Targeting L-type amino acid transporter 1 in urological malignancy: Current status and future perspective.

Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.

Heteromeric Solute Carriers: Function, Structure, Pathology and Pharmacology.

Solute carriers form one of three major superfamilies of membrane transporters in humans, and include uniporters, exchangers and symporters. Following several decades of molecular characterisation, multiple solute carriers that form obligatory heteromers with unrelated subunits are emerging as a distinctive principle of membrane transporter assembly. Here we comprehensively review experimentally established heteromeric solute carriers: SLC3-SLC7 amino acid exchangers, SLC16 monocarboxylate/H+ symporters and basigin/embigin, SLC4A1 (AE1) and glycophorin A exchanger, SLC51 heteromer Ost α-Ost ß uniporter, and SLC6 heteromeric symporters. The review covers the history of the heteromer discovery, transporter physiology, structure, disease associations and pharmacology - all with a focus on the heteromeric assembly. The cellular locations, requirements for complex formation, and the functional role of dimerization are extensively detailed, including analysis of the first complete heteromer structures, the SLC7-SLC3 family transporters LAT1-4F2hc, b0,+AT-rBAT and the SLC6 family heteromer B0AT1-ACE2. We present a systematic analysis of the structural and functional aspects of heteromeric solute carriers and conclude with common principles of their functional roles and structural architecture.

Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.

Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris, and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed.

The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2.

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

Volta Phase Plate Cryo-EM Structure of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.

Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two subunits are linked by a conserved disulfide bridge. Several human diseases are associated with loss of function or overexpression of specific HATs making them drug targets. The human HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is specific for the transport of large neutral L-amino acids and specific amino acid-related compounds. Human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris and pure recombinant protein purified. Here we present the first cryo-electron microscopy (cryo-EM) 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex. The structure could be determined at ~13 Å resolution using direct electron detector and Volta phase plate technologies. The 3D-map displays two prominent densities of different sizes. The available X-ray structure of the 4F2hc ectodomain fitted nicely into the smaller density revealing the relative position of 4F2hc with respect to LAT2 and the membrane plane.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc.

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.

The Multifaceted Role of L-Type Amino Acid Transporter 1 at the Blood-Brain Barrier: Structural Implications and Therapeutic Potential.

L-type amino acid transporter 1 (LAT1) is integral to the transport of large neutral amino acids across the blood-brain barrier (BBB), playing a crucial role in brain homeostasis and the delivery of therapeutic agents. This review explores the multifaceted role of LAT1 in neurological disorders, including its structural and functional aspects at the BBB. Studies using advanced BBB models, such as induced pluripotent stem cell (iPSC)-derived systems and quantitative proteomic analyses, have demonstrated LAT1's significant impact on drug permeability and transport efficiency. In Alzheimer's disease, LAT1-mediated delivery of anti-inflammatory and neuroprotective agents shows promise in overcoming BBB limitations. In Parkinson's disease, LAT1's role in transporting L-DOPA and other therapeutic agents highlights its potential in enhancing treatment efficacy. In phenylketonuria, studies have revealed polymorphisms and genetic variations of LAT1, which could be correlated to disease severity. Prodrugs of valproic acid, pregabalin, and gabapentin help use LAT1-mediated transport to increase the therapeutic activity and bioavailability of the prodrug in the brain. LAT1 has also been studied in neurodevelopment disorders like autism spectrum disorders and Rett syndrome, along with neuropsychiatric implications in depression. Its implications in neuro-oncology, especially in transporting therapeutic agents into cancer cells, show immense future potential. Phenotypes of LAT1 have also shown variations in the general population affecting their ability to respond to painkillers and anti-inflammatory drugs. Furthermore, LAT1-targeted approaches, such as functionalized nanoparticles and prodrugs, show promise in overcoming chemoresistance and enhancing drug delivery to the brain. The ongoing exploration of LAT1's structural characteristics and therapeutic applications reiterates its critical role in advancing treatments for neurological disorders.

