Agnes Rasmuson

Microsomal prostaglandin E synthase 1 determines tumor growth in vivo of prostate and lung cancer cells

There is strong evidence for a role of prostaglandin E2 (PGE2) in cancer cell proliferation and tumor development. In PGE2 biosynthesis, cyclooxygenases (COX-1/COX-2) convert arachidonic acid to PGH2, which can be isomerized to PGE2 by microsomal PGE-synthase-1 (MPGES-1). The human prostate cancer cell line DU145 expressed high amounts of MPGES-1 in a constitutive manner. MPGES-1 expression also was detectable in human prostate cancer tissues, where it appeared more abundant compared with benign hyperplasia. By using shRNA, we established stable and practically complete knockdown of MPGES-1, both in DU145 cells with high constitutive expression and in the non-small cell lung cancer cell line A549, where MPGES-1 is inducible. For microsomes prepared from knockdown clones, conversion of PGH2 to PGE2 was reduced by 85–90%. This resulted in clear phenotypic changes: MPGES-1 knockdown conferred decreased clonogenic capacity and slower growth of xenograft tumors (with disintegrated tissue structure) in nude mice. For DU145 cells, MPGES-1 knockdown gave increased apoptosis in response to genotoxic stress (adriamycin), which could be rescued by exogenous PGE2. The results suggest that MPGES-1 is an alternative therapeutic target in cancer cells expressing this enzyme.

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)

Autocrine Prostaglandin E2 Signaling Promotes Tumor Cell Survival and Proliferation in Childhood Neuroblastoma

Background Prostaglandin E2 (PGE2) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE2 production, the expression pattern and localization of PGE2 receptors and intracellular signal transduction pathways activated by PGE2. Principal Findings A high expression of the PGE2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE2 and stimulation of serum-starved neuroblastoma cells with PGE2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE2 (dmPGE2) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE2. Similarly, PGE2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner. Conclusions These findings demonstrate that PGE2 acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE2 signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.

Low-dose aspirin delays an inflammatory tumor progression in vivo in a transgenic mouse model of neuroblastoma

Tumor-associated inflammation is a driving force in several adult cancers and intake of low-dose aspirin has proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. The present study employs the transgenic TH-MYCN mouse model for neuroblastoma (NB) to evaluate inflammatory patterns paralleling tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Spontaneously arising abdominal tumors were monitored for tumor-associated inflammation ex vivo at various stages of disease and homozygous mice received daily low-dose aspirin (10mg/kg) using oral gavage or no treatment, from 4.5 to 6 weeks of age. Using flow cytometry, a transition from an adaptive immune response predominated by CD8(+) T cell in early neoplastic lesions, towards enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells, dendritic cells and tumor-associated macrophages, was detected during tumor progression. An M1 to M2 transition of tumor-associated macrophages was demonstrated, paralleled by a deterioration of dendritic cell status. Treatment with low-dose aspirin to mice homozygous for the TH-MYCN transgene significantly reduced the tumor burden (P < 0.01), the presence of tumor-associated cells of the innate immune system (P < 0.01), as well as the intratumoral expression of transforming growth factor-β, thromboxane A2 (P < 0.05) and prostaglandin D2 (P < 0.01). In conclusion, tumor-associated inflammation appears as a potential therapeutic target in NB and low-dose aspirin reduces tumor burden in the TH-MYCN transgenic mouse model of NB, hence warranting further studies on aspirin in high-risk NB.

Tumor Development, Growth Characteristics and Spectrum of Genetic Aberrations in the TH-MYCN Mouse Model of Neuroblastoma

Background The TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies. Methods We followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations. Results DNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6–19 weeks (median 9.1) and homozygous mice at 4.0–6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17. Conclusion Hemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.

