Induction of CTH expression in response to amino acid starvation confers resistance to anti-LAT1 therapy in MDA-MB-231 cells.

L type amino acid transporter 1 (LAT1) is an attractive molecular target for cancer therapy because of its overexpression in many cancer cells. JPH203, a selective LAT1 inhibitor, causes amino acid deprivation and suppresses cancer cell proliferation. However, several cancer cells showed resistance to amino acid deprivation. In this study, we aimed to elucidate the molecular mechanism of different sensitivity between 2 breast cancer cells to anti-LAT1 therapy. MDA-MB-231 cells were more resistant to growth suppression effect of JPH203 than T-47D cells (IC50 was 200 ± 12.5 µM for MDA-MB-231, and 5 ± 1.1 µM for T-47D cells; p < 0.05). Transcriptome and biochemical analysis were done in these cells in the presence/absence of JPH203. JPH203 induced intracellular amino acid deprivation stress in both cells, but it upregulated cystathionine γ lyase (CTH), an enzyme for synthesis of antioxidants, only in MDA-MB-231 cells. Moreover, siRNA-mediated CTH knockdown induced oxidative stress in response to JPH203 leading to decreased cell viability in MDA-MB-231 cells. These results suggest that activation of anti-oxidation pathways in response to amino acid deprivation confers resistance to anti-LAT1 therapy.

Targeting SLC1A5 and SLC3A2/SLC7A5 as a Potential Strategy to Strengthen Anti-Tumor Immunity in the Tumor Microenvironment.

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.

[Drug Discovery Targeting an Amino Acid Transporter for Diagnosis and Therapy].

Nutrients are essential for all living organisms. Because growing cancer cells have strong metabolic demands, nutrient transporters are constitutively increased to facilitate the nutrient uptake. Among these nutrient transporters, L-type amino acid transporter 1 (LAT1), which transports large neutral amino acids including essential amino acids, is critical for cancer growth. Therefore, LAT1 has been considered as an attractive target for diagnosis and therapy of cancers. We have developed several lines of compounds for cancer diagnosis and therapy. To diagnose cancer by using positron emission tomography (PET) probes, we have created amino acid derivatives which are selectively transported by LAT1 and accumulated in cancer cells. In addition to amino acid derivatives as the LAT1 inhibitors, we also have made non-amino acid small compounds as anti-cancer drugs which inhibit LAT1 function and suppress tumor growth. The LAT1 targeting anti-cancer drug showed low toxicity but strong effects on various types of cancer cells in animal models. The novel PET probe is approved for clinical research and the new anti-cancer drug has been under clinical trial. Small compounds targeting the amino acid transporter bring us new tools for cancer diagnosis and therapy.

The L-Type Amino Acid Transporter LAT1-An Emerging Target in Cancer.

Chronic proliferation is a major hallmark of tumor cells. Rapidly proliferating cancer cells are highly dependent on nutrients in order to duplicate their cell mass during each cell division. In particular, essential amino acids are indispensable for proliferating cancer cells. Their uptake across the cell membrane is tightly controlled by membrane transporters. Among those, the L-type amino acid transporter LAT1 (SLC7A5) has been repeatedly found overexpressed in a vast variety of cancers. In this review, we summarize the most recent advances in our understanding of the role of LAT1 in cancer and highlight preclinical studies and drug developments underlying the potential of LAT1 as therapeutic target.

The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model.

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.

LAT1-Targeting Thermoresponsive Fluorescent Polymer Probes for Cancer Cell Imaging.

L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[³H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[³H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

System L amino acid transporter inhibitor enhances anti-tumor activity of cisplatin in a head and neck squamous cell carcinoma cell line.

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.

Gene expression profiles in T24 human bladder carcinoma cells by inhibiting an L-type amino acid transporter, LAT1.

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.

Pharmacokinetics of gabapentin during delivery, in the neonatal period, and lactation: does a fetal accumulation occur during pregnancy?

