LAT1 Expression Research Articles

Prognostic Value of L-Type Amino Acid Transporter 1 (LAT1) in Various Cancers: A Meta-Analysis.

The L-type amino acid transporter 1 (LAT1, SLC7A5) is overexpressed in various types of cancer and has been thought to assist cancer progression through its uptake of neutral amino acids. However, the prognostic role of LAT1 in human cancers remains uncharacterized. Therefore, we conducted this meta-analysis to determine the prognostic significance of LAT1 in various cancers. We systematically searched the PubMed, Web of Science, EMBASE, Chinese National Knowledge Infrastructure, and WanFang databases to collect relevant cohort studies investigating the prognostic value of LAT1 expression in patients with cancer. Hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) were pooled to clarify the association between the LAT1 expression and the survival of patients with cancer. Odds ratios (ORs) with 95% CIs were calculated to appraise the correlation between LAT1 and the clinicopathological characteristics in patients with cancer. A total of 32 eligible articles, including 34 cohorts and 6410 patients, were enrolled in this meta-analysis. Our results demonstrated that high LAT1 expression was significantly associated with poor overall survival (HR = 1.66, 95% CI 1.41-1.96, P < 0.001), cancer-specific survival (HR = 1.64, 95% CI 1.31-2.05, P < 0.001), disease-free survival (HR = 1.55, 95% CI 1.31-1.83, P < 0.001), and progression-free survival (HR = 1.18, 95% CI 1.02-1.37, P = 0.026) in patients with cancer. In addition, we found that the elevated expression level of LAT1 was significantly related to certain phenotypes of tumor aggressiveness, such as tumor size, clinical stage, T stage, lymphatic invasion, vascular invasion, tumor differentiation, Ki-67, CD34, CD98, p53, and system ASC amino acid transporter-2. Elevated expression of LAT1 is associated with poor prognosis in human cancers and may serve as a potential prognostic marker and therapeutic target for patients with malignancies.

Uptake of positron emission tomography tracers reflects the tumor immune status in esophageal squamous cell carcinoma.

The relationship between the local immune status and cancer metabolism regarding 18F‐FDG and 18F‐FAMT uptake in esophageal squamous cell carcinoma (ESCC) remains unknown. The present study examined the correlations between tumor immune status, clinicopathological factors, and positron emission tomography (PET) tracer uptake in ESCC. Forty‐one ESCC patients who underwent 18F‐FDG PET and 18F‐FAMT PET before surgery were enrolled in the study. Immunohistochemistry was conducted for programmed death 1 (PD‐1), CD8, Ki‐67, CD34, GLUT1 (18F‐FDG transporter) and LAT1 (18F‐FAMT transporter). ESCC specimens with high tumoral PD‐L1 and high CD8‐positive lymphocytes were considered to have “hot tumor immune status.” High PD‐L1 expression (53.7%) was significantly associated with tumor/lymphatic/venous invasion (P = 0.028, 0.032 and 0.018), stage (P = 0.041), CD8‐positive lymphocytes (P < 0.001), GLUT1 (P < 0.001), LAT1 expression (P = 0.006), Ki‐67 labelling index (P = 0.009) and CD34‐positive vessel counts (P < 0.001). SUVmax of 18F‐FDG was significantly higher in high PD‐L1 cases than in low PD‐L1 cases (P = 0.009). SUVmax of 18F‐FAMT was significantly higher in high PD‐L1 (P < 0.001), high CD8 (P = 0.012) and hot tumor groups (P = 0.028) than in other groups. High SUVmax of 18F‐FAMT (≥4.15) was identified as the only predictor of hot tumor immune status. High PET tracer uptake was significantly associated with cancer aggressiveness and hot tumor immune status in ESCC. PET imaging may be an effective tool to predict tumor immune status in ESCC with respect to immune checkpoint inhibitor sensitivity.

