De Novo Lipogenesis from Glutamine in Human Glioma Cells
Anthony Mancuso¹, Justin R. Cross, Craig B. Thompson
¹Cancer Biology, University of Pennsylvania, Philadelphia, PA, United States

Rapidly growing cancer cells require high rates of phospholipid biosynthesis for the formation of new membranes.  Cancer cells produced fatty acids for lipids de novo, primarily from glucose.  An improved understanding of the pathways involved in de novo lipogenesis could greatly advance the development of new therapeutics that inhibit cancer cell growth.  In this work, FA synthesis from both glucose and glutamine was examined with ¹³C NMR spectroscopy in cultured human glioma cells.  Cells were cultured in T-flasks and extracted for high-resolution analysis.  The results show that glucose is the primary source for de novo lipogenesis while glutamine contributes ~30%.

The Interdependence of Choline Kinase and Phospholipase D: Adaptation Mechanisms in Choline Phospholipid Metabolism of Human Breast Cancer Cells
Balaji Krishnamachary¹, Mayur Gadiya², Noriko Mori¹, Yelena Mironchik¹, Kristine Glunde¹, Zaver M. Bhujwalla^1
1JHU ICMIC Program, Russell H. Morgan Department of Radiology & Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, United States; ²JHU ICMIC Program, Russell H. Morgan Department of Radiology & Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, United States

A hallmark of cancer is an increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis.  Enzymes in choline metabolism present attractive targets that can be exploited for treatment.  Here we have shown that at least two of these enzymes are interdependent.  Downregulation of choline kinase (Chk) with siRNA results in increased phospholipase D1 (PLD1) expression and downregulation of PLD1 results in increased Chk expression, typifying the ability of cancer cells to adapt.  These data support multiple targeting of enzymes in the choline pathway using a multiple siRNA approach.

Down Regulation of HIF-1 Alpha in MDA-MB-231 Human Breast Cancer Cells Alters Choline Phospholipid Metabolism
Tariq Shah¹, Balaji Krishnamachary², Flonne Wildes², Zaver M. Bhujwalla^1
1JHU ICMIC Program, Russell H Morgan Department of Radiology and Radiological Sciences, Johns Hopkins School of Medicine , Baltimore, MD, United States; ²JHU ICMIC Program, Russell H Morgan Department of Radiology and Radiological Sciences, Johns Hopkins School of Medicine, Baltimore, MD, United States

The hypoxia inducible factor (HIF) recognizes and binds to consensus sequences called hypoxia response elements on the promoter regions of several genes, increasing their transcription.  As a result hypoxia plays an important role in the cancer phenotype.  Here we silenced HIF-1 alpha expression in invasive MDA-MB-231 breast cancer cells and characterized metabolic changes using a magnetic resonance compatible cell perfusion system with cells maintained under controlled pH, temperature, and oxygenation conditions.  HIF-1 alpha silenced cells acquired a less aggressive metabolic phenotype with reduced choline kinase expression, together with reduced total choline and phosphocholine, compared to parental cells.

MRS Detection of Altered Choline Metabolism Following HSP90 Inhibition
Alissa Brandes¹, Chris S. Ward¹, Judy S. Hwang¹, Sabrina M. Ronen^1
1Radiology, UCSF, San Francisco, CA, United States

Although most anticancer therapies cause a drop in PC levels,, treatment with the HSP90 inhibitor 17-AAG has been shown to have the unique consequence of increasing PC. Our study investigated the mechanism behind this observed increase by monitoring the uptake and metabolism of [1,2-13C]-choline in live cells and cells extracts using 1H, 31P and 13C MRS and performing assays on the activity of enzymes involved in choline metabolism.  Our data indicate that the observed increase in PC levels in 17-AAG-treated cancer cells is due to an increase in the synthesis of PC from extracellular choline, along with increased breakdown of PtdCho via PLC.

Silencing GDPD5, a Novel Anticancer Target, Increases Glycerophosphocholine in Human Breast Cancer Cells
Mailin Döpkens^1,2, Tiffany R. Blackwell¹, Farhad Vesuna¹, Venu Raman¹, Balaji Krishnamachary¹, Zaver M. Bhujwalla¹, Dieter Leibfritz², Kristine Glunde^1
1JHU ICMIC Program, Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, United States; ²Department of Chemistry and Biology, University of Bremen, Bremen, Germany

Altered choline phospholipid metabolism in breast cancers provides multiple targets for anticancer therapy.  In addition to increasing total choline levels, malignant transformation of breast cancer cells results in a switch from high glycerophosphocholine (GPC) and low phosphocholine (PC) to low GPC and high PC.  The glycerophosphocholine phosphodiesterase (GPC-PDE) genes responsible for the low GPC levels in breast cancer cells have not been identified.  Here we demonstrate that glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5), a gene encoding a GPC-PDE, is at least partially responsible for the low GPC levels in breast cancer cells, and may be a useful therapeutic target.