Evidence for a relationship between genetic polymorphisms of the L-DOPA transporter LAT2/4F2hc and risk of hypertension in the context of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) and hypertension are chronic diseases affecting a large portion of the population frequently coexistent and interdependent. The inability to produce/use adequate renal dopamine may contribute to the development of hypertension and renal dysfunction. The heterodimeric amino acid transporter LAT2/4F2hc (SLC7A8/SLC3A2 genes) promotes the uptake of L-DOPA, the natural precursor of dopamine. We examined the plausibility that SLC7A8/SLC3A2 gene polymorphisms may contribute to hypertensive CKD by affecting the L-DOPA uptake. METHODS: 421 subjects (203 men and 218 women, mean age of 78.9 ± 9.6 years) were recruited and divided in four groups according to presence/absence of CKD, defined as reduced estimated glomerular filtration rate (eGFR < 60 ml/min/m2) calculated using the creatinine-based Berlin Initiative Study-1 (BIS1) equation, and to presence/absence of hypertension (systolic blood pressure ≥ 140 and/or diastolic blood pressure ≥ 90 mmHg). Subjects were analysed for selected SNPs spanning the SLC7A8 and SLC3A2 loci by Sequenom MassARRAY iPLEX platform. RESULTS: The most significant SNP at the SLC3A2 (4F2hc) locus was rs2282477-T/C, with carriers of the C-allele having a lower chance to develop hypertension among CKD affected individuals [OR = 0.33 (CI 0.14-0.82); p = 0.016]. A similar association with hypertensive CKD was found for the SLC7A8 (LAT2) rs3783436-T/C, whose C-allele resulted associated with decreased risk of hypertension among subjects affected by CKD [OR = 0.56 (95% CI 0.35-0.90; p = 0.017]. The two variants were predicted to be potentially functional. CONCLUSIONS: The association between SLC3A2 and SLC7A8 variants to hypertension development in patients with renal failure could be linked to changes in L-DOPA uptake and consequently dopamine synthesis. Although the associations do not survive correction for Bonferroni multiple testing, and additional research is needed, our study opens new avenues for future basic and translational research in the field of hypertensive CKD.

The human LAT1-4F2hc (SLC7A5-SLC3A2) transporter complex: Physiological and pathophysiological implications.

LAT1 and 4F2hc form a heterodimeric membrane protein complex, which functions as one of the best characterized amino acid transporters. Since LAT1-4F2hc is required for the efficient uptake of essential amino acids and hormones, it promotes cellular growth, in part, by stimulating mTORC1 (mechanistic target of rapamycin complex 1) signalling and by repressing the integrated stress response (ISR). Gain or loss of LAT1-4F2hc function is associated with cancer, diabetes, and immunological and neurological diseases. Hence, LAT1-4F2hc represents an attractive drug target for disease treatment. Specific targeting of LAT1-4F2hc will be facilitated by the increasingly detailed understanding of its molecular architecture, which provides important concepts for its function and regulation. Here, we summarize (i) structural insights that help to explain how LAT1 and 4F2hc assemble to transport amino acids across membranes, (ii) the role of LAT1-4F2hc in key metabolic signalling pathways, and (iii) how derailing these processes could contribute to diseases.

Contribution of LAT1-4F2hc in Urological Cancers via Toll-like Receptor and Other Vital Pathways.

Tumor cells are known for their ability to proliferate. Nutrients are essential for rapidly growing tumor cells. In particular, essential amino acids are essential for tumor cell growth. Tumor cell growth nutrition requires the regulation of membrane transport proteins. Nutritional processes require amino acid uptake across the cell membrane. Leucine, one of the essential amino acids, has recently been found to be closely associated with cancer, which activate mTOR signaling pathway. The transport of leucine into cells requires an L-type amino acid transporter protein 1, LAT1 (SLC7A5), which requires the 4F2 cell surface antigen heavy chain (4F2hc, SLC3A2) to form a heterodimeric amino acid transporter protein complex. Recent evidence identified 4F2hc as a specific downstream target of the androgen receptor splice variant 7 (AR-V7). We stressed the importance of the LAT1-4F2hc complex as a diagnostic and therapeutic target in urological cancers in this review, which covered the recent achievements in research on the involvement of the LAT1-4F2hc complex in urinary system tumors. In addition, JPH203, which is a selective LAT1 inhibitor, has shown excellent inhibitory effects on the proliferation in a variety of tumor cells. The current phase I clinical trials of JPH203 in patients with biliary tract cancer have also achieved good results, which is the future research direction for LAT1 targeted therapy drugs.

