J. C. Harris1, A. Russo3, E. Herrero3, J. P. O’Bryan3, B. Chiu2 1Rush University Medical Center,Surgery,Chicago, IL, USA 2University Of Illinois At Chicago,Pediatric Surgery,Chicago, IL, USA 3University Of Illinois At Chicago,Pharmacology,Chicago, IL, USA
Introduction: Neuroblastoma is the most common solid extracranial tumor in childhood and accounts for 15% of all pediatric cancer deaths. Intersectin 1 (ITSN1) protein is involved in phosphoinositide 3-kinase (PI3K) signaling, which has been shown to stabilize the MYCN oncogenic transcription factor. Silencing ITSN1 in vitro significantly reduced the anchorage-independent growth of neuroblastoma cells. We hypothesize that silencing ITSN1 with shRNA in neuroblastoma cells leads to decreased tumor growth in an orthotopic mouse model.
Methods: We have previously created stable SK-N-AS neuroblastoma cell lines with empty vector (pSR) and vector that contains scrambled shRNA (pSCR), that served as control lines, or vector that contains shRNAs to ITSN1 (Sh#1 and Sh#2). Orthotopic neuroblastoma tumors of each cell line were established in three immunocompromised mice by injecting 1×106 cells directly into the adrenal gland. Tumor volume was monitored weekly with ultrasound, using tumor volume >1000mm3 as the end point. Harvested tumors were analyzed with anti-ITSN1 antibody on Western blot. Tumor growth was analyzed using Kaplan Meier curves and student’s T-test, with p<0.05 deemed statistically significant.
Results: Orthotopic tumors were successfully created in all cell lines. At day 25-post injection, tumor size for pSR was 1235.7±761.2mm3, pSCR was 1311.4±296.5mm3, Sh#1 was 574.4± 539.9mm3, Sh#2 was 439.5±95.3mm3. Sh#2 was significantly smaller than pSCR (p=0.027), and there was a trend toward significance that Sh#1 was smaller than pSCR (p=0.13). Overall survival was superior in Sh#2, 31±1.7 days, compared to pSCR, 25±0 days (p=0.025). Again, a trend towards overall survival in Sh#1, 31±7.2 days, compared to pSCR, 25±0 days (p= 0.11). There was no difference in tumor growth between pSR with the other groups, likely due to high variance within this group, overall survival of 29.67±14.1 days. Western blot analysis of the harvested tumor showed decreased ITSN1 expression in experimental groups Sh#1 and Sh#2 compared to the control groups pSR and pSCR.
Conclusion: Silencing ITSN1 expression in neuroblastoma cells leads to decreased tumor growth in vivo. We demonstrate that orthotopic animal models can serve as an authentic ‘read out’ of molecular manipulation of ITSN1 signaling in vitro. Thus, understanding the role of ITSN1 signaling pathways in tumor development may provide new insight into neuroblastoma tumorigenesis.
