C. V. Romain1, J. Qiao1, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA
Introduction: Solid tumors, having outgrown their blood supply, are known to exhibit central area of tumor necrosis and cell death. In the tumor microenvironment, a process of selection occurs where signaling pathways are activated which promote tumor growth and resistance to anti-tumor strategies despite relative hypoxic environment. Hypoxia is known to upregulate cell signaling through tumor necrosis factor receptor (TNFR)-1 and induces aggressive phenotype in certain cancer cells; however, its role in neuroblastoma is largely unknown. Here, we developed an in vitro system to select out a subclonal population of human neuroblastoma cells conditioned by exposure to chronic hypoxia. The purpose of this study was to characterize the exact cell signaling pathways contributing to the survival and growth of virulent hypoxia-conditioned human neuroblastoma cells.
Methods: Human neuroblastoma BE(2)-C cells were plated under normoxic conditions to 70% confluence in 100 mm dish. They were then transferred to hypoxic chamber (1% O2) until > 90% cells had undergone cell death (detached) for a period of 7-10 days. The remaining viable cells, labeled as HS1, were plated and incubated under normoxia and allowed to re-grow to 70% confluence. This cycle was repeated and the recovered subclonal cell population was designated as HS2. Gene expression for death receptor pathway was performed on BE(2)-C parental vs. HS2 cells. Transcription activity of TNFR superfamily member1A (TNFR1) and TNF-α were assessed using RT-PCR for BE(2)-C and HS2 cells under normoxia and hypoxia, as well as for BE(2)-C and HS1 cells after TNFR1 silencing. Cell viability was measured in BE(2)-C and HS1 cells with or without treatment with vincristine (200 nM) or cisplatin (2 μ M).
Results: Focused gene array showed >100-fold increase in the expression of TNFR1 in HS2 cells as compared to control. RT-PCR confirmed nearly 8-fold increase in mRNA levels of TNFR1 and TNF-α in HS1 and HS2 cells as compared to BE(2)-C parental cells. After silencing of TNFR, there were increases in TNF-α in both BE(2)-C/siTNFR1 as well as HS1/siTNFR1 when compared to control cells. An increased HS1 cell proliferation was found when compared to BE(2)-C cells under either normoxic or hypoxic conditions at 24 h with or without the chemotherapeutic agents. This trend continued at 48 h time point under normoxia but was lost under hypoxic conditions.
Conclusion: Upregulation of TNFR1 was observed in HS1/2, subclonal populations of human neuroblastoma cells created via chronic hypoxia cycling. Increased expression of TNFR/TNF-α mRNA in both HS1 and HS2 suggests that activation of this signaling pathway may contribute to more robust cell survival in hypoxia-conditioned subclonal neuroblastoma cells. HS1 cells exhibited refractoriness to chemotherapy agents, as well as enhanced cell proliferation, mimicking chemoresistance patterns often seen in high-risk neuroblastoma.