Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer.

Proteins overexpressed in early-stage cancers may serve as early diagnosis and prognosis markers as well as targets for cancer therapies. In this study, we examined the expression of an essential amino acid carrier SLC7A5 (LAT1, CD98, or 4F2 light chain) in cancer tissue from two well-annotated cohorts of 575 cases of early-stage and 106 cases of late-stage colorectal cancer patients. Immunohistochemistry showed SLC7A5 overexpression in 72.0% of early-stage and 56.6% of late-stage cases. SLC7A5 expression was not influenced by patient gender, age, location, or mismatch repair status, although it appeared to be slightly less prevalent in tumors of mucinous differentiation or with lymphovascular invasion. Statistical analyses revealed a positive correlation between SLC7A5 overexpression and both overall survival and disease-free survival in early-stage but not late-stage cancers. Co-expression analyses of the TCGA and CPTAC colorectal cancer cohorts identified a network of gene transcripts positively related to SLC7A5, with its heterodimer partner SLC3A2 having the highest co-expression score. Network analysis uncovered the SLC7A network to be significantly associated with ncRNA such as tRNA processing and the mitotic cell cycle. Since SLC7A5 is also a marker of activated lymphocytes such as NK, T, and B lymphocytes, SLC7A5 overexpression in early colorectal cancers might trigger a strong anti-tumor immune response which could results in better clinical outcome. Overall, our study provides clear evidence of differential SLC7A5 expression and its prognostic value for early-stage colorectal cancer, although the understanding of its functions in colorectal tumorigenesis and cancer immunity is currently rather limited and awaits further characterization.

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

High expression of SLC7A1 in high-grade serous ovarian cancer promotes tumor progression and is involved in MAPK/ERK pathway and EMT.

Our previous studies have shown that upregulation of SLC7A1 in epithelial ovarian cancer (EOC) tumor cells significantly increases cancer cell proliferation, migration, and cisplatin resistance; however, the molecular mechanism by which SLC7A1 functions in EOC remains unknown. In later studies, we found that SLC7A1 is also highly expressed in the interstitial portion of high-grade serous ovarian cancer (HGSOC), but the significance of this high expression in the interstitial remains unclear. Here, we showed the Interstitial high expression of SLC7A1 in HGSOC by immunohistochemistry. SLC7A1 enriched in cancer-associated fibroblasts (CAFs) was upregulated by TGF-ß1. Transwell assay, scratch assay, cck8 assay and cell adhesion assay showed that SLC7A1 highly expressed in CAFs promoted tumor cells invasion, migration and metastasis in vitro. The effect of SLC7A1 on MAPK and EMT pathway proteins in ovarian cancer (OC) was verified by RNA sequencing and western blotting. Overexpression of SLC7A1 in OC is involved in MAPK/ ERK pathway and EMT. In general, in HGSOC, CAFs overexpressing SLC7A1 supported the migration and invasion of tumor cells; SLC7A1 is highly expressed in ovarian cancer and is involved in ERK phosphorylation and EMT signaling in MAPK signaling pathway. This suggests that SLC7A1 may be a potential therapeutic target for OC metastasis.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

Radiosynthesis and Preclinical Evaluation of m-[18F]FET and [18F]FET-OMe as Novel [18F]FET Analogs for Brain Tumor Imaging.

O-([18F]Fluoroethyl)-l-tyrosine ([18F]FET) is actively transported into the brain and cancer cells by LAT1 and possibly other amino acid transporters, which enables brain tumor imaging by positron emission tomography (PET). However, tumor delivery of this probe in the presence of competing amino acids may be limited by a relatively low affinity for LAT1. The aim of the present work was to evaluate the meta-substituted [18F]FET analog m-[18F]FET and the methyl ester [18F]FET-OMe, which were designed to improve tumor delivery by altering the physicochemical, pharmacokinetic, and/or transport properties. Both tracers could be prepared with good radiochemical yields of 41-56% within 66-90 min. Preclinical evaluation with [18F]FET as a reference tracer demonstrated reduced in vitro uptake of [18F]FET-OMe by U87 glioblastoma cells and no advantage for in vivo tumor imaging. In contrast, m-[18F]FET showed significantly improved in vitro uptake and accelerated in vivo tumor accumulation in an orthotopic glioblastoma model. As such, our work identifies m-[18F]FET as a promising alternative to [18F]FET for brain tumor imaging that deserves further evaluation with regard to its transport properties and in vivo biodistribution.

L-type Amino Acid Transporter 1 as a Therapeutic Target in Pancreatic Cancer.

Metabolic rewiring is a key feature of cancer cells to support the demands of growth and proliferation. The metabolism of amino acids is altered in many cancers, including pancreatic cancer. The cellular uptake of amino acids is regulated by amino acid transporters, such as L-type amino acid transporter 1 (LAT1). Accumulating evidence suggests that LAT1 is overexpressed in pancreatic cancer and confers a poor prognosis. Here we discuss the prospects of utilizing LAT1 as a novel target for pancreatic cancer therapy.

[Effect of L-Type Amino Acid Transporter 1 Expression on Clinicopathological Features and Prognosis of Non-Hodgkin's Lymphoma].

OBJECTIVE: To detect the expression of L-type amino acid transporter 1 (LAT1) in non-Hodgkin's lymphoma (NHL) tissues, and analyze its effect on clinicopathological characteristics and prognosis of patients. METHODS: A total of 92 NHL patients who were treated in our hospital from January 2017 to April 2019 were collected. The expression of LAT1 in NHL tissue was detected by immunohistochemistry and compared between patients with different pathological features (including sex, Ann Arbor stage, extranodal infiltration, Ki-67). The risk factors affecting mortality were analyzed using univariate and multivariate Cox proportional hazards regression. Receiver operating characteristic (ROC) curve was used to detect the predictive value of percentage of LAT1-positive cells in NHL tissue for patient mortality, and analyzing the effect of percentage of LAT1-positive cells on survival rate. RESULTS: LAT1 was positively expressed in NHL tissue. The high expression rate of LAT1 in Ann Arbor stage III and IV groups were higher than that in Ann Arbor stage I group, that in extranodal infiltration group was higher than non-extranodal infiltration group, and that in Ki-67 positive expression group was higher than Ki-67 negative expression group (all P < 0.05). The remission rate after 3 courses of treatment in high-LAT1 expression group was 70.7%, which was lower than 91.2% in low-LAT1 expression group (P < 0.05). Ann Arbor stage III and IV, extranodal invasion, Ki-67 positive expression and increased expression of LAT1 (LAT1-positive cell percentage score ≥2) were risk factors for mortality. The cut-off value of percentage of LAT1-positive cells for predicting NHL death was 45.6%, and the area under the ROC curve was 0.905 (95%CI: 0.897-0.924). The 3-year survival rate of high-LAT1 level group (the percentage of LAT1-positive cells≥45.6%) was 50.00%, which was lower than 78.26% of low-LAT1 level group (P < 0.05). CONCLUSION: The expression level of LAT1 in NHL tissue increases, which affects Ann Arbor stage and extranodal infiltration of patients. LAT1 is a risk factor for death.

Prognostic value of large amino acid transporter type 1 (LAT1) expression in pulmonary adenocarcinoma: A tissue microarray study.

BACKGROUND: Large amino acid transporter type 1 (LAT1) provides cancer cells with essential amino acids for both protein synthesis and cell growth and may predict patient prognosis. Additionally, LAT1 inhibition can be a therapeutic target. This study aimed to examine the prognostic significance of LAT1 expression in lung cancer, paying special attention to adenocarcinoma subtypes. METHODS: Tissue microarrays (TMA) of 1,560 total cores obtained from surgically resected lung cancer specimens between 1995 and 2008 at our hospital were used. Overall, 795 cases of adenocarcinoma were identified, and 717 underwent further evaluation. Immunohistochemical staining of whole slides and TMA cores were assessed to set H-score cutoff value.. Immunohistochemical expression of LAT1 was examined based on the subtypes of adenocarcinoma. Statistical analyses explored the prognostic significance of LAT1. RESULTS: Adenocarcinoma accounted for 71.8% of all cases (n = 795), and 216 cases (27.1%) expressed LAT1. The 795 cases were categorized into five subtypes: lepidic (n = 29, 3.6%), papillary (n = 601, 75.6%), acinar (n = 58, 7.3%), and solid (n = 9, 1.1%); 717 of the 795 cases were further assessed according to the exclusion criteria. The LAT1-positive ratio increased as the architectural grade increased. Notably, in papillary adenocarcinoma, the LAT1-positive group had significantly lower overall survival compared to the negative group (10-year survival: 45.6% vs. 60.8%, p < 0.001). CONCLUSION: LAT1 expression was higher in high-grade subtypes of pulmonary adenocarcinoma. Moreover, LAT1 expression is useful for predicting prognosis, particularly in papillary adenocarcinoma, facilitating prognostic stratification of papillary adenocarcinoma.

