Choline Kinase Activity Research Articles

Abstract 509: Hypoxia and HIF silencing mediated dysregulation of total choline, CD44 expression, and metastatic burden in MDA-MB-231 human breast cancers

Abstract Hypoxic tumors frequently exhibit an aggressive phenotype due to dysregulated gene expression and metabolic changes. Hypoxia results in the stabilization of hypoxia inducible factors (HIF-1/2) that transcriptionally activate genes involved in invasion, metastasis, metabolism, and in the adaptation of cancer cells to their microenvironment. In breast cancer, stem-like breast cancer cells that survive, repopulate and metastasize to distant locations, have elevated expression of CD44. In a previous study, we observed elevated expression of CD44 in hypoxic tumor regions, and identified HIF-1α as a regulator of CD44 expression in breast cancer cells under hypoxic conditions [1]. Hypoxia has also been implicated in increasing the activity of choline kinase (Chk)-alpha, the enzyme responsible for elevated phosphocholine (PC) and total choline (tCho) consistently observed in cancers [2]. In previous studies, lentiviral transduction of MDA-MB-231 breast cancer cells (231 cells) with shRNA against Chk-alpha and the in vivo delivery of the Chk-shRNA virus into tumor bearing mice resulted in decreased CD44 message and expression together with effective silencing of Chk message and expression [3]. Here, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI), we have established the importance of HIF in reducing total choline and metastatic tumor burden, and have identified a role for CD44 in establishing lung metastasis. HIF silencing in MDA-MB-231 cells significantly delayed tumor growth in mice. Both, the in vitro 1H and 31P MR spectra and in vivo 1H MRS images of tumors derived from engineered cells showed decreased tCho levels and distribution. This decrease of tCho was statistically significant in tumors derived from double silenced cells. Western blot analysis of tumors detected a decrease in Chk expression in double silenced (HIF-1 and 2) tumors. Silencing HIF-1α, -2α or both resulted in a significant reduction of metastatic lung burden in mice. Additionally, HIF-2α silencing was more effective at reducing lung colonization than HIF-1α, while silencing both was the most effective. Although metastatic burden decreased in HIF-1α silenced cells, the percentage of cells with high CD44 expression in the metastatic foci was comparable to that in the wild type or empty vector foci. These data identify the importance of targeting HIF and CD44 to prevent lung colonization and disrupt the metastatic cascade. This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for valuable technical assistance.

Sitamaquine-resistance in Leishmania donovani affects drug accumulation and lipid metabolism

This study focuses on the mechanism of sitamaquine-resistance in Leishmania donovani. Sitamaquine accumulated 10 and 1.4 fold more in cytosol than in membranes of wild-type (WT) and of sitamaquine-resistant (Sita-R160) L. donovani promastigotes, respectively. The sitamaquine accumulation was a concentration-dependent process in WT whereas a saturation occurred in Sita-R160 suggesting a reduced uptake or an increase of the sitamaquine efflux. Membrane negative phospholipids being the main target for sitamaquine uptake, a lipidomic analysis showed that sitamaquine-resistance did not rely on a decrease of membrane negative phospholipid rate in Sita-R160, discarding the hypothesis of reduced uptake. However, sterol and phospholipid metabolisms were strongly affected in Sita-R160 suggesting that sitamaquine-resistance could be related to an alteration of phosphatidylethanolamine-N-methyl-transferase and choline kinase activities and to a decrease in cholesterol uptake and of ergosterol biosynthesis. Preliminary data of proteomics analysis exhibited different protein profiles between WT and Sita-160R remaining to be characterized.

Choline transporter‐like proteins CTLs/SLC44 family as a novel molecular target for cancer therapy

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in various cancers. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. Previous studies have demonstrated abnormalities in choline uptake and choline phospholipid metabolism in cancer cells using the imaging of cancer with positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). The aberrant choline metabolism in cancer cells is strongly correlated with their malignant progression. Using quantitative real-time PCR, the mRNA expression of choline transporters was measured, and it was found that choline transporter-like proteins CTLs/SLC44 family are highly expressed in various cancer cell lines. Choline uptake through CTLs is associated with cell viability, and the functional inhibition of CTLs could promote apoptotic cell death. Furthermore, non-neuronal cholinergic systems that include CTLs-mediated choline transport are associated with cell proliferation and their inhibition promotes apoptotic cell death in colon cancer, small cell lung cancer and human leukemic T-cells. The identification of this new CTLs-mediated choline transport system provides a potential new target for cancer therapy.