Prolactin regulates LAT1 expression via STAT5 (signal transducer and activator of transcription 5) signaling in mammary epithelial cells of dairy cows

The l-type amino acid transporter 1 (LAT1; also known as SLC7A5) is a transporter that allows the uptake of large neutral amino acids into mammalian cells. In dairy cows, LAT1 is highly expressed in lactating mammary tissues and involved in milk protein synthesis. Prolactin (PRL) has a lactogenic role and is capable of inducing milk production in ruminants. However, the relationship between PRL stimulation and LAT1 expression in dairy cow mammary gland has not been well understood. In this study, we showed that PRL stimulation increased expression of LAT1 and β-casein in mammary epithelial cells of dairy cows. The stimulatory effect of PRL on milk protein production was inhibited by LAT1-specific inhibitor or LAT1 knockdown, suggesting that PRL-induced milk protein production is involved in LAT1 expression. To determine whether the PRL signaling pathway participates in regulation of LAT1 expression, PRLR (PRL receptor) or STAT5 (signal transducer and activator of transcription 5) was knocked down by short interfering (si)RNA in mammary epithelial cells of dairy cows. Western blot results showed that knockdown of PRLR or STAT5 with siRNA markedly decreased PRL-stimulated LAT1 expression. In addition, we observed a marked increase in plasma membrane expression of LAT1 in PRL-stimulated cells compared with control cells. These observations indicated that PRL signaling can regulate LAT1 expression and activity in mammary epithelial cells of dairy cows, contributing to increased amino acid availability and milk protein synthesis in mammary gland of dairy cow.

LAT1 (SLC7A5) Overexpression in Negative Her2 Group of Breast Cancer: A Potential Therapy Target.

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.

Glioblastoma Exhibits Inter-Individual Heterogeneity of TSPO and LAT1 Expression in Neoplastic and Parenchymal Cells.

Molecular imaging is essential for diagnosis and treatment planning for glioblastoma patients. Positron emission tomography (PET) with tracers for the detection of the solute carrier family 7 member 5 (SLC7A5; also known as the amino acid transporter light chain L system, LAT1) and for the mitochondrial translocator protein (TSPO) is successfully used to provide additional information on tumor volume and prognosis. The current approaches for TSPO-PET and the visualization of tracer ([18F] Fluoroethyltyrosine, FET) uptake by LAT1 (FET-PET) do not yet exploit the full diagnostic potential of these molecular imaging techniques. Therefore, we investigated the expression of TSPO and LAT1 in patient glioblastoma (GBM) samples, as well as in various GBM mouse models representing patient GBMs of different genetic subtypes. By immunohistochemistry, we found that TSPO and LAT1 are upregulated in human GBM samples compared to normal brain tissue. Next, we orthotopically implanted patient-derived GBM cells, as well as genetically engineered murine GBM cells, representing different genetic subtypes of the disease. To determine TSPO and LAT1 expression, we performed immunofluorescence staining. We found that both TSPO and LAT1 expression was increased in tumor regions of the implanted human or murine GBM cells when compared to the neighboring mouse brain tissue. While LAT1 was largely restricted to tumor cells, we found that TSPO was also expressed by microglia, tumor-associated macrophages, endothelial cells, and pericytes. The Cancer Genome Atlas (TCGA)-data analysis corroborates the upregulation of TSPO in a bigger cohort of GBM patient samples compared to tumor-free brain tissue. In addition, AIF1 (the gene encoding for the myeloid cell marker Iba1) was also upregulated in GBM compared to the control. Interestingly, TSPO, as well as AIF1, showed significantly different expression levels depending on the GBM genetic subtype, with the highest expression being exhibited in the mesenchymal subtype. High TSPO and AIF1 expression also correlated with a significant decrease in patient survival compared to low expression. In line with this finding, the expression levels for TSPO and AIF1 were also significantly higher in (isocitrate-dehydrogenase wild-type) IDHWT compared to IDH mutant (IDHMUT) GBM. LAT1 expression, on the other hand, was not different among the individual GBM subtypes. Therefore, we could conclude that FET- and TSPO-PET confer different information on pathological features based on different genetic GBM subtypes and may thus help in planning individualized strategies for brain tumor therapy in the future. A combination of TSPO-PET and FET-PET could be a promising way to visualize tumor-associated myeloid cells and select patients for treatment strategies targeting the myeloid compartment.

The Role of Large Neutral Amino Acid Transporter (LAT1) in Cancer.