Upregulation of ATF4 mediates the cellular adaptation to pharmacologic inhibition of amino acid transporter LAT1 in pancreatic ductal adenocarcinoma cells.

L-type amino acid transporter 1 (LAT1) is recognized as a promising target for cancer therapy; however, the cellular adaptive response to its pharmacological inhibition remains largely unexplored. This study examined the adaptive response to LAT1 inhibition using nanvuranlat, a high-affinity LAT1 inhibitor. Proteomic analysis revealed the activation of a stress-induced transcription factor ATF4 following LAT1 inhibition, aligning with the known cellular responses to amino acid deprivation. This activation was linked to the GCN2-eIF2α pathway which regulates translation initiation. Our results show that ATF4 upregulation counteracts the suppressive effect of nanvuranlat on cell proliferation in pancreatic ductal adenocarcinoma cell lines, suggesting a role for ATF4 in cellular adaptation to LAT1 inhibition. Importantly, dual targeting of LAT1 and ATF4 exhibited more substantial anti-proliferative effects in vitro than individual treatments. This study underscores the potential of combining LAT1 and ATF4 inhibition as a therapeutic strategy in cancer treatment.

Structure-activity characteristics of phenylalanine analogs selectively transported by L-type amino acid transporter 1 (LAT1).

L-type amino acid transporter 1 (LAT1) is a transmembrane protein responsible for transporting large neutral amino acids. While numerous LAT1-targeted compound delivery for the brain and tumors have been investigated, their LAT1 selectivity often remains ambiguous despite high LAT1 affinity. This study assessed the LAT1 selectivity of phenylalanine (Phe) analogs, focusing on their structure-activity characteristics. We discovered that 2-iodo-L-phenylalanine (2-I-Phe), with an iodine substituent at position 2 in the benzene ring, markedly improves LAT1 affinity and selectivity compared to parent amino acid Phe, albeit at the cost of reduced transport velocity. L-Phenylglycine (Phg), one carbon shorter than Phe, was found to be a substrate for LAT1 with a lower affinity, exhibiting a low level of selectivity for LAT1 equivalent to Phe. Notably, (R)-2-amino-1,2,3,4-tetrahydro-2-naphthoic acid (bicyclic-Phe), with an α-methylene moiety akin to the α-methyl group in α-methyl-L-phenylalanine (α-methyl-Phe), a known LAT1-selective compound, showed similar LAT1 transport maximal velocity to α-methyl-Phe, but with higher LAT1 affinity and selectivity. In vivo studies revealed tumor-specific accumulation of bicyclic-Phe, underscoring the importance of LAT1-selectivity in targeted delivery. These findings emphasize the potential of bicyclic-Phe as a promising LAT1-selective component, providing a basis for the development of LAT1-targeting compounds based on its structural framework.

A bis-boron boramino acid PET tracer for brain tumor diagnosis.

PURPOSE: Boramino acids are a class of amino acid biomimics that replace the carboxylate group with trifluoroborate and can achieve the 18F-labeled positron emission tomography (PET) and boron neutron capture therapy (BNCT) with identical chemical structure. METHODS: This study reports a trifluoroborate-derived boronophenylalanine (BBPA), a derived boronophenylalanine (BPA) for BNCT, as a promising PET tracer for tumor imaging. RESULTS: Competition inhibition assays in cancer cells suggested the cell accumulation of [18F]BBPA is through large neutral amino acid transporter type-1 (LAT-1). Of note, [18F]BBPA is a pan-cancer probe that shows notable tumor uptake in B16-F10 tumor-bearing mice. In the patients with gliomas and metastatic brain tumors, [18F]BBPA-PET shows good tumor uptake and notable tumor-to-normal brain ratio (T/N ratio, 18.7 ± 5.5, n = 11), higher than common amino acid PET tracers. The [18F]BBPA-PET quantitative parameters exhibited no difference in diverse contrast-enhanced status (P = 0.115-0.687) suggesting the [18F]BBPA uptake was independent from MRI contrast-enhancement. CONCLUSION: This study outlines a clinical trial with [18F]BBPA to achieve higher tumor-specific accumulation for PET, provides a potential technique for brain tumor diagnosis, and might facilitate the BNCT of brain tumors.

