Cecilia Dyberg

Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)‐dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c‐MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high‐risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.

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.

Planar cell polarity gene expression correlates with tumor cell viability and prognostic outcome in neuroblastoma

BackgroundThe non-canonical Wnt/Planar cell polarity (PCP) signaling pathway is a major player in cell migration during embryonal development and has recently been implicated in tumorigenesis.MethodsTransfections with cDNA plasmids or siRNA were used to increase and suppress Prickle1 and Vangl2 expression in neuroblastoma cells and in non-tumorigenic cells. Cell viability was measured by trypan blue exclusion and protein expression was determined with western blotting. Transcriptional activity was studied with luciferase reporter assay and mRNA expression with real-time RT-PCR. Immunofluorescence stainings were used to study the effects of Vangl2 overexpression in non-tumorigenic embryonic cells. Statistical significance was tested with t-test or one-way ANOVA.ResultsHere we show that high expression of the PCP core genes Prickle1 and Vangl2 is associated with low-risk neuroblastoma, suppression of neuroblastoma cell growth and decreased Wnt/β-catenin signaling. Inhibition of Rho-associated kinases (ROCKs) that are important in mediating non-canonical Wnt signaling resulted in increased expression of Prickle1 and inhibition of β-catenin activity in neuroblastoma cells. In contrast, overexpression of Vangl2 in MYC immortalized neural stem cells induced accumulation of active β-catenin and decreased the neural differentiation marker Tuj1. Similarly, genetically modified mice with forced overexpression of Vangl2 in nestin-positive cells showed decreased Tuj1 differentiation marker during embryonal development.ConclusionsOur experimental data demonstrate that high expression of Prickle1 and Vangl2 reduce the growth of neuroblastoma cells and indicate different roles of PCP proteins in tumorigenic cells compared to normal cells. These results suggest that the activity of the non-canonical Wnt/PCP signaling pathway is important for neuroblastoma development and that manipulation of the Wnt/PCP pathway provides a possible therapy for neuroblastoma.

Transgenic increase of Wnt7b in neural progenitor cells decreases expression of T-domain transcription factors and impairs neuronal differentiation.

Wnt/beta-catenin signaling plays an important role in neural development, instructing both progenitor cell division and differentiation. During early corticogenesis, Wnt7b is expressed in a restricted expression pattern in the ventricular zone progenitor cells. However, its influence on progenitor cell behavior has not been fully studied. We report that transgenic overexpression of Wnt7b in neural progenitor cells impairs neuronal differentiation and the development of forebrain structures at embryonic day 10.5 (E10.5). This was accompanied by a decreased expression of T-domain transcription factors Tbr1 and Tbr2, in both progenitor cells and post-mitotic neurons. However, proliferation, apoptosis and the overall proportion of pax6(+) neural progenitor cells were similar to wild-type litter mates. These results suggest that Wnt signaling may affect early neural progenitor differentiation by regulating the expression of pro-neural transcription factors.

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.

Rho-associated kinase is a therapeutic target in neuroblastoma

Significance Despite intensive therapy, the cure rate for children diagnosed with high-risk neuroblastoma is still below 50%, accentuating the need for more effective therapies. Recurrent somatic mutations are relatively infrequent in neuroblastoma. We show that approximately 30% of neuroblastoma contains mutations in genes regulating Rho/Rac signaling. The mutations may be associated with activation of downstream Rho-associated kinases (ROCKs). High ROCK2 expression is associated with poor patient survival. Inhibition of ROCK activity suppresses the growth of neuroblastoma in preclinical in vivo models. ROCK inhibitors induce differentiation of neuroblastoma cells partly by glycogen synthase kinase 3β-mediated phosphorylation and degradation of MYCN proteins. These findings suggest that inhibitors of ROCK may represent a therapeutic opportunity for children with high-risk 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.

