Omega-3 fatty acids decrease CRYAB, production of oncogenic prostaglandin E2 and suppress tumor growth in medulloblastoma.

AIMS: Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the ω3-long chain polyunsaturated fatty acids (ω3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model. MAIN METHODS: Effects of ω3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography. KEY FINDINGS: ω3-LCPUFA decreased prostaglandin E2 (PGE2) secretion from MB cells, and impaired MB cell viability and colony forming ability and increased apoptosis in a dose-dependent manner. DHA reduced tumor growth in vivo, and both PGE2 and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All ω3-LCPUFA and dihomo-γ-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among ω3-LCPUFA treated tumors. CRYAB was the most significantly altered gene and the downregulation was confirmed by immunohistochemistry. SIGNIFICANCE: Our findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment.

Targeting Fibroblast Growth Factor Receptor (FGFR) and Phosphoinositide 3-kinase (PI3K) Signaling Pathways in Medulloblastoma Cell Lines.

BACKGROUND/AIM: Medulloblastoma (MB) accounts for ~20% of pediatric malignant central nervous system tumors. Treatment strategies, including surgery, radiation therapy and/or chemotherapy, are effective, but recurrence and metastasis frequently occur. Therefore, novel therapies are required. Herein, the effects of fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibitors on MB cells lines were evaluated. MATERIALS AND METHODS: MB cell lines (UW228-3, DAOY, Med8a, D425, D283) were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BYL719) inhibitors by viability, cytotoxicity, apoptosis, and proliferation assays. RESULTS: Single treatments with FGFR and PI3K inhibitors decreased viability and proliferation in a dose-dependent pattern in most cell lines. Combinination of the two type of drugs, increased sensitivity, especially of the most resistant cell line UW228-3. CONCLUSION: Combination treatments with FGFR and PI3K inhibitors were superior to single treatments with FGFR and PI3K inhibitors, especially with BEZ235, for MB cell lines.

In vitro antitumor effects of FGFR and PI3K inhibitors on human papillomavirus positive and negative tonsillar and base of tongue cancer cell lines.

Human papillomavirus positive (HPV+) tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC) have better outcomes than corresponding HPV- negative (HPV-) cancer cases. Our previous study demonstrated that fibroblast growth factor receptor 3 (FGFR3) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit a (PIK3CA) are often mutated in HPV+ cancer. To investigate whether targeted therapy is an option for TSCC/BOTSCC, two HPV+ and one HPV- TSCC/BOTSCC cell lines were tested for their sensitivity towards FGFR and PI3K inhibitors. The HPV+ cell lines UM-SCC-47 and UPCI-SCC-154, and the HPV- cell line UT-SSC-60A were tested by competitive allele-specific TaqMan-PCR for presence/absence of frequently occurring FGFR3 and PIK3CA mutations. All cells were then treated with FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120 alone, or with AZD4547 and BEZ235 in combination. Viability was analyzed using a WST-1 assay, cytotoxicity tested by a CellTox Green cytotoxicity assay, apoptosis analyzed by a Caspase Glo 3/7 assay and proliferation examined with the xCELLigence system. HPV+ UM-SCC-47 and UPCI-SCC-154 cells, and HPV- UT-SSC-60A cells, did not exhibit any common FGFR3 or PIK3CA mutations, but were all sensitive to FGFR inhibitor AZD4547 and PI3K inhibitors BEZ235 and BKM120. Notably, HPV+ UPCI-SCC-154 cells were more sensitive than the other two cell lines. Furthermore, when AZD4547 and BEZ235 treatment was combined in HPV+ UPCI-SCC-154 and HPV- UT-SSC-60A cells, potentiated combination effects were observed. HPV+ UM-SCC-47 and UPCI-SCC-154 cells, and HPV- UT-SSC-60A cells had no common FGFR3 or PIK3CA mutations, but were sensitive to FGFR inhibitor AZD4547, and PI3K inhibitors BEZ235 and BKM120. Furthermore, the latter two cell lines were particularly sensitive to combinations of AZD4547 and BEZ235.

Analyses of FGFR3 and PIK3CA mutations in neuroblastomas and the effects of the corresponding inhibitors on neuroblastoma cell lines.