A mechanism for suppression of the CDP-choline pathway during apoptosis

Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)α is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. In this study we utilized siRNA silencing of caspases in HEK293 cells and caspase 3-deficient MCF7 cells to show that caspase 3 is required for CCTα proteolysis and release from the nucleus during apoptosis. CCTα-Δ28 (a caspase-cleaved mimic) expressed in CCTα-deficient Chinese hamster ovary cells was cytosolic and had increased in vitro activity. However, [³H]choline labeling experiments in camptothecin-treated MCF7 cells and MCF7 cells expressing caspase 3 (MCF7-C3) revealed a global suppression of the CDP-choline pathway that was consistent with inhibition of a step prior to CCTα. In camptothecin-treated MCF7 and MCF7-C3 cells, choline kinase activity was unaffected; however, choline transport into cells was reduced by 30 and 60%, respectively. We conclude that caspase 3-mediated removal of the CCTα NLS contributes minimally to the inhibition of PtdCho synthesis during DNA damage-induced apoptosis. Rather, the CDP-choline pathway is inhibited by caspase 3-independent and -dependent suppression of choline transport into cells.

Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: A target molecule for lung cancer therapy

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na+ from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na+-free conditions was inhibited by dimethylamiloride (DMA), a Na+/H+ exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na+-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H+ gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

HDAC Inhibition Induces Increased Choline Uptake and Elevated Phosphocholine Levels in MCF7 Breast Cancer Cells

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using 13C MRS to monitor [1,2-13C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by 31P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using 1H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Sci-Fri AM: Imaging - 01: Feasibility of estimating choline kinase activity with kinetic modeling of 18F-fluorocholine pet imaging of prostate cancer.

Prostate cancer (PCa) detection and delineation remains a challenge for medical imaging. Studies have shown 18 F-Fluorocholine (FCH) PET imaging to be a promising modality in the detection of recurrent PCa. Detection of denovo PCa is more challenging, as lesions such as benign prostatic hyperplasia (BPH) may adversely affect the sensitivity and specificity of the modality. PCa and BPH have been shown to exhibit similar uptake of FCH, yet it has been shown that phosphocholine levels are much more elevated in PCa compared to BPH. Therefore, it would be useful to measure the activity of phosphorylation via choline kinase (k3 ) in order to differentiate PCa from BPH. This work examines the feasibility of using a compartmental model to estimate k3 with dynamic 18 F-Fluorocholine PET imaging. JSim software [1] was used to simulate the compartmental model for FCH exchange. A simulated tissue curve was generated using predefined parameters and the model's ability to estimate these parameters through fitting of the simulated tissue curve with and without noise was investigated. The fitting procedure was performed using the non-negative least squares algorithm in MATLAB after the equation governing fitting was linearized. In the noiseless case, the model was able to accurately identify the values of each rate parameter. For the noisy case with an SNR of 10:1, the mean estimated k3 for 10,000 runs had a coefficient of variation of 14.9%. The kinetic model shows promise for quantifying k3 , which would allow the differentiation of malignant and benign tumours of the prostate.

