icg-001 and Neuroblastoma

Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.

Topics: Antineoplastic Agents; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Proteomics; Pyrimidinones; RNA Interference; Survival Analysis; Tretinoin; Wnt Proteins; Wnt Signaling Pathway

Neuroblastoma is the most common solid tumor in infancy. We have shown that the neuroblastoma cell line SK-N-SH contains CD133+ cells that are more resistant than 133- cells to Doxorubicin (DOX), a common chemotherapeutic agent. We hypothesize that activation of wnt signaling pathway in CD133+ cells contributes to their chemoresistance. To test this hypothesis, CD133+ cells were positively selected using magnetic micro-beads. Subsequently, CD133+ and negatively selected CD133- cells were treated with 100 ng/ml of DOX for up to 72 h. Then, cells were either lysed for total RNA extraction or fixed for immunostaining. Wnt "SIGNATURE" PCR Array was used to determine if changes in wnt related gene expression levels occurred and to estimate a pathway activity score. Expression of wnt pathway proteins β-Catenin and p-GSK3β (S-9) was determined by immunocytochemistry. Two wnt pathway inhibitors were used to determine the changes in cell viability, using the MTT assay. Results showed that wnt related genes were differentially expressed in CD133+ cells as compared to CD133- cells, both with and without DOX treatment. Pathway activity scores showed that DOX treatment significantly suppressed the wnt pathway activity in CD133- cells. Expression of β-catenin and p-GSK3β (S-9) was significantly greater in DOX treated and untreated CD133+ cells. The presence of wnt inhibitors with DOX decreased the number of live cells in CD133+ group and the percentage of live cells in both groups were equal. These data suggest that higher wnt pathway activity could be responsible for the chemoresistance of CD133+ cells in neuroblastoma cell lines.

Topics: AC133 Antigen; Antibiotics, Antineoplastic; Antigens, CD; beta Catenin; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Proliferation; Doxorubicin; Drug Resistance, Neoplasm; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Heterocyclic Compounds, 3-Ring; Humans; Immunoenzyme Techniques; Immunophenotyping; Neoplastic Stem Cells; Neuroblastoma; Peptides; Pyrimidinones; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Wnt Proteins; Wnt Signaling Pathway