The solute carrier family 7 (SLC7) can be categorically divided into two subfamilies, the L-type amino acid transporters (LATs) including SLC7A5-13, and SLC7A15, and the cationic amino acid transporters (CATs) including SLC7A1-4 and SLC7A14. Members of the CAT family transport predominantly cationic amino acids by facilitating diffusion with intracellular substrates. LAT1 (also known as SLC7A5), is defined as a heteromeric amino acid transporter (HAT) interacting with the glycoprotein CD98 (SLC3A2) through a conserved disulfide to uptake not only large neutral amino acids, but also several pharmaceutical drugs to cells. In this review, we provide an overview of the interaction of the structure-function of LAT1 and its essential role in cancer, specifically, its role at the blood-brain barrier (BBB) to facilitate the transport of thyroid hormones, pharmaceuticals (e.g., I-DOPA, gabapentin), and metabolites into the brain. LAT1 expression increases as cancers progress, leading to higher expression levels in highgrade tumors and metastases. In addition, LAT1 plays a crucial role in cancer-associated reprogrammed metabolic networks by supplying tumor cells with essential amino acids. The increasing understanding of the role of LAT1 in cancer has led to an increase in interest surrounding its potential as a drug target for cancer treatment.

Abstract B15: Therapeutic potential of targeting amino acid metabolism in pancreatic cancer

Abstract Background: A wide range of cancers, including pancreatic cancer, display altered expression of amino acid transporter LAT-1. LAT-1 overexpression has been shown to be an important prognostic factor and predicts chemo resistance in pancreatic cancer (Kaira K, 2012; Altan B, 2018). Radiolabeled amino acids for PET imaging, including 3-18F-l-α-methyl-tyrosine ([18F] FAMT), have successfully leveraged cancer’s aberrant LAT1 expression and amino acid uptake for decades. SM-88 is a denatured D/L racemic form of α-methyl-tyrosine designed for selective uptake and disruption of protein synthesis in cancer cells, as well as disruption of catecholamine production via inhibition of tyrosine hydroxylase. Catecholamines have been reported to potentially have a role in driving pancreatic cancer progression and changes in the tumor immune status (Renz B, Cancer Cell 2018; Calvani M, Oncotarget 2014). Initial clinical and preclinical results with SM-88 indicate that this agent could pose a novel approach in the treatment of pancreatic cancer. Methods and Results: Initial xenograft models of human gastrointestinal cancers using monotherapy SM-88 showed a &amp;gt;50% decrease in tumor growth at three weeks of treatment (p&amp;lt;0.05). Ongoing preclinical experiments are testing the full pharmacodynamic effects of SM-88 on pancreatic cancer cells, as well as potential effects on the tumor microenvironment due to catecholamine interruption. In results from the ongoing phase 2 trial of SM-88 in heavily pretreated metastatic pancreatic cancer patients, SM-88 has shown monotherapy antitumor activity based on RECIST 1.1 criteria. Additionally, 16/24 patients demonstrated a reduction in circulating tumor cells, with the median nadir decline of 63%. LAT1 has been reported to be important in EMT transition and mesenchymal cell survival (Holldorsson S, Cancer letters 2017); examination of the impact of SM-88 on this important emerging biomarker is ongoing. Conclusion: Altered amino acid (LAT1) metabolism has been shown to be a negative prognosticator in pancreatic cancer outcomes and progression. Targeting LAT1 and amino acid pathways has been identified as a therapeutic approach in multiple cancers (Fuchs BC, Semin Ca Biol 2005). Initial and ongoing studies of SM-88 may clarify the potential utility of this approach in the treatment of pancreatic cancer. Citation Format: Martin Fernandez-Zapico, Dae Won Kim, Philip Philip, Alexander Vandell, Jonathan Eckard, Ron Korn, Giuseppe Del Priore, Diane Simeone. Therapeutic potential of targeting amino acid metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B15.

Characterization of the expression of LAT1 as a prognostic indicator and a therapeutic target in renal cell carcinoma

Large neutral amino acid transporter 1 (LAT1, SLC7A5) is abundantly expressed in various types of cancer, and it has been thought to assist cancer progression through its activity for uptake of neutral amino acids. However, the roles of LAT1 in renal cell carcinoma (RCC) prognosis and treatment remain uncharacterized. Therefore, we first retrospectively examined the LAT1 expression profile and its associations with clinical factors in RCC tissues (n = 92). The results of immunohistochemistry showed that most of the tissues examined (92%) had cancer-associated LAT1 expression. Furthermore, the overall survival (OS) and progression-free survival (PFS) were shorter in patients with high LAT1 expression levels than in those with low LAT1 expression levels (P = 0.018 and 0.014, respectively), and these associations were further strengthened by the results of univariate and multivariate analyses. Next, we tested the effects of JPH203, which is a selective LAT1 inhibitor, on RCC-derived Caki-1 and ACHN cells. It was found that JPH203 inhibited the growth of these cell types in a dose-dependent manner. Moreover, JPH203 clearly suppressed their migration and invasion activities. Thus, our results show that LAT1 has a great potential to become not only a prognosis biomarker but also a therapeutic target in RCC clinical settings.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation

Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1β, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1β, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1β stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1β-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to controlskininflammation mediated by the IL-23/IL-1β/IL-17 axis.

P06 - SLFN5 regulates amino acid metabolism by altering LAT1 expression in CRPC

P06 - SLFN5 regulates amino acid metabolism by altering LAT1 expression in CRPC

Abstract 4361: Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer

Abstract The effect of tumor metabolism on the tumor microenvironment is not well established. Our previous data have shown that cisplatin resistant (CR) lung cancer cells increased secretion of thioredoxin-1 (TRX1) and kynurenine (KYN). Interestingly, high TRX1 and KYN levels in tumor microenvironment can enhance immunosuppressive environment. We have demonstrated that CR cells possessed lower levels of hypoxia-inducible factor-1α (HIF1α) due to metabolic re-programming. ARNT or HIF1β is a known binding partner of both HIF1α and aryl hydrocarbon (AHR). Importantly, recent study indicates that KYN can serve as an endogenous ligand for AHR in cancer cells. Thus, we hypothesize that in the absence of HIF1α, ARNT is now available to bind and form a new partner with KYN/AHR and initiate the transcription of genes which favor survival/proliferation of CR cells. Four pairs of NSCLC cell lines and their CR variants (ALC, FC, H460R, A549R) were used. Using immunofluorescence technique, our result showed that AHR localized primary in the nucleus of CR cells when compared with parental cell counterparts. We further determined that KYN was specific to AHR activation in CR cells by inhibiting AHR translocation using 10µM of dimethoxyflavone (DMF; an AHR antagonist). AHR was less accumulated inside nuclease after treatment. Importantly, treatment of DMF also resulted in suppression of Indoleamine 2,3-Dioxygenase-1 (IDO1) activities. To further verify these findings, we assayed the expression of AHR-target gene (LAT1 and CYP1B1) with and without adding KYN. Both genes expressions were higher in CR cells and were further augmented upon an addition of KYN. In contrast, we did not find an increase in LAT1 after exposure to KYN in parental cells. Using flow cytometer, we found that CR cells possessed higher surface-LAT1 levels when compared to parental cell counterparts, and treatment of KYN resulted in significant tryptophan uptake in CR cells. Importantly, treatment with IDO1 inhibitor (5.5uM) significantly suppressed LAT1 and CYP1B1 expression in CR cells and reverse cisplatin resistance in CR from 2.5 to 0.75 μg/ml (3.3 fold). Thus, our data strongly indicate that KYN/AHR/ARNT axis plays a unique modulator role in CR cells metabolism. Overall these results will have potential impact on (i) how one can effectively exploit the KP pathway to treat CR tumors, and (ii) improving understanding on how CR cells evade immune surveillance. Supported by Department of Veterans Affairs (BLR&amp;D Merit review and CDA2) Citation Format: Medhi Wangpaichitr, Dan JM Nguyen, Ying-Ying Li, Chunjing Wu, Lynn G. Feun, Niramol Savaraj. Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4361.

Expression of tryptophan metabolizing enzymes (TMEs) and its transporter, LAT1, in metastatic melanoma (MM): Prognostic and therapeutic implications.

e21014 Background: The ECHO-301/KEYNOTE-252 trial failed to show clinical benefit of adding epacadostat to pembrolizumab in PD-1 inhibitor-naïve MM. We reasoned that the expression of other TMEs and/or LAT1 by melanoma cells plays a more important role. Methods: Melanoma tissues from stage III/IV pts were stained for the 4 TMEs (TPH1, TPH2, TDO2, IDO1) and LAT1 by single-color immunohistochemistry. Tissues were scored for the status of tumor-infiltrating lymphocytes (TILs) and the expression of 5 proteins separately in melanoma cells and TILs (if present). Association between protein expression, TIL status, and melanoma-specific overall survival was performed. Results: Tissues from 87 pts (stage IV, n = 25) were available. Expression of all 4 TMEs and LAT1 was significantly higher in melanoma cells compared to TILs ( p&lt; 0.001); TPH1, TPH2, and TDO2 expression in melanoma cells was significantly higher than IDO1 (p &lt; 0.001). Lower TPH1 expression in melanoma cells was associated with presence of TILs. Multivariate analysis using a Cox proportional hazards model that included expression of the 5 proteins in melanoma cells showed that high IDO1 was a favorable and high LAT1 expression was an adverse prognostic factor. Conclusions: Expression of TMEs and LAT1 by melanoma cells may have more important role in metastatic melanoma by depleting an essential amino acid from the tumor microenvironment. Pharmacologic targeting of TPH1/TPH2 (e.g. telotristat) may be more clinically relevant in MM as opposed to IDO1 inhibition.