Abstract SY42-01: MRI/MRS of tumor microenvironment

Abstract Multi-modality and multi-parametric molecular and functional imaging provide windows into the tumor microenvironment (TME) and into the interactions between cancer cells and stromal cells. Tumors have abnormal physiological environments such as hypoxia and acidic extracellular pH that influence the cancer cell-stromal cell interaction, generate a more aggressive phenotype, and play a major role in the response to treatment. The influence of hypoxia and acidic extracellular pH on the cancer cell phenotype has justifiably received significant attention. Magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and MR spectroscopic imaging (MRSI) techniques have been used for several decades to characterize physiologic parameters such as tumor vasculature, oxygenation, and necrosis, pH and metabolism. The bench to bedside span of noninvasive MR methods to characterize cancer makes them an attractive choice for clinically translatable applications. Recent advances in the development of molecular targeted contrast agents have expanded the traditional strengths of MRI and MRS of characterizing functional tumor parameters such as pH, vascularization, metabolism and cell death, to include visualization of stromal cells, cell surface receptors, molecular pathways, degradative enzyme activity, and the extracellular matrix integrity in preclinical models. The TME is rich with targets to exploit for therapy that can be used in combination with imaging for ‘theranostic’ imaging agents. The use of MR biomarkers such as total choline and perfusion are already being explored clinically for characterizing tumors and following treatment response. The elevation of choline compounds presents a unique target to exploit for molecular targeting; such targeting can be imaged noninvasively with MRS. We are developing molecular and molecular imaging based approaches to target choline metabolism, specifically choline kinase activity, which is the first step in choline phospholipid biosynthesis. New areas that are being developed in our program include targeting choline kinase using siRNA delivered using lentiviral vectors, and performing image-guided targeting of choline kinase using siRNA in combination with a prodrug enzyme cytosine deaminase using a multi-modal imaging platform. The interaction between cancer cells and the tumor microenvironment is providing new insights into the etiology and progression of cancer. Using combined MR and optical imaging of human breast and prostate cancer xenografts engineered to express green fluorescent protein (GFP) or red fluorescent protein (RFP) under hypoxia, we have obtained useful insights into the dynamics between hypoxia and the tumor extracellular matrix (ECM), vascularization, extracellular pH, interstitial fluid transport, and metabolism. MRI and MRSI were used to obtain co-localized maps of vascular volume, permeability, interstitial fluid transport, total choline and lactate/lipid, while optical imaging was used to obtain co-localized maps of hypoxia and ECM fiber distribution. These insights can be exploited to find effective treatment strategies. Tumor recurrence and metastasis continue to be the leading causes of morbidity and mortality from cancer and, despite the tremendous advances in cancer research and treatment, even in the twenty-first century cancer continues to evade cure and frequently control. The discovery of cancer initiating cells or cancer stem cells is generating tremendous excitement and is offering new paradigms for understanding and treating cancer recurrence and metastasis. These stem-like cancer cell populations can form tumors in immune suppressed mice from low cell number inoculums, are more drug resistant, more invasive, and more likely to metastasize. Identifying biomarkers associated with cancer stem cells, and imaging and targeting permissive or preventive microenvironmental niches for cancer stem cells is another area that will have significant impact on cancer research and treatment. A major direction for the future will be to translate preclinical molecular imaging developments for use in the clinic. Exciting new areas that should rapidly develop in the future include targeting specific microenvironments or stromal compartments with theranostic agents. Unlike radiopharmaceuticals, the inherent insensitivity of MR methods will require that most molecular contrast agents be used at concentrations that will be subject to much more stringent FDA control. Challenges for the future will be to increase the sensitivity of detection of such agents through novel chemistry, or instrumentation and the incorporation of multi-modality imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY42-01. doi:1538-7445.AM2012-SY42-01

Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Choline phosphorylation and regulation of transcription of choline kinase α in hypoxia

Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis. Increased phosphorylation of choline is a hallmark characteristic of the malignant phenotype in a variety of neoplasms. However, in hypoxic cancer cells, choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major choline kinase isoform, choline kinase α (ChKα), in hypoxic PC-3 human prostate cancer cells. Hypoxia decreased choline phosphorylation, choline kinase activity, and ChKα mRNA and protein levels. Promoter analysis studies revealed a region upstream of the ChKα gene bearing a conserved DNA consensus binding motif, hypoxia response element-7 (HRE7), at position -222 relative to +1 translation start site, for binding the hypoxia dependent master regulator transcription factor, hypoxia-inducible factor 1α (HIF-1α). Electrophoretic mobility shift competition/supershift assay and chromatin immunoprecipitation assay confirmed binding of HIF-1α to HRE7. A putative promoter of ChKα was isolated from PC-3 genomic DNA and cloned into a luciferase-based reporter vector system. In PC-3 cells, hypoxia decreased the expression of luciferase under the control of the ChKα promoter. Mutation of HRE7 abrogated this hypoxia effect, further demonstrating the involvement of HRE7 in hypoxia-sensitive regulation of ChKα. The results strongly suggest that transcriptional control of choline phosphorylation is largely mediated via HIF-1α binding to the newly identified HRE7.

Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma.

The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

Metabolic consequences of treatment with AKT inhibitor perifosine in breast cancer cells

Activation of the PI3K/Akt pathway is associated with the development of numerous human cancers. As a result, many emerging therapies target this pathway. Previous studies have shown that targeting the PI3K/Akt pathway at the level of PI3K is associated with a drop in phosphocholine (PCho) and a reduction in hyperpolarized lactate production. However, the consequences of targeting downstream of PI3K at the level of Akt have not been investigated. Perifosine is an anticancer alkylphospholipid used in clinical trials. It acts by inhibiting phosphorylation of Akt and has been shown to inhibit CTP-phosphocholine cytidyltransferase (CT). The goal of this study was to identify the MRS-detectable metabolic consequences of treatment with perifosine in MCF-7 breast cancer cells. We found that perifosine treatment led to a 51 ± 5% drop in PCho from 30 ± 5 to 15 ± 1 fmol/cell and a comparable drop in de novo synthesized PCho. This was associated with a drop in choline kinase (ChoK) activity and ChoKα expression. CT inhibition could not be ruled out but likely did not contribute to the change in PCho. We also found that intracellular lactate levels decreased from 2.7 ± 0.5 to 1.5 ± 0.3 fmol/cell and extracellular lactate levels dropped by a similar extent. These findings were consistent with a drop in lactate dehydrogenase expression and associated with a drop in activity of the hypoxia inducible factor (HIF)-1α. The drops in PCho and lactate production following perifosine treatment are therefore mediated downstream of Akt by the drop in HIF-1α, which serves as the transcription factor for both ChoK and lactate dehydrogenase. The metabolic changes were confirmed in a second breast cancer cell line, MDA-MB-231. Taken together, these findings indicate that PCho and lactate can serve as noninvasive metabolic biomarkers for monitoring the effects of inhibitors that target the PI3K/Akt pathway, independent of the step that leads to inhibition of HIF-1α.

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.

A Congenital Muscular Dystrophy with Mitochondrial Structural Abnormalities Caused by Defective De Novo Phosphatidylcholine Biosynthesis

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

Abstract 4076: Biology of altered choline metabolism in ovarian cancer: Prognostic and therapeutic opportunities

Abstract Epithelial Ovarian Cancer (EOC) remains the leading cause of death in women with gynecologic malignancies, due to late diagnosis and early relapse associated with development of chemoresistance. We recently reported alterations of the choline-metabolite magnetic resonance spectral (MRS) profile in EOC cells, characterized by an increase content of phosphocholine (PCho). A multidisciplinary approach revealed that one of the more relevant changes was a significant increase of Choline Kinase (ChoK) activity (consistent with an increased protein and alpha-isoform mRNA expression) in cancer cells as compared to the normal counterpart. ChoK catalyzes the phosphorylation of choline to yield PCho as the first step in the biosynthesis of phosphatidylcholine, required for mammalian cell structural stability and proliferation. ChoK has been extensively related to cell proliferation being at the cross-roads of signalling pathways related to mitogenesis and survival and a prognostic role for ChoK in some cancer types has been described. Aim of the study is the evaluation of the biological relevance of ChoK expression and activity to define its possible role as a non-invasively detectable EOC biomarker and druggable target. We specifically silenced ChoK-alpha expression by transient RNA interference in two different EOC cell lines and evaluated the biological effects. Following ChoK-alpha silencing we observed a 70% reduction of mRNA and protein expression with a similar reduction in PCho accumulation as assessed by HMRS analysis. The ChoK-alpha silencing was accompanied by 20% inhibition of cell growth and similar percentage of cells blocked in the G1-phase of cell cycle. By transcriptome analysis we found CyclinA, related to regulation of cell cycle progression, and cytokines genes (IL-6 and IL-8) related to inflammation and EOC aggressiveness among the most relevant co-repressed genes, whereas among the most up-regulated genes we found Acyl-CoA synthetase and Osteoprotegerin. These observations suggest ChoK-alpha involvement in cell proliferation and survival. Evaluation of the biological effects of long-term Chok-alpha silencing by lentiviral transduction, together with concurrent inhibition of relevant survival signaling pathway (PI3K/AKT) or other enzymes involved in PCho metabolism (PC-PLC) is currently ongoing. To evaluate its possible role as prognostic biomarker, ChoK-alpha mRNA and protein expression is currently being analyzed on well characterized retrospective EOC case materials. The assessment of biological and prognostic relevance of choline metabolites might significantly contribute in the identification of new non-invasive diagnostic modalities and in the development of new targeted therapeutic approaches. (Partially supported by AIRC) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2011-4076