Abstract 2746: Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Neuroblastoma (NB) cells are enriched in the omega-6 fatty acid arachidonic acid, the substrate for the cyclooxygenase (COX) enzymes and prostaglandin biosynthesis. The inducible isoform, COX-2 is overexpressed in NB and NB cells produce prostaglandin E2 (PGE2) that acts as an autocrine and/or paracrine survival and proliferation factor. Downstream of the COX enzymes, specific synthases are responsible for the production of the respective prostaglandins. Microsomal prostaglandin E2 synthase-1 (mPGES-1) specifically converts PGH2 to PGE2 and is thought to primarily couple to COX-2. The aim of this study was to investigate if inhibition of mPGES-1 could represent an alternative therapeutic approach to COX- inhibition in NB. Methods: Western blot and immunohistochemistry were used for protein detection. Cell viability of seven NB cell-lines treated with the mPGES-1 inhibitor CAY1052 was determined by MTT-assay. Stable mPGES-1 knockdown SK-N-BE2 clones were established using shRNA and the clonogenic capacity was analysed by clonogenic assay. To study the in vivo effect of COX inhibition, four week old homozygous TH-MYCN mice were treated with 10mg/L diclofenac for two weeks. Ex vivo analyses of COX-metabolites in tumors were performed by LC-MS/MS. Results: Expression of mPGES-1 in human NB tumors and in NB cell lines could be detected. Inhibition of mPGES-1 reduced NB cell growth in vitro and knockdown of mPGES-1 significantly reduced the clonogenic capacity. Expression of COX-1, COX-2 and mPGES-1 in TH-MYCN tumors could be detected and treatment with the dual COX-inhibitor diclofenac significantly reduced tumour weight, compared to untreated animals. Ex vivo analysis of tumor tissues from treated animals revealed a significantly decreased level of COX metabolites compared to controls. The expression of mPGES-1was not affected by the treatment. Furthermore, cells staining positive for cleaved caspase-3 were more prevalent in treated tumors indicating apoptosis Induction. Conclusion: We found that mPGES-1 is expressed in NB, with a potential role for PGE2 synthesis and tumor growth. mPGES-1 represents an alternative therapeutic target for inhibiting NB growth through specific PGE2 inhibition and the TH-MYCN model is well suited for in vivo studies with this purpose. Citation Format: Anna Kock, Agnes Rasmuson, Marina Korotkova, Helena Idborg, John Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2013-2746

Abstract 1420: Microsomal prostaglandin E2 synthase 1 (mPGES-1) is expressed in neuroblastoma and may represent a novel target for therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Neuroblastoma (NB) is an embryonic tumor of the sympathetic nervous system and it is the most common extracranial tumor affecting young children. Despite a very intensive treatment regimen high-risk neuroblastoma patients still have a poor prognosis. Neuroblastoma cells are enriched in arachidonic acid, over express COX-2 and produce prostaglandin E2 (PGE2) that acts as an autocrine or paracrine survival and proliferation factor by induction of Akt signaling. Downstream of the COX enzymes, specific synthases converts PGH2 into the different prostaglandins. Microsomal prostaglandin E2 synthase-1 (mPGES-1) specifically converts PGH2 to PGE2 and is thought to primarily couple to COX-2. The aim of this study is to investigate if inhibition of mPGES-1 could represent an alternative therapeutic approach to COX- inhibition in neuroblastoma. We could detect expression of mPGES-1 in a panel of seven human neuroblastoma cell lines. Stable knock down of mPGES-1 expression using shRNA significantly reduced the clonogenic capacity and treatment with the mPGES-1 inhibitor CAY1052 inhibited neuroblastoma cell growth in vitro. By immunohistochemistry and western blot we could also detect expression of COX-1, COX-2 and mPGES-1 in tumors from the transgenic TH-MYCN mouse model of neuroblastoma. Treatment of homozygous TH-MYCN mice with 10 mg/L diclofenac for two weeks, starting at the age of four weeks, significantly reduced tumour weight compared to untreated animals. Ex vivo analysis with LC-MS/MS of tumor tissues from diclofenac treated animals revealed a significant decreased level of COX metabolites compared to control. The expression of mPGES-1was not affected by the treatment. Furthermore the number of cells staining positive for cleaved caspase-3 were higher in the treated tumors indicating induced apoptosis. In conclusion, our results show that mPGES-1 is expressed in neuroblastoma therefore mPGES-1 might represent an alternative way of reducing NB growth through specific PGE2 inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1420. doi:1538-7445.AM2012-1420

Abstract LB-496: Low-dose aspirin targets tumor-associated inflammation and delays neuroblastoma tumor growth in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL ABSTRACT Tumor-associated inflammation is a driving force in several adult cancers, and low doses of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin have proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. Our study evaluates inflammatory patterns paralleling neuroblastoma (NB) tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Using the TH-MYCN mouse model of NB, this study demonstrates the presence and progression of tumor-associated inflammation during NB tumor growth. Ex vivo analysis of tumors revealed a transition from an adaptive immune response predominated by CD8+ T-cells in neoplastic lesions from 5 week old homozygous mice, towards an enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells (MDSCs), immature dendritic cells (DCs) and M2 tumor-associated macrophages (TAMs) during tumor progression. An ongoing M1 to M2 transition of TAMs was demonstrated, which was paralleled by a gradual deterioration of DC status. Ten days of consecutive anti-inflammatory treatment with low-dose aspirin significantly reduced tumor burden (p<0.01) and the presence of tumor-associated cells of the innate immune system (p<0.01) in homozygous mice. In conclusion, our study depicts how inflammatory pathways assist tumor progression through sculpturing of the tumor microenvironment, and for the first time demonstrates an in vivo effect of low-dose aspirin on established MYCN amplified NB tumors in a transgenic mouse model, suggesting a potential new treatment option for high-risk NB patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-496. doi:1538-7445.AM2012-LB-496