MON-470 Pharmacological Inhibition of LAT1 Suppresses Proliferation and Hormone Synthesis in Rat Pituitary Tumor GH4 Cells [cc1] Synthesis May Be Better as Only mRNAs Were Measured

Pituitary tumors (PTs) occur in almost 17 % of the population and they represent about 10 % of all intracranial tumors. Growth hormone-producing pituitary tumors account for about 12% of pituitary tumors. At present, many patients with this type of pituitary tumor suffer from symptoms and complications caused by abnormal hormone production and/or mass effects due to limited treatment options. L-type amino acid transporter 1 (LAT1/SLC7A5) delivers essential amino acids into cells, and higher expression of LAT1 has been detected in various types of cancer including gliomas and pancreatic cancers. Furthermore, several lines of evidence suggest that blockade of LAT1 in tumor cells suppresses mammalian target of rapamycin complex (mTORC) 1 activity, resulting in either cell growth arrest or apoptotic cell death. However, LAT1 expression has not been elucidated in normal pituitary glands or PTs. In addition, the effects of LAT1 inhibition on cell growth and hormone synthesis in PTs also remain unknown. Here we show that LAT1, with expression comparable to LAT2, LAT3 and LAT4 in the normal mouse pituitary gland, is predominantly expressed in rat pituitary tumor GH4 cells, which secrete GH and PRL. An LAT1-specific inhibitor, JPH203, provided by J-Pharma Co., Ltd., Yokohama Japan, suppressed growth of these cells as well as GH and PRL synthesis. Water-Soluble Tetrazolium salt (WST)-8 assay revealed that effective doses of JPH203 on cell proliferation were ranging from 5 to 40 μM. Analyzing Annexin V-Fluorescein isothiocyanate (FITC) binding by flow cytometry, we found that JPH203 (40μM) induced apoptosis within three days and growth arrest for at least three weeks in GH4 cells. JPH203 also reduced the amount of GH and PRL mRNAs 40 % and 50 %, respectively. These results indicate that LAT1 is a potential target of therapy for pituitary tumors. Next, the mechanisms of JPH203 action in GH4 cells were investigated. Unexpectedly, JPH203 did not have any apparent effects on the phosphorylation state of mTOR1 and its downstream effectors. In contrast, rapamycin decreased phosphorylation levels of these proteins, resulting in a reduction in cell proliferation. Our findings suggest that in pituitary tumor cells there could be differential amino acid sensing pathways, which regulate both cell growth and hormone synthesis. Further studies will be required to determine the mechanism of action of the LAT1 inhibitor in GH4 cells.

Effects of chronic stress on intestinal amino acid pathways

Major depressive disorder (MDD) is a debilitating mental disorder with a high prevalence and severe impacts on quality of life. However, the pathophysiological mechanisms underlying MDD remain poorly understood. Here, we used high-performance liquid chromatography with ultraviolet detection-based targeted metabolomics to identify amino acid changes in the small intestine, in a rat model of chronic unpredictable mild stress (CUMS). Pearson's correlation analysis was conducted to investigate the correlations between amino acid changes and behavioral outcomes. Western blot analysis was employed to verify intestinal amino acid transport function. Moreover, we performed an integrated analysis of related differential amino acids in the hippocampus, peripheral blood mononuclear cells (PBMCs), urine and cerebellum identified in our previous studies using the CUMS rat model to further our understanding of amino acid metabolism in depression. Decreased concentrations of glutamine and glycine and upregulation of aspartic acid were found in CUMS model rats. These changes were significantly correlated with depressive-like behaviors. Western blot analysis revealed that CUMS rats exhibited a reduction in the expression levels of amino acid transporters ASCT2 and B0AT1, as well as an increase in LAT1 expression. Impaired transport of glycine and glutamine into the small intestine may contribute to a central deficiency. The current findings suggest that the glycine and glutamine uptake systems may be potential therapeutic targets for depression. The integrated analysis strategy used in the current study may provide new insight into the cellular and molecular mechanisms underlying the gut-brain axis, and help to elucidate the pathophysiological changes in central and peripheral systems in depression.

