Targeting dysfunctional developmental signaling cascades as a tool for tailored therapy in embryonal tumors

Neuroblastoma and medulloblastoma are embryonal malignant tumors of the central and peripheral nervous systems, respectively. The overall survival for both diagnoses is 60-75% despite intense multimodal therapy. This warrants the search for novel therapeutic approaches. Hedgehog (HH) and Wingless (WNT) signaling pathways are important regulators of embryogenesis that have been associated with the development of cancer including medulloblastoma. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors. WNT signaling is divided into canonical and noncanonical signaling. Canonical signaling involves the key molecule β-catenin while noncanonical signaling includes the planar cell polarity pathway (PCP). PCP signaling including the Rac/Rho cascade are important for proper migration and differentiation of neural crest cells during neuritogenesis. In this thesis we have assessed the role of HH and WNT signaling in neuroblastoma and medulloblastoma, with the aim to gain insights and develop novel therapeutic approaches based on biological understanding of the diseases. Key molecules within the HH signaling pathway are overexpressed in neuroblastoma. We investigated the effects of inhibiting HH signaling in neuroblastoma and found that inhibition of GLI was more effective in reducing neuroblastoma growth compared to inhibition of SMO located more upstream in the signaling pathway. The GLI inhibitor GANT61 effectively repressed neuroblastoma growth in vitro and in vivo, downregulated c-MYC, GLI1, MYCN and Cyclin D1 expression and augmented the cytotoxic effects when combined with clinically used chemotherapeutic drugs. These findings suggest that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma (Paper I). The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is frequently overexpressed in cancers and is coupled to chemoresistance. In a search for new inhibitors for MGMT we investigated cellular regulators of MGMT expression in multiple cancers including neuroblastoma and medulloblastoma. We found a significant correlation between WNT signaling and MGMT expression that was confirmed by bioinformatic analysis and studies of protein expression of MGMT and β-catenin. Pharmacological as well as genetic inhibition of WNT activity downregulated the MGMT expression and restored chemosensitivity of the DNA-alkylating drug temozolomide in vitro and in vivo. These results have possible therapeutic entailments for chemoresistant cancers, especially of brain tumors where temozolomide is commonly used in treatment (Paper II). Investigating non-canonical WNT signaling in neuroblastoma revealed that overexpression of PCP core genes Prickle1 and Vangl2 led to suppression of neuroblastoma cell growth and reduced Wnt/β-catenin signaling. On the other hand, overexpression of Vangl2 in neural stem cells produced accumulation of active β-catenin and decreased differentiation, suggesting different roles of PCP proteins in tumorigenic cells compared to normal cells (Paper III). Furthermore, genetic analyses demonstrated that neuroblastoma tumors harbor frequent mutations of genes controlling neuritogenesis associated with the Rac/Rho signaling cascade. The majority of these mutations were described to result in inhibition of Rac or activation of Rho. Inhibition of ROCK, a key enzyme downstream of Rho, resulted in differentiation, inhibition of neuroblastoma cell growth and migration and degradation of MYCN protein. Small molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth both in a transgenic and in a xenograft model. This study proposes that manipulation of Rho signaling might offer new therapeutic alternatives for neuroblastoma (Paper IV). Taken together, the work in this thesis demonstrates that the embryonal signaling pathways HH and WNT may offer new therapeutic targets for neuroblastoma and medulloblastoma

Neuroblastoma—A Neural Crest Derived Embryonal Malignancy

Neuroblastoma is a neural crest derived malignancy of the peripheral nervous system and is the most common and deadliest tumor of infancy. It is characterized by clinical heterogeneity with a disease spectrum ranging from spontaneous regression without any medical intervention to treatment resistant tumors with metastatic spread and poor patient survival. The events that lead to the development of neuroblastoma from the neural crest have not been fully elucidated. Here we discuss factors and processes within the neural crest that when dysregulated have the potential to be initiators or drivers of neuroblastoma development. A more precise biological understanding of neuroblastoma causes and cell of origin is highly warranted. This will give valuable information for the development of medicines that specifically target molecules within neuroblastoma cells and also give hint about the mechanisms behind treatment resistance that is frequently seen in neuroblastoma

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

GIT1 protects against breast cancer growth through negative regulation of Notch

Notch signalling is reported to be hyperactivated in oestrogen receptor-negative (ER-) breast cancer. Here the authors show that G protein-coupled receptor kinase-interacting protein 1 (GIT1) negatively regulates Notch signalling and tumour growth in ER- breast cancer by blocking Notch ICD nuclear translocation.Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.</p

Aberrant immunostaining pattern of the CD24 glycoprotein in clinical samples and experimental models of pediatric medulloblastomas.

