Amino Acid Transporter SLC6A14 Research Articles

TRIM35 triggers cardiac remodeling by regulating SLC7A5-mediated amino acid transport and mTORC1 activation in fibroblasts

BackgroundCardiac maladaptive remodeling is one of the leading causes of heart failure with highly complicated pathogeneses. The E3 ligase tripartite motif containing 35 (TRIM35) has been identified as a crucial regulator governing cellular growth, immune responses, and metabolism. Nonetheless, the role of TRIM35 in fibroblasts in cardiac remodeling remains elusive.MethodsHeart tissues from human donors were used to verify tissue-specific expression of TRIM35. Fibroblast-specific Trim35 gene knockout mice (Trim35cKO) were used to investigate the function of TRIM35 in fibroblasts. Cardiac function, morphology, and molecular changes in the heart tissues were analyzed after transverse aortic constriction (TAC) surgery. The mechanisms by which TRIM35 regulates fibroblast phenotypes were elucidated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA sequencing (RNA-Seq). These findings were further validated through the use of adenoviral and adeno-associated viral transfection systems, as well as the mTORC1 inhibitor Rapamycin.ResultsTRIM35 expression is primarily up-regulated in cardiac fibroblasts in both murine and human fibrotic hearts, and responds to TGF-β1 stimulation. Specific deletion of TRIM35 in cardiac fibroblasts significantly improves cardiac fibrosis and hypertrophy. Consistently, the overexpression of TRIM35 promotes fibroblast proliferation, migration, and differentiation. Through paracrine signaling, it induces hypertrophic growth of cardiomyocytes. Mechanistically, we found that TRIM35 interacts with, ubiquitinates, and up-regulates the amino acid transporter SLC7A5, which enhances amino acid transport and activates the mTORC1 signaling pathway. Furthermore, overexpression of SLC7A5 significantly reverses the reduced cardiac fibrosis and hypertrophy caused by conditional knockout of TRIM35.ConclusionOur findings demonstrate a novel role of fibroblast-TRIM35 in cardiac remodeling and uncover the mechanism underlying SLC7A5-mediated amino acid transport and mTORC1 activation. These results provide a potential novel therapeutic target for treating cardiac remodeling.

ATP dependence and antiport/uniport switch of the essential amino acid transporter SLC7A5 (LAT1): molecular basis and pathophysiology

ATP dependence and antiport/uniport switch of the essential amino acid transporter SLC7A5 (LAT1): molecular basis and pathophysiology

Rag-Ragulator is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway.

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates cell growth and metabolism in response to many environmental cues, including nutrients. Amino acids signal to mTORC1 by modulating the guanine nucleotide loading states of the heterodimeric Rag GTPases, which bind and recruit mTORC1 to the lysosomal surface, its site of activation. The Rag GTPases are tethered to the lysosome by the Ragulator complex and regulated by the GATOR1, GATOR2, and KICSTOR multiprotein complexes that localize to the lysosomal surface through an unknown mechanism(s). Here, we show that mTORC1 is completely insensitive to amino acids in cells lacking the Rag GTPases or the Ragulator component p18. Moreover, not only are the Rag GTPases and Ragulator required for amino acids to regulate mTORC1, they are also essential for the lysosomal recruitment of the GATOR1, GATOR2, and KICSTOR complexes, which stably associate and traffic to the lysosome as the "GATOR" supercomplex. The nucleotide state of RagA/B controls the lysosomal association of GATOR, in a fashion competitively antagonized by the N terminus of the amino acid transporter SLC38A9. Targeting of Ragulator to the surface of mitochondria is sufficient to relocalize the Rags and GATOR to this organelle, but not to enable the nutrient-regulated recruitment of mTORC1 to mitochondria. Thus, our results reveal that the Rag-Ragulator complex is the central organizer of the physical architecture of the mTORC1 nutrient-sensing pathway and underscore that mTORC1 activation requires signal transduction on the lysosomal surface.

Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer.

Proteins overexpressed in early-stage cancers may serve as early diagnosis and prognosis markers as well as targets for cancer therapies. In this study, we examined the expression of an essential amino acid carrier SLC7A5 (LAT1, CD98, or 4F2 light chain) in cancer tissue from two well-annotated cohorts of 575 cases of early-stage and 106 cases of late-stage colorectal cancer patients. Immunohistochemistry showed SLC7A5 overexpression in 72.0% of early-stage and 56.6% of late-stage cases. SLC7A5 expression was not influenced by patient gender, age, location, or mismatch repair status, although it appeared to be slightly less prevalent in tumors of mucinous differentiation or with lymphovascular invasion. Statistical analyses revealed a positive correlation between SLC7A5 overexpression and both overall survival and disease-free survival in early-stage but not late-stage cancers. Co-expression analyses of the TCGA and CPTAC colorectal cancer cohorts identified a network of gene transcripts positively related to SLC7A5, with its heterodimer partner SLC3A2 having the highest co-expression score. Network analysis uncovered the SLC7A network to be significantly associated with ncRNA such as tRNA processing and the mitotic cell cycle. Since SLC7A5 is also a marker of activated lymphocytes such as NK, T, and B lymphocytes, SLC7A5 overexpression in early colorectal cancers might trigger a strong anti-tumor immune response which could results in better clinical outcome. Overall, our study provides clear evidence of differential SLC7A5 expression and its prognostic value for early-stage colorectal cancer, although the understanding of its functions in colorectal tumorigenesis and cancer immunity is currently rather limited and awaits further characterization.

How α -lactalbumin and β –casein level in infant formula influence the protein and minerals absorption properties by using Caco-2 cell model

This study investigated the differences in absorption of infant formula with different levels of α-lactalbumin (α-La) and β-casein (β-CN), and how α-La and β-CN (α+β) affect mineral absorption using the Caco-2 cell model. Compared to the control group, the free amino acid content transported to the basal side significantly increased in the fortification of α-La and β-CN, particularly in the α+β (high) group, which reached 11.343 μmol/L. Furthermore, fortification of α-La and β-CN upregulated the expression of the amino acid transporter SLC6A14 and the oligopeptide transporter PEPT1, in particular the expression of SLC6A14 in the α+β (high) group was 103% of that of the control group. In addition, fortification of α-La and β-CN enhanced the absorption of Ca, Mg, and Zn. The absorption rates of Ca, Mg, and Zn in α+β (high) group increased to 31.27%, 13.66%, and 58.59%, respectively, of which α-La contributed the most on Ca absorption. In conclusion, α-La and β-CN fortification not only increased the absorption of amino acids and peptides but also promoted the absorption of minerals, which provide evidence for the benefits of adjustment of α-La and β-CN in the infant formula.

Resistance to Combined Anthracycline-Taxane Chemotherapy Is Associated with Altered Metabolism and Inflammation in Breast Carcinomas.

Approximately 30% of early-stage breast cancer (BC) patients experience recurrence after systemic chemotherapy; thus, understanding therapy resistance is crucial in developing more successful treatments. Here, we investigated the mechanisms underlying resistance to combined anthracycline-taxane treatment by comparing gene expression patterns with subsequent therapeutic responses. We established a cohort of 634 anthracycline-taxane-treated patients with pathological complete response (PCR) and a separate cohort of 187 patients with relapse-free survival (RFS) data, each having transcriptome-level expression data of 10,017 unique genes. Patients were categorized as responders and non-responders based on their PCR and RFS status, and the expression for each gene was compared between the two groups using a Mann-Whitney U-test. Statistical significance was set at p < 0.05, with fold change (FC) > 1.44. Altogether, 224 overexpressed genes were identified in the tumor samples derived from the patients without PCR; among these, the gene sets associated with xenobiotic metabolism (e.g., CYP3A4, CYP2A6) exhibited significant enrichment. The genes ORAI3 and BCAM differentiated non-responders from responders with the highest AUC values (AUC > 0.75, p < 0.0001). We identified 51 upregulated genes in the tumor samples derived from the patients with relapse within 60 months, participating primarily in inflammation and innate immune responses (e.g., LYN, LY96, ANXA1). Furthermore, the amino acid transporter SLC7A5, distinguishing non-responders from responders, had significantly higher expression in tumors and metastases than in normal tissues (Kruskal-Wallis p = 8.2 × 10-20). The identified biomarkers underscore the significance of tumor metabolism and microenvironment in treatment resistance and can serve as a foundation for preclinical validation studies.

Amino acid transporter SLC7A5 regulates cell proliferation and secretary cell differentiation and distribution in the mouse intestine.

The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5ΔIEC reduces mTORC1 signaling. Surprisingly, adult Slc7a5ΔIEC intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in Slc7a5ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.

Nanoengineered sonosensitive platelets for synergistically augmented sonodynamic tumor therapy by glutamine deprivation and cascading thrombosis.

Ultrasound (US)-activated sonodynamic therapy (SDT) stands for a distinct antitumor modality because of its attractive characteristics including intriguing noninvasiveness, desirable safety, and high tissue penetration depth, which, unfortunately, suffers from compromised therapeutic efficacy due to cancer cell-inherent adaptive mechanisms, such as glutathione (GSH) neutralization response to reactive oxygen species (ROS), and glutamine addictive properties of tumors. In this work, we developed a biological sonosensitive platelet (PLT) pharmacytes for favoring US/GSH-responsive combinational therapeutic of glutamine deprivation and augmented SDT. The amino acid transporter SLC6A14 blockade agent α-methyl-DL-tryptophan (α-MT)-loaded and MnO2-coated porphyrinic metal-organic framework (MOF) nanoparticles were encapsulated in the PLTs through the physical adsorption of electrostatic attraction and the intrinsic endocytosis of PLTs. When the sonosensitive PLT pharmacytes reached tumor sites through their natural tendencies to TME, US stimulated the PLTs-loaded porphyrinic MOF to generate ROS, resulting in morphological changes of the PLTs and the release of nanoparticles. Subsequently, intracellular high concentration of GSH and extracellular spatio-temporal controlled US irradiation programmatically triggered the release of α-MT, which enabled the synergistically amplified SDT by inducing amino acid starvation, inhibiting mTOR, and mediating ferroptosis. In addition, US stimulation achieved the targeted activation of PLTs at tumor vascular site, which evolved from circulating PLTs to dendritic PLTs, effectively blocking the blood supply of tumors through thrombus formation, and revealing the encouraging potential to facilitate tumor therapeutics.

SRC2 controls CD4+ T cell activation via stimulating c-Myc-mediated upregulation of amino acid transporter Slc7a5

Tcell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ Tcell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in Tcells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C.rodentium)infection. Adoptive transfer of naive CD4+ Tcells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/recipients. Further, CD4+ Tcells from SRC2fl/fl/CD4Cre mice display defective Tcell proliferation, cytokine production, and differentiation both invitro and invivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for Tcell activation. Slc7a5 fails to be up-regulated in CD4+ Tcells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ Tcells to induce EAE. Therefore, SRC2 is essential for CD4+ Tcell activation and, thus, a potential drug target for controlling CD4+ Tcell-mediated autoimmunity.

Abstract 2785: Carbidopa/α-methyltryptophan as an ideal combination therapy for breast cancer

Abstract Background/Hypothesis: The amino acid transporter SLC6A14 is upregulated in estrogen receptor (ER)-positive breast cancer, a subtype that represents 75-80% of all breast cancers. Obesity/overweight increases the risk of breast cancer due to estrogen generated from fat cells that fuel the growth of the ER-positive breast cancer. Previous studies from our lab have demonstrated that blockade of the amino acid transporter SLC6A14 by the small molecule α-methyltryptophan reduces the growth of ER-positive breast cancer in mouse xenografts of human breast cancer cells and in a spontaneous mouse model of ER-positive breast cancer. We have also discovered that α-methyltryptophan functions as a potent weight-loss agent. We believe that this compound would be effective for the treatment of ER-positive breast cancer, especially in obese/overweight women. α-methyltryptophan is metabolized by aromatic amino acid decarboxylase. Therefore, combination of this compound with carbidopa, a well-known inhibitor of this enzyme, might potentiate the anticancer efficacy of α-methyltryptophan. We tested this idea in the present study using a syngeneic mouse model of ER-positive breast cancer using the mouse breast cancer cell line AT3. Results: A syngeneic tumor-cell transplant mouse model was used for ER-positive breast cancer. AT3 cells were injected into the mammary fat pad of C57BL/6 mice. This allows to monitor the growth of AT3 tumor growth in an immunologically competent mammary tissue environment. Drug treatment in drinking water was started after tumor growth was noticeable (100 mm3). There were four groups: (i) control; (ii) α-methyltryptophan at 0.5 mg/ml; (iii) carbidopa at 0.25 mg/ml; (iv) α-methyltryptophan at 0.5 mg/ml + carbidopa at 0.25 mg/ml. Tumor growth was monitored every three days. The mice were killed after 4 weeks of treatment. Tumors were excised and weighed. These studies showed that α-methyltryptophan and carbidopa were independently effective in reducing the tumor (48% reduction, p&amp;lt;0.05 for α-methyltryptophan; 50% reduction, p&amp;lt;0.05 for carbidopa), but the combination was the most effective (92% reduction, p&amp;lt;0.01). These effects were evident irrespective of whether the monitored parameter was tumor weight or tumor volume. Conclusion: The combination treatment significantly decreased the tumor size and volume compared to carbidopa and α-methyltryptophan alone. We do not know yet if the improved efficacy of the combination of the two drugs is due to a simple additive effect or due to a synergistic effect. Studies have shown that carbidopa promotes degradation of ER, indicating that carbidopa has its own independent anticancer effect that is relevant to ER-positive breast cancer. Further studies are needed to differentiate between the effect of carbidopa as an independent anticancer agent and its ability to potentiate the anticancer efficacy of α-methyltryptophan by preventing its metabolic degradation. Citation Format: Sirin Falconi, Sabarish Ramachandran, Vadivel Ganapathy. Carbidopa/α-methyltryptophan as an ideal combination therapy for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2785.

Abstract 3544: ALK signaling activity stabilizes SLC3A2 protein levels in neuroblastoma tumorigenesis

Abstract Background: Neuroblastoma is the most common childhood tumor that grows in the adrenal glands or sympathetic ganglia. Approximately 10% of pediatric neuroblastoma patients harbor mutations in anaplastic lymphoma kinase (ALK). In a previous study, we identified SLC3A2 as a potential interacting partner with ALK using BioID-based in vivo proximity labeling. SLC3A2 is a multifunctional protein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and regulates polyamine transport. No previous link to ALK signaling has been reported and the underlying mechanisms and functional consequences of a SLC3A2-ALK interaction in ALK-driven neuroblastoma are unclear. Materials &amp; Methods: The interaction of SLC3A2 and ALK initially detected with BioID-based in vivo proximity labeling was validated by immunoprecipitation. The effect of ALK signaling on SLC3A2 expression and protein stability was evaluated by quantitative PCR and immunoblotting. The functional effect of SLC3A2 on cell viability were investigated by siRNA and inhibitor treatments in both human and mouse ALK-driven neuroblastoma cells. Results: SLC3A2 was confirmed to interact with ALK in both anti-ALK and anti-SLC3A2 antibody co-immunoprecipitations from NB1 cells (ALK-wild-type) and CLB-GE (ALK-F1174V). Moreover, this interaction was decreased by ALK inhibitor (lorlatinib) treatment. Upon ALKAL2 ligand treatment, ALK signaling increased the protein levels of SLC3A2 and this was abrogated by lorlatinib treatment in NB1 cells. Furthermore, lorlatinib treatment suppressed SLC3A2 expression and significantly affected protein stability in neuroblastoma cells with different ALK mutations including CLB-BAR (ALK exon4-11 deletion), CLB-GAR (ALK-R1275Q), and CLB-GE (ALK-F1174V) during cycloheximide chase analysis. Knockdown of SLC3A2 significantly inhibited cell growth and down-regulated the amino acid transporter SLC7A5 (LAT1) expression in CLB-GE cells (ALK-F1174V). While mono treatment of either lorlatinib (ALK TKI) or AMXT-1501 (polyamine transport inhibitor) only showed moderate effects, combinatorial treatment exhibited a synergistic effect on cell growth in ALK-driven primary-cultured mouse neuroblastoma #9883 (Th-MYCN; Alk-F1178S) and #111 (Th-MYCN; Rosa26_Alkal2) cells. Conclusions: SLC3A2 protein stability and its interaction with ALK is dependent on ALK signaling in an ALK-driven neuroblastoma context. The synergistic effect of combined ALK and polyamine transport inhibitors suggests a potential therapeutic option for ALK-driven neuroblastoma. Citation Format: Wei-Yun Lai, Tzu-Po Chuang, Marcus Borenäs, Ruth Palmer, Bengt Hallberg. ALK signaling activity stabilizes SLC3A2 protein levels in neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3544.

Competition between Slc7a5 and LMAN2 for modulation of Kv1.2 gating.

The voltage-gated potassium channel Kv1.2 is a prominent ion channel in the CNS, where it regulates electrical activity. Kv1.2 structure and function are well understood, but its regulation by auxiliary proteins has not been widely studied. We identified novel regulators of Kv1.2 by a mass spectrometry approach, including the neutral amino acid transporter Slc7a5, which causes a dramatic hyperpolarizing shift of channel activation. In contrast, LMAN2 is a recently identified candidate regulator that has the opposite effect on gating: large depolarizing voltages are required to activate Kv1.2 channels co-expressed with LMAN2. In this study, we characterized the functional interaction between LMAN2 and Slc7a5 on Kv1.2 gating properties, and identified key structural elements that underlie sensitivity to either regulator. When LMAN2 and Slc7a5 are expressed together, Kv1.2 activation exhibits a bimodal voltage-dependence, suggesting two distinct populations of channels regulated either by LMAN2 or Slc7a5, but not both. Kv1.5 is a close homolog of Kv1.2, but insensitive to both LMAN2 and Slc7a5. Using a Kv1.2:1.5 chimeric approach, we identified the S1 segment of the voltage sensing domain (VSD) as essential for Slc7a5 sensitivity, and the S2-S3 linker of the VSD for LMAN2 sensitivity. By replacing either segment with sequence from Kv1.5, modulation by the corresponding regulator was selectively abolished. Replacement of the S1-S3 segment of Kv1.2 with sequence from Kv1.5 rendered the channel insensitive to both forms of regulation. These results suggest that Slc7a5 and LMAN2 compete for interaction with the Kv1.2 voltage sensor, leading to complex voltage-dependence of channel activity when both regulators are present in the cell. Although the structural basis for this modulation remains uncertain, Kv1.2 clearly exhibits a propensity for modulation over a wide range of voltages.

Modulation of Kv1.2 redox-sensitive gating by the transmembrane lectin LMAN2.

The voltage-gated potassium channel Kv1.2 plays a pivotal role in the nervous system, where it regulates membrane excitability and action potential propagation. Compared to other Kv1 channels, Kv1.2 exhibits dynamic voltage-dependent gating with dramatic cell-to-cell variability that is attributed to regulation by unidentified external factors. Recent progress from our lab has identified extrinsic factors that shift the voltage-dependence of Kv1.2 gating. For example, the neutral amino acid transporter Slc7a5 causes a dramatic hyperpolarizing shift of Kv1.2 channel activation. A counteracting effect is that extracellular reducing conditions promote a depolarizing shift in channel activation. However, the underlying mechanism of redox sensitivity has been challenging to identify. Using a mass-spectrometry approach to identify novel Kv1.2 accessory proteins, followed by screening candidates with whole-cell patch clamp electrophysiology, we observed that co-expression with the transmembrane lectin LMAN2 caused a pronounced depolarizing shift of Kv1.2 voltage-dependence. Overexpression of LMAN2 enhanced Kv1.2 sensitivity to extracellular reducing conditions, while shRNA knockdown of endogenous LMAN2 abrogated the redox effects. Using a chimeric approach between Kv1.2 and the LMAN2 insensitive Kv1.5 subunit, we found the S2-S3 linker of the VSD to be essential for sensitivity to both LMAN2 and reducing conditions. Biotinylation experiments revealed that LMAN2 maturation to the plasma membrane is required for its effects on Kv1.2 gating. Furthermore, extracellular application of the membrane impermeable reducing agent TCEP to dorsal root ganglion (DRG) neurons, decelerated the activation kinetics of isolated Kv1.2 currents and increased excitability. In conclusion, we identified a novel accessory protein (the transmembrane Lectin, LMAN2) that alters Kv1.2 gating properties and contributes to redox sensitivity in heterologous cells and DRG neurons.

