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]

Elevated phosphocholine (PC) and total choline (tCho) metabolites are widely established characteristics of most cancer cells, including breast cancer. Effective silencing of choline kinase (chk), the enzyme that converts choline to PC, is associated with reduced tumor growth. The functional importance and down-regulation of chk using RNA interference has been previously established. Here, we report on the preclinical evaluation of lentiviral vector-mediated downregulation of chk using short hairpin RNA (shRNA) in established tumors derived from human breast cancer cells. Concentrated lentivirus expressing shRNA against chk was injected i.v. in the tail vein of MDA-MB-231 tumor-bearing female severe combined immunodeficient mice. Transduction efficiency in cells and tumors in vivo was assessed optically by enhanced green fluorescent protein expression and additionally from chk mRNA and protein levels. An 80% reduction in chk mRNA and protein was achieved following f90% transduction efficiency in cells. After transduction with chkshRNA, 1 H magnetic resonance spectroscopy (MRS) of cell and tumor extracts showed decreases in PC and tCho levels (P < 0.01 and 0.05, respectively) in comparison with controls. PC levels were monitored noninvasively by 31 P MRS in tumors and by 1 H MRS in cell and tumor tissue extracts. Noninvasive 31 P MR spectra of chk-shRNA-transduced tumors in vivo showed lower PC and phosphomonoester levels that were associated with reduced tumor growth and proliferation. This study shows the use of lentiviral vectors to target chk in a human breast cancer xenograft and noninvasive MRS detection of this targeting. [Cancer Res 2009;69(8):3464-71]

Identifying strategies to increase cancer cell kill while sparing normal tissue is critically important in cancer chemotherapy. Choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC), is elevated in cancer cells and presents a novel target for increasing cell kill. Here, we have examined the effects of transiently down-regulating Chk by small interfering RNA against Chk (siRNA-chk) on PC and total choline-containing compound (tCho) levels and on the viability/proliferation of estrogen receptor-negative and estrogen receptor-positive breast cancer cell lines and a nonmalignant mammary epithelial cell line. We investigated the effects of combination treatment with transient siRNAchk transfection and the anticancer drug 5-fluorouracil (5-FU) in those cell lines. Microarray analysis of the invasive estrogen receptor-negative MDA-MB-231 cell line was done to characterize molecular changes associated with Chk downregulation. Chk down-regulation decreased PC and tCho levels in the malignant cell lines, whereas the cell viability/ proliferation assays detected a decrease in proliferation in these cells. In contrast, Chk down-regulation had an almost negligible effect on PC and tCho levels as well as cell viability/proliferation in the nonmalignant cell line. A combination of siRNA-chk with 5-FU treatment resulted in a larger reduction of cell viability/proliferation in the breast cancer cell lines; this reduction was evident to a much lesser degree in the nonmalignant cells. Microarray analysis showed that Chk down-regulation affected 33 proliferation-related genes and 9 DNA repair-related genes. Chk down-regulation with siRNA-chk may provide a novel alternative to enhance the effect of anticancer drugs in malignant cells. [Cancer Res 2007;67(23):11284-90]