Expression of LAT1 and 4F2hc in Gastroenteropancreatic Neuroendocrine Neoplasms.

BACKGROUND/AIM: Little is known about the expression of L-type amino acid transporter 1 (LAT1) and 4F2hc in gastroenteropancreatic-neuroendocrine neoplasms (GEP-NENs). Hence, we conducted a study to verify the clinicopathological significance of LAT1 and 4F2hc. PATIENTS AND METHODS: Tissues from 126 patients with GEP-NENs were collected between August 2007 and August 2019 at our institution. We evaluated LAT1 and 4F2hc expression by immunohistochemistry, and examined their clinical significance. RESULTS: No statistically significant associations were observed between LAT1 expression and the different NENs. Expression of 4F2hc was significantly different between neuroendocrine tumour (NET)-G1, NET-G2, and NET-G3 (p=0.029), and was significantly associated with vascular invasion (p=0.044) and the Ki-67 index (p=0.042). CONCLUSION: No association between LAT1 expression and malignant features in GEP-NENs was observed. However, an association between 4F2hc expression and the potential of malignancy in GEP-NENs was evident.

Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein.

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

Expression and roles of system L amino acid transporters in human embryonal carcinoma cells.

BACKGROUND: Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive. MATERIALS AND METHODS: Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated. RESULTS: Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation. CONCLUSION: Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs.

Role of Amino Acid Transporter Expression as a Prognostic Marker in Patients With Surgically Resected Colorectal Cancer.

BACKGROUND/AIM: L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers. However, the clinicopathological significance of LAT1 and 4F2 cell surface antigen (4F2hc) in patients with colorectal cancer (CRC) is unknown. The aim of this study was to clarify the prognostic significance of LAT1 expression in CRC patients who underwent surgical resection. MATERIALS AND METHODS: Samples from one hundred and forty-seven patients were examined by immunohistochemistry. The expression of LAT1 and 4F2hc, and the Ki-67 labeling index were assessed using resected tumor specimens. RESULTS: The positive expression of LAT1 and 4F2c was 80% (118/147) and 58% (86/147) (p<0.01), respectively. The expression of LAT1 was identified as an independent significant marker linked to worse prognosis in patients with CRC, and was correlated with tumor cell proliferation, tumor aggressiveness, and metastasis. Moreover, LAT1 was closely associated with the expression of 4F2hc and phosphorylation of the mTOR pathway. CONCLUSION: LAT1 is a significant molecular marker used to predict prognosis after surgical resection of CRC patients.

Expression of amino acid transporter (LAT1 and 4F2hc) in pulmonary pleomorphic carcinoma.

Amino acid transporters are necessary for tumor growth, metastasis, and survival of various neoplasms; however, the clinicopathological significance of L-type amino acid transporter 1 (LAT1) and 4F2 cell surface antigen (4F2hc) in patients with pulmonary pleomorphic carcinoma (PPC) remainsunknown. The aim of this study is to clarify the prognostic impact of these amino acid transporters in PPC. One hundred five patients with surgically resected PPC were assessed by immunohistochemistry. The expression of LAT1 and 4F2hc, and Ki-67 labeling index were investigated using specimens of the resected tumors. LAT1 and 4F2hc were highly expressed in 35% and 53% of all patients (n = 105, P < .01), 25% and 48% of patients with an adenocarcinoma component (n = 48, P = .02), and 44% and 58% of patients with a nonadenocarcinoma component (n = 57, P = .18), respectively. A high LAT1 expression was significantly related to advanced disease, lymphatic permeation, tumor cell proliferation, and 4F2hc expression. By multivariate analysis, LAT1 and 4F2hc were identified as significant independent markers for predicting a worse prognosis. LAT1 is highly expressed in PPC, and high LAT1 expression can serve as a significant predictor linked to a worse prognosis in patients with PPC.

The Ion Transporter NKCC1 Links Cell Volume to Cell Mass Regulation by Suppressing mTORC1.

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2), a known regulator of cell volume, is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity, as well as activation of Akt and Erk, leading to activation of mTORC1 in cells, colonic organoids, and mouse colon. Moreover, NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1, therefore, suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly, by linking ion transport and cell volume regulation to mTORC1 function, NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation.