The CD24 glycoprotein is a mediator of neuronal proliferation, differentiation and immune suppression in the normal CNS, and a proposed cancer biomarker in multiple peripheral tumor types. We performed a comparative analysis of CD24 gene expression in a large cohort of pediatric and adult brain tumors (n = 813), and further characterized protein expression in tissue sections (n = 39), primary brain tumor cultures (n = 12) and a novel orthotopic group 3 medulloblastoma xenograft model. Increased CD24 gene expression was demonstrated in ependymomas, medulloblastomas, anaplastic astrocytomas and glioblastomas, although medulloblastomas displayed higher expression than all other tumor entities. Preferential expression of CD24 in medulloblastomas was confirmed at protein level by immunostaining and computerized image analysis of cryosections. Morphologies and immunophenotyping of CD24(+) cells in tissue sections tentatively suggested disparate functions in different tumor subsets. Notably, protein staining of medulloblastoma cells was associated with prominent cytoplasmic and membranous granules, enabling rapid and robust identification of medulloblastoma cells in clinical tissue samples, as well as in experimental model systems. In conclusion, our results implicate CD24 as a clinically and experimentally useful medulloblastoma immunomarker. Although our results encourage further functional studies of CD24 as a potential molecular target in subsets of brain tumors, the promiscuous expression of CD24 in vivo highlights the importance of specificity in the future design of such targeted treatment

Establishment and characterization of an orthotopic patient-derived Group 3 medulloblastoma model for preclinical drug evaluation

Medulloblastomas comprise a heterogeneous group of tumours and can be subdivided into four molecular subgroups (WNT, SHH, Group 3 and Group 4) with distinct prognosis, biological behaviour and implications for targeted therapies. Few experimental models exist of the aggressive and poorly characterized Group 3 tumours. In order to establish a reproducible transplantable Group 3 medulloblastoma model for preclinical therapeutic studies, we acquired a patient-derived tumour sphere culture and inoculated low-passage spheres into the cerebellums of NOD-scid mice. Mice developed symptoms of brain tumours with a latency of 17-18 weeks. Neurosphere cultures were re-established and serially transplanted for 3 generations, with a negative correlation between tumour latency and numbers of injected cells. Xenografts replicated the phenotype of the primary tumour, including high degree of clustering in DNA methylation analysis, high proliferation, expression of tumour markers, MYC amplification and elevated MYC expression, and sensitivity to the MYC inhibitor JQ1. Xenografts maintained maintained expression of tumour-derived VEGFA and stromal-derived COX-2. VEGFA, COX-2 and c-Myc are highly expressed in Group 3 compared to other medulloblastoma subgroups, suggesting that these molecules are relevant therapeutic targets in Group 3 medulloblastoma

Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment

Additional file 1: of Planar cell polarity gene expression correlates with tumor cell viability and prognostic outcome in neuroblastoma

Knockdown of Prickle1 or Vangl2 alters neuroblastoma cell viability. Transfection of neuroblastoma cells using alternative siRNA’s against Prickle1 and Vangl2 resulted in a significant increase of cell viability compared to control cells transfected with scrambled siRNA sequence (48 h) in SK-N-AS. Also in SK-N-BE (2) an increase were recorded after siRNA against Prickle1 (one-way ANOVA with Bonferroni post-test, SK-N-AS: P < 0.0001 control vs Prickle1 P < 0.0001, control vs Vangl2 P < 0.0001; SK-N-BE (2): P = 0.026: control vs Prickle1 P = 0.017). Figure S2: Correlation between expression of Prickle1 and Vangl2 and survival in neuroblastoma. a) Kaplan-Meier survival estimates of high vs low expression of Prickle1 and Vangl2 in neuroblastoma expression cohorts analyzed using the microarray analysis and visualization platform (http://r2.amc.nl). b Box-plot of Prickle1 and Vangl2 expression correlated to neuroblastoma disease stage (PPTX 486 kb

GIT1 protects against breast cancer growth through negative regulation of Notch

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth