Abiel Orrego

Inhibitors of mammalian target of rapamycin downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in vivo

Mammalian target of rapamycin (mTOR) has been shown to play an important function in cell proliferation, metabolism and tumorigenesis, and proteins that regulate signaling through mTOR are frequently altered in human cancers. In this study we investigated the phosphorylation status of key proteins in the PI3K/AKT/mTOR pathway and the effects of the mTOR inhibitors rapamycin and CCI-779 on neuroblastoma tumorigenesis. Significant expression of activated AKT and mTOR were detected in all primary neuroblastoma tissue samples investigated, but not in non-malignant adrenal medullas. mTOR inhibitors showed antiproliferative effects on neuroblastoma cells in vitro. Neuroblastoma cell lines expressing high levels of MYCN were significantly more sensitive to mTOR inhibitors compared to cell lines expressing low MYCN levels. Established neuroblastoma tumors treated with mTOR inhibitors in vivo showed increased apoptosis, decreased proliferation and inhibition of angiogenesis. Importantly, mTOR inhibitors induced downregulation of vascular endothelial growth factor A (VEGF-A) secretion, cyclin D1 and MYCN protein expression in vitro and in vivo. Our data suggest that mTOR inhibitors have therapeutic efficacy on aggressive MYCN amplified neuroblastomas.

Effects of valganciclovir as an add‐on therapy in patients with cytomegalovirus‐positive glioblastoma: A randomized, double‐blind, hypothesis‐generating study

Cytomegalovirus is highly prevalent in glioblastomas. In 2006, we initiated a randomized, double‐blind, placebo‐controlled, hypothesis‐generating study to examine the safety and potential efficacy of Valganciclovir as an add‐on therapy for glioblastoma. Forty‐two glioblastoma patients were randomized in double‐blind fashion to receive Valganciclovir or placebo in addition to standard therapy for 6 months. Magnetic resonance images were obtained before and immediately and 3 and 6 months after surgery to evaluate treatment efficacy by measuring contrast enhancing tumor volume (primary end point). Survival data were analyzed for patients and controls in explorative analyses to aid the design of future randomized trials. Trends but no significant differences were observed in tumor volumes in Valganciclovir and placebo patients at 3 (3.58 vs. 7.44 cm3, respectively, p = 0.2881) and 6 (3.31 vs. 13.75 cm3, p = 0.2120) months. Median overall survival (OS) was similar in both groups (17.9 vs. 17.4 months, p = 0.430). Patients could take Valganciclovir for compassionate use after the study phase. Explorative analyses showed an OS of 24.1 months (95% CI, 17.4–40.3) in patients receiving >6 months of Valganciclovir (Val > 6M) versus 13.1 months (95% CI, 7.9–17.7, p < 0.0001) in patients receiving Valganciclovir for 0 or <6 months, and 13.7 months (95% CI, 6.9–17.3, p = 0.0031) in contemporary controls. OS at 4 years was 27.3% in Val>6M patients versus 5.9% in controls (p = 0.0466). Prolonged OS in Val>6M patients suggest that future randomized trials are warranted and should evaluate whether continuous antiviral treatment can improve outcome in glioblastoma patients.

Cyclooxygenase-2 Is Expressed in Neuroblastoma, and Nonsteroidal Anti- Inflammatory Drugs Induce Apoptosis and Inhibit Tumor Growth In vivo

Neuroblastoma is the single most common and deadly tumor of childhood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs, inhibitors of COX, induced caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX-1/COX-2 inhibitor diclofenac or the COX-2–specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachidonic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy (1H MRS) of neuroblastoma cells treated with COX inhibitors demonstrated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, 1H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma.

Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma.

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal- and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies.

Human cytomegalovirus infection levels in glioblastoma multiforme are of prognostic value for survival

