Despite the numerous advances in technology and screening breast cancer remains the major form of human cancers (lifetime risk ca. 1/8). A neglected aspect of tumorigenesis is the metabolic profile of cells, which expresses the biochemical phenotype. Transformed cells differ from their normal counterparts by loss of critical regulatory functions that lead to immortalization, avoidance of apoptosis and deregulated cell cycling. A dividing cell must double its contents during each cycle, requiring intake of materials both for biosynthesis, and to provide the energy to drive endergonic anabolic reactions. Thus, cancer cells must increase the activity of metabolic pathways for energy production and anabolism. Cancer cells of different origin should display metabolism characteristic of the cell type, and reflect specific aspects of proliferation, and how these depend on the external conditions such as the supply of nutrients and oxygen. We propose to determine the relative importance of specific cancer-relevant metabolic pathways in the development and progression of breast cancer cells (four lines) both in cell culture and in an orthotopic mouse tumor model (xenografts), using stable isotope assisted metabolomics by NMR and MS, as follows.
Aim 1. To determine the relative importance of energy producing pathways and anabolic metabolism by quantitative isotopomer analysis of normal and cancerous human breast epithelial cells in culture in response to hypoxia and specific nutrients. The precursors ([U-13C]-glucose, [13C-1] and [13C-2] glucose), and 13C/15N glutamine can differentiate relative fluxes through glycolysis, pentose phosphate pathways, citric acid cycle and major anaplerotic reactions, amino acid metabolism, nucleobase and phospholipid biosynthesis. Relative fluxes through these major pathways in non-estrogen-sensitive MDAMB231 and estrogen sensitive ZR-75-1 and MCF-7 cells will be compared with normal human mammary epithelial cells (HMEC).
Aim 2. To determine the extent that the specific pathway activities identified in cell culture are present in actual tumors in a xenograft mouse model. Female nude mice bearing mammary fat pad tumors deriving from non MDAMB231 and ZR-75-1 cells are given 13C glucose or 13C/15N glutamine i.v. and the tumors excised after sufficient time has elapsed for metabolism to occur. Plasma is analyzed to monitor uptake and redistribution of labeled precursors. Metabolites are extracted and analyzed by the same techniques as in Aim 1. The comparison of the tumor model with cell culture shows how the tumor environment and tissue interactions determine the relative importance of different pathways to breast tumor progression. The biochemical information will be used to interpret the metabolic profiles of human breast cancers. The development of these approaches will provide basic biochemical information about breast cancer, and is essential for the design of future studies with human subjects, with the goal of improved diagnosis of early stage tumors.

Public Health Relevance

Despite the numerous advances in technology and screening breast cancer remains the major form of human cancers (lifetime risk ca. 1/8). However, although oncogenes such as Myc and Ras or tumor suppressors such as p53 are frequently upregulated in cancers, the precise roles of these factors in disease initiation and progression is not completely clear. A deeper understanding of the biochemical phenotype of cancerous cells and their normal counterparts under defined conditions is an essential counterpart to genomics analyses of cancer cells. We are developing the technology to measure the activity of biochemical pathways in cancer cells that ultimately can be applied to human tumors in a clinical setting. The comparison of the in vivo tumor model with cell culture will show how the tumor environment and tissue interactions may determine the relative importance of different metabolic pathways to breast tumor progression. This fundamental knowledge of tumor biochemistry then provides a rich source of hypotheses about tumor progression that can be tested directly in actual breast cancer tissue. Application of this approach to a clinical setting will set the stage for identifying sets of markers for early diagnosis, prognosis and ultimately drug responses.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA133668-02
Application #
7876986
Study Section
Tumor Microenvironment Study Section (TME)
Program Officer
Spalholz, Barbara A
Project Start
2009-07-01
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2012-06-30
Support Year
2
Fiscal Year
2010
Total Cost
$162,800
Indirect Cost
Name
University of Louisville
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057588857
City
Louisville
State
KY
Country
United States
Zip Code
40292
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Fan, Teresa W-M; Tan, Jinlian; McKinney, Martin M et al. (2012) Stable Isotope Resolved Metabolomics Analysis of Ribonucleotide and RNA Metabolism in Human Lung Cancer Cells. Metabolomics 8:517-527
Lorkiewicz, Pawel; Higashi, Richard M; Lane, Andrew N et al. (2012) High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS. Metabolomics 8:930-939
Fan, Teresa W-M; Lane, Andrew N (2011) NMR-based stable isotope resolved metabolomics in systems biochemistry. J Biomol NMR 49:267-80
Fan, Teresa W-M; Lane, Andrew N; Higashi, Richard M et al. (2011) Stable isotope resolved metabolomics of lung cancer in a SCID mouse model. Metabolomics 7:257-269
Moseley, Hunter N B; Lane, Andrew N; Belshoff, Alex C et al. (2011) A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on (13)C mass isotopologue profiles under non-steady-state conditions. BMC Biol 9:37