Genetic Disruption of the Multifunctional CD98/LAT1 Complex Demonstrates the Key Role of Essential Amino Acid Transport in the Control of mTORC1 and Tumor Growth.
Cormerais Yann,Giuliano Sandy,LeFloch Renaud,Front Benoît,Durivault Jerome,Tambutté Eric,Massard Pierre-André,de la Ballina Laura Rodriguez,Endou Hitoshi,Wempe Michael F,Palacin Manuel,Parks Scott K,Pouyssegur Jacques
Cancer research
The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex promotes tumorigenesis with CD98 (4F2hc) engaging β-integrin signaling while LAT1 (SLC7A5) imports essential amino acids (EAA) and promotes mTORC1 activity. However, it is unclear as to which member of the heterodimer carries the most prevalent protumoral action. To answer this question, we explored the tumoral potential of each member by gene disruption of CD98, LAT1, or both and by inhibition of LAT1 with the selective inhibitor (JPH203) in six human cancer cell lines from colon, lung, and kidney. Each knockout respectively ablated 90% (CD98 KO: ) and 100% (LAT1 KO: ) of Na(+)-independent leucine transport activity. LAT1 KO: or JPH203-treated cells presented an amino acid stress response with ATF4, GCN2 activation, mTORC1 inhibition, and severe in vitro and in vivo tumor growth arrest. We show that this severe growth phenotype is independent of the level of expression of CD98 in the six tumor cell lines. Surprisingly, CD98 KO: cells with only 10% EAA transport activity displayed a normal growth phenotype, with mTORC1 activity and tumor growth rate undistinguishable from wild-type cells. However, CD98 KO: cells became extremely sensitive to inhibition or genetic disruption of LAT1 (CD98 KO: /LAT1 KO: ). This finding demonstrates that the tumoral potential of CD98 KO: cells is due to residual LAT1 transport activity. Therefore, these findings clearly establish that LAT1 transport activity is the key growth-limiting step of the heterodimer and advocate the pharmacology development of LAT1 transporter inhibitors as a very promising anticancer target. Cancer Res; 76(15); 4481-92. ©2016 AACR.
10.1158/0008-5472.CAN-15-3376
Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter.
Yan Renhong,Li Yaning,Müller Jennifer,Zhang Yuanyuan,Singer Simon,Xia Lu,Zhong Xinyue,Gertsch Jürg,Altmann Karl-Heinz,Zhou Qiang
Cell discovery
LAT1 (SLC7A5) is one of the representative light chain proteins of heteromeric amino acid transporters, forming a heterodimer with its heavy chain partner 4F2hc (SLC3A2). LAT1 is overexpressed in many types of tumors and mediates the transfer of drugs and hormones across the blood-brain barrier. Thus, LAT1 is considered as a drug target for cancer treatment and may be exploited for drug delivery into the brain. Here, we synthesized three potent inhibitors of human LAT1, which inhibit transport of leucine with IC values between 100 and 250 nM, and solved the cryo-EM structures of the corresponding LAT1-4F2hc complexes with these inhibitors bound at resolution of up to 2.7 or 2.8 Å. The protein assumes an outward-facing occluded conformation, with the inhibitors bound in the classical substrate binding pocket, but with their tails wedged between the substrate binding site and TM10 of LAT1. We also solved the complex structure of LAT1-4F2hc with 3,5-diiodo-L-tyrosine (Diiodo-Tyr) at 3.4 Å overall resolution, which revealed a different inhibition mechanism and might represent an intermediate conformation between the outward-facing occluded state mentioned above and the outward-open state. To our knowledge, this is the first time that the outward-facing conformation is revealed for the HAT family. Our results unveil more important insights into the working mechanisms of HATs and provide a structural basis for future drug design.
10.1038/s41421-021-00247-4
Novel prognostic immunohistochemical biomarker panel for estrogen receptor-positive breast cancer.
Ring Brian Z,Seitz Robert S,Beck Rod,Shasteen William J,Tarr Shannon M,Cheang Maggie C U,Yoder Brian J,Budd G Thomas,Nielsen Torsten O,Hicks David G,Estopinal Noel C,Ross Douglas T
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:Patients with breast cancer experience progression and respond to treatment in diverse ways, but prognostic and predictive tools for the oncologist are limited. We have used gene expression data to guide the production of hundreds of novel antibody reagents to discover novel diagnostic tools for stratifying carcinoma patients. PATIENTS AND METHODS:One hundred forty novel and 23 commercial antisera, selected on their ability to differentially stain tumor samples, were used to stain paraffin blocks from a retrospective breast cancer cohort. Cox proportional hazards and regression tree analysis identified minimal panels of reagents able to predict risk of recurrence. We tested the prognostic association of these prospectively defined algorithms in two independent cohorts. RESULTS:In both validation cohorts, the Kaplan-Meier estimates of recurrence confirmed that both the Cox model using five reagents (p53, NDRG1, CEACAM5, SLC7A5, and HTF9C) and the regression tree model using six reagents (p53, PR, Ki67, NAT1, SLC7A5, and HTF9C) distinguished estrogen receptor (ER)-positive patients with poor outcomes. The Cox model was superior and distinguished patients with poor outcomes from patients with good or moderate outcomes with a hazard ratio of 2.21 (P = .0008) in validation cohort 1 and 1.88 (P = .004) in cohort 2. In multivariable analysis, the calculated risk of recurrence was independent of stage, grade, and lymph node status. A model proposed for ER-negative patients failed validation in the independent cohorts. CONCLUSION:A panel of five antibodies can significantly improve on traditional prognosticators in predicting outcome for ER-positive breast cancer patients.
10.1200/JCO.2006.05.6564
Comprehensive Characterization of LAT1 Cholesterol-Binding Sites.
Journal of chemical theory and computation
The human L-type amino acid transporter 1 (LAT1; SLC7A5), is an amino acid exchanger protein, primarily found in the blood-brain barrier, placenta, and testis, where it plays a key role in amino acid homeostasis. Cholesterol is an essential lipid that has been highlighted to play a role in regulating the activity of membrane transporters, such as LAT1, yet little is known about the molecular mechanisms driving this phenomenon. Here we perform a comprehensive computational analysis to investigate cholesterol's role in LAT1 structure and function, focusing on four cholesterol-binding sites (CHOL1-4) identified in a recent LAT1-apo inward-open conformation cryo-EM structure. Through a series of independent molecular dynamics (MD) simulations, molecular docking, MM/GBSA free energy calculations, and other analysis tools, we explored the interactions between LAT1 and cholesterol. Our findings suggest that CHOL3 forms the most stable and favorable interactions with LAT1. Principal component analysis (PCA) and center of mass (COM) distance assessments show that CHOL3 binding stabilizes the inward-open state of LAT1 by preserving the spatial arrangement of the hash and bundle domains. Additionally, we propose an alternative cholesterol-binding site for originally assigned CHOL1. Overall, this study improves the understanding of cholesterol's modulatory effect on LAT1 and proposes candidate sites for the discovery of future allosteric ligands with rational design.
10.1021/acs.jctc.3c01391
Combination effects of amino acid transporter LAT1 inhibitor nanvuranlat and cytotoxic anticancer drug gemcitabine on pancreatic and biliary tract cancer cells.
Cancer cell international
BACKGROUND:Cytotoxic anticancer drugs widely used in cancer chemotherapy have some limitations, such as the development of side effects and drug resistance. Furthermore, monotherapy is often less effective against heterogeneous cancer tissues. Combination therapies of cytotoxic anticancer drugs with molecularly targeted drugs have been pursued to solve such fundamental problems. Nanvuranlat (JPH203 or KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), has novel mechanisms of action to suppress the cancer cell proliferation and tumor growth by inhibiting the transport of large neutral amino acids into cancer cells. This study investigated the potential of the combined use of nanvuranlat and cytotoxic anticancer drugs. METHODS:The combination effects of cytotoxic anticancer drugs and nanvuranlat on cell growth were examined by a water-soluble tetrazolium salt assay in two-dimensional cultures of pancreatic and biliary tract cancer cell lines. To elucidate the pharmacological mechanisms underlying the combination of gemcitabine and nanvuranlat, we investigated apoptotic cell death and cell cycle by flow cytometry. The phosphorylation levels of amino acid-related signaling pathways were analyzed by Western blot. Furthermore, growth inhibition was examined in cancer cell spheroids. RESULTS:All the tested seven types of cytotoxic anticancer drugs combined with nanvuranlat significantly inhibited the cell growth of pancreatic cancer MIA PaCa-2 cells compared to their single treatment. Among them, the combined effects of gemcitabine and nanvuranlat were relatively high and confirmed in multiple pancreatic and biliary tract cell lines in two-dimensional cultures. The growth inhibitory effects were suggested to be additive but not synergistic under the tested conditions. Gemcitabine generally induced cell cycle arrest at the S phase and apoptotic cell death, while nanvuranlat induced cell cycle arrest at the G0/G1 phase and affected amino acid-related mTORC1 and GAAC signaling pathways. In combination, each anticancer drug basically exerted its own pharmacological activities, although gemcitabine more strongly influenced the cell cycle than nanvuranlat. The combination effects of growth inhibition were also verified in cancer cell spheroids. CONCLUSIONS:Our study demonstrates the potential of first-in-class LAT1 inhibitor nanvuranlat as a concomitant drug with cytotoxic anticancer drugs, especially gemcitabine, on pancreatic and biliary tract cancers.
10.1186/s12935-023-02957-z
ALYREF-JunD-SLC7A5 axis promotes pancreatic ductal adenocarcinoma progression through epitranscriptome-metabolism reprogramming and immune evasion.
Cell death discovery
Pancreatic ductal adenocarcinoma (PDAC) is a kind of tumor lacking nutrients due to its poor vascularity and desmoplasia. Recent studies have shown that cancer cells might achieve growth advantage through epitranscriptome reprogramming. However, the role of mC in PDAC was not fully understood. We found that Aly/REF export factor (ALYREF), a reader of mC modification, was overexpressed in PDAC, and associated with bad prognosis. In addition, the ALYREF expression was negatively related to CD8 T cells infiltration in clinical samples. ALYREF knockdown decreased tumor growth in vivo partly dependent of immunity. ALYREF silencing decreased SLC7A5 expression and subsequently inactivated mTORC1 pathway, resulting in decreased tumor proliferation. Mechanically, ALYREF specifically recognized mC sites in JunD mRNA, maintained the stabilization of JunD mRNA and subsequently upregulated transcription of SLC7A5. Since SLC7A5 was a key transporter of large neutral amino acids (LNAAs), overexpression of SLC7A5 on tumor cells depleted amino acid in microenvironment and restricted CD8 T cells function. Moreover, ALYREF-JunD-SLC7A5 axis was overexpressed and negatively related with survival through TMA assays. In conclusion, this research revealed the relationship between mC modification, amino acid transportation and immune microenvironment. ALYREF might be a novel target for PDAC metabolic vulnerability and immune surveillance.
10.1038/s41420-024-01862-2
SLC7A5 correlated with malignancies and immunotherapy response in bladder cancer.
Cancer cell international
BACKGROUND:Metabolic reprogramming contributes to bladder cancer development. This study aimed to understand the role of SLC7A5 in bladder cancer. METHODS:We systematically analyzed the correlation between SLC7A5 and bladder cancer through various approaches, including bioinformatics, western blotting, cell cycle analysis, cell proliferation assays, and invasion experiments. We also investigated the immunological features within the tumor microenvironment (TME), encompassing cancer immune cycles, immune modulators, immune checkpoints, tumor-infiltrating immune cells (TIIC), T cell inflammation scores, and treatment responses. Additionally, for a comprehensive assessment of the expression patterns and immunological roles of SLC7A5, pan-cancer analysis was performed using cancer genomics datasets. RESULTS:SLC7A5 was associated with adverse prognosis in bladder cancer patients, activating the Wnt pathway and promoting bladder cancer cell cycle progression, proliferation, migration, and invasion. Based on the evidence that SLC7A5 positively correlated with immunomodulators, TIIC, the cancer immune cycle, immune checkpoint and T cell inflammation scores, we also found that SLC7A5 was associated with the inflammatory tumor immune microenvironment. EGFR-targeted therapy, cancer immunotherapy, and radiation therapy were effective for patients with high SLC7A5 expression in bladder cancer. Low SLC7A5 patients were, however, sensitive to targeted therapies and anti-angiogenic therapy, such as blocking β-catenin network, PPAR-γ and FGFR3 signaling. Anti-SLC7A5 combined with cancer immunotherapy may have greater effectiveness than either therapy alone. Furthermore, we observed specific overexpression of SLC7A5 in TME of various cancers. CONCLUSION:SLC7A5 can predict therapeutic response to immunotherapy, radiotherapy and chemotherapy in bladder cancer patients. Targeting SLC7A5 in combination with immunotherapy may be a potentially appropriate treatment option.
