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

Sammanfattning: 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.

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