Expression Of SLC5A8 Research Articles

The astrocytic Na+ -HCO3 - cotransporter, NBCe1, is dispensable for respiratory chemosensitivity.

The interoceptive homeostatic mechanism that controls breathing, blood gases and acid-base balance in response to changes in CO2 /H+ is exquisitely sensitive, with convergent roles proposed for chemosensory brainstem neurons in the retrotrapezoid nucleus (RTN) and their supporting glial cells. For astrocytes, a central role for NBCe1, a Na+ -HCO3 - cotransporter encoded by Slc4a4, has been envisaged in multiple mechanistic models (i.e. underlying enhanced CO2 -induced local extracellular acidification or purinergic signalling). We tested these NBCe1-centric models by using conditional knockout mice in which Slc4a4 was deleted from astrocytes. In GFAP-Cre;Slc4a4fl/fl mice we found diminished expression of Slc4a4 in RTN astrocytes by comparison to control littermates, and a concomitant reduction in NBCe1-mediated current. Despite disrupted NBCe1 function in RTN-adjacent astrocytes from these conditional knockout mice, CO2 -induced activation of RTN neurons or astrocytes in vitro and in vivo, and CO2 -stimulated breathing, were indistinguishable from NBCe1-intact littermates; hypoxia-stimulated breathing and sighs were likewise unaffected. We obtained a more widespread deletion of NBCe1 in brainstem astrocytes by using tamoxifen-treated Aldh1l1-Cre/ERT2;Slc4a4fl/fl mice. Again, there was no difference in effects of CO2 or hypoxia on breathing or on neuron/astrocyte activation in NBCe1-deleted mice. These data indicate that astrocytic NBCe1 is not required for the respiratory responses to these chemoreceptor stimuli in mice, and that any physiologically relevant astrocytic contributions must involve NBCe1-independent mechanisms. KEY POINTS: The electrogenic NBCe1 transporter is proposed to mediate local astrocytic CO2 /H+ sensing that enables excitatory modulation of nearby retrotrapezoid nucleus (RTN) neurons to support chemosensory control of breathing. We used two different Cre mouse lines for cell-specific and/or temporally regulated deletion of the NBCe1 gene (Slc4a4) in astrocytes to test this hypothesis. In both mouse lines, Slc4a4 was depleted from RTN-associated astrocytes but CO2 -induced Fos expression (i.e. cell activation) in RTN neurons and local astrocytes was intact. Likewise, respiratory chemoreflexes evoked by changes in CO2 or O2 were unaffected by loss of astrocytic Slc4a4. These data do not support the previously proposed role for NBCe1 in respiratory chemosensitivity mediated by astrocytes.

Nicotine up-regulates SLC7A5 expression depending on TRIM29 in non-small cell lung cancer

Nicotine up-regulates SLC7A5 expression depending on TRIM29 in non-small cell lung cancer

#2685 SLC34A2 EXACERBATES TUBULAR INTERSTITIAL FIBROSIS VIA INDUCTION OF APOPTOSIS AND CELL CYCLE ARREST IN S PHASE

Abstract Background and Aims Chronic kidney disease (CKD) is an urgent public health issue in the world. However, effective treatments for intervention of CKD remain undefined. Phosphate (Pi) is an essential element for synthesis of DNA, RNA, ATP, phospholipid membranes, and for regulation of phosphorylation/dephosphorylation, cellular signaling and metabolic pathways. Solute carrier family 34 (SLC34) is responsible for Pi absorption in the small intestine. However, the role of SLC34 in progression of CKD is still unknown. Method A membrane protein, solute carrier family 34 member 2 (SLC34A2), was discovered by using big data mining from the National Center for Biotechnology Information (NCBI). SLC34A2 was overexpressed in human proximal tubular cells, HK-2, by Lipofectamine 3000. RNA sequencing was carried out to uncover SLC34A2-mediated signaling pathways by using Illumina platform. Apoptosis was evaluated by Annexin V-PI staining and TEUNEL assay in cells and mouse kidneys, respectively. Cell cycle was measured by flow cytometry. Unilateral ureteral obstruction (UUO) and bilateral renal ischemia-reperfusion injury (bIRI) were employed to generate CKD mouse models. Results By using big data mining from the NCBI, we identified a membrane protein, SLC34A2, was upregulated in various types of nephropathies including diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, ANCA-associated vasculitis. Overexpression of SLC34A2 suppressed viability of HK-2 cells. RNA sequencing revealed that apoptosis and cell cycle are high scored pathways modulated by SLC34A2. The results were further verified in HK-2 cells. Ectopic SLC34A2 promoted apoptosis via downregulation of BCL-2. Moreover, SLC34A2 induced cell cycle arrest in S phase via downregulation of CDK2. CKD mouse models of UUO and bIRI were carried out to confirm in vitro findings. In mice kidneys, the expression of Slc34a2 in UUO and bIRI groups was superior to that in sham groups. Enriched Bax and TUNEL-positive cells were observed in the fibrotic kidneys of mice. Moreover, increased expression of p21 and downregulated Cyclin A2 in the fibrotic kidneys further confirmed that Slc34a2 induced cell cycle arrest in S phase. Conclusion Slc34a2 induced apoptosis and halted cell cycle in S phase, resulting in subsequent tubulointerstitial fibrosis in mice. Our results suggest that targeting SLC34A2 might be a promising strategy for intervention of CKD.