10.1186/s12935-024-03365-7
L-type amino acid transporter 1 as a potential molecular target in human astrocytic tumors.
Nawashiro Hiroshi,Otani Naoki,Shinomiya Nariyoshi,Fukui Shinji,Ooigawa Hidetoshi,Shima Katsuji,Matsuo Hirotaka,Kanai Yoshikatsu,Endou Hitoshi
International journal of cancer
L-type amino acid transporter 1 (LAT1) is a Na+-independent neutral amino acid transport agency and essential for the transport of large neutral amino acids. LAT1 has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells. We have investigated for the first time, the expression of the transporter in the human primary astrocytic tumor tissue from 60 patients. LAT1 is unique because it requires an additional single membrane spanning protein, the heavy chain of 4F2 cell surface antigen (4F2hc), for its functional expression. 4F2hc expression was also determined by immunohistochemistry. Kaplan-Meier analyses demonstrated that high LAT1 expression correlated with poor survival for the study group as a whole (p<0.0001) and for those with glioblastoma multiforme in particular (p=0.0001). Cox regression analyses demonstrated that LAT1 expression was one of significant predictors of outcome, independent of all other variables. On the basis of these findings, we also investigated the effect of the specific inhibitor to LAT1, 2-aminobicyclo-2 (2,2,1)-heptane-2-carboxylic acid (BCH), on the survival of C6 glioma cells in vitro and in vivo using a rat C6 glioma model. BCH inhibited the growth of C6 glioma cells in vitro and in vivo in a dose-dependent manner. Kaplan-Meier survival data of rats treated with BCH were significant. These findings suggest that LAT1 could be one of the molecular targets in glioma therapy.
10.1002/ijc.21866
Amino Acid Metabolism Abnormity and Microenvironment Variation Mediated Targeting and Controlled Glioma Chemotherapy.
An Sai,Lu Xiuhong,Zhao Weili,Sun Tao,Zhang Yu,Lu Yifei,Jiang Chen
Small (Weinheim an der Bergstrasse, Germany)
Energy metabolism abnormity is one of the most significant hallmarks of cancer. As a result, large amino acid transporter 1 (LAT1) is remarkably overexpressed in both blood-brain-barrier and glioma tumor cells, leading a rapid and sufficient substrate transportation. 3CDIT and 4CDIT are originally synthesized by modifying the existing most potent LAT1 substrate. 3CDIT is selected as its higher glioma-targeting ability. Since the microenvironment variation in tumor cells is another important feature of cancer, a great disparity in adenosine-5'-triphosphate (ATP) and glutathione (GSH) levels between extracellular and intracellular milieu can provide good possibilities for dual-responsive drug release in tumor cells. Doxorubicin (DOX) is successfully intercalated into the ATP aptamer DNA scaffolds, compressed by GSH-responsive polymer pOEI, and modified with 3CDIT to obtain 3CDIT-targeting pOEI/DOX/ATP aptamer nanoparticles (NPs). Enhanced NP accumulation and rapid GSH & ATP dual-responsive DOX release in glioma are demonstrated both in vitro and in vivo. More efficient therapeutic effects are shown with 3CDIT-targeting pOEI/DOX/ATP aptamer NPs than free DOX and no systemic toxicity is observed. Therefore, glioma-targeting delivery and GSH & ATP dual-responsive release guarantee an adequate DOX accumulation within tumor cells and ensure a safe and efficient chemotherapy for glioma.
10.1002/smll.201601249
L-type amino acid transporters LAT1 and LAT4 in cancer: uptake of 3-O-methyl-6-18F-fluoro-L-dopa in human adenocarcinoma and squamous cell carcinoma in vitro and in vivo.
Haase Cathleen,Bergmann Ralf,Fuechtner Frank,Hoepping Alexander,Pietzsch Jens
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
UNLABELLED:Expression of system L amino acid transporters (LAT) is strongly increased in many types of tumor cells. The purpose of this study was to demonstrate that (18)F-labeled amino acids, for example, 3-O-methyl-6-(18)F-fluoro-L-dopa ((18)F-OMFD), that accumulate in tumors via LAT represent an important class of imaging agents for visualization of tumors in vivo by PET. METHODS:(18)F-OMFD uptake kinetics, transport inhibition, and system L messenger RNA expression were studied in vitro in human adenocarcinoma (HT-29), squamous cell carcinoma (FaDu), macrophages (THP-1), and primary aortic endothelial cells (HAEC) and in vivo in the corresponding mouse tumor xenograft models. RESULTS:Uptake of (18)F-OMFD in all cell lines tested was mediated mainly by the sodium-independent high-capacity LAT. We found higher uptake in FaDu cells (V(max), 10.6 +/- 1.1 nmol/min x mg of cell protein) and in the corresponding FaDu tumor xenografts than in the other cells and corresponding xenograft models studied. Quantitative messenger RNA analysis revealed that tumor cells and xenografts have a higher expression of LAT1 than do HAEC and THP-1 macrophages. However, only in the FaDu tumor model did an increased (18)F-OMFD uptake seem to be explained by increased LAT expression. Furthermore, we demonstrated a high expression of LAT4, a recently identified LAT. CONCLUSION:Our findings support the hypothesis that (18)F-OMFD is a tracer for visualization of tumor cells. (18)F-OMFD particularly seems to be a suitable tracer for diagnostic imaging of amino acid transport in poorly differentiated squamous cell head and neck carcinoma with increased LAT1 and LAT4 expression.
10.2967/jnumed.107.043620
Prognostic significance of L-type amino acid transporter 1 expression in resectable stage I-III nonsmall cell lung cancer.
Kaira K,Oriuchi N,Imai H,Shimizu K,Yanagitani N,Sunaga N,Hisada T,Tanaka S,Ishizuka T,Kanai Y,Endou H,Nakajima T,Mori M
British journal of cancer
The clinical significance of L-type amino acid transporter 1 (LAT1) expression remains unclear, whereas many experimental studies have demonstrated that LAT1 is associated with the proliferation of cancer cells. The purpose of this study was to evaluate the prognostic value of LAT1 in patients with nonsmall cell lung cancer (NSCLC). A total of 321 consecutive patients with completely resected pathologic stage I-III NSCLC were retrospectively reviewed. Expression of LAT1 and proliferative activity, as determined by the Ki-67 labelling index, was also evaluated immunohistochemically and correlated with the prognosis of patients who underwent complete resection of the tumour. Expression of LAT1 was positive in 163 patients (51%) (29% of adenocaricnoma (58 of 200 patients), 91% of squamous cell carcinoma (91 of 100 patients), and 67% of large cell carcinoma (14 of 21 patients)). The 5-year survival rate of LAT1-positive patients (51.8%) was significantly worse than that of LAT1-negative patients (87.8%; P<0.001). L-type amino acid transporter 1 expression was significantly associated with lymph node metastasis and disease stage. Multivariate analysis confirmed that positive expression of LAT1 was an independent factor for predicting a poor prognosis. There was a significant correlation between LAT1 expression and Ki-67 labelling index. LAT1 expression is a promising pathological factor to predict the prognosis in patients with resectable stage I-III NSCLC.
10.1038/sj.bjc.6604235
Targeting tumor highly-expressed LAT1 transporter with amino acid-modified nanoparticles: Toward a novel active targeting strategy in breast cancer therapy.
Li Lin,Di Xingsheng,Wu Mingrui,Sun Zhisu,Zhong Lu,Wang Yongjun,Fu Qiang,Kan Qiming,Sun Jin,He Zhonggui
Nanomedicine : nanotechnology, biology, and medicine
Designing active targeting nanocarriers with increased cellular accumulation of chemotherapeutic agents is a promising strategy in cancer therapy. Herein, we report a novel active targeting strategy based on the large amino acid transporter 1 (LAT1) overexpressed in a variety of cancers. Glutamate was conjugated to polyoxyethylene stearate as a targeting ligand to achieve LAT1-targeting PLGA nanoparticles. The targeting efficiency of nanoparticles was investigated in HeLa and MCF-7 cells. Significant increase in cellular uptake and cytotoxicity was observed in LAT1-targeting nanoparticles compared to the unmodified ones. More interestingly, the internalized LAT1 together with targeting nanoparticles could recycle back to the cell membrane within 3 h, guaranteeing sufficient transporters on cell membrane for continuous cellular uptake. The LAT1 targeting nanoparticles exhibited better tumor accumulation and antitumor effects. These results suggested that the overexpressed LAT1 on cancer cells holds a great potential to be a high-efficiency target for the rational design of active-targeting nanosystems.
10.1016/j.nano.2016.11.012
F-FBPA as a tumor-specific probe of L-type amino acid transporter 1 (LAT1): a comparison study with F-FDG and C-Methionine PET.
Watabe Tadashi,Ikeda Hayato,Nagamori Shushi,Wiriyasermkul Pattama,Tanaka Yoko,Naka Sadahiro,Kanai Yasukazu,Hagiwara Kohei,Aoki Masanao,Shimosegawa Eku,Kanai Yoshikatsu,Hatazawa Jun
European journal of nuclear medicine and molecular imaging
PURPOSE:The purpose of this study was to evaluate the usefulness of L-4-borono-2-F-fluoro-phenylalanine (F-FBPA) as a tumor-specific probe, in comparison to F-FDG and C-methionine (Met), focusing on its transport selectivity by L-type amino acid transporter 1 (LAT1), which is highly upregulated in cancers. METHODS:Cellular analyses of FBPA were performed to evaluate the transportablity and K value. PET studies were performed in rat xenograft models of C6 glioma (n = 12) and in rat models of turpentine oil-induced subcutaneous inflammation (n = 9). The kinetic parameters and uptake values on static PET images were compared using the one-tissue compartment model (K, k) and maximum standardized uptake value (SUVmax). RESULTS:The cellular analyses showed that FBPA had a lower affinity to a normal cell-type transporter LAT2 and induced less efflux through LAT2 among FBPA, Met, and BPA, while the efflux through LAT1 induced by FBPA was similar among the three compounds. The K value of F-FBPA for LAT1 (196.8 ± 11.4 μM) was dramatically lower than that for LAT2 (2813.8 ± 574.5 μM), suggesting the higher selectivity of F-FBPA for LAT1. K and k values were significantly smaller in F-FBPA PET (K = 0.04 ± 0.01 ml/ccm/min and k = 0.07 ± 0.01 /min) as compared to C-Met PET (0.22 ± 0.09 and 0.52 ± 0.10, respectively) in inflammatory lesions. Static PET analysis based on the SUVmax showed significantly higher accumulation of F-FDG in the tumor and inflammatory lesions (7.2 ± 2.1 and 4.6 ± 0.63, respectively) as compared to both F-FBPA (3.2 ± 0.40 and 1.9 ± 0.19) and C-Met (3.4 ± 0.43 and 1.6 ± 0.11). No significant difference was observed between F-FBPA and C-Met in the static PET images. CONCLUSION:This study shows the utility of F-FBPA as a tumor-specific probe of LAT1 with low accumulation in the inflammatory lesions.
10.1007/s00259-016-3487-1
Large amino acid transporter 1 (LAT1) prodrugs of valproic acid: new prodrug design ideas for central nervous system delivery.
Peura Lauri,Malmioja Kalle,Laine Krista,Leppänen Jukka,Gynther Mikko,Isotalo Antti,Rautio Jarkko
Molecular pharmaceutics
Central nervous system (CNS) drug delivery is a major challenge in drug development because the blood-brain barrier (BBB) efficiently restricts the entry of drug molecules into the CNS at sufficient amounts. The brain uptake of poorly penetrating drugs could be improved by utilizing the transporters at the BBB with a prodrug approach. In this study, we designed four phenylalanine derivatives of valproic acid and studied their ability to utilize a large amino acid transporter 1 (LAT1) in CNS delivery with an aim to show that the meta-substituted phenylalanine prodrugs bind to LAT1 with a higher affinity compared with the affinity of the para-substituted derivatives. All of the prodrugs crossed the BBB carrier mediatedly via LAT1 in in situ rat brain perfusion. For the first time, we introduced a novel meta-substituted phenylalanine analogue promoiety which improved the LAT1 affinity 10-fold and more importantly the rat brain uptake of the prodrug 2-fold compared with those of the para-substituted derivatives. Therefore, we have characterized a new prodrug design idea for CNS drug delivery utilizing a transporter-mediated prodrug approach.
