Zaver M. Bhujwalla

Choline metabolism in malignant transformation

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.

Enhancement of chemotherapy by manipulation of tumour pH.

SummaryThe extracellular (interstitial) pH (pHe) of solid tumours is significantly more acidic compared to normal tissues. In-vitro, low pH reduces the uptake of weakly basic chemotherapeutic drugs and, hence, reduces their cytotoxicity. This phenomenon has been postulated to contribute to a ‘physiological’ resistance to weakly basic drugs in vivo. Doxorubicin is a weak base chemotherapeutic agent that is commonly used in combination chemotherapy to clinically treat breast cancers. This report demonstrates that MCF-7 human breast cancer cells in vitro are more susceptible to doxorubicin toxicity at pH 7.4, compared to pH 6.8. Furthermore 31P-magnetic resonance spectroscopy (MRS) has shown that the pHe of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water. The bicarbonate-induced extracellular alkalinization leads to significant improvements in the therapeutic effectiveness of doxorubicin against MCF-7 xenografts in vivo. Although physiological resistance to weakly basic chemotherapeutics is well-documented in vitro and in theory, these data represent the first in vivo demonstration of this important phenomenon.

Natural D -glucose as a biodegradable MRI contrast agent for detecting cancer

Modern imaging technologies such as CT, PET, SPECT, and MRI employ contrast agents to visualize the tumor microenvironment, providing information on malignancy and response to treatment. Currently, all clinical imaging agents require chemical labeling, i.e. with iodine (CT), radioisotopes (PET/SPECT), or paramagnetic metals (MRI). The goal was to explore the possibility of using simple D‐glucose as an infusable biodegradable MRI agent for cancer detection.

Activation of Phosphatidylcholine Cycle Enzymes in Human Epithelial Ovarian Cancer Cells

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based 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 ChoKalpha (but not ChoKbeta) 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 A2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha 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 biosynthesis and degradation, respectively.

Plasmalemmal pH-gradients in drug-sensitive and drug-resistant MCF-7 human breast carcinoma xenografts measured by 31P magnetic resonance spectroscopy

31p Magnetic resonance spectroscopy (MRS) was employed to investigate tumor pH in xenografts of drug-sensitive and drug-resistant MCF-7 human breast carcinoma cells. Measured extracellular pH values were found to be lower than the intracellular pH in all three tumor types investigated. The magnitude of this acid-outside plasmalemmal pH gradient increased with increasing tumor size in tumors of two drug-resistant variants of MCF-7 cells, but not in tumors of the parent (drug-sensitive) cells. The partitioning of weak-base or weak-acid drug molecules across the plasma membrane of a tumor cell is dependent upon the acid-dissociation constant (pKa) of the drug as well as the plasmalemmal pH gradient. A large acid-outside pH gradient, such as those seen in MCF-7 xenografts, can exert a protective effect on the cell from weak-base drugs such as anthracyclines and Vinca alkaloids, which have pKa values of 7.5 to 9.5. The possibility of enhancing the therapeutic efficacy of weak-base drugs by dietary or metabolic manipulation of the extracellular pH, in order to reduce or reverse the plasmalemmal pH gradient, deserves investigation.

Tumor angiogenesis, vascularization, and contrast-enhanced magnetic resonance imaging.

Angiogenesis, the process by which new blood vessels are generated, occurs during wound healing, in the female reproductive system during ovulation and gestation, and during embryonic development. The process is carefully controlled with positive and negative regulators, because several vital physiological functions require angiogenesis. The consequences of abnormal angiogenesis are either excessive or insufficient blood vessel growth. Ulcers, strokes, and heart attacks can result from the absence of angiogenesis normally required for natural healing, whereas excessive blood vessel proliferation may favor tumor growth and dissemination, blindness, and arthritis. In this review, the process of angiogenesis and the characteristics of the resulting tumor vasculature are outlined. Contrast-enhanced magnetic resonance imaging techniques that currently are available for basic research and clinical applications to study various aspects of tumor angiogenesis and neovascularization are discussed.

Imaging of cationic multifunctional liposome-mediated delivery of COX-2 siRNA

Liposomes are a useful means of delivering molecular targeting agents such as small interfering RNA (siRNA) to downregulate specific pathways important in cancer growth and progression. The ability to non-invasively image these carriers is important to ascertain their delivery within the tumor. As cyclooxygenase-2 (COX-2) is an important therapeutic target in cancer, we investigated loading COX-2-specific siRNA into cationic liposomes containing MR contrast agents for imaging delivery in cancer cells and tumors. COX-2 and GAPDH siRNA, as well as Magnevist or Feridex, were loaded directly into the liposomes. These lipoplexes were used for cell transfection of the poorly differentiated and highly metastatic breast cancer cell line MDA-MB-231. PEGylated liposomes loaded with Feridex and fluorescently labeled COX-2 siRNA were used for in vivo delivery of lipoplexes in MDA-MB-231 breast cancer xenografts in female SCID mice. Transient transfection assays demonstrated potent and specific downregulation of the COX-2 protein in cells in culture. Tail vein injections of PEGylated COX-2 lipoplexes resulted in intratumoral delivery of siRNA. Biodistribution studies showed significant localization in the lung, liver and kidney at 24 h. These data demonstrate the feasibility of liposomal-mediated delivery of COX-2-specific siRNA to downregulate COX-2 in cancer cells, and multi-modality imaging of the delivery of specific siRNA in tumors.

Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate

Purpose: Glutamine addiction in c-MYC–overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. Experimental Design: A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress–mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. Results: We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress–mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc–overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. Conclusions: AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOAs effectiveness provide a strong rationale for further clinical development, particularly for c-MYC–overexpressing breast cancers. Clin Cancer Res; 21(14); 3263–73. ©2015 AACR.

Synthesis and Evaluation of GdIII‐Based Magnetic Resonance Contrast Agents for Molecular Imaging of Prostate‐Specific Membrane Antigen

Magnetic resonance (MR) imaging is advantageous because it concurrently provides anatomic, functional, and molecular information. MR molecular imaging can combine the high spatial resolution of this established clinical modality with molecular profiling in vivo. However, as a result of the intrinsically low sensitivity of MR imaging, high local concentrations of biological targets are required to generate discernable MR contrast. We hypothesize that the prostate-specific membrane antigen (PSMA), an attractive target for imaging and therapy of prostate cancer, could serve as a suitable biomarker for MR-based molecular imaging. We have synthesized three new high-affinity, low-molecular-weight Gd(III) -based PSMA-targeted contrast agents containing one to three Gd(III)  chelates per molecule. We evaluated the relaxometric properties of these agents in solution, in prostate cancer cells, and in an in vivo experimental model to demonstrate the feasibility of PSMA-based MR molecular imaging.

Molecular Imaging of Cancer: Applications of Magnetic Resonance Methods

Cancer is a complex disease exhibiting a host of phenotypic diversities. Noninvasive multinuclear magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) provide an array of capabilities to characterize and understand several of the vascular, metabolic, and physiological characteristics unique to cancer. The availability of targeted contrast agents has widened the scope of MR techniques to include the detection of receptor and gene expression. In this paper, we have highlighted the application of several MR techniques in imaging and understanding cancer.