Alzheimer\u2019s Disease Phenotype or Inflammatory Insult Does Not Alter Function of L-Type Amino Acid Transporter 1 in Mouse Blood-Brain Barrier and Primary Astrocytes

PurposeThe study aim was to evaluate the effect of Alzheimer’s disease (AD) and inflammatory insult on the function of L-type amino acid transporter 1 (Lat1) at the mouse blood-brain barrier (BBB) as well as Lat1 function and expression in mouse primary astrocytes.MethodsThe Lat1 function and expression was determined in wildtype astrocytes with and without lipopolysaccharide (LPS)-induced inflammation and in LPS treated AD APP/PS1 transgenic astrocytes. The function of Lat1 at the BBB was evaluated in wildtype mice with and without LPS-induced neuroinflammation and APP/PS1 transgenic mice by in situ brain perfusion.ResultsThere were 2.1 and 1.6 -fold decreases in Lat1 mRNA and protein expression in LPS-treated wildtype astrocytes compared to vehicle-treated astrocytes. In contrast, Lat1 mRNA and protein expression were increased by 1.7 and 1.2 -fold (not statistically significant) in the transgenic cells. A similar trend was observed in the cell uptake of [14C]-L-leucine. There were no statistically significant differences in [14C]-L-leucine BBB permeation between the groups.ConclusionsThe results showed that neither LPS-induced inflammation or the presence of APP/PS1 mutations alters Lat1 function at the mouse BBB as well as Lat1 protein expression and function in mouse primary astrocytes.

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

BackgroundThe 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.MethodsLAT1 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.ResultsLAT1 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.ConclusionsThese preclinical results show that LAT1 inhibition is a novel therapeutic approach in the context of thyroid cancers, and more interestingly in untreatable thyroid cancers.

The effects of L-type amino acid transporter 1 on milk protein synthesis in mammary glands of dairy cows

The mammary gland requires the uptake of AA for milk protein synthesis during lactation. The L-type amino acid transporter 1 (LAT1, encoded by SLC7A5), found in many different types of mammalian cells, is indispensable as a transporter of essential AA to maintain cell growth and protein synthesis. However, the function of LAT1 in regulating milk protein synthesis in the mammary gland of the dairy cow remains largely unknown. For the current study, we characterized the relationship between LAT1 expression and milk protein synthesis in lactating dairy cows and investigated whether the mammalian target of rapamycin complex 1 (mTORC1) signaling controls the expression of LAT1 in their mammary glands. We found that LAT1 and the heavy chain of its chaperone, 4F2, were expressed in mammary tissues of lactating cows, with the expression levels of LAT1 and the 4F2 heavy chain being significantly greater in lactating mammary tissues with high-milk protein content (milk yield, 33.8 ± 2.1 kg/d; milk protein concentration >3%, wt/vol,; n = 3) than in tissues from cows with low-milk protein content (milk yield, 33.7 ± 0.5 kg/d; milk protein concentration <3%, wt/vol; n = 3). Immunofluorescence staining of sectioned mammary tissues from cows with high and low milk protein content showed that LAT1 was located on the whole plasma membrane of alveolar epithelial cells, suggesting that LAT1 provides essential AA to the mammary gland. In cultured mammary epithelial cells from the dairy cows with high-milk protein content, knockdown of LAT1 expression decreased cell viability and β-casein expression; in contrast, overexpression of LAT1 had the opposite effect. Inhibition of mTORC1 by rapamycin attenuated the phosphorylation of molecules related to mTORC1 signaling and caused a marked decrease in LAT1 expression in the cultured cells; expression of β-casein also decreased significantly. These results suggest that LAT1 is involved in milk protein synthesis in the mammary glands of lactating dairy cows and that the mTORC1 signaling pathway might be a control point for regulation of LAT1 expression, which could ultimately be used to alter milk protein synthesis.