Fibroblast growth factor receptor (FGFR)3 and phosphatidylinositol­4,5­bisphosphate 3­kinase, catalytic subunit alpha (PIK3CA) mutations are found in various types of cancer, such as urinary bladder cancer, human papillomavirus­positive tonsillar and base of the tongue squamous cell carcinoma, breast cancer and some childhood sarcomas. Several drugs can target these genes, some of which have been used for the treatment of urinary bladder cancer. Much less is known about childhood cancer. For this reason, the present study investigated the presence of such mutations in neuroblastomas (NBs) and tested NB cell lines for sensitivity to FGFR and phosphoinositide 3­kinase (PI3K) inhibitors. In total, 29 NBs were examined for the presence of the three most common FGFR3 and PIK3CA mutations using a competitive allele­specific TaqMan PCR (CAST­PCR). Furthermore, the SK­N­AS, SK­N­BE(2)­C, SK­N­DZ, SK­N­FI and SK­N­SH NB cell lines (where SK­N­DZ had a deletion of PIK3C2G, none had FGFR mutations according to the Cancer Program's Dependency Map, but some were chemoresistant), were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BKM120) inhibitors by viability, cytotoxicity, apoptosis and proliferation assays. CAST­PCR detected one FGFR3 mutation in 1/29 NBs. Following treatment with FGFR and PI3K inhibitors, a decrease in viability and proliferation was observed in the majority, but not all, the cell lines. Following combination treatment with both drugs, the sensitivity of all cell lines was increased. On the whole, the findings of this study demonstrate that FGFR3 and PIK3CA mutations are uncommon in patients with NB. However, certain NB cell lines are rather sensitive to both FGFR and PI3K inhibitors alone, and even more so when the different drugs are used in combination.

Inhibition of Rho-Associated Kinase Suppresses Medulloblastoma Growth.

Medulloblastoma is one of the most common malignant brain tumor types in children, with an overall survival of 70%. Mortality is associated with metastatic relapsed tumors. Rho-associated kinases (ROCKs), important for epithelial-mesenchymal transition (EMT) and proper nervous system development, have previously been identified as a promising drug target to inhibit cancer growth and metastatic spread. Here, we show that ROCKs are expressed in medulloblastoma, with higher ROCK2 mRNA expression in metastatic compared to non-metastatic tumors. By evaluating three ROCK inhibitors in a panel of medulloblastoma cell lines we demonstrated that medulloblastoma cells were sensitive for pharmacological ROCK inhibition. The specific ROCK inhibitor RKI-1447 inhibited the tumorigenicity in medulloblastoma cells as well as impeded cell migration and invasion. Differential gene expression analysis suggested that ROCK inhibition was associated with the downregulation of signaling pathways important in proliferation and metastasis e.g., TNFα via NFκß, TGFß, and EMT. Expression of key proteins in these pathways such as RHOA, RHOB, JUN, and vimentin was downregulated in ROCK inhibited cells. Finally, we showed that ROCK inhibition by RKI-1447 suppressed medulloblastoma growth and proliferation in vivo. Collectively, our results suggest that ROCK inhibition presents a potential new therapeutic option in medulloblastoma, especially for children with metastatic disease.

ERV1/ChemR23 Signaling Protects Against Atherosclerosis by Modifying Oxidized Low-Density Lipoprotein Uptake and Phagocytosis in Macrophages.

BACKGROUND: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein-coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. METHODS: Lipidomic plasma analysis were performed after EPA supplementation in Apoe-/- mice. Erv1/Chemr23-/- xApoe-/- mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. RESULTS: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe-/- mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1-mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. CONCLUSIONS: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Resolution of Inflammation Through the Lipoxin and ALX/FPR2 Receptor Pathway Protects Against Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a progressive aortic dilation that may lead to rupture, which is usually lethal. This study identifies the state of failure in the resolution of inflammation by means of decreased expression of the pro-resolving receptor A lipoxin/formyl peptide receptor 2 (ALX/FPR2) in the adventitia of human AAA lesions. Mimicking this condition by genetic deletion of the murine ALX/FPR2 ortholog in hyperlipidemic mice exacerbated the aortic dilation induced by angiotensin II infusion, associated with decreased vascular collagen and increased inflammation. The authors also identified key roles of lipoxin formation through 12/15-lipoxygenase and neutrophil p38 mitogen-activated protein kinase. In conclusion, this study established pro-resolving signaling by means of the ALX/FPR2 receptor in aneurysms and vascular inflammation.

Rho-associated kinase is a therapeutic target in neuroblastoma.

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3ß-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.