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.

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.

Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

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.

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.

LAT1-dependent placental methionine uptake is a key player in fetal programming of metabolic disease.

The Developmental Origins of Health and Disease hypothesis sustains that exposure to different stressors during prenatal development prepares the offspring for the challenges to be encountered after birth. We studied the gestational period as a particularly vulnerable window where different stressors can have strong implications for fetal programming of the offspring's life-long metabolic status via alterations of specific placentally expressed nutrient transporters. To study this mechanism, we used a murine prenatal stress model, human preeclampsia, early miscarriage, and healthy placental tissue samples, in addition to in vitro models of placental cells. In stressed mice, placental overexpression of L-type amino acid transporter 1 (Lat1) and subsequent global placental DNA hypermethylation was accompanied by fetal and adult hypothalamic dysregulation in global DNA methylation and gene expression as well as long-term metabolic abnormalities exclusively in female offspring. In human preeclampsia, early miscarriage, and under hypoxic conditions, placental LAT1 was significantly upregulated, leading to increased methionine uptake and global DNA hypermethylation. Remarkably, subgroups of healthy term placentas with high expression of stress-related genes presented increased levels of placental LAT1 mRNA and protein, DNA and RNA hypermethylation, increased methionine uptake capacity, one-carbon metabolic pathway disruption, higher methionine concentration in the placenta and transport to the fetus specifically in females. Since LAT1 mediates the intracellular accumulation of methionine, global DNA methylation, and one-carbon metabolism in the placenta, our findings hint at a major sex-specific global response to a variety of prenatal stressors affecting placental function, epigenetic programming, and life-long metabolic disease and provide a much-needed insight into early-life factors predisposing females/women to metabolic disorders.

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.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

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.

Overexpression of the LAT1 in primary human trophoblast cells increases the uptake of essential amino acids and activates mTOR signaling.

The System L amino acid transporter, particularly the isoform Large Neutral Amino Acid Transporter Small Subunit 1 (LAT1) encoded by SLC7A5, is believed to mediate the transfer of essential amino acids in the human placenta. Placental System L amino acid transporter expression and activity is decreased in pregnancies complicated by IUGR and increased in fetal overgrowth. However, it remains unknown if changes in the expression of LAT1 are mechanistically linked to System L amino acid transport activity. Here, we combined overexpression approaches with protein analysis and functional studies in cultured primary human trophoblast (PHT) cells to test the hypothesis that SLC7A5 overexpression increases the uptake of essential amino acids and activates mTOR signaling in PHT cells. Overexpression of SLC7A5 resulted in a marked increase in protein expression of LAT1 in the PHT cells microvillous plasma membrane and System L amino acid transporter activity. Moreover, mTOR signaling was activated, and System A amino acid transporter activity increased following SLC7A5 overexpression, suggesting coordination of trophoblast amino transporter expression and activity to ensure balanced nutrient flux to the fetus. This is the first report showing that overexpression of LAT1 is sufficient to increase the uptake of essential amino acids in PHT cells, which activates mTOR, a master regulator of placental function. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter system in some cases of fetal overgrowth may directly contribute to changes in fetal amino acid availability and altered fetal growth in these pregnancy complications.

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.

The proliferation of human mucosal-associated invariant T cells requires a MYC-SLC7A5-glycolysis metabolic axis.

Mucosal-associated invariant T (MAIT) cells are an abundant population of innate T cells that recognize bacterial ligands and play a key role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells undergo proliferative expansion and increase their production of effector molecules such as cytokines. In this study, we found that both mRNA and protein abundance of the key metabolism regulator and transcription factor MYC was increased in stimulated MAIT cells. Using quantitative mass spectrometry, we identified the activation of two MYC-controlled metabolic pathways, amino acid transport and glycolysis, both of which were necessary for MAIT cell proliferation. Last, we showed that MAIT cells isolated from people with obesity showed decreased MYC mRNA abundance upon activation, which was associated with defective MAIT cell proliferation and functional responses. Collectively, our data uncover the importance of MYC-regulated metabolism for MAIT cell proliferation and provide additional insight into the molecular basis for the functional defects of MAIT cells in obesity.