Akkermansia muciniphila Protects Against Antibiotic-Associated Diarrhea in Mice.

Probiotics are used to prevent antibiotic-associated diarrhea (AAD) via the restoration of the gut microbiota. However, the precise effects of Akkermansia muciniphila (Akk), which is a promising probiotics, on AAD are unknown. Here, AAD models were established via the administration of lincomycin and ampicillin with or without pasteurized Akk or Amuc_1100 treatment. A diffusion test revealed that Akk was susceptible to the majority of the antibiotics, such as ampicillin. These effects were confirmed by the reduced Akk abundance in AAD model mice. Pasteurized Akk or Amuc_1100 significantly decreased the diarrhea status score and colon injury of AAD model mice. Additionally, these treatments significantly decreased the relative abundance of Citrobacter at genus level and reshaped the metabolic function of gut microbiota. Notably, pasteurized Akk or Amuc_1100 significantly changed the serum metabolome of AAD model mice. In addition, pasteurized Akk or Amuc_1100 suppressed intestinal inflammation by upregulating the expression of GPR109A and SLC5A8 and downregulating the expression of TNFα, IFNγ, IL1β, and IL6. Furthermore, they enhanced water and electrolyte absorption by upregulating AQP4, SLC26A3, and NHE3. Pasteurized Akk or Amuc_1100 also restored intestinal barrier function by ameliorating the downregulation of ZO-1, OCLN, CLDN4, and Muc2 in AAD model mice. In summary, optimizing intestinal health with pasteurized Akk or Amuc_1100 may serve as an approach for preventing AAD.

#5592 THE SODIUM PHOSPHATE COTRANSPORTER NAPI-IIB IS UPREGULATED AND EXPRESSED DE NOVO IN KIDNEYS OF PATIENTS WITH CHRONIC KIDNEYS DISEASE

