Metabolomics of cancer Serkova, Natalie J; Glunde, Kristine
Methods in molecular biology (Clifton, N.J.),
2009, Volume:
520
Journal Article
Metabolomics, one of the "omic" sciences in systems biology, is the global assessment and validation of endogenous small-molecule biochemicals (metabolites) within a biologic system. Initially, ...putative quantitative metabolic biomarkers for cancer detection and/or assessment of efficacy of anticancer treatment are usually discovered in a preclinical setting (using animal and human cell cultures), followed by translational validation of these biomarkers in biofluid or tumor tissue. Based on the tumor origin, various biofluids, such as blood, urine, and expressed prostatic secretions, can be used for validating metabolic biomarkers noninvasively in cancer patients. Metabolite detection and quantification is usually carried out by nuclear magnetic resonance (NMR) spectroscopy, while mass spectrometry (MS) provides another highly sensitive metabolomics technology. Usually, sophisticated statistical analyses are carried out either on spectroscopic or on quantitative metabolic data sets to provide meaningful information about the metabolic makeup of the sample. Various metabolic biomarkers, related to glycolysis, mitochondrial citric cycle acid, choline and fatty acid metabolism, were recently reported to play important roles in cancer development and responsiveness to anticancer treatment using NMR-based metabolic profiling.Carefully designed and validated protocols for sample handling and sample extraction followed by appropriate NMR techniques and statistical analyses, which are required to establish quantitative (1)H-NMR-based metabolomics as a reliable analytical tool in the area of cancer biomarker discovery, are discussed in the present chapter.
An increase of cellular phosphocholine (PC) and total choline (tCho)-containing compounds as well as alterations in lipids have been consistently observed in cancer cells and tissue. These metabolic ...changes are closely related to malignant transformation, invasion, and metastasis. The study of cancer cells in culture plays an important role in understanding mechanisms leading to altered choline (Cho) and lipid metabolism in cancer, as it provides a carefully controlled environment. However, a solid tumor is a complex system with a unique tumor microenvironment frequently containing hypoxic and acidic regions and areas of nutrient deprivation and necrosis. Cancer cell-stromal cell interactions and the extracellular matrix may also alter Cho and lipid metabolism. Human tumor xenograft models in mice are useful to mimic the growth of human cancers and provide insights into the influence of
conditions on metabolism. Here, we have compared metabolites, obtained with high resolution
H MRS of extracts from human breast and prostate cancer cells in a 2-dimensional (2D) monolayer culture and from solid tumor xenografts derived from these cells, as well as the protein expression of enzymes that regulate Cho and lipid metabolism. Our data demonstrate significant differences in Cho and lipid metabolism and protein expression patterns between human breast and prostate cancer cells in culture and in tumors derived from these cells. These data highlight the influence of the tumor microenvironment on Cho and lipid metabolism.
Elevated phosphoethanolamine (PE) is frequently observed in MRS studies of human cancers and xenografts. The role of PE in cell survival and the molecular causes underlying this increase are, ...however, relatively underexplored. In this study, we investigated the roles of ethanolamine kinases (Etnk‐1 and 2) and choline kinases (Chk‐α and β) in contributing to increased PE in human breast and pancreatic cancer cells. We investigated the effect of silencing Etnk‐1 and Etnk‐2 on cell viability as a potential therapeutic strategy. Both breast and pancreatic cancer cells showed higher PE compared with their nonmalignant counterparts. We identified Etnk‐1 as a major cause of the elevated PE levels in these cancer cells, with little or no contribution from Chk‐α, Chk‐β, or Etnk‐2. The increase of PE observed in pancreatic cancer cells in culture was replicated in the corresponding tumor xenografts. Downregulation of Etnk‐1 with siRNA resulted in cell cytotoxicity that correlated with PE levels in breast and pancreatic cancer cells. Etnk‐1 may provide a potential therapeutic target in breast and pancreatic cancers.
Ethanolamine kinase‐1 (Etnk‐1) was identified as a major cause of elevated phosphoethanolamine (PE) in breast cancer and pancreatic cancer cells. Downregulation of Etnk‐1 with siRNA resulted in cell cytotoxicity that correlated with PE levels. Etnk‐1 may provide a potential diagnostic and therapeutic target in breast and pancreatic cancers.
A tribute to Professor Sebastián Cerdán Pacheco‐Torres, Jesus; Glunde, Kristine; Griffiths, John R. ...
