Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Growth factors are potential targets for therapeutic strategies because they are essential for tumor growth and progression. Peptidylglycine ␣-amidating monooxygenase is the enzyme producing ␣-amidated bioactive peptides from their inactive glycine-extended precursors. The high expression of peptidylglycine ␣-amidating monooxygenase mRNA in glioblastoma and glioma cell lines points to the involvement of ␣-amidated peptides in tumorigenic growth processes in the brain. After screening of amidated peptides, it was found that human glioblastoma cell lines express high levels of adrenomedullin (AM) mRNA, and that immunoreactive AM is released into the culture medium. AM is a multifunctional regulatory peptide with mitogenic and angiogenic capabilities among others. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that AM mRNA was correlated to the tumor type and grade, with high expression in all glioblastomas analyzed, whereas a low expression was found in anaplastic astrocytomas and barely detectable levels in low-grade astrocytomas and oligodendrogliomas. In the present study we also demonstrate the presence of mRNA encoding the putative AM receptors, calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CRLR/RAMP2; CRLR/RAMP3) in both glioma tissues and glioblastoma cell lines and further show that exogenously added AM can stimulate the growth of these glioblastoma cells in vitro. These findings suggest that AM may function as an autocrine growth factor for glioblastoma cells. One way to test the autocrine hypothesis is to interrupt the function of the endogenously produced AM. Herein, we demonstrate that a polyclonal antibody specific to AM, blocks the binding of the hormone to its cellular receptors and decreases by 33% (P < 0.001) the growth of U87 glioblastoma cells in vitro. Malignant glioblastomas are highly aggressive tumors with a median patient survival time of 9 to 14 months.

DESBRIERE, RAOUL, VINCENT VUAROQUEAUX, VINCENT ACHARD, SANDRINE BOULLU-CIOCCA, MARTIN LABUHN, ANNE DUTOUR, AND MICHEL GRINO. Increased expression of 11␤-hydroxysteroid dehydrogenase type 1 mRNA in both visceral and subcutaneous adipose tissue of obese patients. Obesity. 2006;14:794 -798. Objective: Data from rodents provide evidence for a causal role of 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD-1) in the development of obesity and its complications. In humans, 11␤-HSD-1 is increased in subcutaneous adipose tissue (SAT) of obese patients, and higher adipose 11␤-HSD-1 was associated with features of the metabolic syndrome. To date, there is no evidence for an increased expression of 11␤-HSD-1 in human visceral adipose tissue (VAT), although VAT is the major predictor for insulin resistance and the metabolic syndrome. Research Methods and Procedures: 11␤-HSD-1 and hexose-6-phosphate dehydrogenase (the enzyme responsible for the synthesis of nicotinamide adenine dinucleotide phosphate, the cofactor required for 11␤-HSD-1 oxoreductase activity) mRNA levels were measured using real-time quantitative reverse transcriptase-polymerase chain reaction in abdominal SAT and VAT biopsies obtained from 10 normal-weight and 12 obese women. Adiponectin mRNA was used as an internal control.Results: 11␤-HSD-1 mRNA concentrations were significantly increased in both SAT and VAT of obese patients (720% and 450% of controls, respectively; p Ͻ 0.05) and correlated with hexose-6-phosphate dehydrogenase mRNA levels. The level of VAT 11␤-HSD-1 mRNA correlated with anthropometric parameters: BMI (r ϭ 0.41, p ϭ 0.05), waist circumference (r ϭ 0.44, p ϭ 0.04), abdominal sagittal diameter (r ϭ 0.51, p ϭ 0.02), and percentage fat (r ϭ 0.51, p ϭ 0.02). Discussion: Our results demonstrate for the first time that 11␤-HSD-1 mRNA expression is increased in VAT from obese patients. They strengthen the importance of 11␤-HSD-1 in human obesity and its associated complications and suggest the need of clinical studies with specific 11␤-HSD-1 inhibitors.

Purpose: We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multicenter study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor-positive, HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy. Experimental Design: Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis, we assessed the predictive power of the individual marker/ marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels. Results: DNA methylation of PITX2 and 14 other genes was correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A four-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in improvement of outcome prediction compared with PITX2 alone. Conclusions: This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, three of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population. Our results show that a well-defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.

After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.