Amino Acid Transporter L-type Amino Acid Transporter 1 Research Articles

Upregulation of ATF4 mediates the cellular adaptation to pharmacologic inhibition of amino acid transporter LAT1 in pancreatic ductal adenocarcinoma cells

L-type amino acid transporter 1 (LAT1) is recognized as a promising target for cancer therapy; however, the cellular adaptive response to its pharmacological inhibition remains largely unexplored. This study examined the adaptive response to LAT1 inhibition using nanvuranlat, a high-affinity LAT1 inhibitor. Proteomic analysis revealed the activation of a stress-induced transcription factor ATF4 following LAT1 inhibition, aligning with the known cellular responses to amino acid deprivation. This activation was linked to the GCN2-eIF2α pathway which regulates translation initiation. Our results show that ATF4 upregulation counteracts the suppressive effect of nanvuranlat on cell proliferation in pancreatic ductal adenocarcinoma cell lines, suggesting a role for ATF4 in cellular adaptation to LAT1 inhibition. Importantly, dual targeting of LAT1 and ATF4 exhibited more substantial anti-proliferative effects in vitro than individual treatments. This study underscores the potential of combining LAT1 and ATF4 inhibition as a therapeutic strategy in cancer treatment.

The association between the amino acid transporter LAT1, tumor immunometabolic and proliferative features and menopausal status in breast cancer.

L-type Amino Acid Transporter 1 (LAT1) facilitates the uptake of specific essential amino acids, and due to this quality, it has been correlated to worse patient outcomes in various cancer types. However, the relationship between LAT1 and various clinical factors, including menopausal status, in mediating LAT1's prognostic effects remains incompletely understood. This is particularly true in the unique subset of tumors that are both obesity-associated and responsive to immunotherapy, including breast cancer. To close this gap, we employed 6 sets of transcriptomic data using the Kaplan-Meier model in the Xena Functional Genomics Explorer, demonstrating that higher LAT1 expression diminishes breast cancer patients' survival probability. Additionally, we analyzed 3'-Deoxy-3'-18F-Fluorothymidine positron emission tomography-computed tomography (18F-FLT PET-CT) images found on The Cancer Imaging Archive (TCIA). After separating all patients based on menopausal status, we correlated the measured 18F-FLT uptake with various clinical parameters quantifying body composition, tumor proliferation, and immune cell infiltration. By analyzing a wealth of deidentified, open-access data, the current study investigates the impact of LAT1 expression on breast cancer prognosis, along with the menopausal status-dependent associations between tumor proliferation, immunometabolism, and systemic metabolism.

Conditional inactivation of the L-type amino acid transporter LAT1 in chondrocytes models idiopathic scoliosis in mice.

Scoliosis, usually diagnosed in childhood and early adolescence, is an abnormal lateral curvature of the spine. L-type amino acid transporter 1 (LAT1), encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types. We previously demonstrated the pivotal role of LAT1 on bone homeostasis in mice, and the expression of LAT1/SLC7A5 in vertebral cartilage of pediatric scoliosis patients; however, its role in chondrocytes on spinal homeostasis and implications regarding the underlying mechanisms during the onset and progression of scoliosis, remain unknown. Here, we identified LAT1 in mouse chondrocytes as an important regulator of postnatal spinal homeostasis. Conditional inactivation of LAT1 in chondrocytes resulted in a postnatal-onset severe thoracic scoliosis at the early adolescent stage with normal embryonic spinal development. Histological analyses revealed that Slc7a5 deletion in chondrocytes led to general disorganization of chondrocytes in the vertebral growth plate, along with an increase in apoptosis and a decrease in cell proliferation. Furthermore, loss of Slc7a5 in chondrocytes activated the general amino acid control (GAAC) pathwaybut inactivated the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the vertebrae. The spinal deformity in Slc7a5-deficient mice was corrected by genetic inactivation of the GAAC pathway, but not by genetic activation of the mTORC1 pathway. These findings suggest that the LAT1-GAAC pathway in chondrocytes plays a critical role in the maintenance of proper spinal homeostasis by modulating cell proliferation and survivability.

