Introduction: 13-cis retinoic acid (13-cisRA) is used clinically in the treatment of high-risk neuroblastoma, but retinoid disease resistance remains a major challenge. Increased retinoid metabolism has been proposed to contribute to retinoid resistance. The CYP26 enzymes (CYP26A1, CYP26B1, and CYP26C1) are the intracellular metabolizers of all-trans retinoic acid, and drugs that inhibit their functions have been developed. However, the efficacy of these drugs alone or in combination with retinoid has not been evaluated in neuroblastoma.
Methods: Retinoid sensitive cell lines SHSY5Y and NB1648 and retinoid-resistant cell lines SK-N-RA and SK-N-AS were grown in the presence of vehicle, CYP26 inhibitor talarozole, nonspecific CYP inhibitor ketoconazole, 13-cisRA, or a combination of CYP inhibitor and 13-cisRA. Cell viability was measured by Alamar blue (ThermoSci), and cell morphology was analyzed with the Incucyte Zoom (Essen). Gene expression of differentiation biomarkers was evaluated by RT-qPCR, and protein expression was evaluated by Western blot. Synergy testing was performed with Compusyn software. Dataset evaluations were done with R2 and Oncogenomics online platforms.
Results: All four cell lines had decreased proliferation and cell viability when treated with talarazole, while none were affected when treated with ketoconazole. Combination treatment of talarazole with retinoic acid resulted in synergistic inhibition of proliferation, although no significant change in cell morphology was noted. Gene and protein expression evaluation demonstrated MYCN expressed decreased while NTRK1 and PBX1 expression increased, consistent with retinoid-induced differentiation. Low expression of CYP26B1 specifically was prognostic of survival in a two datasets of patient tumors (n=88, n=102).
Conclusions: CYP26 inhibition has a synergistic effect with retinoid therapy in vitro, with increased markers of retinoid activity and differentiation. Further evaluations including in in vivo models are warranted to evaluate the potential benefits of combination retinoid and CYP26 inhibitor therapy to improve therapy in neuroblastoma.