10.1021/mp2001878
The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer.
Nature genetics
Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.
10.1038/s41588-020-00753-3
L-type amino acid transporter 1 utilizing prodrugs: How to achieve effective brain delivery and low systemic exposure of drugs.
Puris Elena,Gynther Mikko,Huttunen Johanna,Petsalo Aleksanteri,Huttunen Kristiina M
Journal of controlled release : official journal of the Controlled Release Society
L-type amino acid transporter 1 (LAT1) is selectively expressed in the blood-brain barrier (BBB) and brain parenchyma. This transporter can facilitate brain delivery of neuroprotective agents and additionally give opportunity to minimize systemic exposure. Here, we investigated structure-pharmacokinetics relationship of five newly synthesized LAT1-utilizing prodrugs of the cyclooxygenase inhibitor, ketoprofen, in order to identify beneficial structural features of prodrugs to achieve both targeted brain delivery and low peripheral distribution of the parent drug. Besides, we studied whether pharmacokinetics and bioconversion of LAT1-utilizing prodrugs in vivo can be predicted in early stage experiments. To achieve these goals, we compared the in vitro brain uptake mechanism of prodrugs, rate of BBB permeation of compounds using in situ perfusion technique, their systemic pharmacokinetics and release of parent drug in brain, plasma and liver of mice. The results revealed that both excellent LAT1-binding ability and transporter utilization in vitro can be achieved by conjugating the parent drug to aromatic amino acids such as phenylalanine in comparison to prodrugs with an aliphatic promoiety. The presence of an aromatic promoiety directly conjugated in meta- or para-position to ketoprofen led to LAT1-utilizing prodrugs capable of delivering the parent drug into the brain with higher unbound brain to plasma ratio and reduced liver exposure than with ketoprofen itself. In contrast, the prodrugs with aliphatic promoieties and with an additional carbon attached between the parent drug and phenylalanine aromatic ring did not enhance brain delivery of ketoprofen. Furthermore, we have devised a screening strategy to pinpoint successful candidates at an early stage of development of LAT1-utilizing prodrugs. The screening approach demonstrated that early stage experiments could not replace pharmacokinetic studies in vivo due to the lack of prediction of the intra-brain/systemic distribution of the prodrugs as well as the release of the parent drug. Overall, this study provides essential knowledge required for improvement of targeted brain delivery and reduction of systemic exposure of drugs via the LAT1-mediated prodrug approach.
10.1016/j.jconrel.2017.06.023
The LAT1 inhibitor JPH203 suppresses the growth of castration-resistant prostate cancer through a CD24-mediated mechanism.
Cancer science
L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/β-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.
10.1111/cas.16191
LAT1 (SLC7A5) Overexpression in Negative Her2 Group of Breast Cancer: A Potential Therapy Target.
Bodoor Khaldon,Almomani Rowida,Alqudah Mohammad,Haddad Yazan,Samouri Walaa
Asian Pacific journal of cancer prevention : APJCP
OBJECTIVE:HER2 negative carcinomas of the breast pose a challenge for treatment due to redundancies in potential drug targets and poor patient outcomes. Our aim was to investigate the role of L-type amino acid transporter - LAT1 as a potential prognosticator and a drug target. METHODS:In this retrospective work, we have studied the expression of LAT1 in 145 breast cancer tissues via immunohistochemistry. Overall survival analysis was used to evaluate patient outcome in various groups of our cohort. RESULTS:Positive LAT1 expression was found in 27 (84.4%) luminal A subtype, 27 (64.3%) luminal B/triple positive subtype, 29 (82.9%) triple negative subtype, and 24 (66.7%) HER2-only positive subtype (p=0.1). Interestingly, negative correlation was found between LAT1 and HER2; where positive expression of LAT1 was found in 56 (83.6%) cases in negative HER2 group and 51 (65.4%) cases from positive HER2 group (p=0.01). Unfortunately, we were unable to report significant survival differences when LAT1 expression was studied in the negative HER2 group. Nevertheless, five incidents of mortality (out of 55) were reported in LAT1+/HER2- group compared to none in the LAT1-/HER2- group (N=11). CONCLUSION:Our findings of overexpression of LAT1 in negative HER2 group suggest a role of this protein as prognosticator and drug target in a challenging therapeutic cohort.<br />.
10.31557/APJCP.2020.21.5.1453
Gene expression profiles in T24 human bladder carcinoma cells by inhibiting an L-type amino acid transporter, LAT1.
Baniasadi Shadi,Chairoungdua Arthit,Iribe Yuji,Kanai Yoshikatsu,Endou Hitoshi,Aisaki Ken-ichi,Igarashi Katsuhide,Kanno Jun
Archives of pharmacal research
Inhibition of LAT1 (L-type amino acid transporter 1) activity in tumor cells could be effective in the inhibition of tumor cell growth by depriving tumor cells of essential amino acids. Because of the high level of expression of LAT1 in tumor cells, LAT1 inhibitors would be useful for anticancer therapy in suppressing tumor growth without affecting normal tissues. In recent years, cDNA microarray technique is useful technology for anticancer drug development. It allows identifying and characterizing new targets for developments in cancer drug therapy through the understanding genes involved in drug action. The present study was designed to investigate gene expression profile induced by LAT1 inhibitor using gene chip technology. Human bladder carcinoma cells (T24 cells) were treated with classical system L inhibitor 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid (BCH). Gene chip experiment was applied for treated and untreated cells after 3 and 12 h. Two independent experiments with a high degree of concordance identified the altered expression of 151 and 200 genes after 3 and 12 h BCH treatment. Among these genes, 132 and 13 were up-regulated and 19 and 187 were down-regulated by 3 and 12 h BCH treatment respectively. We found that BCH affected the expression of a large number of genes that are related to the control of cell survival and physiologic behaviors. These data are useful for understanding of intracellular signaling of cell growth inhibition induced by LAT1 inhibitors as candidate for anticancer drug therapy.
Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: a possible target for combination therapy with anti-proliferative aminopeptidase inhibitors.
Fan Xuetao,Ross Douglas D,Arakawa Hiroshi,Ganapathy Vadivel,Tamai Ikumi,Nakanishi Takeo
Biochemical pharmacology
Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC(50) value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.
10.1016/j.bcp.2010.05.021
Aryl hydrocarbon receptor (AHR) regulation of L-Type Amino Acid Transporter 1 (LAT-1) expression in MCF-7 and MDA-MB-231 breast cancer cells.
Tomblin Justin K,Arthur Subha,Primerano Donald A,Chaudhry Ateeq R,Fan Jun,Denvir James,Salisbury Travis B
Biochemical pharmacology
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is regulated by environmental toxicants that function as AHR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). L-Type Amino Acid Transporter 1 (LAT1) is a leucine transporter that is overexpressed in cancer. The regulation of LAT1 by AHR in MCF-7 and MDA-MB-231 breast cancer cells (BCCs) was investigated in this report. Ingenuity pathway analysis (IPA) revealed a significant association between TCDD-regulated genes (TRGs) and molecular transport. Overlapping the TCDD-RNA-Seq dataset obtained in this study with a published TCDD-ChIP-seq dataset identified LAT1 as a primary target of AHR-dependent TCDD induction. Short interfering RNA (siRNA)-directed knockdown of AHR confirmed that TCDD-stimulated increases in LAT1 mRNA and protein required AHR expression. TCDD-stimulated increases in LAT1 mRNA were also inhibited by the AHR antagonist CH-223191. Upregulation of LAT1 by TCDD coincided with increases in leucine uptake by MCF-7 cells in response to TCDD. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays revealed increases in AHR, AHR nuclear translocator (ARNT) and p300 binding and histone H3 acetylation at an AHR binding site in the LAT1 gene in response to TCDD. In MCF-7 and MDA-MB-231 cells, endogenous levels of LAT1 mRNA and protein were reduced in response to knockdown of AHR expression. Knockdown experiments demonstrated that proliferation of MCF-7 and MDA-MB-231 cells is dependent on both LAT1 and AHR. Collectively, these findings confirm the dependence of cancer cells on leucine uptake and establish a mechanism for extrinsic and intrinsic regulation of LAT1 by AHR.
10.1016/j.bcp.2016.02.020
System L amino acid transporter inhibitor enhances anti-tumor activity of cisplatin in a head and neck squamous cell carcinoma cell line.
Yamauchi Kohichi,Sakurai Hiroyuki,Kimura Toru,Wiriyasermkul Pattama,Nagamori Shushi,Kanai Yoshikatsu,Kohno Naoyuki
Cancer letters
LAT1, a subunit of heterodimeric system L transporter responsible for transporting neutral amino acids into cells, has been investigated in several cancers because of its onco-fetal nature. Based on the studies of its functional inhibition, LAT1 has been proposed to be a new molecular target of a cancer therapy. We have shown here that human head and neck cancer cell line, Hep-2, expresses both LAT1 and 4F2hc, another subunit of system L transporter. An inhibitor of system L, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), inhibited leucine uptake by the cells. BCH administration or restriction of essential amino acid leucine decreased viability of Hep-2 cells. Co-administration of cisplatin with BCH reduced the viability of the cells more than either agent alone. When BCH treatment preceded cisplatin administration, reduction in Hep-2 cell viability was additive. In contrast, when BCH was given after cisplatin treatment, synergistic effect in decreasing the number of viable cells was obtained. BCH treatment decreased the phosphorylation of mTOR, p70S6K and 4EBP1, suggesting that BCH enhanced anti-tumor action of cisplatin by inhibiting mTOR pathway. This potentiation may be used to reduce cisplatin exposure to alleviate many unwanted toxicity of the drug.
10.1016/j.canlet.2008.10.035
L-type amino acid transporter 1 (LAT1)-utilizing efflux transporter inhibitors can improve the brain uptake and apoptosis-inducing effects of vinblastine in cancer cells.
Montaser Ahmed,Markowicz-Piasecka Magdalena,Sikora Joanna,Jalkanen Aaro,Huttunen Kristiina M
International journal of pharmaceutics
Efflux transporter-mediated multidrug resistance (MDR) prevents chemotherapeutics to achieve therapeutically relevant concentrations within the cancer cells. Therefore, inhibitors of efflux transporters have been in demand. However, to be safe, these inhibitors are needed to be targeted into the cancer cells. For this purpose, L-type amino acid transporter 1 (LAT1), overexpressed in many different cancer cell types, can be utilized. In the present study, two LAT1-utilizing derivatives of probenecid (PRB) that can inhibit e.g., multiresistance proteins (MRPs) and organic anion transporters (OATs), were studied for their apoptosis-inducing effects in cancer cells alone and a combination with another chemotherapeutic, vinblastine (VBL). Also, their hemocompatibility and off-target toxicity were evaluated. Moreover, the brain uptake rate and extent of VBL together with the most promising LAT1-utilizing efflux inhibitor was studied after in situ rat brain perfusion and after intraperitoneal administration to mice. As a result, these targeted inhibitors increased significantly the apoptosis-inducing effects of VBL and more effectively than PRB itself. They also were hemocompatible and non-toxic in healthy cells with a concentration below 100 µM. Interestingly, the most promising compound doubled the penetration rate of VBL across the rat blood-brain barrier (BBB). This makes it a promising candidate for further studies to improve efflux transporter-related MDR of brain-targeted anti-cancer agents.
10.1016/j.ijpharm.2020.119585
Targeting the cerebrovascular large neutral amino acid transporter (LAT1) isoform using a novel disulfide-based brain drug delivery system.
Killian Dennis M,Hermeling Suzanne,Chikhale Prashant J
Drug delivery
We describe a novel strategy to achieve high affinity recognition for the specific, cerebrovascular large neutral amino acid transporter (LAT1) isoform by covalent coupling of small molecules to the amino acid, L-cysteine (L-Cys). L-Cys (as the carrier) was covalently attached via a disulfide bond to either 6-mercaptopurine or 2-methyl-1-propanethiol (IBM) to form the brain-targeted drug delivery systems (BTDS). BTDS were designed for high affinity recognition by LAT1 at the cerebrovasculature. Using an in situ rat brain perfusion technique, competition between BTDS and the radiotracer [14C]L-Leu demonstrated significant inhibition of [14C]L-Leu brain uptake. BTDS possess affinity for cerebrovascular LAT1 in many distinct brain compartments, and the recognition of BTDS by LAT1 is influenced by hydrophobicity of the side-chain in BTDS. Thus, the BTDS strategy may be utilized for rapid shuttling of various neuropharmaceuticals into brain.