L-type amino acid transporter 1 inhibitor JPH203 prevents the growth of cabazitaxel-resistant prostate cancer by inhibiting cyclin-dependent kinase activity.

L-type amino acid transporter 1 (LAT1, SLC7A5) is an amino acid transporter expressed in various carcinomas, and it is postulated to play an important role in the proliferation of cancer cells through the uptake of essential amino acids. Cabazitaxel is a widely used anticancer drug for treating castration-resistant prostate cancer (CRPC); however, its effectiveness is lost when cancer cells acquire drug resistance. In this study, we investigated the expression of LAT1 and the effects of a LAT1-specific inhibitor, JPH203, in cabazitaxel-resistant prostate cancer cells. LAT1 was more highly expressed in the cabazitaxel-resistant strains than in the normal strains. Administration of JPH203 inhibited the growth, migration, and invasive ability of cabazitaxel-resistant strains in vitro. Phosphoproteomics using liquid chromatography-mass spectrometry to comprehensively investigate changes in phosphorylation due to JPH203 administration revealed that cell cycle-related pathways were affected by JPH203, and that JPH203 significantly reduced the kinase activity of cyclin-dependent kinases 1 and 2. Moreover, JPH203 inhibited the proliferation of cabazitaxel-resistant cells in vivo. Taken together, the present study results suggest that LAT1 might be a valuable therapeutic target in cabazitaxel-resistant prostate cancer.

Identification of autophagy and angiogenesis modulators in colorectal cancer based on bioinformatics analysis.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related death worldwide. The purpose of this study was to discover novel molecular pathways and potential prognosis biomarkers. To achieve this, we acquired five microarray datasets from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes between CRC and adjacent normal tissue samples and further validated them using The Cancer Genome Atlas (TCGA) database. Using various analytical approaches, including the construction of a competing endogenous RNA (ceRNA) network, Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analyses, as well as survival analysis, we identified key genes and pathways associated with the diagnosis and prognosis of CRC. We obtained a total of 185 differentially expressed genes, comprising 17 lncRNAs, 30 miRNAs, and 138 mRNAs. The ceRNA network consisted of 17 lncRNAs, 25 miRNAs, and 7 mRNAs. Among the 7 mRNAs involved in the ceRNA network, SLC7A5 and KRT80 were found to be upregulated, while ADIPOQ, CCBE1, KCNB1, CADM2, and CHRDL1 were downregulated in CRC. Further analysis revealed that ADIPOQ and SLC7A5 are involved in the AMPK and mTOR signaling pathway, respectively. In addition, survival analysis demonstrated a statistically significant relationship between ADIPOQ, SLC7A5, and overall survival rates in CRC patients. In conclusion, our findings suggest that downregulation of ADIPOQ and upregulation of SLC7A5 in tumor cells lead to increased mTORC1 activity, reduced autophagy, enhanced angiogenesis, and ultimately contribute to cancer progression and decreased survival in CRC patients.

Increased sodium fluorescein transport by corticosteroids is inhibited by a LAT-1 specific inhibitor in retinal pigment epithelial cells in vitro.