Effect of light-load resistance exercise on postprandial amino acid transporter expression in elderly men.

An impaired amino acid sensing is associated with age‐related loss of skeletal muscle mass. We tested whether light‐load resistance exercise (LL‐RE) affects postprandial amino acid transporter (AAT) expression in aging skeletal muscle. Untrained, healthy men (age: +65 years) were subjected to 13 h of supine rest. After 2 1/2 h of rest, unilateral LL‐RE was conducted (leg extensions, 10 sets of 36 repetitions) at 16% 1RM. Thereafter, the subjects were randomized into groups that orally ingested 40 g of whey protein either as hourly drinks (4 g per drink) (PULSE, N = 10) or two boluses (28 g at 0 h and 12 g at 7 h) (BOLUS, N = 10), or hourly isocaloric maltodextrin drinks (placebo, N = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from both the resting and exercised leg, from which the membrane protein and mRNA expression of select AATs were analyzed by Western Blot and RT‐PCR, respectively. LAT1 and PAT1 protein expression increased in response to LL‐RE in the PULSE group, and SNAT2 and PAT1 protein expression increased in the BOLUS group when plasma BCAA concentration was low. In all three groups, LL‐RE increased LAT1 mRNA expression, whereas a time course decrease in SNAT2 mRNA expression was observed. LL‐RE increased membrane‐associated AAT protein expression and mRNA expression. Altered AAT protein expression was only seen in groups that ingested whey protein, with the greatest effect observed after hourly feeding. This points toward an importance of AATs in the anabolic response following LL‐RE and protein intake.

Clinical Significance and Phenotype of MTA1 Expression in Esophageal Squamous Cell Carcinoma.

Metastasis-associated gene 1 (MTA1) is considered a potential prognostic factor in esophageal cancer. We investigated the clinical relationship between MTA1, LAT1, and tumor metabolism, as evaluated by positron emission tomography (PET) in esophageal squamous cell carcinoma. We analyzed 142 esophageal squamous cell carcinoma patients who underwent curative resection without preoperative treatment. MTA1 expression was assessed by immuno-zahistochemistry, and tested against standardized uptake values from preoperative PET-CT. The association among MTA1, LAT1, and 18FAMT PET results were analyzed. MTA1 staining was observed in 82 of 142 cancer tissues. Five-year overall survival was 69.9 % in the absence of MTA1, but 50.7% otherwise (p=0.021), while disease-free survival was 66.5% and 49.0% (p=0.071), respectively. Abnormal 18FAMT accumulation was noted in 13 patients without MTA1 and in 18 patients with MTA1 (p=0.079), with maximum standardized uptake value 1.6±1.6 and 2.7±1.6, respectively (p=0.036). MTA1 expression was positively correlated with LAT1 (p=0.013) and CD34 (p=0.034) expression, but not with Ki-67 (p=0.078). MTA1 shows promise as a diagnostic and prognostic marker in esophageal cancer, and we anticipate that the gene will also prove to be a good therapeutic target.

Characterization of 5-(2- 18F-fluoroethoxy)-L-tryptophan for PET imaging of the pancreas.

Purpose: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2- 18F-fluoroethoxy)-L-tryptophan ( 18F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes. Procedures: Uptake of 18F-L-FEHTP was determined in wild-type C57BL/6 mice by ex vivo biodistribution. Both dynamic and static positron emission tomography (PET) images were acquired in wild-type and Akita mice, a model of ER stress-induced diabetes, as well as in mice treated with streptozotocin (STZ). LAT1 expression in both groups of mice was evaluated by immunofluorescence microscopy. Results: Uptake of 18F-L-FEHTP was highest in the pancreas, and static PET images showed highly specific pancreatic signal. Time-activity curves showed significantly reduced 18F-L-FEHTP uptake in Akita mice, and LAT1 expression was also reduced. However, mice treated with STZ, in which beta cell mass was reduced by 62%, showed no differences in 18F-L-FEHTP uptake in the pancreas, and there was no significant correlation of 18F-L-FEHTP uptake with beta cell mass. Conclusions:18F-L-FEHTP is highly specific for the pancreas with little background uptake in kidney or liver. We were able to detect changes in LAT1 in a mouse model of diabetes, but these changes did not correlate with beta cell function or mass. Therefore, 18F-L-FEHTP PET is not a suitable method for the noninvasive imaging of changes in beta cell function during the progression of diabetes.