Noninvasive Monitoring of PI3K Inhibition: Reduced  Hyperpolarized Lactate and PC Are Independent of Genetic Background in Glioblastoma
Humsa S. Venkatesh¹, Charles D. James², Daphne A. Haas-Kogan², Sabrina M. Ronen^1
1Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States; ²Neurological Surgery, University of California, San Francisco, CA, United States

As the PI3K pathway is activated in 88% of glioblastomas, it is the target of several novel therapies. The purpose of this investigation is to study GBM cells with different genetic backgrounds in order to establish hyperpolarized lactate and PC as biomarkers of PI3K inhibition. Two inhibitors of PI3K signaling and agents that do not affect signaling were investigated. Hyperpolarized lactate and PC dropped only when signaling was inhibited and this observation was mechanistically linked to a drop in HIF-1, which controls expression of LDH and choline kinase. This suggests an application for these metabolites as noninvasive biomarkers for PI3K-targeted anticancer treatments.

Hyperpolarized  [1-13C] Pyruvate Metabolism in a Human Prostate Tissue Culture Bioreactor
David J. Joun¹, Mark Albers¹, Kayvan Keshari¹, Robert Bok¹, Christopher Ward, Donna Peehl², Sabrina Ronin, Daniel Vigneron, John Kurhanewicz
¹Radiology, UCSF, San Francisco, CA, United States; ²Urology, University of Stanford, Stanford, CA, United States

We demonstrate for the first time that the pathologic and metabolic integrity of benign and malignant human prostate tissues can be maintained in a NMR compatible 3-D tissue culture bioreactor for 32 hours. After administration of hyperpolarized [1-13C] pyruvate, the generation of labeled hyperpolarized lactate and LDH activity was significantly higher in malignant tissues (N=3) relative to benign human prostate tissues (N=3). Moreover, there was minimal overlap of the labeled hyperpolarized lactate signal between individual cancer and benign tissues suggesting that hyperpolarized lactate will be an accurate biomarker of prostate cancer in patients.

The Glucose Dependent Transcription Factor ChREBP Contributes to Glucose-Dependent Anabolic Synthesis and Cell Proliferation
Xuemei Tong¹, Anthony Mancuso², Fangping Zhao, Joshua J. Gruber, Craig B. Thompson
¹University of Pennsylvania, Philadelphia, PA, United States; ²Cancer Biology, University of Pennsylvania, Philadelphia, PA, United States

Many human tumors display high rates of aerobic glycolysis, de novo fatty acid synthesis and nucleotide biosynthesis.  Although these metabolic alterations might not be initiating events in oncogenesis, blocking them may be a useful strategy for slowing carcinogenesis. The carbohydrate responsive element binding protein (ChREBP) is a critical mediator of glucose-dependent metabolism.  In this study, the metabolic effects of ChREBP knockdown in human colon cancer cells were examined with ¹³C NMR and ¹⁴C scintillation.  The results demonstrated that knockdown reduced aerobic glycolysis and growth-related biosynthesis.  It also increased TCA cycle flux and oxygen consumption, resulting in a less cancerous phenotype.

Metabolic Profiling of Post-Radiation Prostate Biopsy Tissues
Vickie Yi Zhang^1,2, Mark Swanson¹, Laura Tabatabai³, Jeff Simko³, Lynn DeLosSantos¹, Daniel Vigneron¹, John Kurhanewicz^1
1Radiology, University of California, San Francisco, San Francisco, CA, United States; ²Joint Bioengineering Program, University of California, Berkeley/San  Francisco, San Francisco, CA, United States; ³Pathology, University of California, San Francisco, San Francisco, CA, United States

Synopsis: This study used quantitative 1-D 1H HR-MAS spectroscopy of snap frozen prostate biopsies to investigate the metabolic profiles of healthy versus cancer prostate tissues after radiation therapy. Metabolite concentrations were correlated with pathology and Ki-67 immunohistochemistry to identify a metabolic phenotype of proliferating residue cancer.  Significantly higher concentrations of PC+GPC, lactate and glutamate were observed in benign versus residual proliferating cancer tissues after radiation treatment.

Measurements of Mean Nuclear and Cell Sizes Using Ultra-Short Diffusion Times
Junzhong Xu¹, Jingping Xie¹, Ke Li¹, Jerome Jourquin², Mark D. Does¹, Daniel F. Gochberg¹, Vito Quaranta², John C. Gore^1
1Institute of Imaging Science, Vanderbilt University, Nashville, TN, United States; ²Cancer Biology, Vanderbilt University, Nashville, TN, United States

Tumor cell nuclear size usually can be found only by invasive biopsy. In the present work, a novel approach, which employs an oscillating gradient spin echo (OGSE) method, has been developed to measure nuclear size with ultra-short diffusion times (low as ~0.13ms). Both simulations and experiments were performed and the results obtained from OGSE diffusion measurements are consistent with light microscopy, proving the feasibility of our method. This new approach provides structural parameters which may be helpful for the assessment of tumor malignancy, tracking intracellular changes in tissues, and potentially monitoring tumor response to treatment in vivo.