10.1080/10717540600559510
A Selective and Slowly Reversible Inhibitor of l-Type Amino Acid Transporter 1 (LAT1) Potentiates Antiproliferative Drug Efficacy in Cancer Cells.
Huttunen Kristiina M,Gynther Mikko,Huttunen Johanna,Puris Elena,Spicer Julie A,Denny William A
Journal of medicinal chemistry
The l-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is overexpressed in several types of tumors, and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, l-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 μM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective toward LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer.
10.1021/acs.jmedchem.6b00190
MicroRNA-126-3p Inhibits Angiogenic Function of Human Lung Microvascular Endothelial Cells via LAT1 (L-Type Amino Acid Transporter 1)-Mediated mTOR (Mammalian Target of Rapamycin) Signaling.
Arteriosclerosis, thrombosis, and vascular biology
OBJECTIVE:MicroRNA-126-3p (miR-126) is required for angiogenesis during organismal development or the repair of injured arterial vasculature. The role of miR-126 in lung microvascular endothelial cells, which are essential for gas exchange and for lung injury repair and regeneration, remains poorly understood. Considering the significant heterogeneity of endothelial cells from different vascular beds, we aimed to determine the role of miR-126 in regulating lung microvascular endothelial cell function and to elucidate its downstream signaling pathways. Approach and Results: Overexpression and knockdown of miR-126 in primary human lung microvascular endothelial cells (HLMVEC) were achieved via transfections of miR-126 mimics and antisense inhibitors. Increasing miR-126 levels in HLMVEC reduced cell proliferation, weakened tube formation, and increased cell apoptosis, whereas decreased miR-126 levels stimulated cell proliferation and tube formation. Whole-genome RNA sequencing revealed that miR-126 was associated with an antiangiogenic and proapoptotic transcriptomic profile. Using validation assays and knockdown approaches, we identified that the effect of miR-126 on HLMVEC angiogenesis was mediated by the LAT1 (L-type amino acid transporter 1), via regulation of mTOR (mammalian target of rapamycin) signaling. Furthermore, downregulation of miR-126 in HLMVEC inhibited cell apoptosis and improved endothelial tube formation during exposure to environmental insults such as cigarette smoke. CONCLUSIONS:miR-126 inhibits HLMVEC angiogenic function by targeting the LAT1-mTOR signaling axis, suggesting that miR-126 inhibition may be useful for conditions associated with microvascular loss, whereas miR-126 augmentation may help control unwanted microvascular angiogenesis.
10.1161/ATVBAHA.119.313800
The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model.
Journal of experimental & clinical cancer research : CR
BACKGROUND:The L-type amino acid transporter 1 (LAT1/SLC7A5) transports essential amino acids across the plasma membrane. While LAT1 is overexpressed in a variety of human neoplasms, its expression and its role in thyroid cancer is currently unknown. Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy for which no effective therapy exists. The purpose of this study was to explore whether the inhibition of LAT1 in ATC would affect tumor growth both in vitro and in vivo. METHODS:LAT1 was pharmacologically blocked by JPH203 in human ATC and papillary thyroid cancer (PTC) cell lines. The effects on proliferation and mTORC1 activity were addressed in vitro. A genetically engineered mouse model of ATC was used to address the effect of blocking LAT1 on tumor growth in vivo. SLC7A5 transcription was measured in patient-derived ATC samples to address the clinical relevance of the findings. RESULTS:LAT1 block by JPH203 reduced proliferation and mTORC1 signaling in human thyroid cancer cell lines. SLC7A5 transcription was upregulated in ATC tissues derived from a genetically engineered mouse model and in ATC samples recovered from patients. JPH203 treatment induced thyroid tumor growth arrest in vivo in a fully immunocompetent mouse model of thyroid cancer. Additionally, analysis of publicly available datasets of thyroid carcinomas revealed that high LAT1 expression is associated with potentially untreatable PTC presenting reduced NIS/SLC5A5 transcription and with ATC. CONCLUSIONS:These preclinical results show that LAT1 inhibition is a novel therapeutic approach in the context of thyroid cancers, and more interestingly in untreatable thyroid cancers.
10.1186/s13046-018-0907-z
JPH203, a newly developed anti-cancer drug, shows a preincubation inhibitory effect on L-type amino acid transporter 1 function.
Okunushi Kentaro,Furihata Tomomi,Morio Hanae,Muto Yasuhide,Higuchi Kosuke,Kaneko Meika,Otsuka Yusuke,Ohno Yuta,Watanabe Yasuhiro,Reien Yoshie,Nakagawa Kiyoshi,Sakamoto Shinichi,Wakashin Hidefumi,Shimojo Naoki,Anzai Naohiko
Journal of pharmacological sciences
JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 μM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 μM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.
10.1016/j.jphs.2020.06.006
Structural properties for selective and efficient l-type amino acid transporter 1 (LAT1) mediated cellular uptake.
Kärkkäinen Jussi,Gynther Mikko,Kokkola Tarja,Petsalo Aleksanteri,Auriola Seppo,Lahtela-Kakkonen Maija,Laine Krista,Rautio Jarkko,Huttunen Kristiina M
International journal of pharmaceutics
l-Type amino acid transporter 1 (LAT1) is a sodium-independent exchanger transporting large neural amino acids and several amino-acid mimicking drugs across the cell membranes. LAT1 is highly expressed at the blood brain barrier (BBB) and in numerous cancer cells and is therefore a potential drug target. However, structural features affecting the ability to bind to LAT1 and the cellular translocation by LAT1 are unclear. In the present study we determined the binding to and transport through human LAT1 of several compounds into the human breast adenocarcinoma cells (MCF-7). We show that the meta-conjugation of l-phenylalanine increases binding to human LAT1 compared to para-conjugation or aliphatic amino acid moiety. Furthermore, large, rigid and aromatic meta-substituted l-phenylalanine derivative enabled selective and efficient LAT1-mediated cellular uptake. Our results also demonstrates that in addition to binding studies, it is of utmost importance to determine the cellular accumulation of compounds. It provides crucial information on transport efficiency and selectivity of transport mechanisms that the compounds are able to utilize. Overall, these structural findings and the methodology used herein are exploitable to design LAT1-utilizing compounds, such as markers for cancer imaging and drug molecules, enabling more effective and safer treatments for cancer in the future.
10.1016/j.ijpharm.2018.04.025
LAT-1 functions as a promotor in gastric cancer associated with clinicopathologic features.
Wang Junqing,Chen Xuehua,Su Liping,Li Pu,Liu Bingya,Zhu Zhenggang
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
L-type amino-acid transporter 1 (LAT-1) is a member of system L-type transporters, essential for cells maintenance and proliferation. However, the role of LAT-1 remains illegible in gastric cancer (GC). In this study, we found that LAT-1 was aberrantly up-regulated in both GC cell lines (MKN-45, MGC-803 and CRL-5974) and human GC specimens. The expression characteristic of LAT-1 in GC was significantly associated with clinicopathologic features such as tumor size, lymph node metastasis, local invasion and TNM stage. By suppressing the expression of LAT-1 in MKN-45 cells, the cell cycle was arrested in G0/G1 phase, and the ability of cell proliferation was significantly decreased in vitro. Moreover, the cell migration and invasion of MKN-45 cells was significantly impaired by knocking down LAT-1. Thus, our results suggest that LAT-1 may function as an oncogene in GC, which provides us a new biomarker in GC and perhaps a potential target for GC prevention, diagnose and therapeutic treatment.
10.1016/j.biopha.2013.05.003
Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation.
Nature immunology
T lymphocytes must regulate nutrient uptake to meet the metabolic demands of an immune response. Here we show that the intracellular supply of large neutral amino acids (LNAAs) in T cells was regulated by pathogens and the T cell antigen receptor (TCR). T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells. Slc7a5-null T cells were unable to metabolically reprogram in response to antigen and did not undergo clonal expansion or effector differentiation. The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc. Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.
10.1038/ni.2556
Proteomics and phosphoproteomics reveal key regulators associated with cytostatic effect of amino acid transporter LAT1 inhibitor.
Okanishi Hiroki,Ohgaki Ryuichi,Okuda Suguru,Endou Hitoshi,Kanai Yoshikatsu
Cancer science
L-type amino acid transporter 1 (LAT1) is highly expressed in various cancers and plays important roles not only in the amino acid uptake necessary for cancer growth but also in cellular signaling. Recent research studies have reported anticancer effects of LAT1 inhibitors and demonstrated their potential for cancer therapy. Here, we characterized the proteome and phosphoproteome in LAT1-inhibited cancer cells. We used JPH203, a selective LAT1 inhibitor, and performed tandem mass tag-based quantitative proteomics and phosphoproteomics on four biliary tract cancer cell lines sensitive to JPH203. Our analysis identified hundreds to thousands of differentially expressed proteins and phosphorylated sites, demonstrating the broad influence of LAT1 inhibition. Our findings showed various functional pathways altered by LAT1 inhibition, and provided possible regulators and key kinases in LAT1-inhibited cells. Comparison of these changes among cell lines provides insights into general pathways and regulators associated with LAT1 inhibition and particularly suggests the importance of cell cycle-related pathways and kinases. Moreover, we evaluated the anticancer effects of the combinations of JPH203 with cell cycle-related kinase inhibitors and demonstrated their potential for cancer therapy. This is the first study providing the proteome-wide scope of both protein expression and phosphorylation signaling perturbed by LAT1 inhibition in cancer cells.
10.1111/cas.14756
Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2.
Dann Stephen G,Ryskin Michael,Barsotti Anthony M,Golas Jonathon,Shi Celine,Miranda Miriam,Hosselet Christine,Lemon Luanna,Lucas Judy,Karnoub Maha,Wang Fang,Myers Jeremy S,Garza Scott J,Follettie Maximillian T,Geles Kenneth G,Klippel Anke,Rollins Robert A,Fantin Valeria R
The EMBO journal
Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.
10.15252/embj.201488166
Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1.
Geier Ethan G,Schlessinger Avner,Fan Hao,Gable Jonathan E,Irwin John J,Sali Andrej,Giacomini Kathleen M
Proceedings of the National Academy of Sciences of the United States of America
The Large-neutral Amino Acid Transporter 1 (LAT-1)--a sodium-independent exchanger of amino acids, thyroid hormones, and prescription drugs--is highly expressed in the blood-brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood-brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism.
10.1073/pnas.1218165110
Large amino acid transporter 1 mediated glutamate modified docetaxel-loaded liposomes for glioma targeting.
Li Lin,Di Xingsheng,Zhang Shenwu,Kan Qiming,Liu Hao,Lu Tianshu,Wang Yongjun,Fu Qiang,Sun Jin,He Zhonggui
Colloids and surfaces. B, Biointerfaces
The therapeutic outcome of glioma treatment is rigorously limited by blood-brain barrier (BBB) and infiltrating growth of glioma. To tackle the dilemma, more and more attentions were focused on developing nutrient transporters-mediated dual-targeted drug delivery system, in one side for BBB penetration, another for intracranial glioma targeting. Herein, Large amino acid transporter 1 (LAT1), overexpressed both on BBB and glioma cells, was selected as a target. Docetaxel-loaded glutamate-d-α-tocopherol polyethylene glycol 1000 succinate copolymer (Glu-TPGS) functionalized LAT1-targeting liposomes (DTX-TGL) were applied to enhance the BBB penetration and glioma therapy. The in vivo results of the fluorescent image indicated that TGL possessed an effective BBB penetration than that of unmodified ones in mice. The LAT1 targeting effcicacy and cell cytotoxicity of TGL were investigated in C6 glioma cells. Compared with unmodified liposomes, a significant higher cellular uptake and cell cytotoxicity was found in TGL treated group. Our results indicated that LAT1-targeting docetaxel-loaded liposome paves up a new direction using LAT1 transporter as a good target in designing brain glioma-targeting nanosystems.
10.1016/j.colsurfb.2016.01.041
Tyrosine modified irinotecan-loaded liposomes capable of simultaneously targeting LAT1 and ATB for efficient tumor therapy.
