title: PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.
abstract: Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: S-J
authors.initials: SJ
authors.lastname: Fan
authors.affiliation: Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: C
authors.initials: C
authors.lastname: Snell
authors.affiliation: Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: H
authors.initials: H
authors.lastname: Turley
authors.affiliation: Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: J-L
authors.initials: JL
authors.lastname: Li
authors.affiliation: Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: R
authors.initials: R
authors.lastname: McCormick
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: S M W
authors.initials: SM
authors.lastname: Perera
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: S
authors.initials: S
authors.lastname: Heublein
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: S
authors.initials: S
authors.lastname: Kazi
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: A
authors.initials: A
authors.lastname: Azad
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: C
authors.initials: C
authors.lastname: Wilson
authors.affiliation: Molecular Oncology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
authors.forename: A L
authors.initials: AL
authors.lastname: Harris
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: D C I
authors.initials: DC
authors.lastname: Goberdhan
mesh_terms: D026905:Amino Acid Transport Systems / Q000096:biosynthesis / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism*; D045325:HCT116 Cells; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016513:Mice, SCID; D046912:Multiprotein Complexes / Q000378:metabolism*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D016896:Treatment Outcome
chemical_list: D026905:Amino Acid Transport Systems; D000596:Amino Acids; D046912:Multiprotein Complexes; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus