The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma.

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.

A druggable cascade links methionine metabolism to epigenomic reprogramming in squamous cell carcinoma.

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.

ZAP70, too little, too much can lead to autoimmunity.

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self- from agonist-peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self-reactive TCRs a greater chance to escape negative selection. Such 'forbidden clones' may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self-ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases.

LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner.

Amino acid transporters play a vital role in cellular homeostasis by maintaining protein synthesis. L-type amino acid transporter 1 (LAT1/SLC7A5/CD98lc) is a major transporter of large neutral amino acids in cancer cells because of its predominant expression. Although amino acid restriction with various amino acid analog treatments is known to induce mitotic defects, the involvement of amino acid transporters in cell division remains unclear. In this study, we identified that LAT1 is responsible for mitotic progression in a transport activity-independent manner. LAT1 knockdown activates the spindle assembly checkpoint, leading to a delay in metaphase. LAT1 maintains proper spindle orientation with confinement of the lateral cortex localization of the NuMA protein, which mediates the pulling force against the mitotic spindle toward the lateral cortex. Unexpectedly, JPH203, an inhibitor of LAT1 amino acid transport activity, does not affect mitotic progression. Moreover, the transport activity-deficient LAT1 mutant maintains the proper spindle orientation and mitotic progression. LAT1 forms a heterodimer with CD98 (SLC3A2/CD98hc) both in interphase and mitosis. Although CD98 knockdown decreases the plasma membrane localization of LAT1, it does not affect mitotic progression. LAT1 is localized to the Golgi and ER not only at the plasma membrane in interphase, and promotes Golgi unlinking during the mitotic entry, leading to centrosome maturation. These results suggest that LAT1 supports mitotic progression in an amino acid transport activity-independent manner and that Golgi-localized LAT1 is important for mitotic progression through the acceleration of Golgi unlinking and centrosome maturation. These findings reveal a novel LAT1 function in mitosis.

L-type amino acid transporter 1 inhibitor JPH203 prevents the growth of cabazitaxel-resistant prostate cancer by inhibiting cyclin-dependent kinase activity.

L-type amino acid transporter 1 (LAT1, SLC7A5) is an amino acid transporter expressed in various carcinomas, and it is postulated to play an important role in the proliferation of cancer cells through the uptake of essential amino acids. Cabazitaxel is a widely used anticancer drug for treating castration-resistant prostate cancer (CRPC); however, its effectiveness is lost when cancer cells acquire drug resistance. In this study, we investigated the expression of LAT1 and the effects of a LAT1-specific inhibitor, JPH203, in cabazitaxel-resistant prostate cancer cells. LAT1 was more highly expressed in the cabazitaxel-resistant strains than in the normal strains. Administration of JPH203 inhibited the growth, migration, and invasive ability of cabazitaxel-resistant strains in vitro. Phosphoproteomics using liquid chromatography-mass spectrometry to comprehensively investigate changes in phosphorylation due to JPH203 administration revealed that cell cycle-related pathways were affected by JPH203, and that JPH203 significantly reduced the kinase activity of cyclin-dependent kinases 1 and 2. Moreover, JPH203 inhibited the proliferation of cabazitaxel-resistant cells in vivo. Taken together, the present study results suggest that LAT1 might be a valuable therapeutic target in cabazitaxel-resistant prostate cancer.

The LAT1 inhibitor JPH203 suppresses the growth of castration-resistant prostate cancer through a CD24-mediated mechanism.

L-type amino acid transporter 1 (LAT1) is specifically expressed in many malignancies, contributes to the transport of essential amino acids, such as leucine, and regulates the mammalian target of rapamycin (mTOR) signaling pathway. We investigated the expression profile and functional role of LAT1 in prostate cancer using JPH203, a specific inhibitor of LAT1. LAT1 was highly expressed in castration-resistant prostate cancer (CRPC) cells, including C4-2 and PC-3 cells, but its expression level was low in castration-sensitive LNCaP cells. JPH203 significantly inhibited [14C] leucine uptake in CRPC cells but had no effect in LNCaP cells. JPH203 inhibited the proliferation, migration, and invasion of CRPC cells but not of LNCaP cells. In C4-2 cells, Cluster of differentiation (CD) 24 was identified by RNA sequencing as a novel downstream target of JPH203. CD24 was downregulated in a JPH203 concentration-dependent manner and suppressed activation of the Wnt/ß-catenin signaling pathway. Furthermore, an in vivo study showed that JPH203 inhibited the proliferation of C4-2 cells in a castration environment. The results of this study indicate that JPH203 may exert its antitumor effect in CRPC cells via mTOR and CD24.

Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR).

The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, nor actin remodeling following CAR activation. However, CAR T cells still require LAT for an optimal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.

A viral lncRNA tethers HSV-1 genomes at the nuclear periphery to establish viral latency.

IMPORTANCE: Herpes simplex virus 1 (HSV-1) establishes lifelong latency in neuronal cells. Following a stressor, the virus reactivates from latency, virus is shed at the periphery and recurrent disease can occur. During latency, the viral lncRNA termed the latency-associated transcript (LAT) is known to accumulate to high abundance. The LAT is known to impact many aspects of latency though the molecular events involved are not well understood. Here, we utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to identify the molecular-binding partners of the LAT during latency. We found that the LAT binds to both the cellular protein, TMEM43, and HSV-1 genomes in LUHMES cells. Additionally, we find that knockdown of TMEM43 prior to infection results in a decreased ability of HSV-1 to establish latency. This work highlights a potential mechanism for how the LAT facilitates the establishment of HSV-1 latency in human neurons.

L-type Amino Acid Transporter 1 as a Therapeutic Target in Pancreatic Cancer.

Metabolic rewiring is a key feature of cancer cells to support the demands of growth and proliferation. The metabolism of amino acids is altered in many cancers, including pancreatic cancer. The cellular uptake of amino acids is regulated by amino acid transporters, such as L-type amino acid transporter 1 (LAT1). Accumulating evidence suggests that LAT1 is overexpressed in pancreatic cancer and confers a poor prognosis. Here we discuss the prospects of utilizing LAT1 as a novel target for pancreatic cancer therapy.

Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors.

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

Inhibition of amino acid transporter LAT1 in cancer cells suppresses G0/G1-S transition by downregulating cyclin D1 via p38 MAPK activation.

L-type amino acid transporter 1 (LAT1, SLC7A5) is upregulated in various cancers and associated with disease progression. Nanvuranlat (Nanv; JPH203, KYT-0353), a selective LAT1 inhibitor, suppresses the uptake of large neutral amino acids required for rapid growth and proliferation of cancer cells. Previous studies have suggested that the inhibition of LAT1 by Nanv induces the cell cycle arrest at G0/G1 phase, although the underlying mechanisms remain unclear. Using pancreatic cancer cells arrested at the restriction check point (R) by serum deprivation, we found that the Nanv drastically suppresses the G0/G1-S transition after release. This blockade of the cell cycle progression was accompanied by a sustained activation of p38 mitogen-activated protein kinase (MAPK) and subsequent phosphorylation-dependent proteasomal degradation of cyclin D1. Isoform-specific knockdown of p38 MAPK revealed the predominant contribution of p38α. Proteasome inhibitors restored the cyclin D1 amount and released the cell cycle arrest caused by Nanv. The increased phosphorylation of p38 MAPK and the decrease of cyclin D1 were recapitulated in xenograft tumor models treated with Nanv. This study contributes to delineating the pharmacological activities of LAT1 inhibitors as anti-cancer agents and provides significant insights into the molecular basis of the amino acid-dependent cell cycle checkpoint at G0/G1 phase.

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.

Identification of tumor-suppressive miRNAs that target amino acid transporter LAT1 and exhibit anti-proliferative effects on cholangiocarcinoma cells.

Amino acid transporter LAT1 is highly upregulated in various cancer types, including cholangiocarcinoma (CHOL), and contributes to the rapid proliferation of cancer cells and disease progression. However, the molecular mechanisms underlying the pathological upregulation of LAT1 remain largely unknown. This study pursued the possibility of miRNA-mediated regulation of the LAT1 expression in CHOL cells. Using online target prediction methods, we extracted five candidate miRNAs commonly predicted to regulate the LAT1 expression. Three of them, miR-194-5p, miR-122-5p, and miR-126-3p, were significantly downregulated in CHOL cancer compared to normal tissues. Correlation analysis revealed weak-to-moderate negative correlations between the expression of these miRNAs and LAT1 mRNA in CHOL cancer tissues. We selected miR-194-5p and miR-122-5p for further analyses and found that both miRNAs functionally target 3'UTR of LAT1 mRNA by a luciferase-based reporter assay. Transfection of the miRNA mimics significantly suppressed the LAT1 expression at mRNA and protein levels and inhibited the proliferation of CHOL cells, with a trend of affecting intracellular amino acids and amino acid-related signaling pathways. This study indicates that the decreased expression of these LAT1-targeting tumor-suppressive miRNAs contributes to the upregulation of LAT1 and the proliferation of CHOL cells, highlighting their potential for developing novel cancer therapeutics and diagnostics.

