Alpha-1-Adrenergic receptors as new targets in Neuroblastoma

High-risk neuroblastoma (NB) is an aggressive childhood tumor that originates from progenitor neural crest cells. Even if the therapeutic protocol for NB is articulate and aggressive, the outcome remains dismal, with the 5-year disease-free overall survival below rating 50%. A novel drug combination strategy can possibly provide a new solution to this unmet therapeutic need. 13-cis retinoic acid (13-cis-RA, isotretinoin) is an anti-proliferative and pro-differentiative agent currently used in the post-consolidation phase of NB therapy. To identify molecules able to potentiate the anti-proliferative activity of 13-cis-RA, NB cells were treated with a library of 169 naturally occurring polyphenols in combination with the retinoid. This in vitro screen led to the identification of isorhamnetin as a synergistic partner of 13-cis-RA, producing an 80% reduction in cell viability. At the molecular level, this synergistic effect is followed by a marked increase in the expression of a member of the catecholamine receptor superfamily: the adrenergic receptor alpha-1B (ADRA1B) suggesting that this receptor might represent a key mediator of the synergistic effect of 13-cis-RA and isorhamnetin observed in vitro. This finding redirected our attention to the class of adrenergic receptors (ARs) as novel targets in NB. To investigate the role of ADRA1B in the synergism, we generated CHP134 NB cell lines knocked-out (KO) for the receptor and observed that exposure of CHP134 KO cell to 13-cis-RA leads to a reduction of cell viability and neural differentiation. We, therefore, substituted the genetic KO strategy with the alpha-1B adrenergic antagonist, L765,314, obtaining the same results. Subsequently, we extended the analysis on the role of adrenergic receptors (AR) performing a biased screen using two libraries of AR-ligands. The screen results confirm that the molecules working as alpha-1-ARs antagonists are those that greatly increase cell sensitivity to 13-cis-RA with reduction of cell viability and increase in differentiation. We confirmed our observation in NB xenograft mice models in vivo, treating mice with a combination of 13-cis-RA and the FDA approved alpha-1 AR antagonist doxazosin. The proposed pharmacological treatment was effective in slowing tumor growth, leading to tumors of smaller size. From our results, we can conclude that the deletion or inhibition of alpha-1-AR sensitizes NB cells to 13-Cis-RA, both in terms of induction of apoptosis and neural differentiation. Since NB is a catecholamine-rich tumor, we propose that antagonization of alpha-1-AR disrupts the established autocrine pro-survival circuit generated by catecholamines in NB and restores the ability of the cells to follow the pro-differentiative and pro-apoptotic programs endorsed by 13-cis-RA. Considering the druggable nature of the alpha-1-AR receptors, we indicate this class of receptors as a novel pharmacological target for the treatment of neuroblastoma.