Ultrastructural immunohistochemical study of L-type amino acid transporter 1-4F2 heavy chain in tumor microvasculatures of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced rat bladder carcinoma.

Angiogenesis is essential for tumor growth, and an enhanced vasculature supplying nutrients and oxygen might reflect malignant potential. L-type amino acid transporter 1 (LAT1/4F2hc) comprises a major nutrient transport system responsible for the Na+-independent transport of large neutral amino acids. Seventy five to seventy eight percent N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinoma cells showed high LAT1/4F2hc expression. While the intracarcinoma microvasculatures of fenestrated endothelial cells highly expressing LAT1/4F2hc might progressively transport essential amino acids from the microvasculatures to the extracellular matrix, non-fenestrated endothelial cells and pericytes did not. The present study revealed that the tumor angiogenesis is one of target anti-L-type amino acid transporter 1 drug.

Downregulation of LAT1 expression suppresses cholangiocarcinoma cell invasion and migration.

Currently, there is no effective treatment for cholangiocarcinoma (CCA), which is the most prevalent in the northeastern part of Thailand. A new molecular target for the treatment of CCA is, therefore, urgently needed. Although L-type amino acid transporter 1 (LAT1) is highly expressed in CCA cells, its role in malignant phenotypes of CCA cells remains unclear. This study aimed to investigate the impact of LAT1 on proliferation, migration, and invasion of KKU-M213 cells, the CCA cells derived from Thai patients with intrahepatic cholangiocarcinoma. Results showed that KKU-M213 cells expressed all LAT isoforms (LAT1, LAT2, LAT3 and LAT4). The expressions of LAT1 and its associated protein 4F2hc were highest whereas those of LAT2 and LAT4 were extremely low. Treatment with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced L-leucine uptake concomitant with an inhibition of cell motility and, to a lesser extent, on cell proliferation. It also induced a time dependent up-regulation of LAT1 and 4F2hc expressions. Similarly, cell migration and invasion, but not proliferation, were reduced in LAT1 knockdown KKU-M213 cells. In addition, silencing of LAT1 inhibited the expressions of 4F2hc mRNA and protein whereas the expression of microRNA-7, the 4F2hc down-regulator, was increased. Furthermore, the phosphorylation levels of ERK1/2 and p70S6K were reduced after LAT1 knockdown. Collectively, these results suggest that suppression of cell invasion and migration in LAT1 knockdown KKU-M213 cells may be partly mediated through the inhibition of the 4F2hc-signaling pathway by the up-regulation of microRNA-7. Based on this finding, LAT1 may be a potential therapeutic target for treating CCA.

Significance of system L amino acid transporter 1 (LAT-1) and 4F2 heavy chain (4F2hc) expression in human developing intestines.

To clarify the significance of expression of system L amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in the developing intestine, immunohistochemical investigation and molecular analysis were performed in the human embryonic and/or fetal intestines, ranging from 28-30 days to 34-35 weeks gestation. The molecular analysis for the expression of LAT1 and 4F2hc mRNAs was done in the pure epithelial cell samples prepared after laser assisted microdissection. The immunoreactivities against LAT1 and 4F2hc were detected along the basolateral cell membrane of the primitive gut epithelium at 28-30 days gestation. According to advance in gestational age of up to 24-25 weeks gestation, the immunoreactivity of LAT1 was predominantly observed in the supranuclear cytoplasmic localization with a granular or dot-like staining pattern. Up to 8-9 weeks gestation, the immunoreactivity of 4F2hc showed almost the same as that of LAT1. However, after the age of 12-13 weeks gestation, the immunoreactivity of 4F2hc was predominantly localized along the cell membrane of apical surface of the epithelial cells. No apical and linear membranous localization of LAT1 was observed until nearly 20 weeks gestation. In the late gestational stage, both the immunoreactivities against LAT1 and 4F2hc were localized along the apical surface of the epithelial cells. In conclusion, the expression of LAT1 and 4F2hc in early developing intestine suggests they have a more important role in cell proliferation rather than functional differentiation. The predominant cytoplasmic localization of LAT1 during mid-fetal life seems to be largely inactive as amino acid transporter. On the other hand, the apical and linear membranous co-localization of LAT1 and 4F2hc in the late fetal life suggests that these molecules may play a role as a functional amino acid transporter in the fetal intestinal epithelium.