BACKGROUND Patients with glioblastoma multiforme (GBM) generally live 12-15 months after diagnosis, despite maximal surgical resection, adjuvant radiotherapy, and chemotherapy. HCMV has been detected in 90-100% of GBMs. We recently found that low grade HCMV infection in GBM tumours was highly associated with survival over 18 months (case-control study). Here, we sought to determine whether low-grade HCMV infection in GBMs is associated with prolonged survival in a consecutive patient cohort, analysed retrospectively. STUDY DESIGN Tumour samples from 75 consecutive GBM patients treated surgically at Karolinska University Hospital in 2004-2005 were examined by immunohistochemistry (IHC) and in situ hybridization for HCMV proteins and DNA, respectively. Tumours were graded 1-4, depending on the percentage of positive cells by IHC. Low-grade HCMV was defined as grade 1 (< 25% of HCMV infected tumour cells). Time to tumour progression (TTP) and survival data were analysed with Cox regression and Kaplan-Meier models. RESULTS HCMV infection was detected in 74 of 75 tumours (99%). In patients with low-grade HCMV infection, median survival was 20 months longer than in patients with high-grade infections (P = 0.036, HR: 2.2), and TTP was 8 months longer (P = 0.1, HR: 1.8). Two-year survival was much higher in patients with low-grade HCMV infection (63.6% vs. 17.2%, P = 0.003). CONCLUSION The longer survival in patients whose tumours had low-grade HCMV infection suggests that the level of HCMV infection in GBMs has a prognostic value and that HCMV may contribute to the pathogenesis of GBM.

Tumor-growth–promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets

Prostaglandin E(2) (PGE(2)) has been shown to play important roles in several aspects of tumor development and progression. PGE(2) is synthesized from arachidonic acid by cyclooxygenases (COX) and prostaglandin E synthases (PGES) and mediates its biological activity through binding to the four prostanoid receptors EP(1) through EP(4). In this study, we show for the first time that medulloblastoma (MB), the most common malignant childhood brain tumor, expresses high levels of COX-2, microsomal prostaglandin E synthase-1, and EP(1) through EP(4) and secretes PGE(2). PGE(2) and the EP(2) receptor agonist butaprost stimulated MB cell proliferation. Treatment of MB cells with COX inhibitors suppressed PGE(2) production and induced caspase-dependent apoptosis. Similarly, specific COX-2 silencing by small interfering RNA inhibited MB cell growth. EP(1) and EP(3) receptor antagonists ONO-8713 and ONO-AE3-240, but not the EP(4) antagonists ONO-AE3-208 and AH 23848, inhibited tumor cell proliferation, indicating the significance of EP(1) and EP(3) but not EP(4) for MB growth. Administration of COX inhibitors at clinically achievable nontoxic concentrations significantly inhibited growth of established human MB xenografts. Apoptosis was increased, proliferation was reduced, and angiogenesis was inhibited in MBs treated with COX inhibitors. This study suggests that PGE(2) is important for MB growth and that therapies targeting the prostanoid metabolic pathway are potentially beneficial and should be tested in clinical settings for treatment of children with MB.

Low levels of Human Cytomegalovirus Infection in Glioblastoma multiforme associates with patient survival; -a case-control study

BackgroundGlioblastoma multiforme (GBM) represent the most aggressive brain tumor with a median overall survival of about 12-15 months. Over 90% of GBM tumors have recently been shown to be infected with human cytomegalovirus (HCMV). In this case-control study, we evaluated whether there was an association between the grade of HCMV infection and long-term survival (> 18 months) in GBM patients.Material and methodsBrain tumor tissue sections from consecutive GBMs patients who survived more than 18 months (n = 40), and an equal number of GBM patients, matched to date of diagnosis and surgery, operated at Karolinska University Hospital in 2000-2005 were selected. HCMV infection grade was determined by estimation of the number of HCMV positive cells (scored negative or grade 1-4) in tumor tissue specimens. Using Chi-Square test and logistic regression analysis, we analyzed whether there was an association between long-term survival and HCMV low-grade infection or other clinical parameters known to be associated with prolonged survival of GBM patients; age under 50 years, radical surgery or low recursive partition analysis (RPA) subclass.ResultsHCMV infection was detected in tumor samples from 79 of 80 patients (99%). Among patients surviving > 18 months, HCMV infection grade 1 in the GBM tumor was predominant. A low grade HCMV infection was found in 19 patients, of these 16 survived > 18 months. Thus, 16 of 40 (40%) GBM patients who lived > 18 months had low-grade HCMV infection while only 3 of 40 (8%) GBM patients who lived < 18 months did (p .0006, Chi-Square test). Multiple logistic regression analyses yielded an odds ratio estimate of 6.604 with 95% confidence interval (1.36-32.1) (p .019) for low grade HCMV after adjustment for RPA class III and IV, radical surgery, age and gamma knife treatment.ConclusionIn conclusion, we found that low-grade HCMV infection was strongly associated with long-term survival in GBM patients.

Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy

The metabolism of arachidonic acid by the cyclooxygenase (COX) or lipoxygenase (LO) pathways generates eicosanoids that have been implicated in the pathogenesis of a variety of human diseases, including cancer. In this study, we examined the expression and significance of components within the 5‐LO pathway in human neuroblastoma, an embryonal tumor of the sympathetic nervous system. High expression of 5‐LO, 5‐LO‐activating protein (FLAP), leukotriene A4 hydrolase, leukotriene C4 synthase, and leukotriene receptors was detected in a majority of primary neuro‐blastoma tumors and all cell lines investigated. Expression of 5‐LO and FLAP was evident in tumor cells but not in nonmalignant adrenal medulla where neuroblastomas typically arise. Moreover, neuroblastoma cells produce leukotrienes, and stimulation of neuroblastoma cells with leukotrienes increased neuroblastoma cell viability. Inhibitors of 5‐LO (AA‐861), FLAP (MK‐886), or the leukotriene receptor antagonist montelukast inhibited neuroblastoma cell growth by induction of G1‐cell cycle arrest and apoptosis. Similarly, specific 5‐LO and leukotriene receptor silencing by small interfering RNA decreased neuroblastoma cell growth. These findings provide new insights into the pathobiology of neuroblastoma, and the use of leukotriene pathway inhibitors as a novel adjuvant therapy for children with neuroblastoma warrants further consideration.—Sveinbjörnsson, B., Rasmuson, A., Baryawno, N., Wan, M., Ingvild Pettersen, I., Frida Ponthan, F., Orrego, A., Haeggström, J. Z., Johnsen, J. I., Kogner, P. Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy. FASEB J. 22, 3525–3536 (2008)

Frequency of epithelioid granulomas in colonoscopic biopsy specimens from paediatric and adult patients with Crohn's colitis.

Aims: To test the assumption that epithelioid granulomas found in colonoscopic biopsy specimens in patients with Crohn’s colitis are markers of a different clinical behaviour. Methods: Sections from colonoscopic biopsy specimens from 352 consecutive patients (119 children and 233 adults) were investigated. Results: A total of 1117 colonoscopies were performed: 293 in children (mean 2.46 per patient) and 824 in adults (mean 3.53 per patient) (p<0.05). Granulomas at initial colonoscopy were recorded in 67.2% (43/64) of children and 65.9% (27/41) of adults (p>0.6), and at subsequent colonoscopies in 53.8% (64/119) of children and 17.6% (41/233) of adults (p<0.05). Surgical intervention was required in 6.3% (4/64) of the children having previous granuloma, but also in 14.5% (8/55) of those without previous granuloma, the rate for operated adults being 26.8% (11/41) and 24.5% (47/192), respectively (p>0.6). Conclusions: Granulomas in entry and/or in subsequent colonoscopic biopsy specimens in patients with Crohn’s colitis did not predict the need for subsequent surgical intervention. The fact that the frequency of granulomas was significantly higher in children than in adults with Crohn’s colitis (despite a higher mean number of colonoscopic biopsies in adults), and that granulomas were present in colonoscopic biopsy specimens but not in the subsequent surgical specimens from 50% of the paediatric and 36% of the adult patients strengthen the conviction that granulomas in Crohn’s colitis might evolve or regress at different time intervals during the course of the disease. This behaviour would reflect a particular immunological reaction, an epiphenomenon from immature tissues—as in children—when challenged by the so far elusive aetiological agent responsible for Crohn’s disease.

Expression of PROX1 Is a common feature of high-grade malignant astrocytic gliomas.

PROX1 is a prospero-related transcription factor that plays a critical role in the development of various organs including the mammalian lymphatic and central nervous systems; it controls cell proliferation and differentiation through different transcription pathwaysand has both oncogenic and tumor-suppressive functions. We investigated PROX1 expression patterns in 56 human astrocytic gliomas of different grades using immunohistochemistry. An average of 79% of cells in World Health Organization Grade IV (glioblastoma, n = 15) and 57% of cells in World Health Organization Grade III (anaplastic astrocytoma, n = 13) were strongly PROX1 positive; low-grade diffuse astrocytomas (Grade II, n = 13) had 21% of cells that were strongly positive; Grade I tumors (n = 15) had 1.5%; and non-neoplastic brain tissue (n = 15) had 3.7% of cells that were PROX1 positive. Double immunolabeling showed that PROX1+ cells in glioblastomas frequently coexpressed early neuronal proteins MAP2 and &bgr;III-tubulin but not the mature neuronal marker protein NeuN. Analyses of coexpression with proliferation markers suggest that PROX1+ cells have a marginally lower rate of proliferation than other tumor cells but are still mitotically active. We conclude that PROX1 may constitute a useful tool for the diagnosis and grading ofastrocytic gliomas and for distinguishing Grade III and Grade IV tumors from Grade I and Grade II tumors.