BACKGROUND: The CXCR4 chemokine receptor and its ligand CXCL12 are one of the most well studied chemokine systems in tumor biology. In gliomas CXCL12/CXCR4 axis regulates multiple mechanisms that sustain tumor growth as migration, proliferation, angiogenesis and is emerging as a novel biologically relevant target for brain tumor therapy. Previous studies, in our laboratory highlights a role of the phosphatydilcholine-specific phospholipase C (PC-PLC) in the regulation and expression of growth factor membrane receptors, suggesting a relationship between the metabolism of phosphatydilcholine (PC) and cell receptor status. AIM: Investigate the crosstalk between the chemokine receptor CXCR4 and PC-PLC and its implications in glioma cells growth. MATERIALS AND METHODS: U87MG, a glioblastoma cell line was treated with a conventional CXCR4 antagonist, AMD3100 (10 µM), or with a PC-PLC inhibitor, D609 (Xantate) (100 µM). Confocal Laser Scanning Microscopy (CLSM) and Western Blotting (WB) experiments were performed on cells to investigate protein expression and intracellular localization. Cell metabolism on both treated or untreated U87MG cells was evaluated by in vitro Nuclear Magnetic Resonance Spectroscopy (MRS). RESULTS: CLSM analyses highlighted for the first time an extensive co-localization of CXCR4 and PC-PLC on the plasma membrane of U87MG cells. Furthermore we observed a down-modulation of CXCR4 from cell membrane in response to both inhibitors, AMD3100 and D609, however D609 only induced a decrease in CXCR4 total protein content. Moreover, D609 treatment reduced PC-PLC expression, induced a reduction of pAKT and exerted a significative anti-proliferative effect on U87MG cells (49% inhibition). MRS analyses revealed alterations on U87MG metabolism, showing in D609-treated cells an increase of choline-containing metabolites (Glycerophosphocholine, GPCho; Choline, Cho) (increase of 77%) that could be correlated with activation of lipases and phospholipases that can induce an accumulation of neutral lipids associated with the inhibition of cell proliferation. D609 treatment also induced a significative decrease of intracellular concentration of Lactate (38%), and preliminary analyses on lactate deydrogenase (LDH) showed a decrement of the enzyme activity (30%) suggesting that inactivation of PC-PLC could impaired the glycolysis pathway in U87MG. CONCLUSIONS: In this study we identified for the first time an interaction between the chemokine receptor CXCR4 and the enzyme PC-PLC on a glioma cell line. The specific inhibition of PC-PLC modulates CXCR4 receptor localization on cellular membrane and protein expression. Our findings suggest that the inhibition of PC-PLC could represent a new molecular approach in CXCR4-overexpressing glioma cells.

preclinical models of HER-2 overexpressing ovarian cancer Rossella Canese, Alessandro Ricci, Maria Elena Pisanu, Luisa Paris, Luisa Altabella, Emiliano Surrentino, Marina Bagnoli, Ludmila Liliac, Anna Granata, Silvana Canevari, Delia Mezzanzanica, Egidio Iorio, and Franca Podo Istituto Superiore di Sanità, Rome, RM, Italy, Fondazione IRCCS Istituto Nazionale dei Tumori;, Milan, Mi, Italy, Morphofunctional Sciences Histology, University of Medicine and Pharmacy, Iasi, Iasi, Romania

Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by 1H-magnetic spectroscopy analysis; (III) a 35–36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