Abstract Background and Aims Renal reabsorption of phosphate is mediated by 3 main transporters located in the luminal brush border membrane of the proximal tubule, namely NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit2 (SLC20A2). The expression of all three proteins has been shown to be reduced in animal models of kidney disease [1]. Recently, we demonstrated that NaPi-IIb, a paralogue of the SLC34 subfamily that is mostly expressed in small intestine and lungs, is also expressed in the loop of Henle. Its mRNA expression is enhanced in rodent models of kidney injury. However, the expression of NaPi-IIb has not been characterized in humans and it is unknown whether NaPi-IIb is also upregulated in chronic kidney disease (CKD). Method In this study, we examined the localization of NaPi-IIa, IIb, and IIc in kidneys of control healthy individuals (kidney donors) (eGFR: 111.6 ± 22.9 ml/min/1.73m2) and patients with diabetic nephropathy (eGFR: 31.9 ± 7.7 ml/min/1.73m2), anti-glomerular basement membrane nephritis (anti-GMB) (eGFR: 27.2 ± 22.6 ml/min/1.73m2), and hypertensive nephropathy (eGFR: 31.5 ± 5.3 ml/min/1.73m2) by immunofluorescence. Moreover, we analyzed data from various transcriptome data bases from healthy individuals and patients with CKD. Results In kidneys from healthy donors, NaPi-IIb staining was restricted to thin and thick limbs of the loop of Henle, while AQP1 was observed in proximal tubules and descending thin limbs. NaPi-IIc was abundantly found in proximal tubule cells. In biopsies from patients, NaPi-IIb signal was also found in addition to thin and thick limbs in cells of the collecting duct and in many proximal tubules. In contrast, the staining of NaPi-IIc was reduced in all groups of patients with CKD. RNA expression data from the European Renal cDNA Bank (ERCB) confirmed our immunofluorescence findings. SLC34A2 (NaPi-IIb) mRNA was progressively more expressed in kidneys of patients with kidney disease of tubulointerstitial origin from CKD stages 1 to 5, while it was strongly upregulated from stage 4 onwards in CKD of glomerular origin. Single cell RNA sequencing data of patients with acute kidney injury and CKD [2] showed elevation of SLC34A2 expression in injured thick ascending limb, degenerative proximal tubule cells (14x higher, adj. p≪0.0001) (de novo expression), and descending thin limb (3.7x higher, adj. p≪0.0001). SLC34A1 (NaPi-IIa) expression levels were reduced in whole kidneys (ERCB data) and SLC34A1 and SLC34A3 expression also decreased in proximal tubule cells of patients with CKD. Conclusion The phosphate transporter NaPi-IIb is highly upregulated in kidneys from patients with various etiologies of CKD and its expression spreads to proximal tubule and collecting duct. In contrast, the expression of the main physiological phosphate transporters NaPi-IIa and NaPi-IIc is reduced in CKD. These findings may have implications for the development and maintenance of hyperphosphatemia in CKD patients and limit the use of specific NaPi-IIa and/or NaPi-IIc inhibitors in patients with CKD.

ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

Differential genomic and transcriptomic analysis of invasive lobular and ductal carcinomas.

579 Background: Invasive lobular carcinoma (ILC) is more aggressive than hormone receptor (HR)-positive invasive ductal carcinoma (IDC). However, in practice, ILC and IDC are often treated in a similar fashion with endocrine therapy and chemotherapy. Identifying novel biomarkers, genetic alterations, transcriptomic features, and tumor microenvironment (TME) variations could initiate the development of personalized treatment plans for patients with ILC. Methods: We collected ILC and luminal (non-basal/non-HER2) IDC samples from four datasets (TCGA, METABRIC, RATHER PMC4700448, and UQCCR PMC31263747) and performed differential expression and gene set enrichment analyses, revealing novel genomic, transcriptomic, and TME differences. Using methods from Bagaev et al., we quantified the activity of 29 functional gene expression signatures with single sample gene set enrichment analysis before clustering the samples into five TME subtypes; statistical significance was measured with the Mann-Whitney U test. Differential expression analysis of RNA-Seq data was completed using DESeq2. Further, we analyzed the frequency of specific biomarkers to identify potential therapeutic options. Mutations and biomarker enrichment were assessed using the chi-squared test. Results: We analyzed 1,735 samples (1,442 luminal IDCs and 293 ILCs). CDH1 mutations were more prevalent in ILC samples (56%) compared to IDC samples (6%). Of the 44% of ILC samples with wild-type CDH1, 90% had low CDH1 expression. Inference models showed differences in transcription factors expression between ILC and IDC. ILC had significantly higher expression of TFAP2B, SOCS2, NOSTRIN, THBS4, SCUBE2, and GDF9 and lower expression of CDCA4, PSMG1, LMOD1, and SLC7A5 (adj p < 0.0001 for all genes). Analysis of the TME showed that 44% of ILC samples were immune enriched with high PDL1, CTLA4, and LAG3 expression. In comparison, approximately 30% of ILC samples contained enhanced vascularization and expressed high VEGFA, PDGFRA, and PDGFRB. Finally, compared to luminal IDC, ILC tend to have a statistically significant higher TROP2 expression, similar to that seen in basal subtype. Conclusions: ILC and IDC expressed distinct genomic alterations, gene expression, transcriptomic features, TMEs, and biomarkers. These differences can be used as a blueprint to tailor ILC phenotype-specific interventional clinical trials.

Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes

BackgroundIndoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated...

A pan-cancer analysis of SLC1A5 in human cancers

BackgroundThe alanine-serine-cysteine transporter 2, ASCT2 (solute carrier family 1 member 5, SLC1A5), is a major transporter of the amino acid, glutamine. Although SLC1A5 has been reported to be associated with some types of cancer, less pan-cancer analysis, which would give a comprehensive understanding of SLC1A5 across human cancers, has been carried out. MethodsWe used the TCGA and GEO databases to investigate the oncogenic role of SLC1A5. We examined gene and protein expression, survival, genetic mutations, protein phosphorylation, immunocyte infiltration and the related genes correlated pathways. In HCT116 cells, SLC1A5 was silenced by siRNAs and the mRNA and protein was checked by Q-PCR and WB, respectively and the cellular function was assessed by CCK8, cell cycle and apoptosis. ResultsWe found that SLC1A5 was over-expressed in multiple types of cancer and that elevated expression of SLC1A5 was associated with poor survival in many cancers. The missense mutation of R330 H/C was associated with poor survival, especially in uterine carcinosarcoma. Furthermore, we found enhanced phosphorylation of S503 in uterine corpus endometrial carcinoma and lung adenocarcinoma. In addition, elevated SLC1A5 expression was associated with immune cell infiltration in many cancers. KEGG and GO analysis showed that SLC1A5 and its related genes were involved in central carbon metabolism in cancer, due to their amino acid transport activity. The cellular function indicated that SLC1A5 may influence the cell proliferation by affecting DNA synthesis. ConclusionsOur findings highlighted the important role of SLC1A5 in tumorigenesis and provided insights into potential cancer treatment strategies.

A metabolic shift toward glycolysis enables cancer cells to maintain survival upon concomitant glutamine deprivation and V-ATPase inhibition.

It is widely known that most cancer cells display an increased reliance on glutaminolysis to sustain proliferation and survival. Combining glutamine deprivation with additional anti-cancer therapies is an intensively investigated approach to increase therapeutic effectiveness. In this study, we examined a combination of glutamine deprivation by starvation or pharmacological tools, with the anti-cancer agent archazolid, an inhibitor of the lysosomal V-ATPase. We show that glutamine deprivation leads to lysosomal acidification and induction of pro-survival autophagy, which could be prevented by archazolid. Surprisingly, a combination of glutamine deprivation with archazolid did not lead to synergistic induction of cell death or reduction in proliferation. Investigating the underlying mechanisms revealed elevated expression and activity of amino acid transporters SLC1A5, SLC38A1 upon starvation, whereas archazolid had no additional effect. Furthermore, we found that the export of lysosomal glutamine derived from exogenous sources plays no role in the phenotype as knock-down of SLC38A7, the lysosomal glutamine exporter, could not increase V-ATPase inhibition-induced cell death or reduce proliferation. Analysis of the cellular metabolic phenotype revealed that glutamine deprivation led to a significant increase in glycolytic activity, indicated by an elevated glycolytic capacity and reserve, when V-ATPase function was inhibited concomitantly. This was confirmed by increased glutamine uptake, augmented lactate production, and an increase in hexokinase activity. Our study, therefore, provides evidence, that glutamine deprivation induces autophagy, which can be prevented by simultaneous inhibition of V-ATPase function. However, this does not lead to a therapeutic benefit, as cells are able to circumvent cell death and growth inhibition by a metabolic shift toward glycolysis.

Identification of SLC2A3 as a prognostic indicator correlated with the NF-κB/EMT axis and immune response in head and neck squamous cell carcinoma

ABSTRACT SLC2A3 is an important member of the glucose transporter superfamily. It has been recently suggested that upregulation of SLC2A3 is associated with poor survival and acts as a prognostic marker in a variety of tumors. Unfortunately, the prognostic role of SLC2A3 in head and neck squamous cell carcinoma (HNSC) is less known. In the present study, we analyzed SLC2A3 expression in HNSC and its correlation with prognosis using TCGA and GEO databases. The results showed that SLC2A3 mRNA expression was higher in HNSC compared with adjacent normal tissues, which was validated with our 9 pairs of HNSC specimens. Moreover, high SLC2A3 expression predicted poor prognosis in HNSC patients. Mechanistically, GSEA revealed that high expression of SLC2A3 was enriched in epithelial-mesenchymal transition (EMT) and NF-κB signaling. In HNSC cell lines, SLC2A3 knockdown inhibited cell proliferation and migration. In addition, NF-κB P65 and EMT-related gene expression was suppressed upon SLC2A3 knockdown, indicating that SLC2A3 may play a preeminent role in the progression of HNSC through the NF-κB/EMT axis. Meanwhile, the expression of SLC2A3 was negatively correlated with immune cells, suggesting that SLC2A3 may be involved in the immune response in HNSC. The correlation between SLC2A3 expression and drug sensitivity was further assessed. In conclusion, our study demonstrated that SLC2A3 could predict the prognosis of HNSC patients and mediate the progression of HNSC via the NF-κB/EMT axis and immune responses.

Anthocyanin-Containing Purple Sweet Potato (Ipomoea batatas L.) Synbiotic Yogurt Inhibited 3T3-L1 Adipogenesis by Suppressing White Adipocyte-Specific Genes.

We unravel the effect of anthocyanin-containing purple sweet potato synbiotic yogurt (PSPY) on 3T3-L1 adipocyte differentiation and its fundamental molecular mechanisms. Molecular docking simulation was performed to observe and identify the affinity and interaction between bioactive compounds and targeted proteins. MDI (isobutylmethylxanthine, dexamethasone, and insulin)-containing medium, a cocktail that stimulates adipogenesis, was used in this study. The toxic effect possibility of the yogurt product was evaluated using 3-[4, 5-dimethylthiazol-2-yl]-2.5 diphenyl tetrazolium bromide (MTT) assay. A 0.25%, 0.5%, 1%, and 5% (v/v) plain or purple sweet potato yogurt supernatant was given to 3T3-L1 preadipocyte culture medium from 24 h after seeding until day 11 of MDI-induced differentiation. The mRNA expression and lipid accumulation were analyzed using RT-qPCR and Oil red O staining, respectively, on day 11 after differentiation induction. In silico study suggested that anthocyanin-derived compounds have the potential to inhibit peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator for white adipogenesis. Anthocyanin-containing PSPY significantly suppressed the expression of Pparg, Adipoq, Slc2a4, and Pgc1a. PSPY significantly suppressed Pparg with 1% and 5% concentrations, while with a concentration of 0.25%, PSPY significantly suppressed Adipoq expression as compared to control. Significant inhibition of Slc2a4 and Pgc1a was observed starting from a 0.25% concentration of PSPY. The suppression of adipogenic genes was also observed with the treatment of plain yogurt; however, the effects were relatively lower than the PSPY. The group treated with 1% and 5% of PSPY also showed inhibition effects on lipid accumulation. This study demonstrated PSPY inhibition effect on white adipocyte differentiation through suppression of Pparg and its downstream genes, Adipoq and Slc2a4, indicating the potential of this yogurt as a functional food for obesity management and prevention.

Postmortem Brains from Subjects with Diabetes Mellitus Display Reduced GLUT4 Expression and Soma Area in Hippocampal Neurons: Potential Involvement of Inflammation.