Preclinical study of treatment response in HCT‐116 cells and xenografts with 1H‐decoupled 31P MRS

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN-38 have been shown to induce G(2) /M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT-116). Subsequent treatment of these G(2) /M-arrested cells with the cyclin-dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT-116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton ((1) H)-decoupled phosphorus ((31) P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT-116 xenografts following treatment, which were evidenced within twenty-four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT-116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN-38 alone underwent 83 ± 5% G(2) /M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN-38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo (1) H-decoupled (31) P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies.

Imaging Lipid Synthesis in Hepatocellular Carcinoma with [Methyl-11C]Choline: Correlation with In Vivo Metabolic Studies

PET with [methyl-(11)C]-choline (11C-choline) can be useful for detecting well-differentiated hepatocellular carcinoma (HCC) that is not 18F-FDG-avid. This study was designed to examine the relationship between choline metabolism and choline tracer uptake in HCC for PET with 11C-choline. Dynamic PET scans of 11C-choline were acquired using the woodchuck models of HCC. After imaging, [methyl-(14)C]-choline was injected, and metabolites from both HCC tissue samples and the surrounding hepatic tissues were extracted and analyzed by radio-high-performance liquid chromatography. The enzymatic activities of choline kinase and choline-phosphate cytidylyltransferase were assayed for correlation with the imaging and metabolism data. PET with 11C-choline showed an HCC detection rate of 9 of 10. The tumor-to-liver ratio for the 9 detected HCCs was 1.89±0.55. Hematoxylin-eosin staining confirmed that all spots with high tracer uptake were well-differentiated HCCs. Variation of radioactivity distribution within HCCs indicated a heterogeneous uptake of choline. The activities of both choline kinase and choline-phosphate cytidylyltransferase were found to be significantly higher in HCC than in the surrounding hepatic tissues. The major metabolites of 11C-choline were phosphocholine in HCC and betaine and choline in the surrounding hepatic tissues at 12 min after injection; in HCC, phosphocholine rapidly converted to phosphatidylcholine at 30 min after injection. HCCs display enhanced uptake of radiolabeled choline despite a moderate degree of physiologic uptake in the surrounding hepatic tissues. Initially, increased radiolabeled choline uptake in HCCs is associated with the transport and phosphorylation of choline; as time passes, the increased uptake of radiolabeled choline reflects increased phosphatidylcholine synthesis derived from radiolabeled cytidine 5'-diphosphocholine (CDP-choline) in HCCs. In contrast, the surrounding hepatic tissues exhibit extensive oxidation of radiolabeled choline via the phosphatidylethanolamine methylation pathway, a major contributor to the observed physiologic uptake.

Phosphatidylcholine metabolic transformation and progression signature as a pharmacodynamic biomarker.

Phosphatidylcholine metabolic transformation and progression signature as a pharmacodynamic biomarker.

544 Aberrant choline metabolism in epithelial ovarian cancer: relevance of choline kinase activity and expression

544 Aberrant choline metabolism in epithelial ovarian cancer: relevance of choline kinase activity and expression