BACKGROUND:Limited knowledge is available on alterations induced by cytostatic drugs on magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters of human cancers, in absence of apoptosis or cytotoxicity. We here investigated the effects of a cytostatic cisplatin (CDDP) treatment on (1)H MRS and MRI of HER2-overexpressing epithelial ovarian cancer (EOC) cells and in vivo xenografts.METHODS:High-resolution MRS analyses were performed on in vivo passaged SKOV3.ip cells and cell/tissue extracts (16.4 or 9.4 T). In vivo MRI/MRS quantitative analyses (4.7 T) were conducted on xenografts obtained by subcutaneous implantation of SKOV3.ip cells in SCID mice. The apparent diffusion coefficient (ADC) and metabolite levels were measured.RESULTS:CDDP-induced cytostatic effects were associated with a metabolic shift of cancer cells towards accumulation of MRS-detected neutral lipids, whereas the total choline profile failed to be perturbed in both cultured cells and xenografts. In vivo MRI examinations showed delayed tumour growth in the CDDP-treated group, associated with early reduction of the ADC mean value.CONCLUSION:This study provides an integrated set of information on cancer metabolism and physiology for monitoring the response of an EOC model to a cytostatic chemotherapy, as a basis for improving the interpretation of non-invasive MR examinations of EOC patients.

S89Although advanced prostate cancer is typically regarded as not expressing detectable p63 levels, we have unexpectedly found low expression levels of the DNp63a variant in the metastatic PC3 cell line.Surprisingly, expression of DNp63a increases when cells are grown in stem cell-favoring conditions.Furthermore, FACS purification and analysis of the CD44/CD133-positive cancer stem cell population reveals that expression of DNp63a is highest in these cells.Conclusion: Together, this work describes how DNp63a is a critical mediator of normal prostate stem cell function.Surprisingly, we have also uncovered expression of DNp63a in a subset of cancer stem cells in prostate cancer that suggests that tumor cells may utilize the stem cell-promoting function of p63 to facilitate tumor maintenance and metastatic colonization.

Abnormalities in phospholipid metabolism represent major hallmarks of cancer cells. Changes in the MRS profiles of aqueous precursors and catabolites of phosphatidylcholine (PtdCho) in cancer lesions allow non invasive monitoring of tumor progression and response to conventional and targeted anti-cancer therapies. Advances and limitations of our present understanding of molecular mechanisms underlying these anomalous metabolic profiles will be here discussed in the light of altered expression and activity of enzymes of the PtdCho cycle and links to dysregulated cell signaling pathways responsible for oncogenesis. An overview will also be provided of a) the role of choline metabolites as possible pharmacodynamic biomarkers of targeted therapies and b) current efforts to identify PtdCho cycle enzymes as possible targets for therapy.

Carcinogenesis is a complex, multi-stage process depending on both endogenous and exogenous factors. In the past years, DNA mutations provided important clues to the comprehension of the molecular pathways involved in numerous cancers. Recently, post-transcriptional modification events, such as RNA editing, are emerging as new players in several human diseases, including tumours. A-to-I RNA editing changes the nucleotide sequence of target RNAs, introducing A-to-I/G “mutations”. Since ADAR enzymes catalyse this nucleotide conversion, their expression/activity is essential and finely regulated in normal cells.This review summarizes the available knowledge on A-to-I RNA editing in the cancer field, giving a new view on how ADARs may play a role in carcinogenesis.

Phospholipase A2 catalyzes the hydrolysis of membrane glycerophospholipids leading to the production of metabolites observable by both 1H and 31P magnetic resonance spectroscopy. The signal of choline-containing compounds (Cho) observed by 1H magnetic resonance spectroscopy is constituted of metabolites of phosphatidylcholine, especially phosphocholine (PCho) and glycerophosphocholine (GPCho). The phosphomonoester (PME) and phosphodiester (PDE) signals observed by 31P magnetic resonance spectroscopy are, respectively, precursors and catabolites of phospholipids. A large number of brain diseases have been reported to cause variations in the intensity of the Cho, PME and PDE signals. Changes in the activity of phospholipase A2 have been measured in many brain diseases. In this review, the relationships between the results of 1H and 31P magnetic resonance spectroscopy and the phospholipase A2 assays are analyzed. In many brain diseases, the variation in the Cho signal intensity can be correlated with a stimulation or inhibition of the phospholipase A2 activity.

