FTY720-induced endocytosis of yeast and human amino acid transporters is preceded by reduction of their inherent activity and TORC1 inhibition

Abstract

FTY720 is a sphingoid base analog that acts as an anticancer agent in animal models. Its effect on tumor cells stems largely from its ability to trigger endocytosis of several nutrient transporters. The observation that FTY720 similarly stimulates downregulation of amino acid permeases in yeast suggests that the cellular mechanisms it targets, which are still poorly characterized, are evolutionarily conserved. We here report that adding FTY720 to yeast cells results in rapid inhibition of the intrinsic activity of multiple permeases. This effect is associated with inhibition of the TORC1 kinase complex, which in turn promotes ubiquitin-dependent permease endocytosis. Further analysis of the Gap1 permease showed that FTY720 elicits its ubiquitylation via the same factors that promote this modification when TORC1 is inhibited by rapamycin. We also show that FTY720 promotes endocytosis of the LAT1/SLC7A5 amino acid transporter in HeLa cells, this being preceded by loss of its transport activity and by mTORC1 inhibition. Our data suggest that in yeast, TORC1 deactivation resulting from FTY720-mediated inhibition of membrane transport elicits permease endocytosis. The same process seems to occur in human cells even though our data and previous reports suggest that FTY720 promotes transporter endocytosis via an additional mechanism insensitive to rapamycin.

Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6

“…While we and others have previously shown that a subset of sphingolipids downregulate glucose and amino acid transporters (Barthelemy et al, 2017;Bultynck et al, 2006;Chung et al, 2001;Guenther et al, 2008;Kim et al, 2016;Romero Rosales et al, 2011;Skrzypek et al, 1998;Summers et al, 1998), the molecular mechanism is incompletely defined. As restricting nutrient access is a key mode of action for both endogenous and synthetic antineoplastic sphingolipids, understanding the molecular mechanism underlying this phenotype will provide insight into normal cell biology and clarify which tumor cells will be sensitive and resistant to therapeutic approaches that involve sphingolipid delivery or generation.…”

“…Natural and synthetic sphingolipids trap cell surface nutrient transporters in a recycling compartment CD98 (4F2hc, SLC3A2) is a chaperone protein for LAT1 (SLC7A5) and xCT (SLC7A11), two amino acid transporter proteins whose overexpression in cancer cells is correlated with poor prognosis (Selwan et al, 2016). FTY720 downregulates CD98 and LAT1 in mammalian cells (Barthelemy et al, 2017;Kim et al, 2016;Romero Rosales et al, 2011). Surface levels of CD98 are readily quantified using flow cytometry while antibodies recognizing the exofacial domains of other nutrient transporters are not generally available, most likely because glycosylation of the extracellular regions of these proteins sterically hinders antibody binding.…”

“…Using synthetic, sphingolipid-like molecules can help to overcome this problem. Similar to the endogenous sphingolipids phytosphingosine and ceramide, the FDA-approved sphingolipid drug FTY720 triggers transporter downregulation and induces a starvation-like response in both yeast and mammalian cells (Barthelemy et al, 2017;Romero Rosales et al, 2011;Welsch et al, 2004). FTY720 is a pro-drug, and FTY720 phosphate produced intracellularly is responsible for the therapeutic, immunosuppressive actions via stimulation of sphingosine-1phosphate (S1P) receptors (Brinkmann et al, 2010).…”

“…However, FTY720 and 893 produce identical disruptions in intracellular trafficking, and their IC50 values are closely matched, suggesting that effects on trafficking, not S1P receptors, are responsible for the anti-cancer activity of FTY720 (Kim et al, 2016;Romero Rosales et al, 2011). FTY720 and 893 disrupt the trafficking of transporters for glucose (GLUT1, also known as SLC2A1), pyruvate, lactate and acetate (MCT1, also known as SLC16A1; MCT4, also known as SLC16A3), glutamine (ASCT2, also known as SLC1A5) and leucine (LAT1, also known as SLC7A5) (Barthelemy et al, 2017;Kim et al, 2016). Downregulation of multiple mammalian nutrient transporters by sphingolipids is consistent with the observation that both phytosphingosine and FTY720 promote internalization of permeases for multiple amino acids (tryptophan, leucine, histidine and proline) and uracil in yeast (Barthelemy et al, 2017;Bultynck et al, 2006;Chung et al, 2000Chung et al, , 2001Skrzypek et al, 1998).…”

“…FTY720 and 893 disrupt the trafficking of transporters for glucose (GLUT1, also known as SLC2A1), pyruvate, lactate and acetate (MCT1, also known as SLC16A1; MCT4, also known as SLC16A3), glutamine (ASCT2, also known as SLC1A5) and leucine (LAT1, also known as SLC7A5) (Barthelemy et al, 2017;Kim et al, 2016). Downregulation of multiple mammalian nutrient transporters by sphingolipids is consistent with the observation that both phytosphingosine and FTY720 promote internalization of permeases for multiple amino acids (tryptophan, leucine, histidine and proline) and uracil in yeast (Barthelemy et al, 2017;Bultynck et al, 2006;Chung et al, 2000Chung et al, , 2001Skrzypek et al, 1998). In summary, synthetic molecules like FTY720 and 893 phenocopy the growth suppressive effects of endogenous sphingolipids on endocytic trafficking while minimizing the confounding effects of sphingolipid metabolism.…”