Determining The Regulatory Role of UFD1 in MYCN-driven Neuroblastoma

Determining The Regulatory Role of UFD1 in MYCN-driven Neuroblastoma Results Discussion Tumor Progression Images For tumor onset, the difference between the control group (3471-) and the UFD1 mutant group (3471+) was not statistically significant. For week 5 the P-value was 0.8182. For tumor progression, the difference between the two groups was statistically significant (P <0.05) every week that the fish were imaged. The P-value for week 0 was 0.0298. Week 2 was 0.0262, week 4 was 0.0270, week 6 was 0.0133, week 8 was 0.0076, week 10 was 0.0210, and week 12 was 0.0060. These P-values indicate that the allelic loss of UFD1 in the experimental group (3471+) played a statistically significant role in mitigating the growth of tumors during progression. This is crucial in our understanding of how we can better control the progression of neuroblastoma. Since the control group (3471-) fish contained two copies of UFD1 and demonstrated more significant tumor growth, we can conclude that the addition of UFD1 plays in an evident role in accelerating disease. Although there was no statistically significant difference between the control fish (3471-) and the UFD1 knocked group (3471+) during tumor onset, this piece information is still quite valuable. The lack of significance tells us that presence of a second allele of UFD1 does not affect initial development of tumors. In short, from birth to the start of progression, tumor development appeared to be similar in both the control group and UFD1 knocked group. Future studies should investigate how the presence of UFD1 effects tumor development in other animal models such as a murine model. Furthermore, UFD1 should be examined as a potential target for other cancers. Lastly, gene editing with UFD1 should be studied as a new treatment angle, as the incredibly significant results of this study warrant more research in this area. Introduction Despite the extensive research on pediatric neuroblastoma, little is known about the mechanism and role that the ubiquitin fusion degradation 1 (UFD1) gene plays in tumorigenesis. UFD1 is part of the endoplasmic reticulum (ER)-associated degradation (ERAD) complex and reduces cell stress by expediting the destruction of misfolded proteins from the ER. 40% of patients experience MYCN- amplified neuroblastoma and it has been shown that MYCN transcriptionally upregulates UFD1 in aggressive disease. Previous patient data has shown that patients with MYCN-amplified neuroblastoma experience a more aggressive form of the disease and poor prognosis compared to patients with MYCN non-amplified tumors. MYCN 3471- MYCN 3471+ The left column images show a control The right column images show a (3471-) zebrafish at week 0, 4, 8, 12 of UFD1 knocked (3471+) zebrafish at progression. week 0, 4, 8, 12 of progression. Methods For this project, a neuroblastoma zebrafish model dβh:MYCN;dβh:EGFP that recaptures human disease to investigate the role of UFD1 in vivo. The tumor fish were then crossed to UFD1 heterozygous fish resulting in an allelic loss of UFD1. These fish were then monitored weekly for overall survival and tumor onset and progression through fluorescent microscopy. ImageJ was used to quantify and analyze tumor progression and onset of the two groups of fish—the homozygous group for UFD1 and the heterozygous group for UFD1. For onset, tumor size was calculated by multiplying the area of the tumor by the mean. Tumor intensity was calculated by subtracting the background measurements. Relative tumor size was calculated using a ratio of the size of the tumor divided by the size of the eye of each fish. In order for fish to be classified as “tumor fish” their tumors had to meet 2 criteria. Firstly, their tumors had to be over 15 in size. Secondly, the tumor had to be larger than the size of the eye. Once each fish met these criteria, their onset was complete and they were transferred to progression tanks where they were then imaged every two weeks. Data for tumor size during onset and progression were compiled for both the UFD1 fish and the control group. T-tests were run to compare the UFD1 knocked fish to the control fish. Tumor Onset Images At onset, 3471- and 3471+ fish appear nearly identical as demonstrated below. The difference between the two groups for week 5 in the onset curve was not statistically significant, as the P-value was 0.8182 MYCN 3471+ MYCN 3471-