Diabetes mellitus (DM) is an important risk factor for dementia, which is a common neurodegenerative disorder. DM is known to activate inflammation, oxidative stress, and advanced glycation end products (AGEs) generation, all capable of inducing neuronal dysfunctions, thus participating in the neurodegeneration progress. In that process, disturbed neuronal glucose supply plays a key role, which in hippocampal neurons is controlled by the insulin-sensitive glucose transporter type 4 (GLUT4). We investigated the expression of GLUT4, nuclear factor NF-kappa B subunit p65 [NFKB (p65)], carboxymethyllysine and synapsin1 (immunohistochemistry), and soma area in human postmortem hippocampal samples from control, obese, and obese+DM subjects (41 subjects). Moreover, in human SH-SY5Y neurons, tumor necrosis factor (TNF) and glycated albumin (GA) effects were investigated in GLUT4, synapsin-1 (SYN1), tyrosine hydroxylase (TH), synaptophysin (SYP) proteins, and respective genes; NFKB binding activity in the SLC2A4 promoter; effects of increased histone acetylation grade by histone deacetylase 3 (HDAC3) inhibition. Hippocampal neurons (CA4 area) of obese+DM subjects displayed reduced GLUT4 expression and neuronal soma area, associated with increased expression of NFKB (p65). Challenges with TNF and GA decreased the SLC2A4/GLUT4 expression in SH-SY5Y neurons. TNF decreased SYN1, TH, and SYP mRNAs and respective proteins, and increased NFKB binding activity in the SLC2A4 promoter. Inhibition of HDAC3 increased the SLC2A4 expression and the total neuronal content of CRE-binding proteins (CREB/ICER), and also counterbalanced the repressor effect of TNF upon these parameters. This study revealed reduced postmortem human hippocampal GLUT4 content and neuronal soma area accompanied by increased proinflammatory activity in the brains of DM subjects. In isolated human neurons, inflammatory activation by TNF reduced not only the SLC2A4/GLUT4 expression but also the expression of some genes related to neuronal function (SYN1, TH, SYP). These effects may be related to epigenetic regulations (H3Kac and H4Kac status) since they can be counterbalanced by inhibiting HDAC3. These results uncover the improvement in GLUT4 expression and/or the inhibition of HDAC3 as promising therapeutic targets to fight DM-related neurodegeneration.

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.

Prognostic value of SLC4A4 and its correlation with the microsatellite instability in colorectal cancer.

To explore new biomarkers related to microsatellite instability in order to better predict prognosis and guide medication. The "limma" R package was used to identify differentially expressed genes in GSE24514, and then weighted correlation network analysis was used to select key genes. Different cell types in the tumor microenvironment were identified and analyzed by single-cell sequencing, with a Lasso regression model used to screen prognostic variables. Furthermore, the correlation between microsatellite instability and potential prognostic variables was explored, as well as the expression characteristics and clinical characteristics of the prognostic variables in the TCGA, UALCAN, and HPA databases. PCR assay was used to investigate the expression of SLC4A4 in colorectal cancer cell lines. Finally, we further verified the expression of SLC4A4 by immunohistochemistry. First, 844 differentially expressed genes in GSE24514 were identified. Subsequently, weighted co-expression network analysis (WGCNA) of GSE24514 obtained all the genes significantly associated with microsatellite instability (MSI), a total of 1452. Analysis of GSE166555 single cell sequencing data set yielded 1564 differentially expressed genes. The gene sets obtained from the above three analysis processes were intersected, and 174 genes were finally obtained. The Lasso regression model revealed two potential prognostic genes, TIMP1 and SLC4A4, of which, there was a stronger correlation between microsatellite instability and SLC4A4. The mRNA and protein expression of SLC4A4 was significantly decreased in tumors, and patients with low SLC4A4 expression had a poor prognosis. In addition, SLC4A4 was specifically expressed in epithelial cells. In the microenvironment of colorectal cancer, malignant cells have a strong interaction with different stromal cells. PCR showed that SLC4A4 was significantly down-regulated in colorectal cancer cell lines Caco-2, HCT116 and HT29 compared with normal control NCM460 cell lines. Immunohistochemistry also showed low expression of SLC4A4 in colorectal cancer. SLC4A4, as a tumor suppressor gene, is significantly downregulated and positively correlated with microsatellite instability, thus it may be combined with microsatellite instability to guide colorectal cancer treatment.

L-Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis.

Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified l-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor-expressing (LepR-expressing) neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by solute carrier transporter 7a5, Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1/mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.

Abstract 3800: The disruption of miR-125b-SLC1A5 cascade defines the oncogenicity and differential immune profile in oral carcinoma