NMR in biomedicine,
November 2022, Volume:
35, Issue:
11
Journal Article
Inactivation of the tumor suppressor genes tumor protein p53 (
) and cyclin-dependent kinase inhibitor 2A (
) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a ...paucity of GEJ-specific disease models, cancer-promoting consequences of
and
inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated
and
knockout (
) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of
GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically,
dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated
transcription by binding to the
promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with
inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.
Collagen I (Col1) fibers are a major structural component in the extracellular matrix of human breast cancers. In a preliminary pilot study, we explored the link between Col1 fiber density in primary ...human breast cancers and the occurrence of lymph node metastasis. Col1 fibers were detected by second harmonic generation (SHG) microscopy in primary human breast cancers from patients presenting with lymph node metastasis (LN+) versus those without lymph node metastasis (LN-). Col1 fiber density, which was quantified using our in-house SHG image analysis software, was significantly higher in the primary human breast cancers of LN+ (fiber volume=29.22%±4.72%, inter-fiber distance=2.25±0.45 μm) versus LN- (fiber volume=20.33%±5.56%, inter-fiber distance=2.88±1.07 μm) patients. Texture analysis by evaluating the co-occurrence matrix and the Fourier transform of the Col1 fibers proved to be significantly different for the parameters of co-relation and energy, as well as aspect ratio and eccentricity, for LN+ versus LN- cases. We also demonstrated that tissue fixation and paraffin embedding had negligible effect on SHG Col1 fiber detection and quantification. High Col1 fiber density in primary breast tumors is associated with breast cancer metastasis and may serve as an imaging biomarker of metastasis.
In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, ...such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 M – H− (m/z 742.5) and PI 38:4 M – H− (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 M – H− (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.
Metabolic reprogramming is a hallmark of cancer, enabling cancer cells to rapidly proliferate, invade, and metastasize. We show that creatine levels in metastatic breast cancer cell lines and ...secondary metastatic tumors are driven by the ubiquitous mitochondrial creatine kinase (CKMT1). We discover that, while CKMT1 is highly expressed in primary tumors and promotes cell viability, it is downregulated in metastasis. We further show that CKMT1 downregulation, as seen in breast cancer metastasis, drives up mitochondrial reactive oxygen species (ROS) levels. CKMT1 downregulation contributes to the migratory and invasive potential of cells by ROS-induced upregulation of adhesion and degradative factors, which can be reversed by antioxidant treatment. Our study thus reconciles conflicting evidence about the roles of metabolites in the creatine metabolic pathway in breast cancer progression and reveals that tight, context-dependent regulation of CKMT1 expression facilitates cell viability, cell migration, and cell invasion, which are hallmarks of metastatic spread.
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•CKMT1 has context-dependent roles in primary breast tumor growth and metastasis•CKMT1 upregulation promotes breast cancer cell viability and primary tumor growth•CKMT1 downregulation elevates ROS levels, which increases cell migration and invasion•CKMT1 knockdown-induced ROS release upregulates adhesion and degradative factors
Ayyappan et al. investigated the role of CKMT1 in breast cancer progression and showed that CKMT1 upregulation increases tumor cell growth and viability, while downregulation promotes metastasis. This context-dependent role is tied to CKMT1-mediated regulation of the mitochondrial permeability transition pore, which modulates reactive oxygen species release.
Lactacidosis is a common feature of ischaemic brain tissue, but its role in ischaemic neuropathology is still not fully understood. Na+/H+ exchange, a mechanism involved in the regulation of ...intracellular pH (pHi), is activated by low pHi. The role of Na+/H+ exchange subtype 1 was investigated during extracellular acidification and subsequent pH recovery in the absence and presence of (4‐isopropyl‐3‐methylsulphonyl‐benzoyl)‐guanidine methanesulfonate (HOE642, Cariporid), a new selective and powerful inhibitor of the Na+/H+ exchanger subtype 1 (NHE‐1). It was compared for normoxia and hypoxia in two glioma cell lines (C6 and F98). pHi was monitored by fluorescence spectroscopy using the intracellularly␣trapped␣pH‐sensitive dye 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). Alterations in glial cell metabolism were characterized using high‐resolution 1H, 13C and 31P NMR spectroscopy of perchloric acid extracts. NHE‐1 contributed to glial pH regulation, especially at pathologically low pHi values. NHE‐1 inhibition with HOE642 during acidification caused exacerbated metabolic disorders which were prolonged during extracellular pH recovery. However, NHE‐1 inhibition during hypoxia protected the energy state of glial cells.
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