To investigate whether aldosterone (ALD) and hydrocortisone (HC) change the gene expression of SLC7A5, which encodes the large neutral amino acid transporter small subunit 1 (LAT1), and the transport activity of LAT1 in the retinal pigment epithelium (RPE) in vitro. ARPE-19 cells were grown to confluence. After withdrawing the serum, ALD or HC was added with several doses and incubated, and SLC7A5 gene expression was measured. The influx and efflux transport of sodium fluorescein (Na-F) were evaluated using the Transwell culture system. SLC7A5 gene expression was upregulated by ALD and downregulated by HC in a dose-dependent manner. Both ALD and HC significantly increased the influx and efflux Na-F transport of RPE cells at a dose that did not change the expression of SLC7A5. JPH203, a specific inhibitor of LAT1, significantly reduced accelerated Na-F transport. Both ALD and HC increased the gene expression of zonula occludin-1 (ZO-1) although they did not change the immunoreactivity of ZO-1 in RPE cells. LAT1 may play an important role in increasing Na-F transport associated with ALD and HC administration. A specific LAT1 inhibitor may effectively regulate the increased material transport of RPE induced by ALD and HC.

MicroRNA-626 inhibits mTOR pathways activity of retinal pigment epithelial cells by targeting SLC7A5 in human ARPE-19 Cells.

Recent studies have shown that miRNAs are associated with the pathological process involved in age-related macular degeneration (AMD). However, the microRNA-mediated post-transcriptional regulation in human retinal pigment epithelium (RPE) cells has not been adequately investigated. We investigated how miR-626 inhibits mTOR activity pathways and pathway-related genes in retinal pigment epithelial cells by targeting the solute carrier family seven-member 5 (SLC7A5) in ARPE19 cells.    We transfected mir-626 mimic, mir-626 inhibitör and siRNA in human retinal pigment epithelial cell line was examined using RT-PCR and western blot, respectively. We knocked down mir-626 levels and overexpression by mir-626-siRNA transfection of human RPE cell lines, and using an MTT assay, we assessed the role of SLC7A5 on RPE cell proliferation. We additionally measured the expression of mTOR, Akt1, caspase 3, Bax, SLC17A7, SLC17A8, Creb1, Pten, HIF1A, HIFI. The findings demonstrate that mir-626 inhibits SLC7A5 gene expression and proliferation of ARPE-19 cells. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-626, has the same effect on ARPE-19 cells. We identified how miR-626 causes apoptosis and macula degeneration in RPE cells by targeting SLC7A5 through the mTOR signaling pathway. miR-626 was an essential regulator of the expression of the Slc7a5 gene. Importantly, we determined that miR-626 is essential to play a role in AMD. This research project shows that SLC7A5 is a direct target of mir-626 in ARPE-19 cells for the first time.

Gene purging and the evolution of Neoave metabolism and longevity.

Maintenance of the proteasome requires oxidative phosphorylation (ATP) and mitigation of oxidative damage, in an increasingly dysfunctional relationship with aging. SLC3A2 plays a role on both sides of this dichotomy as an adaptor to SLC7A5, a transporter of branched-chain amino acids (BCAA: Leu, Ile, Val), and to SLC7A11, a cystine importer supplying cysteine to the synthesis of the antioxidant glutathione. Endurance in mammalian muscle depends in part on oxidation of BCAA; however, elevated serum levels are associated with insulin resistance and shortened lifespans. Intriguingly, the evolution of modern birds (Neoaves) has entailed the purging of genes including SLC3A2, SLC7A5, -7, -8, -10, and SLC1A4, -5, largely removing BCAA exchangers and their interacting Na+/Gln symporters in pursuit of improved energetics. Additional gene purging included mitochondrial BCAA aminotransferase (BCAT2), pointing to reduced oxidation of BCAA and increased hepatic conversion to triglycerides and glucose. Fat deposits are anhydrous and highly reduced, maximizing the fuel/weight ratio for prolonged flight, but fat accumulation in muscle cells of aging humans contributes to inflammation and senescence. Duplications of the bidirectional α-ketoacid transporters SLC16A3, SLC16A7, the cystine transporters SLC7A9, SLC7A11, and N-glycan branching enzymes MGAT4B, MGAT4C in Neoaves suggests a shift to the transport of deaminated essential amino acid, and stronger mitigation of oxidative stress supported by the galectin lattice. We suggest that Alfred Lotka's theory of natural selection as a maximum power organizer (PNAS 8:151,1922) made an unusually large contribution to Neoave evolution. Further molecular analysis of Neoaves may reveal novel rewiring with applications for human health and longevity.

SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 has distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect)-driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2∗SLC7A5 heterodimer with amino-acid/Na+ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na+/K+ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.

