E. J. Rellinger1, H. Song2, S. Park2, P. Paul1, B. T. Craig1, J. Qiao1, V. Athanasios2, D. R. Gius2, D. H. Chung1 1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA 2Northwestern University,Radiation Oncology,Chicago, IL, USA
Introduction: Neuroblastoma (NB) is a pediatric solid tumor that arises from failed differentiation of neural crest progenitors. Infants and children with high-risk NB have a poor prognosis despite our most aggressive therapies, highlighting the need for novel drug targets. Sirtuins are a family of seven NAD+-dependent histone deacetylases that have emerged as key regulators of cell fate, DNA damage repair, neuronal protection, and tumorigenesis. However, the role of Sirtuins in NB tumorigenicity is unknown. The purpose of this study was to evaluate the exact role of Sirtuins in regulating NB cell proliferation and differentiation.
Methods: Sirtuin inhibition in BE(2)-C human NB cells was performed using nicotinamide (NAM), a nonspecific Sirtuin inhibitor, or shRNA transfection (shSIRT1, shSIRT2, shSIRT3, shSIRT6, shSIRT7). Cell viability was determined using CCK-8 assays. Confocal microscopy and immunoblotting with neuron specific enolase (NSE) and neurofilament-M (NF-M) were employed to assess for neural differentiation. Rescue experiments in shSirt6 cells were completed with pCDH-SIRT6 transfection. Sirt6 expression in human tumor sections was assessed using immunohistochemistry and counterstained with H&E to score histologic grade. Cell cycle analyses were completed using flow cytometry. Anchorage-independent soft agar colony growth was used for in vitro representation of tumor invasiveness.
Results: NAM treatment decreased cell viability, induced morphologic differentiation, and increased protein expression of p21 (CDK inhibitor) and NSE, our respective markers of cell cycle arrest and neural differentiation. Only transfection with shSirt6 similarly decreased proliferation and induced cell morphology and neural marker expression patterns consistent with neural differentiation in BE(2)-C cells (Fig. A). Silencing Sirt6 induced NB cell arrest at G0/G1 cell cycle phase and inhibited DNA synthesis and soft agar colony formation. Rescue of cell proliferation, morphologic differentiation, cell cycle arrest, and protein expression was achieved using pCDH-SIRT6 transfection (Fig. B). Sirt6 expression was decreased in differentiated human NB sections. Retinoic acid treatment diminished Sirt6 expression and induced differentiation synergistically with Sirt6 silencing.
Conclusion: Targeted inhibition of Sirt6 in NB downregulates tumor growth and promotes neural differentiation synergistically with retinoic acid treatment. These findings suggest that Sirt6 is a novel therapeutic target in NB and highlights clinical strategies to optimize its efficacy.