Amino acid availability acts as a metabolic rheostat to determine the magnitude of ILC2 responses.

Group 2 innate lymphoid cells (ILC2) are functionally poised, tissue-resident lymphocytes that respond rapidly to damage and infection at mucosal barrier sites. ILC2 reside within complex microenvironments where they are subject to cues from both the diet and invading pathogens-including helminths. Emerging evidence suggests ILC2 are acutely sensitive not only to canonical activating signals but also perturbations in nutrient availability. In the context of helminth infection, we identify amino acid availability as a nutritional cue in regulating ILC2 responses. ILC2 are found to be uniquely preprimed to import amino acids via the large neutral amino acid transporters Slc7a5 and Slc7a8. Cell-intrinsic deletion of these transporters individually impaired ILC2 expansion, while concurrent loss of both transporters markedly impaired the proliferative and cytokine-producing capacity of ILC2. Mechanistically, amino acid uptake determined the magnitude of ILC2 responses in part via tuning of mTOR. These findings implicate essential amino acids as a metabolic requisite for optimal ILC2 responses within mucosal barrier tissues.

Excretion of excess nitrogen and increased survival by loss of SLC6A19 in a mouse model of ornithine transcarbamylase deficiency.

Protein catabolism ultimately yields toxic ammonia, which must be converted to urea by the liver for renal excretion. In extrahepatic tissues, ammonia is temporarily converted primarily to glutamine for subsequent hepatic extraction. Urea cycle disorders (UCDs) are inborn errors of metabolism causing impaired ureagenesis, leading to neurotoxic accumulation of ammonia and brain glutamine. Treatment includes dietary protein restriction and oral "ammonia scavengers." These scavengers chemically combine with glutamine and glycine to yield excretable products, creating an alternate pathway of waste nitrogen disposal. The amino acid transporter SLC6A19 is responsible for >95% of absorption and reabsorption of free neutral amino acids in the small intestine and kidney, respectively. Genetic SLC6A19 deficiency causes massive neutral aminoaciduria but is typically benign. We hypothesized that inhibiting SLC6A19 would open a novel and effective alternate pathway of waste nitrogen disposal. To test this, we crossed SLC6A19 knockout (KO) mice with spfash mice, a model of ornithine transcarbamylase (OTC) deficiency. Loss of SLC6A19 in spfash mice normalized plasma ammonia and brain glutamine and increased median survival in response to a high protein diet from 7 to 97 days. While induced excretion of amino acid nitrogen is likely the primary therapeutic mechanism, reduced intestinal absorption of dietary free amino acids, and decreased muscle protein turnover due to loss of SLC6A19 may also play a role. In summary, the results suggest that SLC6A19 inhibition represents a promising approach to treating UCDs and related aminoacidopathies.

Blockade of the amino acid transporter SLC6A14 suppresses tumor growth in colorectal Cancer

BackgroundThe amino acid transporter SLC6A14, which transports 18 of the 20 proteinogenic amino acids, is too low to be detected in healthy normal tissues but is significantly increased in some solid cancers. However, little is known about the roles of SLC6A14 in colorectal cancer (CRC).MethodsThe mRNA and protein levels of SLC6A14 were detected using TCGA database, real-time polymerase chain reaction, western blot, and tissue microarrays, respectively. Amino acids concentration was determined by LC-MS/MS. Cell proliferation and apoptosis were determined using MTT assay and flow cytometry. Transwell invasion assay and wound healing assay were employed to analyze cell migration and invasion. The protein levels of Akt-mTOR signaling pathway and MMPs proteins were detected by western blot.ResultsBoth of the mRNA and protein levels of SLC6A14 were upregulated in CRC tissues, and the protein levels of SLC6A14 were closely related to the tumor cells differentiation: the higher the expression of SLC6A14 was, the poorer the differentiation of the tumor cells was. Further knockdown SLC6A14 with siRNA or treatment with α-MT in CRC cell lines reduced cell proliferation and migration in vitro and inhibited xenograft tumor growth in vivo. Mechanistically, SLC6A14 was demonstrated to regulate the expression and phosphorylation of Akt-mTOR, which mediates the promoting tumor growth function of SLC6A14. Blockade of SLC6A14 with α-MT inhibited the activation of mTOR signaling.ConclusionSLC6A14 was upregulated in CRC and could promote tumor progression by activating the Akt-mTOR signaling pathway, which may serve as an effective molecular target for the treatment of CRC.