PURPOSE: To study the pharmacokinetics of gabapentin (GBP) during delivery, lactation, and in the neonatal period. METHODS: GBP concentrations in plasma and breast milk were determined with high-performance liquid chromatography in samples from six women treated with GBP and in their offspring. Blood samples were obtained at delivery from mothers, from the umbilical cord, and from the newborns on three occasions during 2 days after delivery. GBP concentration also was determined in breast milk and in blood collected from five of the mothers and suckling infants 2 weeks to 3 months after birth. RESULTS: The umbilical cord/maternal plasma concentration ratios ranged from 1.3 to 2.1 (mean, 1.7). GBP plasma concentrations in the neonates declined with an estimated half-life of 14 h. Mean GBP plasma concentrations in the infants were 27% of the cord plasma levels (range, 12-36%) 24 h postpartum. The mean milk/maternal plasma concentration ratio was 1.0 (range, 0.7-1.3) from 2 weeks to 3 months. The infant dose of GBP was estimated to 0.2-1.3 mg/kg/day, equivalent to 1.3-3.8% of the weight-normalized dose received by the mother. The plasma concentrations in the breast-fed infants were approximately 12% of the mother's plasma levels, but no adverse effects were observed. CONCLUSIONS: Our limited observations suggest an active transplacental transport of GBP, with accumulation in the fetus as a consequence. We suggest that this could be by the specific L-type amino acid transporter 1 (LAT-1), which is expressed in the placenta. Newborns seem to have a slightly lower capacity to eliminate GBP than do adults. Transfer of GBP to breast milk is extensive, but plasma concentrations appear to be low in suckling infants. No adverse effects were observed in the newborn. Although more data are needed, our observations suggest that breastfeeding in conjunction with GBP treatment is safe.

System L-amino acid transporters are differently expressed in rat astrocyte and C6 glioma cells.

The system L-amino acid transporter is a major nutrient transport system that is responsible for Na+-independent transport of neutral amino acids including several essential amino acids. We have compared and examined the expressions and functions of the system L-amino acid transporters in both rat astrocyte cultures and C6 glioma cells. The rat astrocyte cultures expressed the l-type amino acid transporter 2 (LAT2) with its subunit 4F2hc, whereas the l-type amino acid transporter 1 (LAT1) was not expressed in these cells. The C6 glioma cells expressed LAT1 but not LAT2 with 4F2hc. The [14C]l-leucine uptakes by the rat astrocyte cultures and C6 glioma cells were Na+-independent and were completely inhibited by the system l selective inhibitor, BCH. These results suggest that the transport of neutral amino acids including several essential amino acids into rat astrocyte cultures and C6 glioma cells are for the most part mediated by LAT2 and LAT1, respectively. Therefore, the rat astrocyte cultures and C6 glioma cells are excellent tools for examining the properties of LAT2 and LAT1, respectively. Moreover, the specific inhibition of LAT1 in cancer cells might be a new rationale for anti-cancer therapy.

The influx of neutral amino acids into the porcine brain during development: a positron emission tomography study.

Pigs of three different age groups (newborns, 1 week old, 6 weeks old) were used to study the transport of the large neutral amino acids 6-[18F]fluoro-L-DOPA ([18F]FDOPA) and 3-O-methyl-6-[18F]fluoro-L-DOPA ([18F]OMFD) across the blood-brain barrier (BBB) with positron emission tomography (PET). Compartmental modeling of PET data was used to calculate the blood-brain clearance (K1) and the rate constant for the brain-blood transfer (k2) of [18F]FDOPA and [18F]OMFD after i.v. injection. A 40-70% decrease of K1(OMFD), K1(FDOPA) and k2(OMFD) from newborns to juvenile pigs was found whereas k2(FDOPA) did not change. Generally, K1(OMFD) and k2(OMFD) are lower than K1(FDOPA) and k2(FDOPA) in all regions and age groups. The changes cannot be explained by differences in brain perfusion because the measured regional cerebral blood flow did not show major changes during the first 6 weeks after birth. In addition, alterations in plasma amino acids cannot account for the described transport changes. In newborn and juvenile pigs, HPLC measurements were performed. Despite significant changes of single amino acids (decrease: Met, Val, Leu; increase: Tyr), the sum of large neutral amino acids transported by LAT1 remained unchanged. Furthermore, treatment with a selective inhibitor of the LAT1 transporter (BCH) reduced the blood-brain transport of [18F]FDOPA and [18F]OMFD by 35% and 32%, respectively. Additional in-vitro studies using human LAT1 reveal a much lower affinity of FDOPA compared to OMFD or L-DOPA. The data indicate that the transport system(s) for neutral amino acids underlie(s) developmental changes after birth causing a decrease of the blood-brain barrier permeability for those amino acids during brain development. It is suggested that there is no tight coupling between brain amino acid supply and the demands of protein synthesis in the brain tissue.

Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells.

PURPOSE: To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina. METHODS: RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL-[14C]ornithine. RESULTS: An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of DL-[14C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y+LAT1, and 4F2hc were expressed in RPE cells. CONCLUSIONS: The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.