Wang Zhenjie,Chi Dongxu,Wu Xingchen,Wang Yingli,Lin Xinxin,Xu Zhaochu,Liu Hongzhuo,Sun Jin,He Zhonggui,Wang Yongjun
Journal of controlled release : official journal of the Controlled Release Society
As the demand for nutrients in malignant proliferation of tumors increases, the L-type amino acid transporter 1(LAT1) and amino acid transporter B (ATB) of tumor cells are more highly expressed than normal cells which can be used as new targets for active targeting of cancer. However, drug delivery systems often require multi-target design to achieve simultaneous targeting of different receptors or transporters due to the heterogeneity of the tumor. Here we utilized triethylamine-sucrose octasulfate gradient to actively encapsulate irinotecan into the introliposomal aqueous phase. Targeted ability was achieved through inserting different amino acids modified polyethylene glycol monostearate into the liposomes, and found that glutamate-liposomes can be targeted to LAT1, lysine-liposomes can be targeted to ATB, and inspiringly, tyrosine-liposomes can be simultaneously targeted to LAT1 and ATB. The tyrosine-modified liposomes showed the highest cellular uptake in BxPC-3 and MCF-7 cells which were highly expressed both LAT1 and ATB. Moreover, we validated their targeting capabilities and elucidated the transport mechanism of LAT1 and ATB-mediated endocytosis. The tumor inhibition rate of tyrosine-modified liposomes greatly increased from 39% to 87% compared with commercially available liposomes loaded CPT-11(Onivyde®). Overall, it showed a good application prospect for efficient tumor therapy and industrial production.
10.1016/j.jconrel.2019.10.037
LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation.
Milkereit Ruth,Persaud Avinash,Vanoaica Liviu,Guetg Adriano,Verrey Francois,Rotin Daniela
Nature communications
Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA.
10.1038/ncomms8250
Hemocompatible LAT1-inhibitor can induce apoptosis in cancer cells without affecting brain amino acid homeostasis.
Markowicz-Piasecka Magdalena,Huttunen Johanna,Montaser Ahmed,Huttunen Kristiina M
Apoptosis : an international journal on programmed cell death
Increased amounts of amino acids are essential for cancer cells to support their sustained growth and survival. Therefore, inhibitors of amino acid transporters, such as L-type amino acid transporter 1 (LAT1) have been developed. In this study, a previously reported LAT1-inhibitor (KMH-233) was studied for its hemocompatibility and toxicity towards human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (AoSMCs). Furthermore, the cytotoxic effects against human breast adenocarcinoma cells (MCF-7) and its ability to affect mammalian (or mechanistic) target of rapamycin (mTOR) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling were evaluated. Moreover, the effects of this inhibitor to modulate LAT1 function on the cell surface and the brain amino acid homeostasis were evaluated after intraperitoneal (i.p.) administration of LAT1-inhibitor (23 µmol/kg) in mice. The results showed that LAT1-inhibitor (KMH-233) is hemocompatible at concentrations below 25 µM and it does not affect coagulation in plasma. However, it can reduce the total protein amount of mTOR and NF-κB, resulting in increased apoptosis in LAT1-expressing cancer cells. Most importantly, the inhibitor did not affect mouse brain levels of L-Leu, L-Tyr or L-Trp or modulate the function of LAT1 on the MCF-7 cell surface. Therefore, this inhibitor can be considered as a safe but effective anti-cancer agent. However, due to the compensative mechanism of cancer cells for their increased amino acid demand, this compound is most effective inducing apoptosis when used in combinations with other chemotherapeutics, such as protease inhibitor, bestatin, as demonstrated in this study.
10.1007/s10495-020-01603-7
Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc.
Lee Yongchan,Wiriyasermkul Pattama,Jin Chunhuan,Quan Lili,Ohgaki Ryuichi,Okuda Suguru,Kusakizako Tsukasa,Nishizawa Tomohiro,Oda Kazumasa,Ishitani Ryuichiro,Yokoyama Takeshi,Nakane Takanori,Shirouzu Mikako,Endou Hitoshi,Nagamori Shushi,Kanai Yoshikatsu,Nureki Osamu
Nature structural & molecular biology
The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.
10.1038/s41594-019-0237-7
MicroRNA-126 inhibits cell proliferation in gastric cancer by targeting LAT-1.
Wang Junqing,Chen Xuehua,Su Liping,Li Pu,Cai Qu,Liu Bingya,Wu Weize,Zhu Zhenggang
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
MicroRNA-126 (miR-126) is a pivotal post-transcriptional regulator, which has been validated as a suppressor in gastric cancer (GC). However, the downstream of its tumor inhibiting function has not been totally clear. L-type amino-acid transporter 1 (LAT-1) is a novel member of system L-type transporters involving in cell proliferation, and we have previously validated that LAT-1 played a role of promotor in GC. In this study, we further detected and confirmed that LAT-1 was exactly targeted by miR-126 in GC. We found LAT-1 was significantly downregulated in GC MKN-45 cell lines by using miR-126 mimics, along with an impairment on cell proliferation and cell cycle. Additionally, by overexpressing LAT-1 in MKN-45 cells which was firstly treated with miR-126 mimics, the ability of cell proliferation in MKN-45 cells was definitely rescued. Thus, our results suggests and consolidates the standpoint that miR-126 plays a pivotal role in GC suppressing the process of GC cell, and this function is at least partly taken to implement by miR-126s's post-transcriptional effect on LAT-1. This might provide us likely potential biomarkers and targets for GC prevention, diagnosis and therapeutic treatment.
10.1016/j.biopha.2015.04.001
Enhanced glutamine uptake influences composition of immune cell infiltrates in breast cancer.
Ansari Rokaya El,Craze Madeleine L,Althobiti Maryam,Alfarsi Lutfi,Ellis Ian O,Rakha Emad A,Green Andrew R
British journal of cancer
BACKGROUND:Cancer cells must alter their metabolism to support proliferation. Immune evasion also plays a role in supporting tumour progression. This study aimed to find whether enhanced glutamine uptake in breast cancer (BC) can derive the existence of specific immune cell subtypes, including the subsequent impact on patient outcome. METHODS:SLC1A5, SLC7A5, SLC3A2 and immune cell markers CD3, CD8, FOXP3, CD20 and CD68, in addition to PD1 and PDL1, were assessed by using immunohistochemistry on TMAs constructed from a large BC cohort (n = 803). Patients were stratified based on SLC protein expression into accredited clusters and correlated with immune cell infiltrates and patient outcome. The effect of transient siRNA knockdown of SLC7A5 and SLC1A5 on PDL1 expression was evaluated in MDA-MB-231 cells. RESULTS:High SLCs were significantly associated with PDL1 and PD1 +, FOXP3 +, CD68 + and CD20 + cells (p < 0.001). Triple negative (TN), HER2 + and luminal B tumours showed variable associations between SLCs and immune cell types (p ≤ 0.04). The expression of SLCs and PDL1, PD1 +, FOXP3 + and CD68 + cells was associated with poor patient outcome (p < 0.001). Knockdown of SLC7A5 significantly reduced PDL1 expression. CONCLUSION:This study provides data that altered glutamine pathways in BC that appears to play a role in deriving specific subtypes of immune cell infiltrates, which either support or counteract its progression.
10.1038/s41416-019-0626-z
Reevaluating the Substrate Specificity of the L-Type Amino Acid Transporter (LAT1).
Chien Huan-Chieh,Colas Claire,Finke Karissa,Springer Seth,Stoner Laura,Zur Arik A,Venteicher Brooklynn,Campbell Jerome,Hall Colton,Flint Andrew,Augustyn Evan,Hernandez Christopher,Heeren Nathan,Hansen Logan,Anthony Abby,Bauer Justine,Fotiadis Dimitrios,Schlessinger Avner,Giacomini Kathleen M,Thomas Allen A
Journal of medicinal chemistry
The L-type amino acid transporter 1 (LAT1, SLC7A5) transports essential amino acids across the blood-brain barrier (BBB) and into cancer cells. To utilize LAT1 for drug delivery, potent amino acid promoieties are desired, as prodrugs must compete with millimolar concentrations of endogenous amino acids. To better understand ligand-transporter interactions that could improve potency, we developed structural LAT1 models to guide the design of substituted analogues of phenylalanine and histidine. Furthermore, we evaluated the structure-activity relationship (SAR) for both enantiomers of naturally occurring LAT1 substrates. Analogues were tested in cis-inhibition and trans-stimulation cell assays to determine potency and uptake rate. Surprisingly, LAT1 can transport amino acid-like substrates with wide-ranging polarities including those containing ionizable substituents. Additionally, the rate of LAT1 transport was generally nonstereoselective even though enantiomers likely exhibit different binding modes. Our findings have broad implications to the development of new treatments for brain disorders and cancer.
10.1021/acs.jmedchem.8b01007
Insights into the Transport Cycle of LAT1 and Interaction with the Inhibitor JPH203.
International journal of molecular sciences
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.
10.3390/ijms24044042
Polymeric ligands comprising sulfur-containing amino acids for targeting tumor-associated amino acid transporters.
Biomaterials
Various cancer cells overexpress L-type amino acid transporter 1 (LAT1) to take up a large number of neutral amino acids such as phenylalanine and methionine, and LAT1 transporter should be a promising target for cancer diagnosis and therapy. However, only a few studies reported drug delivery systems targeting LAT1 probably due to limited knowledge about the interaction between LAT1 and its substrate. Here, we developed polymers having methionine (Met)- or cysteine (Cys)-like structures on their side chains to examine their affinity with LAT1. While both the Met- and Cys-modified polymers exhibited efficient cellular uptake selectively in cancer cells, the Met-modified polymers exhibited higher cellular uptake efficiency in an LAT1-selective manner than the Cys-modified polymers. In the in vivo study, the intraperitoneally injected Met-modified polymers showed appreciable tumor-selective accumulation in the peritoneal dissemination model, and importantly, Met-modified polymers conjugated with photosensitizers exhibited significant therapeutic effects upon photoirradiation with reduced photochemical damage to normal organs. Our results may provide important knowledge about the polymer-LAT1 interaction, and the Met-modified polymers should offer a new concept for designing LAT1-targeting drug delivery systems.
10.1016/j.biomaterials.2022.121987
Endocytosis-Independent and Cancer-Selective Cytosolic Protein Delivery via Reversible Tagging with LAT1 substrate.
Advanced materials (Deerfield Beach, Fla.)
Protein drugs targeting intracellular machineries have shown profound therapeutic potentials, but their clinical utilities are greatly hampered by the lack of efficient cytosolic delivery techniques. Existing strategies mainly rely on nanocarriers or conjugated cell-penetrating peptides (CPPs), which often have drawbacks such as materials complexity/toxicity, lack of cell specificity, and endolysosomal entrapment. Herein, a unique carrier-free approach is reported for mediating cancer-selective and endocytosis-free cytosolic protein delivery. Proteins are sequentially modified with 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl carbonate as the H O -responsive domain and 3,4-dihydroxy-l-phenylalanine as the substrate of l-type amino acid transporter 1 (LAT1). Thus, the pro-protein can be directly transported into tumor cells by overexpressed LAT1 on cell membranes, bypassing endocytosis and endolysosomal entrapment. In the cytosol, overproduced H O restores the protein structure and activity. Using this technique, versatile proteins are delivered into tumor cells with robust efficiency, including toxins, enzymes, CRISPR-Cas9 ribonucleoprotein, and antibodies. Furthermore, intravenously injected pro-protein of saporin shows potent anticancer efficacy in 4T1-tumor-bearing mice, without provoking systemic toxicity. Such a facile and versatile pro-protein platform may benefit the development of protein pharmaceuticals.
10.1002/adma.202110560
Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime?
Fuchs Bryan C,Bode Barrie P
Seminars in cancer biology
Relative to other neutral amino acid transporters, the expression levels of ASCT2 and LAT1, are coordinately elevated in a wide spectrum of primary human cancers, suggesting that they are frequently co-opted to support the "tumor metabolome". Each has recently been shown to play important roles in the growth and survival of cancer cell lines, making them potential targets for cancer therapy. The properties and putative relationship of these two amino acid exchangers are discussed in the context of their demonstrated utility in cancer biology, including cellular growth and survival signaling and integrated links to the mammalian target-of-rapamycin (mTOR) kinase.
10.1016/j.semcancer.2005.04.005
Novel strategies to improve tumour therapy by targeting the proteins MCT1, MCT4 and LAT1.
Wang Yang,Qin Liuxin,Chen Weiwei,Chen Qing,Sun Jin,Wang Gang
European journal of medicinal chemistry
Poor selectivity, potential systemic toxicity and drug resistance are the main challenges associated with chemotherapeutic drugs. MCT1 and MCT4 and LAT1 play vital roles in tumour metabolism and growth by taking up nutrients and are thus potential targets for tumour therapy. An increasing number of studies have shown the feasibility of including these transporters as components of tumour-targeting therapy. Here, we summarize the recent progress in MCT1-, MCT4-and LAT1-based therapeutic strategies. First, protein structures, expression, relationships with cancer, and substrate characteristics are introduced. Then, different drug targeting and delivery strategies using these proteins have been reviewed, including designing protein inhibitors, prodrugs and nanoparticles. Finally, a dual targeted strategy is discussed because these proteins exert a synergistic effect on tumour proliferation. This article concentrates on tumour treatments targeting MCT1, MCT4 and LAT1 and delivery techniques for improving the antitumour effect. These innovative tactics represent current state-of-the-art developments in transporter-based antitumour drugs.