L-Type Amino Acid Transporter 1 (LAT1) Promotes PMA-Induced Cell Migration through mTORC2 Activation at the Lysosome.

The mTOR signaling pathway integrates signaling inputs from nutrients, including glucose and amino acids, which are precisely regulated by transporters depending on nutrient levels. The L-type amino acid transporter 1 (LAT1) affects the activity of mTORC1 through upstream regulators that sense intracellular amino acid levels. While mTORC1 activation by LAT1 has been thoroughly investigated in cultured cells, the effects of LAT1 expression on the activity of mTORC2 has scarcely been studied. Here, we provide evidence that LAT1 recruits and activates mTORC2 on the lysosome for PMA-induced cell migration. LAT1 is translocated to the lysosomes in cells treated with PMA in a dose- and time-dependent manner. Lysosomal LAT1 interacted with mTORC2 through a direct interaction with Rictor, leading to the lysosomal localization of mTORC2. Furthermore, the depletion of LAT1 reduced PMA-induced cell migration in a wound-healing assay. Consistent with these results, the LAT1 N3KR mutant, which is defective in PMA-induced endocytosis and lysosomal localization, did not induce mTORC2 recruitment to the lysosome, with the activation of mTORC2 determined via Akt phosphorylation or the LAT1-mediated promotion of cell migration. Taken together, lysosomal LAT1 recruits and activates the mTORC2 complex and downstream Akt for PMA-mediated cell migration. These results provide insights into the development of therapeutic drugs targeting the LAT1 amino acid transporter to block metastasis, as well as disease progression in various types of cancer.

Advanced Boron Neutron Capture Therapy Targeting Cancer Stem Cells by Selective Induction of LAT1 Overexpression.

This study conducted fundamental research to develop a more effective BNCT targeting cancer stem cells. We constructed plasmids that induced the overexpression of L-type amino acid transporter 1 (LAT1) tagged with tdTomato on the cytoplasmic membranes of CD133 expressing cancer cells. After transfection of the plasmids into a glioblastoma cell line (T98G), several clones overexpressing LAT1-tdTomato in the hypoxic microenvironment of the spheroids formed from each clone were obtained. Confocal laser microscopic observation confirmed that signals from LAT1-tdTomato overlapped with immunofluorescence signals from the second antibody binding to CD133 in the hypoxic microenvironment of the spheroids. As CD133-positive cells in the hypoxic microenvironment of T98G spheroids have cancer stem cell characteristics, LAT1 seems to be selectively overexpressed in cancer stem cell-like cells. An RI tracer method showed that cells overexpressing LAT1-tdTomato in the hypoxic microenvironment of spheroids incorporate 14C-BPA much more than cells that do not overexpress LAT1-tdTomato. Neutron radiation experiments showed a more significant regression in spheroids formed with clones than in spheroids formed with parental cells when spheroids were treated with 10BPA. These results suggest that BNCT combined with gene therapy targeting cancer stem cells is more effective in glioblastoma therapy.

Insights into the Transport Cycle of LAT1 and Interaction with the Inhibitor JPH203.

The large Amino Acid Transporter 1 (LAT1) is an interesting target in drug discovery since this transporter is overexpressed in several human cancers. Furthermore, due to its location in the blood-brain barrier (BBB), LAT1 is interesting for delivering pro-drugs to the brain. In this work, we focused on defining the transport cycle of LAT1 using an in silico approach. So far, studies of the interaction of LAT1 with substrates and inhibitors have not considered that the transporter must undergo at least four different conformations to complete the transport cycle. We built outward-open and inward-occluded conformations of LAT1 using an optimized homology modelling procedure. We used these 3D models and the cryo-EM structures in outward-occluded and inward-open conformations to define the substrate/protein interaction during the transport cycle. We found that the binding scores for the substrate depend on the conformation, with the occluded states as the crucial steps affecting the substrate affinity. Finally, we analyzed the interaction of JPH203, a high-affinity inhibitor of LAT1. The results indicate that conformational states must be considered for in silico analyses and early-stage drug discovery. The two built models, together with the available cryo-EM 3D structures, provide important information on the LAT1 transport cycle, which could be used to speed up the identification of potential inhibitors through in silico screening.