In ongoing collaborative studies with the Sreekumar laboratory at the Baylor College of Medicine, our group has been investigating the metabolome of breast tumors. For discovery, we used the services of the company, Metabolon, to measure the abundance of 352 known and 184 unknown metabolites in 67 human breast tumors and 65 adjacent non-cancerous tissues with an untargeted mass spectrometry-based profiling approach. Quantitative differences for several key metabolites were validated in 70 estrogen receptor-negative tumors and 36 adjacent non-cancerous tissues with Multiple Reaction Monitoring-based targeted assays by the Sreekumar laboratory (validation set). Our current findings indicate that breast tumors have intrinsic metabolite signatures that persist with disease progression. We did not find that metabolites classify tumors into the gene expression-defined subtypes (luminal A &B, basal-like, HER2-enriched), suggesting that the breast tumor metabolome may describe different traits of the disease, as was suggested by others. Because our study population was ethnically diverse and was race/ethnicity-matched for tumor ER status and triple-negative/basal-like disease, we analyzed tissue metabolite differences comparing the African-American patients (n = 32) with European-American patients (n = 35). All patients were ancestry-typed with an average West African ancestry of 83.2% among the African-American patients and an average European ancestry of 97.6% among the European-American patients. Using unsupervised hierarchical clustering based on the abundance of 296 measured metabolites, both estrogen receptor (ER)-negative tumors and triple-negative tumors separated into subclusters that did not randomly represent the two patient groups. Instead, distinct clusters emerged that were enriched for either African-American or European-American patients, which was most striking for the triple-negative/basal-like tumor category. Notable, many mitochondrial metabolites, and also lyso(phospho)lipids, carnitines, and arachidonic acid, were more abundant in tumors from African-American patients while histamine was markedly decreased in them. Our data indicate that metabolic differences may exist between tumors from African-American and European-American patients. However, these findings need confirmation. We are currently collecting additional breast tumors from various sources for independent validation. Future research will perform a more in-depth analysis of the metabolite differences between the two patient groups, including a description of the key pathways that are affected by these differences, and how they may influence disease outcome and response to therapy. The Sreekumar laboratory has also access to human transplant tumors in nude mice. This system will be used to examine the relationship between the transplant metabolome and response to therapy and allows for experimental tissue metabolome modifications to test whether the changes improve the therapy response. Another interesting facet in our data was the elevated level of 2-hydroxyglutarate (2HG) in a subset of ER-positive and ER-negative tumors, with negligible levels in most adjacent noncancerous tissue samples. 2HG accumulates in gliomas and leukemias with hotspot mutations in either the cytosolic or mitochondrial isocitrate dehydrogenase (IDH1 and IDH2), which cause the enzyme to produce 2HG. Sequencing of the breast tumors did not reveal the presence of mutations in the catalytic domains of IDH1 and IDH2 mutations, consistent with other studies that failed to find these mutations in human breast tumors. Because aberrantly accumulated 2HG alters DNA methylation, we examined the global DNA methylation pattern in the 67 tumors using the Illumina Human Methylation 450 BeadChips. The analysis led to the discovery of a novel poor outcome tumor subtype with a high tissue 2HG concentration, a distinct genome-wide DNA methylation signature, and a stem cell-like transcriptional signature. Our data describe this molecular subtype as a class of tumors that tends to have high 2HG and includes a disproportionately high number of African-American patients. Thus, African-American patients may develop more frequently a methylation-defined subgroup III tumor than European-American patients, consistent with one earlier study that observed DNA methylation differences between these two patient groups. Our future research will concentrate on the relationship between 2HG and the development of the methylation-defined breast cancer subtype with poor outcome (subgroup III). In a second project, we will assess the influence of stress-related exposures on tumor biology in breast cancer patients, pursuing the hypothesis that exposures to stress, social isolation, and discrimination are associated with a prognostic gene expression signature and increased tumor catecholamine levels. We also hypothesize that this stress-induced signature is more prevalent in breast tumors of African-American than European-American patients and is a biomarker for catecholamine signaling that can be used to select patients for intervention therapy (beta-blocker, stress management). The cultural, race/ethnic, and sociodemographic diversity of Baltimore city and of the patients coming to the University of Maryland make the area an ideal study location to evaluate the influence of discrimination and stress on breast cancer biology, as proposed by our study, and to establish a link between stress-related exposures and the development of specific tumor characteristics (e.g., increased TAM infiltration and microvessel density in presence of a distinct poor outcome gene expression signature) that may affect African-American patients more frequently than European-American patients. We will analyze tumors from breast cancer patients who had surgical resection of their cancerous breast. These patients will complete the study questionnaire (evaluating stress) prior to the surgery. Per current design, we will initially analyze 80 fresh-frozen breast tumors from 40 African-American and 40 European-American breast cancer patients who have TNM stage I or stage II disease.
We aim to include 40 ER-positive and 40 ER-negative tumors. The tumors will be selected from a projected patient pool of up to 150 breast cancer patients that are being recruited into the study at the University of Maryland Medical Center. Currently, we have completed recruitment of 18 patients. We will obtain clinicopathological information from pathology and medical records including tumor receptor status (ER, PR, HER2/neu) and survival follow up through the National Death Index. We will determine adrenaline and noradrenaline concentrations and perform gene expression profiling and immunohistochemical analysis of tumors and adjacent normal tissue. We will stratify patients by perceived stress, social isolation, and discrimination, will identify gene signatures associated with stress-related exposures and tumor catecholamine levels, and evaluate these gene signature(s) as predictor of patient survival and/or response to therapy using the many publically available datasets for breast cancer. We will also examine the relationship of stress-related exposures with selected protein markers (macrophage infiltration [CD68 as marker] and tumor microvessel density [CD31 as marker]). It is the strength of our design that we can use the measurement of tumor catecholamine levels as a validation tool for self-reported stress exposures. Our clinical studies will be supported by laboratory research that evaluates the influence of noradrenaline on cell phenotypes, gene expression, and DNA methylation patterns in human breast cancer cells. If we find that noradrenaline can shape DNA methylation patterns, we will analyze the relationship between DNA methylation and stress exposures in the breast tumors.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Wang, Shengfeng; Huo, Dezheng; Ogundiran, Temidayo O et al. (2017) Association of breast cancer risk and the mTOR pathway in women of African ancestry in 'The Root' Consortium. Carcinogenesis 38:789-796
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