L. Gaston1, E. Corwin1, D. Bashllari1, B. Cummings1, A. Shiang1, X. Topalli1, V. Castle1, E. Newman1 1University Of Michigan,Pediatrics, Obstetrics And Gynecology, And Pediatric Surgery,Ann Arbor, MI, USA
Introduction: Neuroblastoma (Nb) is a neoplasm of neural crest origin that accounts for 8% of childhood malignancies and 15% of cancer deaths in this same population. This discrepancy may be attributed to high-risk patients presenting with widely disseminated disease, of which only 25% survive despite cytotoxic therapies. Metastatic Nb cells display increased chemoresistance and a hypoxic environment has been shown to dedifferentiate Nb cells to an immature neuroblastic state with rapid growth and cell survival advantage. Our preliminary data demonstrates that hypoxia induces expression of DNA Ligase III (lig3), a mediator of an alternate nonhomologus end-joining (altNHEJ) DNA repair program in Nb cells. Additional work from our laboratory has shown increased expression of this efficient yet error prone repair pathway in tumorigenic Nb cell lines and in tumors of patients with poor survival outcomes. Given this, we hypothesized that induction of altNHEJ confers a survival advantage to Nb cells in hypoxic microenvironments and to standard chemotherapeutic agents.
Methods: S-type Nb cells (SHEP) were divided into three treatment groups and cultured for 1) 72h at 21% O2, 2) 72h at 1% O2, and 3) 72h at 1% O2 followed by 24h reperfusion. Cells were fixed and analyzed by immunocytochemistry (ICC) for gH2AX and lig3 with DAPI mounting solution. Images were generated with fluorescent microscopy at 40x. Quantitative analysis was carried out with ImageJ software, expressed as mean number of foci/nucleus±SD. SHEP and IMR32 Nb cells were then cultured under the above conditions with doxorubicin, a standard cytotoxic utilized in Nb treatment (0, 1, 5, and 10µg/mL). Cell viability was determined at 72h by trypan blue exclusion. Lig3 expression was analyzed by ICC as described above. All experiments were repeated in triplicate and statistical analysis was performed with ANOVA.
Results: In hypoxic conditions, Nb cells acquired more DNA damage compared to normoxic controls (0.080±0.014 vs. 0.025±0.021 gH2AX positive foci/nucleus, p<.05) with decreased overall survival (38%±4 vs 78%± 12, p<.01). Hypoxic Nb cells had relative resistance to increasing doses of doxorubicin compared to normoxic controls at 1, 5, and 10µg/mL( p<.05). Lig3 expression increased in surviving hypoxic, doxorubicin-treated Nb cells in a dose-dependent manner (p<.0001).
Conclusion: Lig3, a major DNA repair factor in altNHEJ is activated in hypoxic conditions and confers Nb cell survival advantage to Doxorubicin. Lig3 or its interacting altNHEJ partners may prove promising therapeutic targets for Nb patients with high-risk disease.