Observational study on the identification of left atrial tachycardia circuits using Local Activation Time Histogram.

BACKGROUND: Mapping of atypical atrial arrythmias arising in the left atrium is often challenging. The Local Activation Time (LAT) Histogram, a new function of the 3D color mapping system CARTO version 7, may help improve identification of atrial tachycardia circuits. We aimed to assess the effectiveness of the LAT Histogram for identification of left atrial tachycardia circuits. METHODS: This retrospective study compared 25 consecutive cases of left atrial tachycardia that were treated before use of LAT Histogram (unused group) and 25 consecutive cases that were treated after introduction of LAT Histogram (used group) at Nagano Chuo Hospital. We evaluated whether we could identify the circuit of left atrial tachycardia from the electrophysiology lab data during ablation and the CARTO system data and whether we could perform effective ablation. RESULTS: Door-to-door time, skin-to-skin time, and fluoroscopy time (p ≤ .011) were all shorter in the LAT Histogram used group versus unused group, while mapping analysis times were longer in the used group (p ≤ .019). A significantly greater number of cases in the LAT Histogram used compared with the unused group had ablation for entrance or exit points (19 vs. 10 cases; p = .001 for first map). Ablation resulted in a return to sinus rhythm and changed cycle length at the first mapping in 20 cases (80%) in the LAT Histogram unused group and in 24 cases (96%) in the used group. CONCLUSIONS: LAT Histogram may provide a simple and effective method to identify entrance and exit locations in left atrial tachycardia.

Prolactin upregulates amino acids uptake in dairy cow mammary epithelial cells via LAT1.

The uptake of AA in mammary tissues is affected by prolactin (PRL). To investigate whether PRL-induced AA uptake is involved in L-type AA transporter 1 (LAT1), we analyzed the changes of AA in the medium of dairy cow mammary epithelial cells in the presence of PRL or PRL plus BCH, an inhibitor of LAT1. Then Western blot and luciferase assay were used to detect the regulation mechanism of PRL on LAT1 expression and function. Our results showed that Thr, Val, Met, Ile, Leu, Tyr, Lys, Phe, and His are LAT1 substrates and could be transported into mammary epithelial cells via LAT1. PRL stimulation increased the uptake of most AA into mammary epithelial cells of dairy cows, however, inhibition of LAT1 transport activity reduced PRL-induced AA uptake, suggesting that the effect of PRL on AA transport depends on LAT1 expression and function. PRL stimulation upregulated LAT1 expression and plasma membrane location not only in dairy cow mammary epithelial cells, but also in mouse mammary epithelial cell line HC11. Western blot showed that PI3K-AKT-mTOR signaling could be activated in PRL-stimulated mammary epithelial cells. Treatment of cells with LY294002 decreased PI3K-AKT-mTOR activation, as well LAT1 expression, that in turn decreased milk protein synthesis. Luciferase assay showed PRL treatment increased the promoter activity of LAT1 promoter fragment -419∼-86 bp. Treatment of cells with LY294002, an inhibitor of PI3K, or SC79, an activator of AKT abolished or promoted the transcriptional activity of this promoter fragment in the presence of PRL. These results suggested that the -419∼-86 bp fragment of LAT1 promoter mediates the action of PI3K-AKT-mTOR signaling on LAT1 transcription in mammary epithelial cells of dairy cows, which in turn increased LAT1 expression and AA uptake.

Leucine drives LAT1-related SNAIL upregulation in glucose-starved pancreatic cancer cells.

Pancreatic cancer, a highly fibrotic and hypovascular tumor, is thought to have unique metabolic characteristics in surviving and proliferating in malnutritional microenvironments. In this study, we compared the differences in the ability of pancreatic cancer cells to adapt to glucose-free conditions with liver cancer cells, which are representative of hypervascular tumors. Three pancreatic cancer cells and two liver cancer cells were used to examine the transcriptional expression levels of molecules involved in intracellular amino acid uptake, epithelial-mesenchymal transition (EMT), and cancer stemness under glucose deprivation. The results showed that the proliferative activity of pancreatic cancer cells under glucose deprivation was significantly lower than that of liver cancer cells, but the expression levels of amino acid transporters were significantly higher. Among them, L-type amino acid transporter 1 (LAT1) upregulation was unique in concert with increased expression of the EMT regulator SNAIL and the cancer stemness marker doublecortin-like kinase 1. LAT1 knockdown canceled the upregulation of SNAIL in glucose-starved pancreatic cancer cells, suggesting a mechanistic link between the two molecules. When LAT1 was stimulated by its substrate leucine, the SNAIL expression was upregulated dose-dependently. Collectively, pancreatic cancer cells reprogrammed metabolism to adapt to energy crises involving leucine-induced SNAIL upregulation.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18-/- syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Herpes Simplex Virus Type 1 Preferentially Enhances Neuro-Inflammation and Senescence in Brainstem of Female Mice.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons. The latency associated transcript (LAT) is the only viral gene abundantly expressed during latency. Wild-type (WT) HSV-1 reactivates more efficiently than LAT mutants because LAT promotes establishment and maintenance of latency. While sensory neurons in trigeminal ganglia (TG) are important sites for latency, brainstem is also a site for latency and reactivation from latency. The principal sensory nucleus of the spinal trigeminal tract (Pr5) likely harbors latent HSV-1 because it receives afferent inputs from TG. The locus coeruleus (LC), an adjacent brainstem region, sends axonal projections to cortical structures and is indirectly linked to Pr5. Senescent cells accumulate in the nervous system during aging and accelerate neurodegenerative processes. Generally senescent cells undergo irreversible cell cycle arrest and produce inflammatory cytokines and chemokines. Based on these observations, we hypothesized HSV-1 influences senescence and inflammation in Pr5 and LC of latently infected mice. This hypothesis was tested using a mouse model of infection. Strikingly, female but not age-matched male mice latently infected with a LAT null mutant (dLAT2903) exhibited significantly higher levels of senescence markers and inflammation in LC, including cell cycle inhibitor p16, NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3), IL-1α, and IL-ß. Conversely, Pr5 in female but not male mice latently infected with WT HSV-1 or dLAT2903 exhibited enhanced expression of important inflammatory markers. The predilection of HSV-1 to induce senescence and inflammation in key brainstem regions of female mice infers that enhanced neurodegeneration occurs. IMPORTANCE HSV-1 (herpes simplex virus 1), an important human pathogen, establishes lifelong latency in neurons in trigeminal ganglia and the central nervous system. In contrast to productive infection, the only viral transcript abundantly expressed in latently infected neurons is the latency associated transcript (LAT). The brainstem, including principal sensory nucleus of the spinal trigeminal tract (Pr5) and locus coeruleus (LC), may expedite HSV-1 spread from trigeminal ganglia to the brain. Enhanced senescence and expression of key inflammatory markers were detected in LC of female mice latently infected with a LAT null mutant (dLAT2903) relative to age-matched male or female mice latently infected with wild-type HSV-1. Conversely, wild-type HSV-1 and dLAT2903 induced higher levels of senescence and inflammatory markers in Pr5 of latently infected female mice. In summary, enhanced inflammation and senescence in LC and Pr5 of female mice latently infected with HSV-1 are predicted to accelerate neurodegeneration.

HSV-1 latency-associated transcript miR-H3 and miR-H4 target STXBP1 and GABBR2 genes.

