Proton magnetic resonance spectroscopy (¹H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-¹³C]choline, using ¹H and ¹³C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-¹³C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased ¹³C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.

Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pH(e)) resulted in a distinct shift of lysosomes from the perinuclear region to the cell periphery irrespective of the HMECs' degree of malignancy. With decreasing pH, larger lysosomal vesicles were observed more frequently in highly metastatic breast cancer cells, whereas smaller lysosomes were observed in poorly metastatic breast cancer cells and HMECs. The number of lysosomes decreased with acidic pH values. The displacement of lysosomes to the cell periphery driven by extracellular acidosis may facilitate exocytosis of these lysosomes and increase secretion of degradative enzymes. Filopodia formations, which were observed more frequently in highly metastatic breast cancer cells maintained at acidic pH(e), may also contribute to invasion.

The extracellular matrix (ECM) in primary breast cancers is significantly altered compared with normal breast tissue and may provide new biomarkers for cancer diagnosis,¹ 2. 3. ^{– 4} metastasis prediction,^5,6 and prognosis of survival.⁷ Cancer metastasis is a multistep process in which the tumor microenvironment (TME) facilitates cancer cell–ECM interactions that are necessary for metastasis to occur.⁸ The ECM in breast tumors is composed of a complex meshwork of fibrillar collagens, glycoproteins, and proteoglycans,⁹ which profoundly affect metastasis, as well as proliferation, angiogenesis, adhesion, migration, invasion, macromolecular transport, and drug delivery.⁹ 10. 11. 12. ^{– 13} Multiple molecular mechanisms, such as ECM degradation by proteases secreted by cancer cells and dysregulated ECM synthesis by tumor-associated stromal cells, have been implicated in the formation of the significantly altered ECM of breast tumors⁸ that include structural changes in the fiber patterns of fiber-forming ECM components. Collagen I (Col1) fibers are the major structural ECM component in breast tumors,^5,6,9,14,15 and increased stromal Col1 has been found to facilitate breast tumor formation, invasion, and metastasis.^5,6

Abnormal choline metabolism continues to be identified in multiple cancers. Molecular causes of abnormal choline metabolism are changes in choline kinase-α, ethanolamine kinase-α, phosphatidylcholi...