Authors' A Section of Rome, Ital Medicine 3Cogentec 4JHU ICMI Radiologic Baltimore,

Introduction New pharmacological therapies are based on selective molecular targeting of mechanisms able to deregulate pathways of cellular proliferation, survival and apoptosis and activate angiogenesis and metastasis. Recent evidence indicates an important role for CXCL12 (a CXC chemokine) and its receptor (CXCR4) in the metastatic homing of tumor cells (Ottaiano A et al. Cancer Immunol Immunother. 2005). MRS is a powerful approach to detect metabolic alterations of phosphatidylcholine metabolism (PC) associated with tumor cell growth and progression and to investigate the underlying molecular mechanisms. In particular, it has been shown that the inhibition of some PC-cycle enzymes could represent molecular targets for new anticancer therapy (Glunde K. Mol Pharm. 2006; Iorio E et al. Cancer Res. 2010). Purpose of this study was to investigate alterations of CXCR4 expression in a human T-lymphoblastoid cell line (CEM) exposed to a selective inhibitor of the PC-specific phospholipase C (PC-plc). In fact, PC-PLC activation/deactivation status has been reported to modulate the expression of membrane receptors and proteins crucial for specific cell functions, such as HER2 in breast cancer and CD16 in Natural Killer cells (Paris L et al. Breast Cancer Res. 2010; Cecchetti S et al. Eur J Immunol. 2007). Methods: For MRS experiments CEM cells were grown in complete medium and incubated in the absence (CTRL) or presence of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609, 25 μg/ml) for 24 h. H MRS experiments were performed on ethanolic cell extracts on a Bruker Avance 400 spectrometer using a H-X multinuclear inverse probehead. Flow cytometry analyses were performed on a FACScan apparatus (BD Biosciences) using Alexa fluor 488 conjugated secondary Abs. Results 1) Cytofluorimetric analyses performed on unfixed, viable cells, showed overexpression of both CXCR4 and PC-PLC on the outer plasma membrane of CEM cells. Western blot analyses of anti-CXCR4 or anti-PC-PLC immunoprecipitates (IP) blotted with the mutual antibodies (anti-PC-PLC or anti-CXCR4, respectively) showed for the first time the existence of a physical interaction between PC-PLC and the CXCR4 receptor (Fig. 1). 2) Cell exposure to the PC-PLC inhibitor D609 induced a strong down-modulation of CXCR4 (up to 40-50% from 3h to 24h of treatment) from the plasma membrane, suggesting a linkage between PC catabolism and the CXCR4/CXCL12 axis (Fig. 2). 3) MRS analyses on aqueous extracts showed a significant decrease (by about 42 %) in the intracellular PCho content of D609-treated CEM cells compared with the untreated controls (Fig. 3). Conclusions and future directions -Inhibition of the investigated CXC receptor-chemokine axis is currently being investigated as a possible therapeutic strategy for anticancer treatment. The clinical development for these targeted therapies requires identification of pharmacodynamic markers of treatment effectiveness. The results of this study showed for the first time the existence of a physical interaction between the phosphatidylcholine-cycle enzyme PCPLC and CXCR4 on the plasma membrane of a T-lymphoblastoid cell line (CEM). Specific inhibition of PC-PLC activity induced down-modulation of CXC4 from the plasma membrane of CEM cells. 1H MRS profiling of cell extracts showed that a decrease in the PCho signal may act in these cells as a significant marker of simultaneous PCPLC inhibition and CXCR4 downmodulation. Further clarification of these molecular mechanisms may contribute to the development of non invasive MRS approaches to monitor the effects of the CXCR4-CXCL12 axis in these and in other tumor cells.