Abstract Head and neck squamous carcinoma (HNSCC) including oral SCC (OSCC) is a worldwide malignancy that requires vigorous efforts for interception. The metabolic reprogramming sustains malignant tumor cells to overcome stressful microenvironments. As the glutamine is the one of the essential components which helps cells to erase reactive oxygen species and energy production, the increased uptake of glutamine is a common metabolic hallmark in cancers. Bioinformatic analysis of TCGA HNSCC datasets and RNAseq results in our cohort indicated that solute carrier family 1 member 5 (SLC1A5) is the most eminently dysregulated transporter member among the seven glutamate or neutral amino acid transporters in SLC1A family. This work identified that the knockdown of SLC1A5 expression decreased in vitro oncogenicity, xenografic tumorigenesis and glutamine uptake; increased oxidative stress, cell cycle arrest, and sensitivity to cisplatin and ferroptosis inducer in OSCC cells. By way of contrast, the upregulation of endogenous SLC1A5 expression mediated by CRISPR/dCas9 activation system drove the OSCC oncogenesis. The reporter assay and functional analysis demonstrated that suppressor miRNA miR-125b targeted and attenuated SLC1A5, while NEAT1 lncRNA sponged and ablated miR-125b-SLC1A5 axis. The analysis of TCGA and GEO datasets confirmed the concordant upregulation of NEAT1 and downregulation of miR-125b along with the SLC1A5 upregulation in tumors. Besides, SLC1A5 upregulation defined the worse HNSCC patient survival. In our tumor cohort, OSCC harboring higher SLC1A5 expression had lower CD4+ immune score, and higher scores in M0 and M1 macrophage. GSEA algorithm further deciphered that tumors having higher SLC1A5 expression manifested the repression in glycolysis, apoptosis and p53 signaling. This study concludes that the frequent disruption of NEAT1-miR-125b-SLC1A5 cascade modulates the pluripotent oncogenic activities, the treatment efficacy and the immune landscape in oral carcinoma. Citation Format: Ying-Chieh Liu, So-Yu Liu, Yu-Cheng Lin, Chung-Ji Liu, Kuo-Wei Chang, Shu-Chun Lin. The disruption of miR-125b-SLC1A5 cascade defines the oncogenicity and differential immune profile in oral carcinoma. [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 3800.

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 & 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.

Abstract 3671: Targeting SLC2A1 and SLC38A2-mediated metabolic shift in SMARCA4/A2-dual deficient cancer

Abstract SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodelling complexes. These two ATPases are frequently inactivated in cancers and cancer cells deficient in either of them have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4 and SMARCA2 occurs in a subset of cancers that are often associated with very poor patient outcomes. Here, we uncover that dual loss of SMARCA4/A2 represses the expression of SLC2A1, encoding the glucose transporter GLUT1, which results in reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/A2-dual deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/A2-dual deficient cells and tumours are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-dual deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activities in SMARCA4/A2-dual deficient cancer cell lines and patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/A2-dual loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers. * Authors contributed equally# Co-corresponding Citation Format: Xianbing Zhu, Zheng Fu, Shary Chen, Dionzie Ong, Giulio Aceto, Rebecca Ho, Jutta Steinberger, Eunice Li, David Huntsman, William Foulkes, Sidong Huang, Yemin Wang. Targeting SLC2A1 and SLC38A2-mediated metabolic shift in SMARCA4/A2-dual deficient 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 3671.

Fatty Acid Induced Hypermethylation in the Slc2a4 Gene in Visceral Adipose Tissue Is Associated to Insulin-Resistance and Obesity.

De novo lipogenesis (DNL) in visceral adipose tissue (VAT) is associated with systemic insulin sensitivity. DNL in VAT is regulated through ChREBP activity and glucose uptake through Glut4 (encoded by Slc2a4). Slc2a4 expression, ChREBP activity, and DNL are decreased in obesity, the underlying cause however remains unidentified. We hypothesize that increased DNA methylation in an enhancer region of Slc2a4 decreases Slc2a4 expression in obesity and insulin resistance. We found that SLC2A4 expression in VAT of morbidly obese subjects with high HbA1c (>6.5%, n = 35) is decreased, whereas DNA methylation is concomitantly increased compared to morbidly obese subjects with low HbA1c (≤6.5%, n = 65). In diet-induced obese (DIO) mice, DNA methylation of Slc2a4 persistently increases with the onset of obesity and insulin resistance, while gene expression progressively decreases. The regulatory impact of DNA methylation in the investigated enhancer region on SLC2A4 gene expression was validated with a reporter gene assay. Additionally, treatment of 3T3 pre-adipocytes with palmitate/oleate during differentiation decreased DNA methylation and increased Slc2a4 expression. These findings highlight a potential regulation of Slc2a4 by DNA methylation in VAT, which is induced by fatty acids and may play a role in the progression of obesity and insulin resistance in humans.