedicals, both consistent with increased hydrostatic pressure in nephrons following IRI-AKI. TEM revealed shortened proximal tubule microvilli in IRI kidneys with diminished lamina propia. TEM showed dramatic loss of mitochondria in distal tubule epithelia of IRI kidneys and emergence of multivesicular bodies of endosomes indicating ongoing cellular death. Collectively, the data define ultrastructural changes to nephrons and altered kidney metabolism associated with IRI-AKI.

Influence of media selection on NMR based metabolic profiling of human cell lines.

INTRODUCTION: Comparative metabolic profiling of different human cancer cell lines can reveal metabolic pathways up-regulated or down-regulated in each cell line, potentially providing insight into distinct metabolism taking place in different types of cancer cells. It is noteworthy, however, that human cell lines available from public repositories are deposited with recommended media for optimal growth, and if cell lines to be compared are cultured on different growth media, this introduces a potentially serious confounding variable in metabolic profiling studies designed to identify intrinsic metabolic pathways active in each cell line. OBJECTIVES: The goal of this study was to determine if the culture media used to grow human cell lines had a significant impact on the measured metabolic profiles. METHODS: NMR-based metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, AsPC-1, MiaPaCa-2 and Panc-1, were compared after culture on Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI-1640) medium. RESULTS: Comparisons of the same cell lines cultured on different media revealed that the concentrations of many metabolites depended strongly on the choice of culture media. Analyses of different cell lines grown on the same media revealed insight into their metabolic differences. CONCLUSION: The choice of culture media can significantly impact metabolic profiles of human cell lines and should be considered an important variable when designing metabolic profiling studies. Also, the metabolic differences of cells cultured on media recommended for optimal growth in comparison to a second growth medium can reveal critical insight into metabolic pathways active in each cell line.

RUNX1 mutations mitigate quiescence to promote transformation of hematopoietic progenitors in Fanconi anemia.

Many inherited bone marrow failure syndromes (IBMFSs) present a high risk of transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). During transformation of IBMFSs, hematopoietic stem and progenitor cells (HSPCs) with poor fitness gain ectopic, dysregulated self-renewal secondary to somatic mutations via undefined mechanisms. Here, in the context of the prototypical IBMFS Fanconi anemia (FA), we performed multiplexed gene editing of mutational hotspots in MDS-associated genes in human induced pluripotent stem cells (iPSCs) followed by hematopoietic differentiation. We observed aberrant self-renewal and impaired differentiation of HSPCs with enrichment of RUNX1 insertions and deletions (indels), generating a model of IBMFS-associated MDS. We observed that compared to the failure state, FA MDS cells show mutant RUNX1-mediated blunting of the G1/S cell cycle checkpoint that is normally activated in FA in response to DNA damage. RUNX1 indels also lead to activation of innate immune signaling, which stabilizes the homologous recombination (HR) effector BRCA1, and this pathway can be targeted to abrogate viability and restore sensitivity to genotoxins in FA MDS. Together, these studies develop a paradigm for modeling clonal evolution in IBMFSs, provide basic understanding of the pathogenesis of MDS, and uncover a therapeutic target in FA-associated MDS.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Head and Neck Cancer Susceptibility and Metabolism in Fanconi Anemia.

Fanconi anemia (FA) is a rare inherited, generally autosomal recessive syndrome, but it displays X-linked or dominant negative inheritance for certain genes. FA is characterized by a deficiency in DNA damage repair that results in bone marrow failure, and in an increased risk for various epithelial tumors, most commonly squamous cell carcinomas of the head and neck (HNSCC) and of the esophagus, anogenital tract and skin. Individuals with FA exhibit increased human papilloma virus (HPV) prevalence. Furthermore, a subset of anogenital squamous cell carcinomas (SCCs) in FA harbor HPV sequences and FA-deficient laboratory models reveal molecular crosstalk between HPV and FA proteins. However, a definitive role for HPV in HNSCC development in the FA patient population is unproven. Cellular metabolism plays an integral role in tissue homeostasis, and metabolic deregulation is a known hallmark of cancer progression that supports uncontrolled proliferation, tumor development and metastatic dissemination. The metabolic consequences of FA deficiency in keratinocytes and associated impact on the development of SCC in the FA population is poorly understood. Herein, we review the current literature on the metabolic consequences of FA deficiency and potential effects of resulting metabolic reprogramming on FA cancer phenotypes.

Energy metabolism during anchorage-independence. Induction by osteopontin-c.

The detachment of epithelial cells, but not cancer cells, causes anoikis due to reduced energy production. Invasive tumor cells generate three splice variants of the metastasis gene osteopontin, the shortest of which (osteopontin-c) supports anchorage-independence. Osteopontin-c signaling upregulates three interdependent pathways of the energy metabolism. Glutathione, glutamine and glutamate support the hexose monophosphate shunt and glycolysis and can feed into the tricarboxylic acid cycle, leading to mitochondrial ATP production. Activation of the glycerol phosphate shuttle also supports the mitochondrial respiratory chain. Drawing substrates from glutamine and glycolysis, the elevated creatine may be synthesized from serine via glycine and supports the energy metabolism by increasing the formation of ATP. Metabolic probing with N-acetyl-L-cysteine, L-glutamate, or glycerol identified differential regulation of the pathway components, with mitochondrial activity being redox dependent and the creatine pathway depending on glutamine. The multiple skewed components in the cellular metabolism synergize in a flow toward two mechanisms of ATP generation, via creatine and the respiratory chain. It is consistent with a stimulation of the energy metabolism that supports anti-anoikis. Our findings imply a coalescence in cancer cells between osteopontin-a, which increases the cellular glucose levels, and osteopontin-c, which utilizes this glucose to generate energy.