10.1016/j.ejmech.2021.113806
L-Type Amino Acid Transporter 1 (LAT1) Promotes PMA-Induced Cell Migration through mTORC2 Activation at the Lysosome.
Cells
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.
10.3390/cells12202504
Amino acid transporters as emerging therapeutic targets in cancer.
Saito Yasuhiro,Soga Tomoyoshi
Cancer science
Amino acids are indispensable nutrients for both normal and cancer cells. Cancer cells are unable to synthesize essential amino acids as well as some non-essential amino acids adequately to support rapid proliferation, and must take up amino acids from the surroundings. To meet the increased demand for the amino acid needed for proliferation, high levels of amino acid transporters are expressed on the surface of cancer cells. Cancer cells utilize amino acids to synthesize proteins and nucleotides, as well as to obtain energy. In addition, amino acids are known to play pathological roles in cancer cells. Interestingly, breast cancer cells limit the use of amino acids for cell proliferation based on amino acid availability, which depends on estrogen receptor status. Here, we present a summarized literature review of novel amino acid functions in cancer cells. This review organizes the available knowledge on 2 amino acid transporters, SLC7A5 and SLC7A11, which are considered essential for breast cancer cell growth in a cell-dependent manner. In particular, we propose the glutamine recycling model to clarify the mechanism underlying aberrant SLC7A5 activation. Finally, we overview the pathological significances of SLC7A5 and SLC7A11 in cancer tissues.
10.1111/cas.15006
Potent inhibitors of human LAT1 (SLC7A5) transporter based on dithiazole and dithiazine compounds for development of anticancer drugs.
Napolitano Lara,Scalise Mariafrancesca,Koyioni Maria,Koutentis Panayiotis,Catto Marco,Eberini Ivano,Parravicini Chiara,Palazzolo Luca,Pisani Leonardo,Galluccio Michele,Console Lara,Carotti Angelo,Indiveri Cesare
Biochemical pharmacology
The LAT1 transporter is acknowledged as a pharmacological target of tumours since it is strongly overexpressed in many human cancers. The purpose of this work was to find novel compounds exhibiting potent and prolonged inhibition of the transporter. To this aim, compounds based on dithiazole and dithiazine scaffold have been screened in the proteoliposome experimental model. Inhibition was tested on the antiport catalysed by hLAT1 as transport of extraliposomal [H]histidine in exchange with intraliposomal histidine. Out of 59 compounds tested, 8 compounds, showing an inhibition higher than 90% at 100µM concentration, were subjected to dose-response analysis. Two of them exhibited IC lower than 1µM. Inhibition kinetics, performed on the two best inhibitors, indicated a mixed type of inhibition with respect to the substrate. Furthermore, inhibition of the transporter was still present after removal of the compounds from the reaction mixture, but was reversed on addition of dithioerythritol, a S-S reducing agent, indicating the formation of disulfide(s) between the compounds and the protein. Molecular docking of the two best inhibitors on the hLAT1 homology structural model, highlighted interaction with the substrate binding site and formation of a covalent bond with the residue C407. Indeed, the inhibition was impaired in the hLAT1 mutant C407A confirming the involvement of that Cys residue. Treatment of SiHa cells expressing hLAT1 at relatively high level, with the two most potent inhibitors led to cell death which was not observed after treatment with a compound exhibiting very poor inhibitory effect.
10.1016/j.bcp.2017.07.006
The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.
Nature communications
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). 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.
10.1038/s41467-024-47948-4
Expression profile of the amino acid transporters SLC7A5, SLC7A7, SLC7A8 and the enzyme TDO2 in basal cell carcinoma.
Tina E,Prosén S,Lennholm S,Gasparyan G,Lindberg M,Göthlin Eremo A
The British journal of dermatology
BACKGROUND:The incidence of basal cell carcinoma (BCC) is increasing and the costs for care rising. Therefore, the need for simplified and cost-effective treatment choices is substantial. Aberrant signalling in several pathways, induced by ultraviolet radiation, is of importance in the development of BCC. Alterations in tumour metabolic activity are part of general carcinogenesis; however, these alterations are only partially recognized in skin cancer. OBJECTIVES:To study expression profiles in BCCs compared with individually matched nontumour skin, with a focus on finding differences associated with tumour metabolism. MATERIALS AND METHODS:Gene expression in biopsies from BCCs (n = 14) compared with biopsies from nontumour gluteal skin was analysed with microarrays (n = 4 + 4) and/or quantitative real-time polymerase chain reaction (qPCR, n = 14 + 14). Protein expression and localization was assessed using immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded BCC samples. RESULTS:Microarray analysis revealed increased expression of the amino acid transporters SLC7A5, SLC7A7 and SLC7A8 as well as the cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) 2 in BCC. Higher expression of SLC7A5 (P < 0·001), SLC7A8 (P < 0·001) and TDO2 (P = 0·002), but not SLC7A7 (P = 0·50), was confirmed by qPCR, and IHC demonstrated correlating tumour cell protein expression of SLC7A5 and SLC7A8. Protein expression of SLC7A7 was observed in the stratum granulosum, and TDO2 in immune cells. CONCLUSIONS:This study highlights the upregulation of SLC7A5, SLC7A8 and TDO2 in BCC compared with nontumour skin. Our findings imply that amino acid transporters may be further explored as potential targets for future medical treatment.
10.1111/bjd.16905
HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.
Elorza Ainara,Soro-Arnáiz Inés,Meléndez-Rodríguez Florinda,Rodríguez-Vaello Victoria,Marsboom Glenn,de Cárcer Guillermo,Acosta-Iborra Bárbara,Albacete-Albacete Lucas,Ordóñez Angel,Serrano-Oviedo Leticia,Giménez-Bachs Jose Miguel,Vara-Vega Alicia,Salinas Antonio,Sánchez-Prieto Ricardo,Martín del Río Rafael,Sánchez-Madrid Francisco,Malumbres Marcos,Landázuri Manuel O,Aragonés Julián
Molecular cell
The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.
10.1016/j.molcel.2012.09.017
TRIM35 triggers cardiac remodeling by regulating SLC7A5-mediated amino acid transport and mTORC1 activation in fibroblasts.
Cell communication and signaling : CCS
BACKGROUND:Cardiac maladaptive remodeling is one of the leading causes of heart failure with highly complicated pathogeneses. The E3 ligase tripartite motif containing 35 (TRIM35) has been identified as a crucial regulator governing cellular growth, immune responses, and metabolism. Nonetheless, the role of TRIM35 in fibroblasts in cardiac remodeling remains elusive. METHODS:Heart tissues from human donors were used to verify tissue-specific expression of TRIM35. Fibroblast-specific Trim35 gene knockout mice (Trim35) were used to investigate the function of TRIM35 in fibroblasts. Cardiac function, morphology, and molecular changes in the heart tissues were analyzed after transverse aortic constriction (TAC) surgery. The mechanisms by which TRIM35 regulates fibroblast phenotypes were elucidated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA sequencing (RNA-Seq). These findings were further validated through the use of adenoviral and adeno-associated viral transfection systems, as well as the mTORC1 inhibitor Rapamycin. RESULTS:TRIM35 expression is primarily up-regulated in cardiac fibroblasts in both murine and human fibrotic hearts, and responds to TGF-β1 stimulation. Specific deletion of TRIM35 in cardiac fibroblasts significantly improves cardiac fibrosis and hypertrophy. Consistently, the overexpression of TRIM35 promotes fibroblast proliferation, migration, and differentiation. Through paracrine signaling, it induces hypertrophic growth of cardiomyocytes. Mechanistically, we found that TRIM35 interacts with, ubiquitinates, and up-regulates the amino acid transporter SLC7A5, which enhances amino acid transport and activates the mTORC1 signaling pathway. Furthermore, overexpression of SLC7A5 significantly reverses the reduced cardiac fibrosis and hypertrophy caused by conditional knockout of TRIM35. CONCLUSION:Our findings demonstrate a novel role of fibroblast-TRIM35 in cardiac remodeling and uncover the mechanism underlying SLC7A5-mediated amino acid transport and mTORC1 activation. These results provide a potential novel therapeutic target for treating cardiac remodeling.
10.1186/s12964-024-01826-0
Targeted delivery of liposomal Ribociclib to SLC7A5 transporters in breast cancer cells.
Investigational new drugs
This study aimed to prepare SLC7A5 transporters targeted liposomes of Ribociclib (RB) by stear(o)yl conjugation of Phe, Asp, Glu amino acids to liposomes as targeting moieties. The liposomes were optimized for their formulations. Cell analysis on two cell lines of MCF-7 and NIH-3T3 were done including; cell viability test by MTT assay, cellular uptake, and cell cycle arrest by flow cytometry. The optimal liposomes showed the particle size of 123.6 ± 1.3 nm, drug loading efficiency and release efficiency of 83.87% ± 1.33% and 60.55% ± 0.46%, respectively. The RB loaded liposomes showed no hemolysis activity. Targeted liposomes increased cytotoxicity on MCF-7 cells more significantly than NIH-3T3 cells. Cell flow cytometry indicated that targeted liposomes uptake was superior to plain (non-targted) liposomes and free drug. Free drug and RB-loaded liposomes interrupted cell cycle in G1. However, amino acid-targeted liposomes arrested cells more than the free drug at this stage. Targeted liposomes reduced cell cycle with more interruption in the G2/M phase compared to the negative control.
10.1007/s10637-023-01409-9
Identify metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 as potential prognostic markers and correlate with immune infiltrates in head and neck squamous cell carcinoma.
Frontiers in immunology
Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of head and neck squamous cell carcinoma (HNSCC) with poor prognosis. Metabolic reprogramming may regulate the tumor microenvironment (TME) by adapting quickly to cellular stress and regulating immune response, but its role in HSCC has not been reported. We used the nCounter Metabolic Pathways Panel to investigate metabolic reprogramming, cellular stress, and their relationship in HSCC tissues and adjacent normal tissues. Metabolism-related pathways nucleotide synthesis and glycolysis pathways were significantly upregulated, while amino acid synthesis and fatty acid oxidation pathways were significantly downregulated in HSCC tissues compared to adjacent normal tissues. There is a significant correlation between metabolism-related pathways and cellular stress pathways. Enrichment of immune cell and tumor infiltrating lymphocyte (TIL) analysis showed changes in immune responses between HSCC tissues and adjacent normal tissues. Overall survival analysis showed that upregulated genes CD276, LDHB, SLC3A2, EGFR, SLC7A5, and HPRT1 are potential unfavorable prognostic markers in HNSCC, while downregulated genes EEA1, IDO1, NCOA2, REST, CCL19, and ALDH2 are potential favorable prognostic markers in HNSCC. Moreover, metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 are correlated with immune infiltrates in HNSCC. These results suggest that metabolic reprogramming occurs and correlates with cellular stress and immune response in HSCC, which may help researchers understand mechanisms of metabolic reprogramming and develop effective immunotherapeutic strategies in HNSCC.
10.3389/fimmu.2022.955614
The amino acid transporter SLC7A5 confers a poor prognosis in the highly proliferative breast cancer subtypes and is a key therapeutic target in luminal B tumours.
Breast cancer research : BCR
BACKGROUND:Breast cancer (BC) is a heterogeneous disease characterised by variant biology and patient outcome. The amino acid transporter, SLC7A5, plays a role in BC although its impact on patient outcome in different BC subtypes remains to be validated. This study aimed to determine whether the clinicopathological and prognostic value of SLC7A5 is different within the molecular classes of BC. METHODS:SLC7A5 was assessed at the genomic level, using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data (n = 1980), and proteomic level, using immunohistochemical analysis and tissue microarray (TMA) (n = 2664; 1110 training and 1554 validation sets) in well-characterised primary BC cohorts. SLC7A5 expression correlated with clinicopathological and biological parameters, molecular subtypes and patient outcome. RESULTS:SLC7A5 mRNA and protein expression were strongly correlated with larger tumour size and higher grade. High expression was observed in triple negative (TN), human epidermal growth factor receptor 2 (HER2)+, and luminal B subtypes. SLC7A5 mRNA and protein expression was significantly associated with the expression of the key regulator of tumour cell metabolism, c-MYC, specifically in luminal B tumours only (p = 0.001). High expression of SLC7A5 mRNA and protein was associated with poor patient outcome (p < 0.001) but only in the highly proliferative oestrogen receptor (ER)+/ luminal B (p = 0.007) and HER2+ classes of BC (p = 0.03). In multivariate analysis, SLC7A5 protein was an independent risk factor for shorter breast-cancer-specific survival only in ER+ high-proliferation tumours (p = 0.02). CONCLUSIONS:SLC7A5 appears to play a role in the aggressive highly proliferative ER+ subtype driven by MYC and could act as a potential therapeutic target. Functional assessment is necessary to reveal the specific role played by this transporter in the ER+ highly proliferative subclass and HER2+ subclass of BC.
