The PI3-kinase (PI3K)/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signalling cascades promote normal growth and are frequently hyperactivated in tumour cells. mTORC1 is also regulated by local nutrients, particularly amino acids, but the mechanisms involved are poorly understood. Unexpectedly, members of the proton-assisted amino acid transporter (PAT or SLC36) family emerged from in vivo genetic screens in Drosophila as transporters with uniquely potent effects on mTORC1-mediated growth. Here we show the two human PATs that are widely expressed in normal tissues and cancer cell lines, PAT1 and PAT4, behave similarly to fly PATs when expressed in Drosophila. siRNA knockdown reveals that these molecules are required for activation of mTORC1 targets and for proliferation in human MCF-7 breast cancer and HEK-293 embryonic kidney cell lines. Furthermore, activation of mTORC1 in starved HEK-293 cells stimulated by amino acids requires PAT1 and PAT4, and is elevated in PAT1-overexpressing cells. Importantly, in HEK-293 cells, PAT1 is highly concentrated in intracellular compartments, including endosomes, where mTOR shuttles upon amino acid stimulation. Our data are therefore consistent with a model in which PATs modulate mTORC1's activity not by transporting amino acids into the cell, but by modulating the intracellular response to amino acids.

Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

Purpose: Endometrioid ovarian carcinoma (ENOC) is generally associated with a more favorable prognosis compared with other ovarian carcinomas. Nonetheless, current patient treatment continues to follow a “one-size-fits-all” approach. Even though tumor staging offers stratification, personalized treatments remain elusive. As ENOC shares many clinical and molecular features with its endometrial counterpart, we sought to investigate The Cancer Genome Atlas–inspired endometrial carcinoma (EC) molecular subtyping in a cohort of ENOC. Experimental Design: IHC and mutation biomarkers were used to segregate 511 ENOC tumors into four EC-inspired molecular subtypes: low-risk POLE mutant (POLEmut), moderate-risk mismatch repair deficient (MMRd), high-risk p53 abnormal (p53abn), and moderate-risk with no specific molecular profile (NSMP). Survival analysis with established clinicopathologic and subtype-specific features was performed. Results: A total of 3.5% of cases were POLEmut, 13.7% MMRd, 9.6% p53abn, and 73.2% NSMP, each showing distinct outcomes (P < 0.001) and survival similar to observations in EC. Median OS was 18.1 years in NSMP, 12.3 years in MMRd, 4.7 years in p53abn, and not reached for POLEmut cases. Subtypes were independent of stage, grade, and residual disease in multivariate analysis. Conclusions: EC-inspired molecular classification provides independent prognostic information in ENOC. Our findings support investigating molecular subtype–specific management recommendations for patients with ENOC; for example, subtypes may provide guidance when fertility-sparing treatment is desired. Similarities between ENOC and EC suggest that patients with ENOC may benefit from management strategies applied to EC and the opportunity to study those in umbrella trials.