Abstract 3266: Inhibition of Rho-associated kinase 2 suppresses MYCN expression and induces differentiation of neuroblastoma

Background: Neuroblastoma is an embryonic tumor of the peripheral sympathetic nervous system that originates from cells within the neural crest. The non-canonical Wnt/planar cell polarity (PCP) signaling pathway regulates cytoskeletal organization, migration and maturation of neural crest cells during neuritogenesis. We and others have shown that high-risk neuroblastoma exhibit frequent mutations of genes controlling the activity of the Wnt/PCP signaling cascade leading to inhibition of Rac and activation of Rho resulting in downstream activation of the serine/threonine kinases Rho-Associated Coiled-Coil Containing Protein Kinases (ROCK1 and ROCK2) Methods: We performed whole-exome sequencing for 33 neuroblastoma samples of all clinical subgroups to search for mutations and genetic aberrations. Cytotoxic activity of ROCK inhibitors was studied in cell viability assays. Morphology and invasion were studied with microscopy. The molecular mechanisms were characterized using cell- and molecular biology techniques. In vivo studies (xenografts and the transgenic mouse model TH-MYCN) in mice were carried out to validate the therapeutic effects and toxicity. Results: Exome sequencing detected frequent mutations and gene aberrations in genes controlling the activity of PCP signalling. Analysis ofgene signatures and immunohistochemistry showed that high expression of ROCK1 and ROCK2 correlated with poor patient survival. Several mediators of Rho/Rac were differentially expressed in cell lines and patient samples. Using compounds blocking ROCK1 and ROCK2 activity revealed that the ROCK2 inhibitor HA-1077 effectively repressed proliferation and reduced cell viability in neuroblastoma. HA-1077 inhibited migration and induced differentiation through inhibition of neural outgrowth and inhibited MYCN protein expression, effects that were mimicked using siRNA against ROCK. Finally, HA-1077 reduced the growth of established neuroblastoma xenografts in nude mice and repressed tumor progression in a MYCN-driven mouse model of neuroblastoma (T-MYCN) Conclusions: Our results show the significance of the non-canonical/PCP signaling cascade in neuroblastoma development and progression. Furthermore, ROCK inhibition suppress the growth of neuroblastoma in preclinical models and provide a novel option for improved treatment of high-risk neuroblastoma. Citation Format: Cecilia Dyberg, David Forsberg, Susanne Fransson, Jessika Lannerholm Palm, Bjorn Helge Haug, Baldur Sveinbjornsson, Tommy Martinsson, Per Kogner, John Inge Johnsen, Malin Wickstrom. Inhibition of Rho-associated kinase 2 suppresses MYCN expression and induces differentiation of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3266. doi:10.1158/1538-7445.AM2015-3266

Abstract 3110: Targeting the Wnt/PCP signaling through ROCK: A new neuroblastoma drug target

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The non-canonical Wnt/planar cell polarity (PCP) pathway regulates cytoskeletal organization, migration and neuritogenesis. Signaling is characterized by activation of the GTPases Rho and Rac, and the downstream Rho-associated protein kinases (ROCKs). Neuroblastoma is a malignant embryonal tumor of the sympathetic nervous systems, often with poor prognosis. The need for novel therapeutic approaches is great. Genetic analyses have revealed mutations and aberrations in the regulators Rho/Rac in neuroblastoma. The aim of this study was to characterize the Wnt/PCP signaling and the effects of ROCK inhibition in neuroblastoma. Methods: Cytotoxic activity of ROCK inhibitors was studied in cell viability assays. Morphology and invasion were studied with microscopy. The molecular mechanisms were characterized using cell- and molecular biology techniques. In vivo studies in mice were carried out to validate the therapeutic effects and toxicity. Results: Several mediators in the pathway were differentially expressed in cell lines and patient samples. Using compounds blocking ROCK1 and ROCK2 activity revealed that the ROCK2 inhibitor HA-1077 effectively repressed proliferation and reduced cell viability in neuroblastoma. Additionally, HA-1077 inhibited migration and induced differentiation through initiating neural outgrowth. Furthermore, HA-1077 reduced the growth of established neuroblastoma xenografts in nude mice. Conclusions: These results suggest that non-canonical Wnt signaling in general and ROCK in particular is a promising new therapeutic target for high-risk neuroblastoma. Citation Format: Cecilia Dyberg, David Forsberg, Panos Papachristou, Jessika Lannerholm-Palm, Bjorn Helge Haug, Baldur Sveinbjornsson, Hugo Lagercrantz, Per Kogner, John inge Johnsen, Malin Wickstrom. Targeting the Wnt/PCP signaling through ROCK: A new neuroblastoma drug target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3110. doi:10.1158/1538-7445.AM2014-3110

Abstract A84: Stereotactic injections of primary medulloblastomas as a representative model of the different subgroups for preclinical drug screening

Established tumor cell lines are extensively used for screening of novel therapeutic compounds. However, during long-term culturing cells adjust to the conditions and acquire additional genetic aberrations, which may cause them to differ significantly from the original tumor. In vitro conditions may also fail to recapitulate aspects of the tumor microenvironment that may have an impact on cell fate, for example, drug clearance by the circulation, which would lead to renewed cell cycling and the heterogeneity of the tumor cell population versus the homogenous population of cells in culture. Predisposed genetic mouse models more closely recapitulate most aspects of human disease but they usually require complex breeding schemes and may suffer from incomplete tumor penetrance and a variable age of tumor onset. In order to establish a new medulloblastoma model for therapeutic studies, we have acquired primary cultures of medulloblastoma tumors representing the four different subtypes of medulloblastomas (Wnt, Shh, group 3 and group 4). Cells were grown as neurospheres and characterized regarding stem cell markers and immunological status. Early passages were collected and for each primary line, two NOD scid mice were orthotopically injected with 20.000 and 200.000 cells, respectively. Mice were closely monitored for symptoms of brain tumor burden (weight loss, hunched posture, uncoordinated movement, lethargy, poor balance and/or paralysis) during six months. Mice injected with cells from the SHH subgroup and group 3 medulloblastomas developed symptoms of brain tumors with a latency of 13-25 weeks. A positive correlation was observed between tumor latency and number of injected cells. Brains were collected and processed for histopathological confirmation of tumor prevalence, whole genome sequencing and gene expression analysis. Neurosphere cultures were also re-established to create a reproducible model for pre-clinical therapeutic trials on human medulloblastoma by serial orthotopic transplantation. Citation Format: Cecilia Krona, Cecilia Dyberg, Emma Sanden, Anna Darabi, Malin Wickstrom, Paul Northcott, John Inge Johnsen, Peter Siesjo. Stereotactic injections of primary medulloblastomas as a representative model of the different subgroups for preclinical drug screening. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A84.

Abstract 2751: Wnt/β-catenin regulates the expression of MGMT through TCF/LEF transcription: new strategy for DNA alkylators in cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects DNA from the biological effects of alkylating agents. MGMT has been implicated in resistance to alkylating drugs such as temozolomide used in the clinic against brain tumors. Temozolomide has been shown to be ineffective in tumors expressing elevated levels of MGMT. Finding agents that induce MGMT deficiency and increase the susceptibility to alkylating chemotherapeutic agents is highly warranted. Methods: The correlation between Wnt activity and MGMT expression was investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/β-catenin activity and MGMT expression using immunoblotting and real-time RT-PCR. siRNA against β-catenin, forced overexpression of β-catenin, and MGMT promotor reporter plasmids were used to study β-catenin mediated regulation on MGMT. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. In vivo, nude NMRI mice carrying xenografts were treated with celecoxib in combination with temozolomide. Results: Wnt signaling activity was shown to be correlated to increased MGMT expression levels both in primary tumors and cell lines. Transfection experiments revealed that β-catenin regulates MGMT expression. The clinically available COX-2 inhibitor celecoxib augmented the cytotoxic effect of temozolomide in cells with elevated MGMT expression through a reduction of Wnt/β-catenin activity and a subsequent reduction in MGMT expression. Conclusions: This study demonstrates that celecoxib reduces the transcription of the MGMT gene through a prostaglandin E2-Wnt/β-catenin route and thus increases the susceptibility to temozolomide. These finding are the first reported that Wnt/β-catenin pathway regulates the expression of MGMT and thus provides a rational approach for improved efficacy of temozolomide therapy in the treatment of medulloblastoma and other cancers. Citation Format: Malin Wickstrom, Ninib Baryawno, Cecilia Dyberg, Jelena Milosevic, Baldur Sveinbjornsson, Paul A. Northcott, Michael Taylor, Marcel Kool, Per Kogner, John I. Johnsen. Wnt/β-catenin regulates the expression of MGMT through TCF/LEF transcription: new strategy for DNA alkylators in cancer therapy. [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 2751. doi:10.1158/1538-7445.AM2013-2751