Proteomics and phosphoproteomics reveal key regulators associated with cytostatic effect of amino acid transporter LAT1 inhibitor.

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.

Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

BackgroundTumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis.MethodsExpression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells.ResultsLAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells.ConclusionsThese results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.

Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging.

3‐18F‐l‐α‐methyl‐tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer‐specificity and lower physiologic background. FAMT‐PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L‐type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter‐mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l‐methionine, a well‐studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l‐[14C]methionine was taken up by most of the transporters. Km of LAT1‐mediated [14C]FAMT transport was 72.7 μM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer‐type amino acid transporter LAT1, which explains the cancer‐specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer‐specific expression of LAT1. This study has established FAMT as a LAT1‐specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.

Up-Regulation of LAT1 during Antiandrogen Therapy Contributes to Progression in Prostate Cancer Cells

Cancer cells require massive amounts of amino acids for survival. LAT1 (L-type amino acid transporter 1) transports essential amino acids, including leucine, which trigger the downstream mTOR (mammalian target of rapamycin) pathway. We examined the association between androgen receptor and LAT1, and the association between LAT1 expression and the acquisition of castration resistance. Western blot and real-time polymerase chain reaction were performed to study protein and mRNA expression. siRNA was used to knock down target genes. A total of 92 prostate biopsy specimens of patients who underwent androgen deprivation therapy were used for immunohistochemical analyses. Cox hazard proportional models and the Kaplan-Meier method were used for statistical analyses. LAT1 was highly expressed in hormone resistant prostate cancer cell lines. Knockdown of LAT1 in LNCaP and C4-2 cells significantly suppressed cell proliferation, migration and invasion. Androgen receptor siRNA or androgen receptor blocking through bicalutamide (10 μM) or MDV3100 (10 μM) significantly increased LAT1 expression (p <0.01). Treatment with dihydrotestosterone (0.1 to 10 nM) reduced LAT1 expression in a dose dependent manner (p<0.01). Bicalutamide/MDV3100 plus siLAT1 synergistically suppressed prostate cancer cell proliferation compared to single inhibition by androgen receptor or LAT1 (p<0.01). High LAT1 expression correlated with significantly shorter prostate specific antigen recurrence-free survival in patients receiving androgen deprivation therapy (p <0.0001). LAT1 expression was an independentpredictor of castration resistance on multivariate analysis (HR 3.56, p=0.0133). The current data may indicate a novel mechanism to acquire castration resistance through activation of the amino acid transporter LAT1.

Abstract 5286: Expression of cancer type amino acid transporter LAT1 is a prognosis prediction factor in breast carcinoma: comparison between triple-negative and non-triple-negative types

