MYCN In Cells Research Articles

The tumor suppressor CREBBP and the oncogene MYCN cooperate to induce malignant brain tumors in mice

The tumor suppressor and chromatin modifier cAMP response element-binding protein binding protein (CREBBP) and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), a member of the MYC oncogene family, are critically involved in brain development. Both genes are frequently mutated in the same tumor entities, including high-grade glioma and medulloblastoma. Therefore, we hypothesized that alterations in both genes cooperate to induce brain tumor formation. For further investigation, hGFAP-cre::CrebbpFl/Fl::lsl-MYCN mice were generated, which combine Crebbp deletion with overexpression of MYCN in neural stem cells (NSCs). Within eight months, these animals developed aggressive forebrain tumors. The first tumors were detectable in the olfactory bulbs of seven-day-old mice. This location raises the possibility that presumptive founder cells are derived from the ventricular-subventricular zone (V-SVZ). To examine the cellular biology of these tumors, single-cell RNA sequencing was performed, which revealed high intratumoral heterogeneity. Data comparison with reference CNS cell types indicated the highest similarity of tumor cells with transit-amplifying NSCs or activated NSCs of the V-SVZ. Consequently, we analyzed V-SVZ NSCs of our mouse model aiming to confirm that the tumors originate from this stem cell niche. Mutant V-SVZ NSCs showed significantly increased cell viability and proliferation as well as reduced glial and neural differentiation in vitro compared to control cells. In summary, we demonstrate the oncogenic potential of a combined loss of function of CREBBP and overexpression of MYCN in this cell population. hGFAP-cre::CrebbpFl/Fl::lsl-MYCN mice thus provide a valuable tool to study tumor-driving mechanisms in a key neural stem/ progenitor cell niche.

Abstract B49: Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells

Abstract Neuroblastoma (NB) is a pediatric malignancy derived from the sympaticoadrenal lineage of neural crest. It is the most commonly occurring extra-cranial nervous system malignancy of infants and children under the age of 5. Long-term survival is less than 40% in patients with high risk NB and over half of the survivors relapse. Emerging evidence supports the notion that there exists within tumors a small population of cells exhibiting stem-like properties, termed cancer stem-like cells (CSC), that is responsible for clinical relapse; therefore, investigating CSC targets may lead to improved treatments. Recent studies have demonstrated that deubiquitinating enzymes (DUBs) of the ubiquitin proteasome system are important players in cancer-associated pathways. These enzymes could be potential effective therapeutic targets. Previous work has identified expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1), a DUB, in the CSC pool of neuroblastoma cells. Our studies are focused on the potential pathways and mechanism by which UCHL1 is acting in the CSC pool. We performed in vitro analyses on NB cell lines SK-N-BE(2), SK-N-AS, and SK-N-DZ. These cells differ in their MYCN amplification and P53 status. The CSC pool was enriched using the sphere formation assay. The small molecule inhibitor, LDN-57444, was used to impair UCHL1 activity. Furthermore, site-directed mutagenesis was used to generate the C90S mutant, which also blocks UCHL1 activity. The effect of blocking UCHL1 activity on cellular proliferation and CSC self-renewal was determined by the MTS assay and sphere formation assay respectively. We further confirmed the effect of impairing UCHL1 activity on the CSC population using flow cytometry by assessing the CD133 and side population. The soft agar assay was used to study the anchorage independent growth of UCHL1 impaired cells. Droplet digital PCR was used to investigate the effect of impaired UCHL1 activity on genes in the Wnt signaling pathway. Immunoprecipitation assays were used to confirm presence of C90S mutant in transfected cells and to determine potential interaction between UCHL1 and Wnt signaling proteins such as β-catenin. UCHL1 inhibition with LDN-57444 demonstrated a dose-dependent decrease in cellular proliferation and impaired sphere formation capability (read-out for self-renewal). We observed differential gene expression levels of MYC and MYCN in cells with UCHL1 inhibited activity via LDN-57444 treatment. C90S mutant transfected cells also demonstrated impaired self-renewal. C90S mutant cells showed decreased gene expression of Wnt signaling proteins and differential expression of MYC and MYCN. Immunoprecipitation studies and CD133 and side population analyses for assessing the CSC population are ongoing. Our data indicates that UCHL1 is potentially an important player in the regulation of CSC self-renewal. Our results also suggest that UCHL1 may mediate self-renewal through the MYC family of transcription factors. The MYC family has been shown to be promising targets in NB. Reduced MYC or MYCN gene expression in NB leads to cell cycle arrest and/or apoptosis. Further investigation of the effect of UCHL1 impaired activity on Wnt signaling will clarify whether UCHL1 is affecting MYC transcription through this pathway. Our studies indicated that UCHL1 might potentially be an effective therapeutic target for the CSC population to supplement current treatments. Citation Format: Judy C. Chang, Jaclyn Y. Hung. Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells. [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 B49.

Targeted Expression of Human MYCN Selectively Causes Pancreatic Neuroendocrine Tumors in Transgenic Zebrafish

The zebrafish model organism has been used extensively for studies of genetic pathways in development, indicating its potential applicability to cancer. Here we show that targeted expression of MYCN in cells of the pancreatic islet induces neuroendocrine carcinoma. Four transgenic fish developed abdominal tumors between 4 and 6 months of age, and histologic analysis revealed lobulated arrangements of neoplastic cells with expression of the MYCN transgene. The tumors also expressed insulin mRNA, and pancreatic exocrine cells and ducts were identified within the neoplasms, indicating a pancreatic origin for the tumor. Transmission electron microscopy revealed cytoplasmic, endocrine-dense core granules, analogous to those found in human neuroendocrine tumors. Our studies establish a zebrafish transgenic model of pancreatic neuroendocrine carcinoma, setting the stage to evaluate molecular pathways downstream of MYCN in this vertebrate forward genetic model system.