A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue’s metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs—elicited by the loss of Rag GTPases—inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.

Alloferon Affects the Chemosensitivity of Pancreatic Cancer by Regulating the Expression of SLC6A14.

Pancreatic cancer (PCa), one of the most malignant solid tumors, has a high mortality rate. Although there have been many trials of chemotherapeutic drugs such as gemcitabine, the mortality rates remain significantly higher than for other types of cancer. Therefore, more effective ways of improving conventional therapy for PCa are needed. Cancer cells take up large amounts of glutamine to drive their rapid proliferation. Recent studies show that the amino acid transporter SLC6A14 is upregulated in some cancers alongside glutamine metabolism. Alloferon, a peptide isolated from the insect immune system, exerts anti-viral and anti-inflammatory effects via its immunomodulatory function. In addition, it has anti-tumoral effects, although the underlying mechanisms are largely unknown. Therefore, we investigated the effects of alloferon on the PCa cell lines Panc-1 and AsPC-1. Exposure of these cells to alloferon for 3 weeks led to the downregulation of SLC6A14 expression and decreased glutamine uptake. Given that SLC6A14 plays a role in tumor progression and survival by promoting glutamine uptake into cancer cells, alloferon could be a potential adjuvant for the chemotherapeutic drug gemcitabine.

Polarity protein SCRIB interacts with SLC3A2 to regulate proliferation and tamoxifen resistance in ER+ breast cancer

Estrogen receptor (ER) positive breast cancer represents 75% of all breast cancers in women. Although patients with ER+ cancers receive endocrine therapies, more than 30% develop resistance and succumb to the disease, highlighting the need to understand endocrine resistance. Here we show an unexpected role for the cell polarity protein SCRIB as a tumor-promoter and a regulator of endocrine resistance in ER-positive breast cancer cells. SCRIB expression is induced by estrogen signaling in a MYC-dependent manner. SCRIB interacts with SLC3A2, a heteromeric component of leucine amino acid transporter SLC7A5. SLC3A2 binds to the N-terminus of SCRIB to facilitate the formation of SCRIB/SLC3A2/LLGL2/SLC7A5 quaternary complex required for membrane localization of the amino acid transporter complex. Both SCRIB and SLC3A2 are required for cell proliferation and tamoxifen resistance in ER+ cells identifying a new role for the SCRIB/SLC3A2 complex in ER+ breast cancer.

The effect of alloferon on chemosensitivity of pancreatic cancer through the regulation of SLC6A14 expression

Abstract Pancreatic cancer is one of the most malignant solid tumors with a high mortality rate. Although there have been many trials for effective therapies using chemotherapeutic drugs, such as gemcitabine, pancreatic cancer still shows significantly higher mortality than other types of cancer. Therefore, it is needed to examine the effective therapeutic ways to improve conventional therapy for pancreatic cancer. It has been previously reported that cancer cells possess a high rate of glutamine uptake needed for their rapid proliferation. In addition, it has recently been reported that amino acid transporter SLC6A14 is up-regulated in some cancers in close relation to glutamine metabolism. Alloferon is a peptide isolated from the insect immune system and shows anti-viral and anti-inflammatory effects based on its immune modulating function. In addition, its anti-tumor effect was recently discovered, but the related mechanisms are largely unknown. For this reason, I investigated the effect of alloferon on pancreatic cancer cell lines Panc-1 and AsPC-1. When Panc-1 and AsPC-1 were exposed to alloferon for 3 weeks, I found down-regulation of SLC6A14 expression and decreased glutamine uptake. Given that SLC6A14 is involved in tumor progression and survival by promoting glutamine uptake into cancer cells, alloferon could be an effective way with conventional chemotherapeutic drug, gemcitabine. Therefore, alloferon could be a useful adjuvant for an effective therapeutic way with conventional chemotherapeutic drug, gemcitabine. This study was funded by the NRF of Korea (No.:2017R1A2B2010948, 2017R1A6A3A11032576, 2020R1C1C1009334)