Abstract Purpose: Amino acid transporters play important roles in cell survival and proliferation. L-Type amino acid transporter 1 (LAT1) is one of the major Na+-independent neutral amino acid transporters. LAT1 has an aspect of oncofetal protein. Recently, high levels of LAT1 expression have been reported in various tumors, such as prostate cancer, pancreas cancer, bile duct cancer, lung cancer and stomach cancer.We revealed LAT1 expression is an independent prognosis marker in stomach cancer, prostate cancer and pancreas cancer. The purpose of the present study was to investigate the LAT1 expression in breast carcinoma and relationship between LAT1 expression and prognosis. Materials &amp; Methods: We enrolled 290 patients with invasive breast carcinoma surgically resected between 2006 and 2010 at Kitasato University Hospital and Toho University Omori Medical Center, Japan. We investigated LAT1 expression in the enrolled breast carcinoma cases by immunohistochemical staining. All cases were divided into two groups, 78 cases of triple negative carcinoma (TNBC) and 212 cases of non-triple negative carcinoma (NTNBC), with immunohistochemistry of estrogen and progesterone receptors and Her2. After LAT1 expression was compared between TNBC and NTNBC, correlations among LAT1 expression, Ki-67 labeling index (LI), CD98 expression, and clinicopathological findings was assessed. Results: TNBC showed significantly higher LAT1 expression than NTNBC. TNBC also showed significantly higher CD98 expression rather than NTNBC. LAT1 expression of extraductal invasive cancer cells was significantly higher than those of intraductal carcinoma cells. A significantly positive correlation was observed between LAT1 expression and CD98 expression. Further, a positive correlation between LAT1 expression and Ki-67 LI was found. Log-rank test in overall breast cancers revealed that high LAT1 expression group showed significantly poor prognosis, compared to low LAT1 group. In NTNBC, high LAT1expression cases possessed poorer prognosis than low LAT1 expression cases. Conclusion: In invasive ductal carcinoma of breast, LAT1 expression has a potential for a prognosis prediction factor. LAT1 offers a potential target for anti-cancer therapy by its inhibitor JPH203 which has been developed by us. Citation Format: Masaaki Ichinoe, Tetsuo Mikami, Kiyomi Hana, Nobuyuki Yanagisawa, Hitoshi Endou, Isao Okayasu, Yoshiki Murakumo. Expression of cancer type amino acid transporter LAT1 is a prognosis prediction factor in breast carcinoma: comparison between triple-negative and non-triple-negative types. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5286. doi:10.1158/1538-7445.AM2015-5286

Abstract 4543: Uptake of 18F-FDOPA analogue 3H-L-DOPA is mediated by large neutral amino acid transporter LAT1 in human glioblastoma

Abstract INTRODUCTION: The L-Type amino acid transporter 1 (LAT1) transports large neutral amino acids across the plasma membrane in a variety of cells. Positron emission tomography (PET) imaging with the amino acid tracer 6-18F-fluoro-L-dopa (18F-FDOPA) provides useful histological information in human gliomas in addition to providing better localizing information then CT or MRI alone. This study is the first to correlate LAT1 expression with uptake of the 18F-FDOPA analog, 3H-L-DOPA in human glioblastomas. METHODS: Endogenous expression of LAT1 was evaluated in two established glioblastoma multiforme (GBM) cell lines and three primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of FDOPA was assessed in vitro using 3H-L-DOPA as an analog to 18F-FDOPA. Knockdown of LAT1 expression was achieved by lentiviral expression of anti-LAT1 shRNA in GBM lines with high LAT1 expression. The effect of LAT1 knockdown on 3H-L-DOPA uptake was then quantified. RESULTS: Results from Western blotting and qRT-PCR demonstrate that LAT1 is variably expressed in GBM. Levels of 3H-L-DOPA uptake were directly related to LAT1 expression (p&amp;lt;0.001) across the 5 lines. Knockdown of LAT1 was achieved in T98 cells (knockdown efficiency = 82.8%) and xenograft GBM 28 (knockdown efficiency = 73.7%) as confirmed by qRT-PCR and Western blotting. Knockdown of LAT1 reduced 3H-L-DOPA uptake by 57.2% (p&amp;lt;0.0001) in T98 and 52.1% in GBM 28 (p&amp;lt;0.001). CONCLUSIONS: This is the first report of 3H-L-DOPA uptake being driven by LAT1 in human GBM. Our findings suggest LAT1 as a transporter of 18F-FDOPA in GBM. Additional studies are underway to correlate LAT1 expression with PET 18F-FDOPA uptake in patients with GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4543. doi:1538-7445.AM2012-4543

Establishment of Stable Cell Lines With High Expression of Heterodimers of Human 4F2hc and Human Amino Acid Transporter LAT1 or LAT2 and Delineation of Their Differential Interaction With α-Alkyl Moieties

System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that l-[14C]alanine is an appropriate substrate to examine the function of LAT2, whereas l-[14C]leucine is used for LAT1. By using l-[14C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-l-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.

