Metformin action on Nuclear transport

Abstract

The nuclear pore complex (NPC), the sole gateway between the nucleus and the cytoplasm for macromolecules, facilitates protein transport between the two compartments. In young, healthy cells, there is a size threshold below which proteins can diffuse across the NPC via passive transport with little impedance. In aging and disease, the latter is known to deteriorate, leading to much more promiscuous transport of larger molecules that are typically restricted from passage through the NPC in a healthier, younger cell. However, this deterioration of the NPC can be halted or even reversed by metformin, a member of the biguanide class of drugs commonly prescribed in type 2 diabetes. We previously demonstrated that metformin action in aging and cancer requires intact function of the NPC, that is, that mutations in or reduced function of key nucleoporins (proteins that make up the NPC), lead to resistance to the prolongevity and anticancer effects of biguanides, including metformin and the related compound phenformin. To fully understand the molecular mechanism for this finding, we studied the dynamics of nucleocytoplasmic transport in transformed cells with biguanide treatment. We leveraged fluorescently-labeled dextran molecules in HeLa cells as a model system and examined passive nuclear import through the NPC in digitonin-permeabilized cells. Phenformin leads to a dose- and time-dependent restriction of passive nucleocytoplasmic transport. We find that biguanide action on mitochondria is likely to be responsible for the effects of the drugs on nucleocytoplasmic transport. Pharmacologic inhibition of the electron transport chain is sufficient to prompt restriction in passive nucleocytoplasmic transport. Further, HeLa cells lacking mitochondria (Rho0) constitutively demonstrate restricted nucleocytoplasmic transport, and this effect is not additively enhanced by phenformin. Mechanistically, biguanide-mediated reduction of nucleocytoplasmic transport is mediated by a reduced O-GlcNacylation of Nucleoporin 62 (Nup62). This is likely the result of increased mRNA expression of OGA, the sole enzyme that removes O-GlcNAc modifications from protein and a parallel decrease in OGT protein, the sole enzyme that places O-GlcNAc onto proteins. Chemical inhibition of OGA prohibits phenformin mediated nucleocytoplasmic transport restriction, indicating that the effects of biguanide effects on the NPC require reductions in O-GlcNAcylated Nup62 in order to restrict dysregulated passive nucleocytoplasmic trafficking in neoplastic cells. This suggests that O-GlcNAc modification of NUPs may be a therapeutic target in cancer and aging-related diseases.