Understanding and targeting the architecture in cancer : novel therapies in neuroblastoma and medulloblastoma

Cancer is the second leading cause of death worldwide after cardiovascular diseases. In Sweden, childhood cancer is the most common cause of death in children 1-14 years of age. Owing to advances in treatment and a better understanding of tumor biology, survival rates have increased to over 80% in most Western countries. However, neuroblastoma and medulloblastoma, two embryonal childhood cancers that arise in neural tissues, do not have equally satisfactory survival rates, especially not in the high-risk patient groups. Neuroblastoma and medulloblastoma are cancers considered to arise as undifferentiated cells during embryonal development. An orchestra of inductive signals occur during embryonal development that are important to induce cells from totipotent to differentiated normal cells. One of the pathways that is essential during embryogenesis is the Wingless (Wnt) signaling pathway. While Wnt is necessary during early development, dysregulated Wnt signaling may interfere with the differentiation process and participate in the transformation into cancer. The overall aim of this thesis was to investigate the importance of Rho/Rac signaling (a part of Wnt signaling), in neuroblastoma and medulloblastoma. We especially aimed to gain insights in the function of Rho/Rac signaling in the differentiation process, in search for better understanding of the cancers and new therapies. The first two papers focused on the protein Rho Associated Coiled Coil Kinase proteins (ROCK1 and ROCK2), located downstream of Rho signaling. The teneurin family of proteins have been reported to have reoccurring genetic alterations in neuroblastoma and are suggested to be associated with Rho/Rac signaling. The third paper is exploring the role of teneurins in neuroblastoma tumorigeneses. In paper I, we investigated mutations in neuroblastoma. We showed that 27.5% of neuroblastoma patients harbor at least one somatic protein changing alteration in a gene involved in neuritogenesis, related to the Rho/Rac signaling cascade. Furthermore, RhoA and ROCK2 were found to be upregulated and more active in high-risk neuroblastoma compared to non-high-risk. In addition, higher expression of ROCK2 was associated with poor patient survival. Pharmacological or genetic inhibition of ROCK caused neuroblastoma cells to differentiate and repressed neuroblastoma cell proliferation, migration, and invasion. Furthermore, downregulation of ROCK induced degradation of the MYCN protein. Finally, studies in two different neuroblastoma mouse models demonstrated that ROCK inhibition with the drug HA1077 significantly delayed tumor growth and may hence be a new therapeutic target in neuroblastoma. In paper II, we continued studying ROCK inhibitors, but selected a more specific and potent pan-ROCK-inhibitor, RKI-1447. We demonstrated that ROCKs are present in medulloblastoma, with higher ROCK2 mRNA expression in metastatic compared to nonmetastatic tumors. Treatment with RKI-1447 inhibited medulloblastoma proliferation as well as repressed cell migration and invasion. Inhibition of ROCK through RKI-1447 also led to downregulation of genes associated with key signaling pathways in proliferation and metastasis e.g., TNFα and epithelial mesenchymal transition according to differential gene expression analysis. Lastly, we demonstrated that ROCK inhibition by RKI-1447 repressed medulloblastoma growth in vivo. Our findings propose that ROCK inhibition is a possible new therapeutic option in medulloblastoma, particularly for children with metastatic disease. In paper III, we investigated the function of teneurins (TENM1-4). TENMs have been found to have genetic alternations in neuroblastoma and are important proteins during the embryonal development in the nervous system of many species. We identified a significant role of TENM4 in neuroblastoma tumorigenicity and differentiation. Silencing TENM4 with transient knockdown led to an upregulation of genes associated with neuronal differentiation and downregulation of genes associated to pathways related to cancer. Consistent with this, a knockout model of TENM4 of the MYCN-amplified cell line SK-N-BE(2)C induced an evident morphological change consistent with a neuronal like differentiation in the knockout cells. The TENM4 knockout showed an impaired growth rate and decreased MYCN expression compared to wild type cells. Furthermore, the TENM4 knockout cells did not form tumors when injected subcutaneously in mice, in contrast to wild type cells that developed tumors within four weeks. Moreover, we detected a significantly higher protein and mRNA expression of TENM4 in high-risk vs. non-high-risk and MYCN-amplified vs. non-MYCN-amplified human tumors. Our data proposes that a subpopulation of neuroblastomas with MYCN-amplification expresses TENM4, and that TENM4 exhibits functions in neuroblastoma development. Consequently, TENM4 may be a potential therapeutic target in neuroblastoma

Carbopol hydrogel/sorbitan monostearate-almond oil based organogel biphasic formulations: Preparation and characterization of the bigels

Purpose: To obtain and evaluate carbopol hydrogel/sorbitan monostearate-almond oil-based organogel biphasic formulations (bigels) as a semi-solid vehicle for medicated topical applications.Methods: Bigel formulations were obtained under mild conditions at a hydrogel/organogel ratio of 80/20, 70/30, and 60/40 (w/w). Their stability, viscosity, spreadability, microarchitecture, and acute skin toxicity were evaluated.Results: Two formulations, prepared at ratios of 80/20 and 70/30, were stable based on intermediate stability testing, and had a similar viscosity and spreadability (38.0 ± 1.0 mm and 37.3 ± 0.6 mm, p > 0.05, respectively). Both of these formulations had a bimodal droplet size distribution and very similar values for the droplet mean diameter (0.33 ± 0.05 μm and 2.35 ± 0.44; and 0.34 ± 0.04 μm and 2.59 ± 0.21 μm). The formulation obtained at a ratio of 60/40 was unstable during storage. The in vivo results did not reveal any signs of skin toxicity.Conclusion: Considering their beneficial properties, the developed bigels are a potential semi-solid vehicle for topical application and exhibit a moisturizing effect.Keywords: Almond oil, Bigels, Carbopol hydrogel, Moisturizing effect, Organogel, Sorbitan monostearat

Omega-3 fatty acids decrease CRYAB, production of oncogenic prostaglandin E-2 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 omega 3-long chain polyunsaturated fatty acids (omega 3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model.Main methods: Effects of omega 3-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: omega 3-LCPUFA decreased prostaglandin E2 (PGE(2)) 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 PGE(2) and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All omega 3-LCPUFA and dihomo-gamma-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among omega 3-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

Rho-associated kinase is a therapeutic target in neuroblastoma

Source at: http://doi.org/10.1073/pnas.1706011114 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 3β-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

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 3β-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

Comparison of the results of serum total protein concentration measured by 3 methods: Preliminary results

The present study provides the results from a comparative study of the 3 commonly used methods for total protein (TP) measurement. The experiments were carried out with 6 dogs (4-7 year-old, weighing 12.8 ± 1.4 kg). Five blood samples were obtained by saphena venepuncture from all dogs, during the time course of the experimentally induced infection with Staphylococcus intermedius, administered subcutaneously at a dose rate of 5 ml of 1.109 CFU/ml within 14 days. TP concentration was measured by 2 macro protein techniques - biuret method (commonly used) and method of Lowry, and a modified version of biuret method (micro protein technique), suggested by Popov. Serum TP concentration determined by the method of Lowry was significantly (P < 0.001) higher than the ones obtained by standard biuret and Popov's methods. The mean differences between TP values obtained by standard biuret technique and Lowry's method and, Lowry's and Popov's method were 18.6 g/l and 23.5 g/l, respectively. There was no statistically significant difference between standard biuret method and its modified version suggested by Popov

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

Summary: 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. Key Words: abdominal aortic aneurysms, cardiovascular disease, eicosanoids, inflammation, lipoxygenas