10.1186/s13058-018-0946-6
Fat mass and obesity-associated protein (FTO) mediated mA modification of circFAM192A promoted gastric cancer proliferation by suppressing SLC7A5 decay.
Molecular biomedicine
Gastric cancer (GC) is a common malignant tumor worldwide, especially in East Asia, with high incidence and mortality rate. Epigenetic modifications have been reported to participate in the progression of gastric cancer, among which mA is the most abundant and important chemical modification in RNAs. Fat mass and obesity-associated protein (FTO) is the first identified RNA demethylase but little is known about its role in gastric cancer. In our study, data from TCGA and clinical samples showed that FTO was highly expressed in gastric cancer tissues. Kaplan-Meier plotter suggested that patients with the high level of FTO had a poor prognosis. In vitro and in vivo experiments confirmed the role of FTO in promoting gastric cancer cell proliferation. Mechanistically, we found that FTO bound to circFAM192A at the specific site and removed the mA modification in circFAM192A, protecting it from degradation. CircFAM192A subsequently interacted with the leucine transporter solute carrier family 7 member 5 (SLC7A5) and enhancing its stability. As a result, an increased amount of SLC7A5 was on the membrane, which facilitated leucine uptake and activated the mTOR signaling pathway. Therefore, our study demonstrated that FTO promoted gastric cancer proliferation through the circFAM192A/SLC7A5 axis in the mA-dependent manner. Our study shed new light on the role of FTO in gastric cancer progression.
10.1186/s43556-024-00172-4
[Effect of SLC7A5 on the proliferation of tumor cells and its relationship with transforming growth factor-β1
signal pathway].
Zhao Ting,Li Dan,Liu Xiaowen,Liu Zhiyong,Wu Daobing,Zhou Shihua,Duan Hongyan
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
OBJECTIVE:To explore the biological effects of amino acid transporter gene SLC7A5 (solute carrier family 7, member 5) on tumor cells and the regulatory mechanism at transcriptional level.
Methods: The expression of SLC7A5 was examined in human normal tissues and corresponding tumor tissues by Gene Expression Omnibus (GEO) database. The recombinant plasmid of SLC7A5 gene was constructed, and the effect of the SLC7A5 gene on tumor cell proliferation was investigated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry. SLC7A5 gene promoter and transcription factor binding sites were predicted through bioinformatics analysis, and the gene promoter recombinant plasmid was constructed. Then the dual luciferase reporter gene assay and reverse transcription polymerase chain reaction (RT-PCR) were used to explore the regulation of transforming growth factor-β1 (TGF-β1) signal on SLC7A5 gene expression.
Results: The GEO database analysis showed that the distribution of SLC7A5 was tissue specific, and its expression level was significantly higher in the tumor tissues than that in the corresponding normal tissues. The results of MTT and flow cytometry showed that SLC7A5 could promote cell proliferation. Results from the promoter analysis, reporter gene assay and RT-PCR confirmed that TGF-β1 could up-regulate the activity of SLC7A5 promoter and promote the expression of the SLC7A5 gene.
Conclusion: SLC7A5 gene plays a role in promoting tumor development, which is regulated by the TGF-β1 signaling pathway.
10.11817/j.issn.1672-7347.2017.05.001
TP63 transcriptionally regulates SLC7A5 to suppress ferroptosis in head and neck squamous cell carcinoma.
Frontiers in immunology
Background:Most head and neck squamous cell carcinoma (HNSCC) patients are diagnosed at an advanced local stage. While immunotherapy has improved survival rates, only a minority of patients respond durably to targeted immunotherapies, posing substantial clinical challenges. We investigated the heterogeneity of the tumor microenvironment in HNSCC cohorts before and after immunotherapy by analyzing single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing datasets retrieved from public databases. Methods:We constructed a single-cell transcriptome landscape of HNSCC patients before and after immunotherapy and analyzed the cellular composition, developmental trajectories, gene regulatory networks, and communication patterns of different cell type subpopulations. Additionally, we assessed the expression levels of relevant indicators in HNSCC cells via western blot, ELISA, and fluorescent probe techniques. Results:At the single-cell level, we identified a subpopulation of TP63 SLC7A5 HNSCC that exhibited a ferroptosis-resistant phenotype. This subpopulation suppresses ferroptosis in malignant cells through the transcriptional upregulation of SLC7A5 mediated by high TP63 expression, thereby promoting tumor growth and resistance to immunotherapy. The experimental results demonstrated that the overexpression of TP63 upregulated the expression of SLC7A5 and suppressed the concentrations of Fe and ROS in HNSCC cells. By integrating bulk transcriptome data, we developed a clinical scoring model based on TP63 and SLC7A5, which are closely associated with tumor stage, revealing the significant prognostic efficacy of the TP63 SLC7A5 HNSCC-mediated ferroptosis mechanism in HNSCC patients. Conclusion:Our research elucidates the TME in HNSCC before and after immunotherapy, revealing a novel mechanism by which TP63 SLC7A5 HNSCC inhibits ferroptosis and enhances tumor resistance via TP63-induced SLC7A5 upregulation. These insights lay the foundation for the development of more effective treatments for HNSCC.
10.3389/fimmu.2024.1445472
Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth.
Nature communications
Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.
10.1038/s41467-023-42458-1
Structure of the human LAT1-4F2hc heteromeric amino acid transporter complex.
Yan Renhong,Zhao Xin,Lei Jianlin,Zhou Qiang
Nature
The L-type amino acid transporter 1 (LAT1; also known as SLC7A5) catalyses the cross-membrane flux of large neutral amino acids in a sodium- and pH-independent manner. LAT1, an antiporter of the amino acid-polyamine-organocation superfamily, also catalyses the permeation of thyroid hormones, pharmaceutical drugs, and hormone precursors such as L-3,4-dihydroxyphenylalanine across membranes. Overexpression of LAT1 has been observed in a wide range of tumour cells, and it is thus a potential target for anti-cancer drugs. LAT1 forms a heteromeric amino acid transporter complex with 4F2 cell-surface antigen heavy chain (4F2hc; also known as SLC3A2)-a type II membrane glycoprotein that is essential for the stability of LAT1 and for its localization to the plasma membrane. Despite extensive cell-based characterization of the LAT1-4F2hc complex and structural determination of its homologues in bacteria, the interactions between LAT1 and 4F2hc and the working mechanism of the complex remain largely unknown. Here we report the cryo-electron microscopy structures of human LAT1-4F2hc alone and in complex with the inhibitor 2-amino-2-norbornanecarboxylic acid at resolutions of 3.3 Å and 3.5 Å, respectively. LAT1 exhibits an inward open conformation. Besides a disulfide bond association, LAT1 also interacts extensively with 4F2hc on the extracellular side, within the membrane, and on the intracellular side. Biochemical analysis reveals that 4F2hc is essential for the transport activity of the complex. Together, our characterizations shed light on the architecture of the LAT1-4F2hc complex, and provide insights into its function and the mechanisms through which it might be associated with disease.
10.1038/s41586-019-1011-z
Regulation of Melanogenesis by the Amino Acid Transporter SLC7A5.
Gaudel Céline,Soysouvanh Frédéric,Leclerc Justine,Bille Karine,Husser Chrystel,Montcriol François,Bertolotto Corine,Ballotti Robert
The Journal of investigative dermatology
Integration of chromatin immunoprecipitation-sequencing and microarray data enabled us to identify previously unreported MITF-target genes, among which the amino acid transporter SLC7A5 is also included. We reported that small interfering RNA-mediated SLC7A5 knockdown decreased pigmentation in B16F10 cells but neither affected morphology nor dendricity. Treatment with the SLC7A5 inhibitors 2-amino-2-norbornanecarboxylic acid (BCH) or JPH203 also decreased melanin synthesis in B16F10 cells. Our findings indicated that BCH was as potent as reference depigmenting agent, kojic acid, but acted through a different pathway not affecting tyrosinase activity. BCH also decreased pigmentation in human MNT1 melanoma cells or normal human melanocytes. Finally, we tested BCH on a more physiological model, using reconstructed human epidermis and confirmed a strong inhibition of pigmentation, revealing the clinical potential of SLC7A5 inhibition and positioning BCH as a depigmenting agent suitable for cosmetic or dermatological intervention in hyperpigmentation diseases.
10.1016/j.jid.2020.03.941
Co-Expression Effect of SLC7A5/SLC3A2 to Predict Response to Endocrine Therapy in Oestrogen-Receptor-Positive Breast Cancer.
Alfarsi Lutfi H,El-Ansari Rokaya,Craze Madeleine L,Masisi Brendah K,Mohammed Omar J,Ellis Ian O,Rakha Emad A,Green Andrew R
International journal of molecular sciences
The majority of breast cancers are oestrogen-receptor-positive (ER+) and are subject to endocrine therapy; however, an unpredictable subgroup of patients will develop resistance to endocrine therapy. The SLC7A5/SLC3A2 complex is a major route for the transport of large neutral essential amino acids through the plasma membrane. Alterations in the expression and function of those amino-acid transporters lead to metabolic reprogramming, which contributes to the tumorigenesis and drug resistance. This study aims to assess the effects and roles of SLC7A5/SLC3A2 co-expression in predicting responses to endocrine therapy in patients with ER+ breast cancer. The biological and clinical impact of SLC7A5/SLC3A2 co-expression was assessed in large annotated cohorts of ER+/HER2- breast cancer with long-term follow-up at the mRNA and protein levels. In vitro experiments were conducted to investigate the effect of SLC7A5/SLC3A2 knockdown in the proliferation of cancer cells and to the sensitivity to tamoxifen. We found that proliferation-related genes are highly expressed in a subgroup of patients with high SLC7A5/SLC3A2, and knockdown of SLC7A5/SLC3A2 decreased proliferation of ER+ breast cancer cells. In patients treated with endocrine therapy, high SLC7A5/SLC3A2 co-expression was associated with poor patient outcome, and depletion of SLC7A5/SLC3A2 using siRNA increased the sensitivity of breast cancer cells to tamoxifen. On the basis of our findings, SLC7A5/SLC3A2 co-expression has the potential of identifying a subgroup of ER+/HER2- breast cancer patients who fail to benefit from endocrine therapy and could guide the choice of other alternative therapies.
10.3390/ijms21041407
Role of SLC7A5 in Metabolic Reprogramming of Human Monocyte/Macrophage Immune Responses.
Yoon Bo Ruem,Oh Yoon-Jeong,Kang Seong Wook,Lee Eun Bong,Lee Won-Woo
Frontiers in immunology
Amino acids (AAs) are necessary nutrients which act not only as building blocks in protein synthesis but also in crucial anabolic cellular signaling pathways. It has been demonstrated that SLC7A5 is a critical transporter that mediates uptake of several essential amino acids in highly proliferative tumors and activated T cells. However, the dynamics and relevance of SLC7A5 activity in monocytes/macrophages is still poorly understood. We provide evidence that SLC7A5-mediated leucine influx contributes to pro-inflammatory cytokine production mTOR complex 1 (mTORC1)-induced glycolytic reprograming in activated human monocytes/macrophages. Moreover, expression of SLC7A5 is significantly elevated in monocytes derived from patients with rheumatoid arthritis (RA), a chronic inflammatory disease, and was also markedly induced by LPS stimulation of both monocytes and macrophages from healthy individuals. Further, pharmacological blockade or silencing of SLC7A5 led to a significant reduction of IL-1β downstream of leucine-mediated mTORC1 activation. Inhibition of SLC7A5-mediated leucine influx was linked to downregulation of glycolytic metabolism as evidenced by the decreased extracellular acidification rate, suggesting a regulatory role for this molecule in glycolytic reprograming. Furthermore, the expression of SLC7A5 on circulating monocytes from RA patients positively correlated with clinical parameters, suggesting that SLC7A5-mediated AA influx is related to inflammatory conditions.