사람 암세포주들에서 아미노산 수송체 LAT1 mRNA 발현과 아미노산 L - Leucine 수송의 상관관계

사람의 여러 암세포주들에서 아미노산 수송체 LAT1 및 그 보조인자 4F2hc mRNA의 발현과 LAT1의 대표적 기질인 L-leucine 수송을 조사하여 이들 사이의 상관관계를 밝히기 위해, 암세포주들에서 northern blot analysis 및 uptake 실험 등을 시행하여 다음과 같은 결과를 얻었다. 조사한 26 종류의 사람 암 유래 세포주들에서 LAT1 mRNA의 뚜렷한 발현을 확인할 수 있었으나 그 발현양에는 차이가 있었다. 조사한 26종류의 사람 암 유래 세포주들에서 4F2hc mRNA의 뚜렷한 발현을 확인할 수 있었으며, LAT1의 경우에서와 같이 발현하는 정도가 조금씩 다름을 확인할 수 있었다. 사람의 암 유래 세포주들에서 아미노산 transporter에 의한 L-[¹⁴C]leucine 수송을 확인할 수 있었다. 사람의 암세포주들에서 LAT1 mRNA의 발현정도가 높을수록 L-[¹⁴C]leucine의 수송능력이 증가함을 알 수 있었다. 사람의 암세포주들에서 4F2hc mRNA의 발현정도가 높을수록 L-[¹⁴C]leucine 수송 능력이 증가하는 경향은 보였으나, 통계적인 유의성은 없었다. 본 연구의 결과에 의해 사람 암세포주들에서 아미노산 transporter LAT1의 발현과 아미노산 L-leucine의 수송 사이와의 상관관계를 확인할 수 있었으며, 아미노산 transporter LAT1의 특이적 억제에 의한 암세포의 성장 억제에 관한 또 하나의 방향성을 제시할 수 있을 것으로 사료된다.

Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance.

In the last decade, a lot of amino acid transporters were identified by molecular cloning and assigned to the classically characterized amino acid transport systems. Among them, ones which belong to the heterodimeric amino acid transporter family are unique because of their broad substrate selectivity and their pathological implications as well as their structural features. The heterodimeric amino acid transporter family is a subfamily of SLC7 solute transporter family which includes 14-transmembrane cationic amino acid transporters as well as 12-transmembrane heterodimeric amino acid transporters. The members of heterodimeric amino acid transporter family are linked via a disulfide bond to single membrane spanning type II membrane glycoproteins such as 4F2hc (4F2 heavy chain) and rBAT (related to b(0,+)-amino acid transporter). Six members are associated with 4F2hc and one is linked to rBAT. The neutral amino acid transporter of this family seems to rely on the hydrophobic interactions for their substrate recognition which can explain their broad substrate selectivity. Because of this characteristic, they can permeate amino-acid-related drugs and contribute to the pharmacokinetics of these drugs. A neutral amino acid transporter LAT1 (L-type amino acid transporter 1) has actually been shown to be present at the blood-brain-barrier. Because the members of the heterodimeric amino acid transporter family exhibit variety of substrate selectivity, it is proposed that this family members have been diverged from the prototype neutral amino acid transporter such as LAT1 by acquiring the mechanisms for the recognition of electric charges on the substrate amino acid side chains. The dysfunction or hyperfunction of the members of the heterodimeric amino acid transporter family are involved in some diseases and pathologic conditions. The genetic defects of the renal and intestinal transporters BAT1/b(0,+) AT (b(0,+)-type amino acid transporter 1/b(0,+)-type amino acid transporter) and y+ LAT1 (y+ L-type amino acid transporter 1) result in the amino aciduria with sever clinical symptoms such as cystinuria and lysinuric protein intolerance, respectively. LAT1 is proposed to be involved in the progression of malignant tumor. xCT (x- C-type transporter) functions to protect cells against oxidative stress, while its over-function may be damaging neurons leading to the exacerbation of brain damage after, brain ischemia. Therefore, these transporters would be candidates for therapeutic targets based on new strategies. Through the interaction with the associating proteins, the transporters of this family would be endowed with more possibility to be regulated via intracellular and extracellular signalling pathways, which is critical to tune the transporter functions to meet the metabolic requirements of cells.