Abnormal choline metabolism is emerging as a metabolic hallmark that is associated with oncogenesis and tumour progression. Following transformation, the modulation of enzymes that control anabolic and catabolic pathways causes increased levels of choline-containing precursors and breakdown products of membrane phospholipids. These increased levels are associated with proliferation, and recent studies emphasize the complex reciprocal interactions between oncogenic signalling and choline metabolism. Because choline-containing compounds are detected by non-invasive magnetic resonance spectroscopy (MRS), increased levels of these compounds provide a non-invasive biomarker of transformation, staging and response to therapy. Furthermore, enzymes of choline metabolism, such as choline kinase, present novel targets for image-guided cancer therapy.
Proton magnetic resonance spectroscopy ( 1 H 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 [1,2-13 C]choline, using 1 H and 13 C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-13 C]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 13 C-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.
Choline kinase is overexpressed in breast cancer cells and activated by oncogenes and mitogenic signals, making it a potential target for cancer therapy. Here, we have examined, for the first time, the effects of RNA interference (RNAi)-mediated down-regulation of choline kinase in nonmalignant and malignant human breast epithelial cell lines using magnetic resonance spectroscopy (MRS) as well as molecular analyses of proliferation and differentiation markers. RNAi knockdown of choline kinase reduced proliferation, as detected by proliferating cell nuclear antigen and Ki-67 expression, and promoted differentiation, as detected by cytosolic lipid droplet formation and expression of galectin-3. The functional importance of RNAi-mediated choline kinase down-regulation on choline phospholipid metabolism was confirmed by the significant reduction of phosphocholine detected by MRS. These results strongly support the targeting of choline kinase in breast cancer cells with RNAi and show the potential ability of noninvasive MRS to detect and evaluate future treatments incorporating such strategies. (Cancer Res 2005; 65(23): 11034-43)
Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide cholinebased imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKα (but not ChoKβ) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A 2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKα mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC byosinthesis and degradation, respectively. Cancer Res; 70(5); 2126-35. ©2010 AACR.
The experience of treating cancer over the past several decades overwhelmingly demonstrates that the disease continues to evade the vast array of drugs and treatment modalities available in the twenty-first century. This is not surprising in view of the complexity of this disease, and the multiplicities of pathways available to the cancer cell to enable its survival. Although the progression of cancer arrives at a common end point of cachexia, organ failure, and death, common pathways are rare in cancer. Identifying and targeting common pathways that would act across these levels of multiplicity is essential for the successful treatment of this disease. Over the past decade, one common characteristic consistently revealed by magnetic resonance spectroscopic studies is the elevation of phosphocholine and total choline-containing compounds in cancer cells and solid tumors. This elevation has been observed in almost every single cancer type studied with NMR spectroscopy and can be used as an endogenous biomarker of cancer. In this article, we have summarized some of the observations on the choline phospholipid metabolism of cancer cells and tumors, and make a case for targeting the aberrant choline phospholipid metabolism of cancer cells.
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 intensity of the total choline (tCho) signal in spectroscopic images of tumors is spatially heterogeneous. The likewise heterogeneous physiologic tumor microenvironment may contribute to this heterogeneity. We therefore investigated the relationship between hypoxia, choline metabolites, and choline kinase (Chk) in a human prostate cancer model. Human PC-3 prostate cancer cells were engineered to express enhanced green fluorescent protein (EGFP) under hypoxic conditions. These PC-3-5HRE-EGFP cells were characterized in culture and as tumors transplanted in mice using 1 H magnetic resonance spectroscopy (MRS) and MRS imaging (MRSI) combined with EGFP fluorescence microscopy and imaging. Hypoxic EGFP-fluorescing tumor regions colocalized with regions of high tCho in combined MRSI and optical imaging studies. Cellular phosphocholine (PC) and tCho concentrations as well as Chk expression levels significantly increased following exposure of PC-3 cells to hypoxia. A putative promoter region located 5 ¶ of the translation start site of the human chk-a gene was cloned and luciferase (Luc)-based reporter vector constructs were generated. Luc reporter assays provided evidence that some of the putative hypoxia response elements (HRE) within this putative chk-a promoter region functioned in vitro. Chromatin immunoprecipitation assays using an antibody against hypoxia-inducible factor (HIF)-1A showed that HIF-1 can directly bind this region of the endogenous chk-a promoter in hypoxic PC-3-5HRE-EGFP cells. These data suggest that HIF-1 activation of HREs within the putative chk-a promoter region can increase Chk-A expression within hypoxic environments, consequently increasing cellular PC and tCho levels within these environments. [Cancer Res 2008;68(1):172-80]
Magnetic resonance studies from the last 10 years have conclusively demonstrated that choline metabolism is altered in a wide variety of cancers. In cancer, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-containing compounds. This elevation is increasingly being used as an endogenous biomarker of cancer. Importantly, the enzymes and pathways resulting in these distinct alterations in phosphocholine and total choline may provide novel molecular targets for specific, targeted anticancer therapies. In this article, we have summarized some of the magnetic resonance spectroscopy and positron emission tomography techniques that are currently available, or will be in the near future, for choline metabolism-based diagnosis, staging and therapy assessment in cancer patients. This review also outlines currently known molecular alterations that cause the aberrant choline metabolite profile in cancers and concludes with a summary of recent research findings that may, in the future, lead to novel anticancer therapies targeting choline metabolism.
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