10.3389/fimmu.2018.00053
Slc7a5 regulates Kv1.2 channels and modifies functional outcomes of epilepsy-linked channel mutations.
Baronas Victoria A,Yang Runying Y,Morales Luis Carlos,Sipione Simonetta,Kurata Harley T
Nature communications
Kv1.2 is a prominent voltage-gated potassium channel that influences action potential generation and propagation in the central nervous system. We explored multi-protein complexes containing Kv1.2 using mass spectrometry followed by screening for effects on Kv1.2. We report that Slc7a5, a neutral amino acid transporter, has a profound impact on Kv1.2. Co-expression with Slc7a5 reduces total Kv1.2 protein, and dramatically hyperpolarizes the voltage-dependence of activation by -47 mV. These effects are attenuated by expression of Slc3a2, a known binding partner of Slc7a5. The profound Slc7a5-mediated current suppression is partly explained by a combination of gating effects including accelerated inactivation and a hyperpolarizing shift of channel activation, causing channels to accumulate in a non-conducting state. Two recently reported Slc7a5 mutations linked to neurodevelopmental delay exhibit a localization defect and have attenuated effects on Kv1.2. In addition, epilepsy-linked gain-of-function Kv1.2 mutants exhibit enhanced sensitivity to Slc7a5.
10.1038/s41467-018-06859-x
Targeting glutamine metabolic reprogramming of SLC7A5 enhances the efficacy of anti-PD-1 in triple-negative breast cancer.
Frontiers in immunology
Background:Triple-negative breast cancer (TNBC) is a heterogeneous disease that is characterized by metabolic disruption. Metabolic reprogramming and tumor cell immune escape play indispensable roles in the tumorigenesis that leads to TNBC. Methods:In this study, we constructed and validated two prognostic glutamine metabolic gene models, Clusters A and B, to better discriminate between groups of TNBC patients based on risk. Compared with the risk Cluster A patients, the Cluster B patients tended to exhibit better survival outcomes and higher immune cell infiltration. In addition, we established a scoring system, the glutamine metabolism score (GMS), to assess the pattern of glutamine metabolic modification. Results:We found that solute carrier family 7 member 5 (SLC7A5), an amino acid transporter, was the most important gene and plays a vital role in glutamine metabolism reprogramming in TNBC cells. Knocking down SLC7A5 significantly inhibited human and mouse TNBC cell proliferation, migration, and invasion. In addition, downregulation of SLC7A5 increased CD8 T-cell infiltration. The combination of a SLC7A5 blockade mediated via JPH203 treatment and an anti-programmed cell death 1 (PD-1) antibody synergistically increased the immune cell infiltration rate and inhibited tumor progression. Conclusions:Hence, our results highlight the molecular mechanisms underlying SLC7A5 effects and lead to a better understanding of the potential benefit of targeting glutamine metabolism in combination with immunotherapy as a new therapy for TNBC.
10.3389/fimmu.2023.1251643
FBW7/GSK3β mediated degradation of IGF2BP2 inhibits IGF2BP2-SLC7A5 positive feedback loop and radioresistance in lung cancer.
Journal of experimental & clinical cancer research : CR
BACKGROUND:The development of radioresistance seriously hinders the efficacy of radiotherapy in lung cancer. However, the underlying mechanisms by which radioresistance occurs are still incompletely understood. The N-Methyladenosine (mA) modification of RNA is involved in cancer progression, but its role in lung cancer radioresistance remains elusive. This study aimed to identify mA regulators involved in lung cancer radiosensitivity and further explore the underlying mechanisms to identify therapeutic targets to overcome lung cancer radioresistance. METHODS:Bioinformatic mining was used to identify the mA regulator IGF2BP2 involved in lung cancer radiosensitivity. Transcriptome sequencing was used to explore the downstream factors. Clonogenic survival assays, neutral comet assays, Rad51 foci formation assays, and Annexin V/propidium iodide assays were used to determine the significance of FBW7/IGF2BP2/SLC7A5 axis in lung cancer radioresistance. Chromatin immunoprecipitation (ChIP)-qPCR analyses, RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)-qPCR analyses, RNA pull-down analyses, co-immunoprecipitation analyses, and ubiquitination assays were used to determine the feedback loop between IGF2BP2 and SLC7A5 and the regulatory effect of FBW7/GSK3β on IGF2BP2. Mice models and tissue microarrays were used to verify the effects in vivo. RESULTS:We identified IGF2BP2, an mA "reader", that is overexpressed in lung cancer and facilitates radioresistance. We showed that inhibition of IGF2BP2 impairs radioresistance in lung cancer both in vitro and in vivo. Furthermore, we found that IGF2BP2 enhances the stability and translation of SLC7A5 mRNA through mA modification, resulting in enhanced SLC7A5-mediated transport of methionine to produce S-adenosylmethionine. This feeds back upon the IGF2BP2 promoter region by further increasing the trimethyl modification at lysine 4 of histone H3 (H3K4me3) level to upregulate IGF2BP2 expression. We demonstrated that this positive feedback loop between IGF2BP2 and SLC7A5 promotes lung cancer radioresistance through the AKT/mTOR pathway. Moreover, we found that the ubiquitin ligase FBW7 functions with GSK3β kinase to recognize and degrade IGF2BP2. CONCLUSIONS:Collectively, our study revealed that the mA "reader" IGF2BP2 promotes lung cancer radioresistance by forming a positive feedback loop with SLC7A5, suggesting that IGF2BP2 may be a potential therapeutic target to control radioresistance in lung cancer.
10.1186/s13046-024-02959-3
CircARID1A binds to IGF2BP3 in gastric cancer and promotes cancer proliferation by forming a circARID1A-IGF2BP3-SLC7A5 RNA-protein ternary complex.
Journal of experimental & clinical cancer research : CR
BACKGROUND:Gastric cancer (GC) is one of the most common malignant tumors in China. Circular RNAs (circRNAs) are novel non-coding RNAs with important regulatory roles in cancer progression. IGF2BP3 has been found to play oncogenic roles in various cancers including GC, while the exact mechanism of IGF2BP3 is largely unknown. METHODS:The expression of IGF2BP3 in GC was evaluated by Western Blot and bioinformatics analysis. CircRNA expression profiles were screened via IGF2BP3 RIP-seq in GC. Sanger sequencing, RNase R digestion, nucleo-plasmic separation and RNA-FISH assays were used to detect the existence and expression of circARID1A. RNA ISH assay was employed to test the expression of circARID1A in paraffin-embedded GC tissues. Moreover, the function of circARID1A on cellular proliferation was assessed by CCK-8, plate colony formation, EdU assays and GC xenograft mouse model in vivo. Furthermore, the location or binding of circARID1A, IGF2BP3 protein and SLC7A5 in GC was evaluated by RNA-FISH/IF or RNA pull-down assays. RESULTS:We identified a novel circRNA, circARID1A, that can bind to IGF2BP3 protein. CircARID1A was significantly upregulated in GC tissues compared with noncancerous tissues and positively correlated with tumor length, tumor volume, and TNM stage. CircARID1A knockdown inhibited the proliferation of GC cells in vitro and in vivo and circARID1A played an important role in the oncogenic function of IGF2BP3. Mechanistically, circARID1A served as a scaffold to facilitate the interaction between IGF2BP3 and SLC7A5 mRNA, finally increasing SLC7A5 mRNA stability. Additionally, circARID1A was able to directly bind SLC7A5 mRNA through complementary base-pairing and then formed the circARID1A-IGF2BP3-SLC7A5 RNA-protein ternary complex and promoted the proliferation of GC via regulating AKT/mTOR pathway. CONCLUSIONS:Altogether, our data suggest that circARID1A is involved in the function of IGF2BP3 and GC proliferation, and the circARID1A-IGF2BP3-SLC7A5 axis has the potential to serve as a novel therapeutic target for GC.
10.1186/s13046-022-02466-3
Targeting SLC1A5 and SLC3A2/SLC7A5 as a Potential Strategy to Strengthen Anti-Tumor Immunity in the Tumor Microenvironment.
Nachef Marianna,Ali Alaa Kassim,Almutairi Saeedah Musaed,Lee Seung-Hwan
Frontiers in immunology
Cancer cells are metabolically vigorous and are superior in the uptake of nutrients and in the release of the tumor microenvironment (TME)-specific metabolites. They create an acidic, hypoxic, and nutrient-depleted TME that makes it difficult for the cytotoxic immune cells to adapt to the metabolically hostile environment. Since a robust metabolism in immune cells is required for optimal anti-tumor effector functions, the challenges caused by the TME result in severe defects in the invasion and destruction of the established tumors. There have been many recent developments in NK and T cell-mediated immunotherapy, such as engineering them to express chimeric antigen receptors (CARs) to enhance tumor-recognition and infiltration. However, to defeat the tumor and overcome the limitations of the TME, it is essential to fortify these novel therapies by improving the metabolism of the immune cells. One potential strategy to enhance the metabolic fitness of immune cells is to upregulate the expression of nutrient transporters, specifically glucose and amino acid transporters. In particular, the amino acid transporters SLC1A5 and SLC7A5 as well as the ancillary subunit SLC3A2, which are required for efficient uptake of glutamine and leucine respectively, could strengthen the metabolic capabilities and effector functions of tumor-directed CAR-NK and T cells. In addition to enabling the influx and efflux of essential amino acids through the plasma membrane and within subcellular compartments such as the lysosome and the mitochondria, accumulating evidence has demonstrated that the amino acid transporters participate in sensing amino acid levels and thereby activate mTORC1, a master metabolic regulator that promotes cell metabolism, and induce the expression of c-Myc, a transcription factor essential for cell growth and proliferation. In this review, we discuss the regulatory pathways of these amino acid transporters and how we can take advantage of these processes to strengthen immunotherapy against cancer.
10.3389/fimmu.2021.624324
Amino acid transporter LAT1 (SLC7A5) as a molecular target for cancer diagnosis and therapeutics.
Kanai Yoshikatsu
Pharmacology & therapeutics
Cancer cells require a massive supply of nutrients, including sugars and amino acids-the upregulation of transporters for each nutrient contributes to meet the demand. Distinct from glucose transporters, amino acid transporters include ones whose expression is specific to cancer cells. For example, LAT1 (SLC7A5) displays protein expression mostly limited to the plasma membrane of cancer cells. The exceptions are the placental barrier and the blood-brain barrier, where immunohistochemical and mass spectrometric studies have shown LAT1 expression, although their levels are supposed to be lower than those in cancers. The expression of LAT1 has been reported in cancers from various tissue origins, where high LAT1 expression is related to the poor prognosis of patients. LAT1 is essential for cancer cell growth because the pharmacologic inhibition and knockdown/knockout of LAT1 suppress the proliferation of cancer cells and the growth of xenograft tumors. The inhibition of LAT1 suppresses protein synthesis by downregulating the mTORC1 signaling pathway and mobilizing the general amino acid control (GAAC) pathway in cancer cells. LAT1 is, thus, a candidate molecular target for the diagnosis and therapeutics of cancers. F-labeled 3-fluoro-l-α-methyl-tyrosine (FAMT) is used as a LAT1-specific PET probe for cancer detection due to the LAT1 specificity of α-methyl aromatic amino acids. FAMT accumulation is cancer-specific and avoids non-cancer lesions, including inflammation, confirming the cancer-specific expression of LAT1 in humans. Due to the cancer-specific nature, LAT1 can also be used for cancer-specific delivery of anti-tumor agents such as l-para-boronophenylalanine used for boron neutron capture therapy and α-emitting nuclide-labeled LAT1 substrates developed for nuclear medicine treatment. Based on the importance of LAT1 in cancer progression, high-affinity LAT1-specific inhibitors have been developed for anti-tumor drugs. JPH203 (KYT0353) is such a compound designed based on the structure-activity relationship of LAT1 ligands. It is one of the highest-affinity inhibitors with less affecting other transporters. It suppresses tumor growth in vivo without significant toxicity in preclinical studies at doses enough to suppress tumor growth. In the phase-I clinical trial, JPH203 appeared to provide promising activity. Because the mechanisms of action of LAT1 inhibitors are novel, with or without combination with other anti-tumor drugs, they could contribute to the treatment of cancers that do not respond to current therapy. The LAT1-specific PET probe could also be used as companion diagnostics of the LAT1-targeting therapies to select patients to whom therapeutic benefits could be expected. Recently, the cryo-EM structure of LAT1 has been solved, which would facilitate the understanding of the mechanisms of the dynamic interaction of ligands and the binding site, and further designing new compounds with higher activity.
10.1016/j.pharmthera.2021.107964