Metabolomic changes accompanying transformation and acquisition of metastatic potential in a syngeneic mouse mammary tumor model

Abstract

Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials. Our results suggest a two-step metabolic progression hypothesis during the acquisition of tumorigenic and metastatic abilities. Metabolite changes accompanying tumor progression are identified in the intracellular and secreted forms in several pathways, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, fatty acid and nucleotide biosynthesis, and the GSH-dependent antioxidative pathway. These results suggest possible biomarkers of breast cancer progression as well as opportunities of interrupting tumor progression through the targeting of metabolic pathways.

FIGURE 1.

Metabolomic profiling clustering mouse mammary cells with different tumorigenic potentials. A, six mouse cell lines (represented with numbers throughout the figure) with distinct tumorigenic and metastatic abilities. ID, cell line identification. B, hierarchical clustering of metabolomic profiles of the six cell lines identified subsets of metabolites that correlated with tumorigenicity and metastatic ability. Each cell line has four biological replicates labeled above the heat map with numbers as indicated in A. Red, green, and blue bars indicate three patterns of metabolites examined in Fig. 2, A, B, and D, respectively.

FIGURE 2.

Characteristic metabolic changes during transformation and metastasis. A, metabolites increased in transformed cells. B, metabolites decreased in transformed and metastatic cells. C, GSH and GSSG levels in six cell lines. Data represent average ± S.D. on log₂ scale. D, metabolites increased in cells with metastatic potential. E, simplified schematic of glycolysis and tricarboxylic acid cycle (TCA cycle) showing in red the metabolites that are elevated in metastatic cell lines. Six cell lines were labeled with numbers in the same scheme as in Fig. 1. PEP, phosphoenolpyruvate.

FIGURE 3.

Characterization of metabolite changes in conditioned media. A, hierarchical clustering of metabolomic profiles of the conditioned media identified subsets of metabolites that correlated with metastatic ability. Each cell line has four biological replicates (except 4TO7 with two samples) labeled above the heat map with numbers as indicated in Fig. 1A. Number 0 represents the unconditioned media. Blue, orange, green, and purple bars indicate patterns further examined. B, metabolites increased in most of the metastatic cells. C, metabolites increased in more metastatic cells when compared with the non-tumorigenic NMuMG and the non-metastatic 67NR (represented by 1 and 2) as well as the weakly metastatic 168FARN (represented by 3). D, metabolites with lower abundance in conditioned media of cells with metastatic potential. E, schematic of the two-step metabolic progression hypothesis. PPP, pentose phosphate pathway.