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Abstract L-type amino acid transporter 1 (LAT1, SLC7A5), overexpressed in various cancers, mediates the uptake of essential amino acids crucial for tumor growth. It has emerged as a promising target for cancer therapy. Nanvuranlat (JPH203/KYT-0353), a LAT1 inhibitor, has shown antitumor activity in preclinical studies and efficacy in biliary tract cancer during clinical trials. This study provides a comprehensive pharmacological characterization of nanvuranlat and its N-acetyl metabolite, including structural insights into their LAT1 interactions. Both compounds demonstrated high selectivity for LAT1 over LAT2 and other amino acid transporters. Nanvuranlat acts as a competitive, non-transportable LAT1 inhibitor (K i = 38.7 nM), while its N-acetyl metabolite retains selectivity but with reduced affinity (K i = 1.68 µM). Nanvuranlat exhibited a sustained inhibitory effect on LAT1 even after its removal, indicating the potential for prolonged therapeutic effects. Both compounds showed comparable dissociation rates, suggesting that N-acetylation does not affect the interaction responsible for slow dissociation. The U-shaped conformation adopted by nanvuranlat when bound to LAT1 likely contributes to its high affinity, selectivity, sustained inhibitory effect, and non-transportable nature observed in this study. These insights into nanvuranlat’s mechanism and metabolic impact provide essential information for understanding its clinical efficacy and advancing LAT1-targeted cancer therapies.

Abstract L-type amino acid transporter 1 (LAT1) is upregulated in various cancers and contributes to the growth and proliferation of cancer cells. Previous clinicopathological studies have revealed associations between the high expression of LAT1 and the poor prognosis in multiple cancer types. However, in those studies, the expression of LAT1 has been evaluated solely by immunohistochemistry on resected tumors or tissue biopsies. Cancer cell-derived exosomes are attracting increasing attention as a resource of diagnostic, prognostic, and therapeutic biomarkers. In this study, we revealed the expression of LAT1 on exosomes from pancreatic cancer T3M-4 cells by western blotting, immunoprecipitation, and immuno-transmission electron microscopy. LAT1 was generally detected by western blotting and ELISA on exosomes from several pancreatic, biliary tract, and ovarian cancer cells. Notably, similar trends existed between the abundance of exosomal LAT1 and the cellular LAT1 expression levels. The expression of LAT1 on exosomes in vivo was verified using the peritoneal wash from intraperitoneal T3M-4 tumor-bearing mice. These results indicate that exosomal LAT1 released from cancer cells holds significant potential as a novel biomarker for diagnosis and prognosis.

Abstract L-type amino acid transporter 1 (LAT1) is upregulated in various cancer types and contributes to disease progression. Previous studies have demonstrated or suggested that hypoxia-inducible factors (HIFs), the key transcription factors in hypoxic responses, control the expression of LAT1 gene in several types of cancer cells. However, this regulatory relationship has not been investigated yet in colorectal cancer (CRC), one of the cancer types in which the increased LAT1 expression holds prognostic significance. In this study, we found that neither LAT1 mRNA nor protein is induced under hypoxic condition (1% O2) in CRC HT-29 cells in vitro, regardless of the prominent HIF-1/2α accumulation and HIFs-dependent upregulation of glucose transporter 1. The hypoxic treatment generally did not increase either the mRNA or protein expression of LAT1 in eight CRC cell lines tested, in contrast to the pronounced upregulation by amino acid restriction. Interestingly, knockdown of von Hippel-Lindau ubiquitin ligase to inhibit the proteasomal degradation of HIFs caused an accumulation of HIF-2α and increased the LAT1 expression in certain CRC cell lines. This study contributes to delineating the molecular mechanisms responsible for the pathological expression of LAT1 in CRC cells, emphasizing the ambiguity of HIFs-dependent transcriptional upregulation of LAT1 across cancer cells.

Abstract L-type amino acid transporter 1 (LAT1) is a transmembrane protein responsible for transporting large neutral amino acids. While numerous LAT1-targeted compound delivery for the brain and tumors have been investigated, their LAT1 selectivity often remains ambiguous despite high LAT1 affinity. This study assessed the LAT1 selectivity of phenylalanine (Phe) analogs, focusing on their structure–activity characteristics. We discovered that 2-iodo-l-phenylalanine (2-I-Phe), with an iodine substituent at position 2 in the benzene ring, markedly improves LAT1 affinity and selectivity compared to parent amino acid Phe, albeit at the cost of reduced transport velocity. l-Phenylglycine (Phg), one carbon shorter than Phe, was found to be a substrate for LAT1 with a lower affinity, exhibiting a low level of selectivity for LAT1 equivalent to Phe. Notably, (R)-2-amino-1,2,3,4-tetrahydro-2-naphthoic acid (bicyclic-Phe), with an α-methylene moiety akin to the α-methyl group in α-methyl-l-phenylalanine (α-methyl-Phe), a known LAT1-selective compound, showed similar LAT1 transport maximal velocity to α-methyl-Phe, but with higher LAT1 affinity and selectivity. In vivo studies revealed tumor-specific accumulation of bicyclic-Phe, underscoring the importance of LAT1-selectivity in targeted delivery. These findings emphasize the potential of bicyclic-Phe as a promising LAT1-selective component, providing a basis for the development of LAT1-targeting compounds based on its structural framework.

Abstract Metastasis is the leading cause of mortality in cancer patients. L-type amino acid transporter 1 (LAT1, SLC7A5) is a Na+-independent neutral amino acid transporter highly expressed in various cancers to support their growth. Although high LAT1 expression is closely associated with cancer metastasis, its role in this process remains unclear. This study aimed to investigate the effect of LAT1 inhibition on cancer metastasis using B16-F10 melanoma mouse models. Our results demonstrated that nanvuranlat (JPH203), a high-affinity LAT1-selective inhibitor, suppressed B16-F10 cell proliferation, migration, and invasion. Similarly, LAT1 knockdown reduced cell proliferation, migration, and invasion. LAT1 inhibitors and LAT1 knockdown diminished B16-F10 lung metastasis in a lung metastasis model. Furthermore, nanvuranlat and LAT1 knockdown suppressed lung, spleen, and lymph node metastasis in an orthotopic metastasis model. We discovered that the LAT1 inhibitor reduced the cell surface expression of integrin αvβ3. Our findings revealed that the downregulation of the mTOR signaling pathway, induced by LAT1 inhibitors, decreased the expression of integrin αvβ3, contributing to the suppression of metastasis. These results highlight the critical role of LAT1 in cancer metastasis and suggest that LAT1 inhibition may serve as a potential target for anti-metastasis cancer therapy.