M3 Muscarinic Acetylcholine Receptors Inhibit Autophagy through Activation of mTORC1 in Human Neuroblastoma Cells.

Abstract

Activation of Gq/11‐coupled muscarinic acetylcholine receptors (mAChRs) has been shown to promote the growth and survival of different types of cancers derived from epithelial and endothelial cells [1]. Previous studies in human neuroblastoma cells have reported that M3 mAChRs prevent apoptotic cell death induced by a number of cytotoxic agents [2,3]. However, there is little information on the effect of mAChRs on autophagy, an evolutionary conserved self‐degradation process that may act as a tumor‐suppressor pathway. In the present study we show that in human SH‐SY5Y neuroblastoma cells activation of M3 mAChRs inhibits autophagy triggered by serum and L‐glutamine removal from the growth medium. Cell exposure to either the cholinergic agonist carbachol (CCh) (30 μM) or the mAChR agonist oxotremorine‐M (100 μM) inhibited the formation of the autophagosome marker LC3‐II and increased the accumulation of the autophagy substrate p62. These effects were blocked by either atropine or the M3 mAChR‐preferring antagonist darifenacin, but not MT7, a selective M1 mAChR antagonist. The inhibitory effect of CCh on autophagy was prevented by blockade of Gq/11 with YM254890 and by inhibition of protein kinase C (PKC), and was mimicked by the PKC activator phorbol 12‐myristate 13‐acetate (PMA). Both CCh and PMA induced mTORC1 activation and enhanced the phosphorylation/inactivation of ULK1, a mTORC1 substrate and a key initiator of autophagy. Inhibition of mTORC1 by rapamycin prevented CCh‐induced ULK1 phosphorylation and autophagy inhibition. The data indicate that in SH‐SY5Y cells Gq/11‐coupled M3 mAChRs inhibit autophagy by stimulating the PKC‐mTORC1 signaling pathway. The attenuation of the self‐degradation process induced by autophagy may contribute to the growth promoting effects of M3 mAChRs in human neuroblastoma cells.Support or Funding InformationSupported by Regione Autonoma della Sardegna, L.R.7,‐2012‐CRP‐60567