Introduction Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecologic malignancy among women in developed countries. Although the prognosis in cases detected at early stage is quite favorable, the vast majority of cases are diagnosed at an advanced stage when five-year survival rates are only 30-40%. The poor prognosis of ovarian cancer is due to a combination of the aggressive characteristics of the disease and a lack of effective therapy, further compounded by the key problems of late detection of the disease and resistance of most tumors to current treatments. New therapeutic strategies are urgently needed to improve survival rates and to eventually cure patients. Since ovarian cancer cells typically have high choline kinase compared to nonmalignant epithelial cells (1), choline kinase may be a novel target for ovarian cancer treatment. Treatment effects can also be monitored with H MRSI. To evaluate treatment strategies it is important to use models that closely mimic tumor growth in humans. We therefore orthotopically implanted ovarian cancer tissue in the ovary of female mice and demonstrated that tumor growth can be followed by MRI using T1-weighted images combined with diffusion-weighted images (DWI), and tumor metabolism with H MRSI. Significant differences were observed between total choline and PC levels in orthotopic versus subcutaneous tumors that emphasize the importance of using orthotopic tumors for evaluating metabolic targeting.

Background and Purpose: Transmissible spongiform encephalopathy (TSE) diseases are fatal, progressive neurodegenerative disorders affecting both humans and animals. Clinical signs typically appear after years and even decades of silent disease progression. This study was aimed at investigating whether altered brain MRI patterns may precede clinical signs in a TSE rodent model. Methods: In vivo T2-weighted (T2W) MRI examinations (4.7 T) were performed on Golden Syrian hamsters (GSH) intracerebrally, orally, or intraperitoneally (i.p.) infected with the 263K scrapie strain. Histopathological analyses were performed on i.p. infected GSH at the end of one-day or longitudinal MRI sessions. Results: T2W-MRI hyperintensity was detected in the thalamic nuclei of GSH with clinical signs, irrespective of the infection route. Hyperintensity in the thalamus was also observed in pre-clinical animals, between 106 and 121 days post-infection (dpi), while normal T2W intensity was detected in four animals examined between 72 and 96 dpi. Pathological prion protein deposition (but no astrogliosis and only occasionally, weak spongiosis) was detected between 106 and 121 dpi. Conclusions: The altered T2W-MRI pattern detected in the thalamus of asymptomatic i.p. infected GSH provides a useful basis for evaluating the effectiveness of possible therapeutic approaches at early stages of TSE disease.

The clinical use of breast magnetic resonance (MR) imaging is increasing, especially for applications requiring paramagnetic contrast-agent injection. This document presents a synthetic list of acceptable indications with potential advantages for women according to evidence from the literature and the expert opinion of the panel that developed this statement. We generally recommend that breast MR imaging be performed in centres with experience in conventional breast imaging [mammography and ultrasonography (US)] and needle-biopsy procedures (under stereotactic or US guidance) as well as in breast MR imaging and second-look US for findings not revealed by conventional imaging performed before MR imaging. In our opinion, there is no evidence in favour of breast MR imaging as a diagnostic tool to characterise equivocal findings at conventional imaging when needle-biopsy procedures can be performed, nor for the study of asymptomatic, non-high-risk women with negative conventional imaging. After a description of technical and methodological requirements, we define the indications and limitations of breast MR imaging for surveillance of high-risk women, local staging before surgery, evaluation of the effect of neoadjuvant chemotherapy, breast previously treated for carcinoma, carcinoma of unknown primary syndrome, nipple discharge and breast implants.

This literature review assesses the clinical potential of proton (H-1) magnetic resonance spectroscopy (MRS) of breast lesions. We here illustrate the basic principles of spectrum acquisition for volumes of interest, determined on the basis of dynamic magnetic resonance imaging (MRI) and of MRS postprocessing. We discuss the criteria for interpreting the spectrum with particular reference to the metabolic significance of the peak of total choline containing compounds at 3.2 ppm, a marker that is correlated with malignancy. We then summarise the findings obtained in lesion characterisation (with a possible gain in specificity with respect to dynamic MRI), the assessment of the effects of neoadjuvant chemotherapy and the correlation reported at high-field between the tumour tissue concentration of choline-containing compounds and the presence of lymph node metastases. Lastly, we outline the clinical use of this technique as the final phase of a complete breast MR examination after intravenous administration of paramagnetic contrast material for the dynamic study, with reference to its use by radiologists dedicated to breast imaging.