During latent infection, the HSV-1 virus generates only a single transcript, LAT, which encodes six miRNAs. The GABAergic pathway signaling system is an essential cell signaling pathway influenced by various therapeutic targets and some brain disorders, such as epilepsy. This study found that miRNAs encoding LAT might target the STXBP1 and GABBR2 genes, which are among the significant genes in the GABAergic pathway. Bioinformatic analysis utilizing TargetScan version 5.2 and the RNA22 tools uncovered miRNAs encoding LAT that can influence STXBP1 and GABBR2 transcripts. To evaluate the targeting effect of candidate microRNAs encoding LAT, namely, miR-H3 and miR-H4, LAT constructs were transfected into HEK 293T cells. The expression levels of microRNAs encoding LAT, as well as STXBP1 and GABBR2, were assayed by real-time PCR. Finally, the targeting potential of STXBP1 and GABBR2 3'UTR by LAT-encoded microRNAs was evaluated by the luciferase assay. In the current study, the bioinformatic tool TargetScan demonstrated that miR-H3 has the potential to target the transcripts of the STXBP1 and GABBR2 genes, whereas miR-H4 solely targeted GABBR2. On the other hand, the bioinformatic tool RNA22 validated the potential targeting of STXBP1 and GABBR2 by miR-H3 and miR-H4. Our findings showed that overexpression of miR-H4, miR-H3, or LAT significantly decreased STXBP1 gene expression by an average of 0.0593-fold, 0.237-fold, and 0.84-fold, respectively. Similarly, overexpression of miR-H3 or miR-H4 decreased GABBR2 expression by an average of 0.055- or 0.687-fold, respectively. Notably, targeting the GABBR2 3'UTR with the LAT transcript had no detectable effect. The evaluation of the targeting potential of STXBP1 and GABBR2 3'UTR by microRNAs encoded by LAT was conducted with a luciferase assay. Our results showed that miR-H3 overexpression reduces Renilla expression in psiCHECK2 plasmids with STXBP1 or GABBR2 3'UTR genes by 0.62- and 0.55-fold, respectively. miR-H4 reduced Renilla gene expression regulated by GABBR2's 3'UTR plasmid but had no effect on the Renilla gene expression regulated by STXBP1's 3'UTR. When the LAT transcript was overexpressed, there was a decrease in Renilla expression by 0.44-fold because of the regulation of STXBP1's 3'UTR. However, there was no significant effect observed through the control of GABBR2's 3'UTR.

Combination effects of amino acid transporter LAT1 inhibitor nanvuranlat and cytotoxic anticancer drug gemcitabine on pancreatic and biliary tract cancer cells.

BACKGROUND: Cytotoxic anticancer drugs widely used in cancer chemotherapy have some limitations, such as the development of side effects and drug resistance. Furthermore, monotherapy is often less effective against heterogeneous cancer tissues. Combination therapies of cytotoxic anticancer drugs with molecularly targeted drugs have been pursued to solve such fundamental problems. Nanvuranlat (JPH203 or KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), has novel mechanisms of action to suppress the cancer cell proliferation and tumor growth by inhibiting the transport of large neutral amino acids into cancer cells. This study investigated the potential of the combined use of nanvuranlat and cytotoxic anticancer drugs. METHODS: The combination effects of cytotoxic anticancer drugs and nanvuranlat on cell growth were examined by a water-soluble tetrazolium salt assay in two-dimensional cultures of pancreatic and biliary tract cancer cell lines. To elucidate the pharmacological mechanisms underlying the combination of gemcitabine and nanvuranlat, we investigated apoptotic cell death and cell cycle by flow cytometry. The phosphorylation levels of amino acid-related signaling pathways were analyzed by Western blot. Furthermore, growth inhibition was examined in cancer cell spheroids. RESULTS: All the tested seven types of cytotoxic anticancer drugs combined with nanvuranlat significantly inhibited the cell growth of pancreatic cancer MIA PaCa-2 cells compared to their single treatment. Among them, the combined effects of gemcitabine and nanvuranlat were relatively high and confirmed in multiple pancreatic and biliary tract cell lines in two-dimensional cultures. The growth inhibitory effects were suggested to be additive but not synergistic under the tested conditions. Gemcitabine generally induced cell cycle arrest at the S phase and apoptotic cell death, while nanvuranlat induced cell cycle arrest at the G0/G1 phase and affected amino acid-related mTORC1 and GAAC signaling pathways. In combination, each anticancer drug basically exerted its own pharmacological activities, although gemcitabine more strongly influenced the cell cycle than nanvuranlat. The combination effects of growth inhibition were also verified in cancer cell spheroids. CONCLUSIONS: Our study demonstrates the potential of first-in-class LAT1 inhibitor nanvuranlat as a concomitant drug with cytotoxic anticancer drugs, especially gemcitabine, on pancreatic and biliary tract cancers.