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.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation.

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

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.

[Effect of L-Type Amino Acid Transporter 1 Expression on Clinicopathological Features and Prognosis of Non-Hodgkin's Lymphoma].

OBJECTIVE: To detect the expression of L-type amino acid transporter 1 (LAT1) in non-Hodgkin's lymphoma (NHL) tissues, and analyze its effect on clinicopathological characteristics and prognosis of patients. METHODS: A total of 92 NHL patients who were treated in our hospital from January 2017 to April 2019 were collected. The expression of LAT1 in NHL tissue was detected by immunohistochemistry and compared between patients with different pathological features (including sex, Ann Arbor stage, extranodal infiltration, Ki-67). The risk factors affecting mortality were analyzed using univariate and multivariate Cox proportional hazards regression. Receiver operating characteristic (ROC) curve was used to detect the predictive value of percentage of LAT1-positive cells in NHL tissue for patient mortality, and analyzing the effect of percentage of LAT1-positive cells on survival rate. RESULTS: LAT1 was positively expressed in NHL tissue. The high expression rate of LAT1 in Ann Arbor stage III and IV groups were higher than that in Ann Arbor stage I group, that in extranodal infiltration group was higher than non-extranodal infiltration group, and that in Ki-67 positive expression group was higher than Ki-67 negative expression group (all P < 0.05). The remission rate after 3 courses of treatment in high-LAT1 expression group was 70.7%, which was lower than 91.2% in low-LAT1 expression group (P < 0.05). Ann Arbor stage III and IV, extranodal invasion, Ki-67 positive expression and increased expression of LAT1 (LAT1-positive cell percentage score ≥2) were risk factors for mortality. The cut-off value of percentage of LAT1-positive cells for predicting NHL death was 45.6%, and the area under the ROC curve was 0.905 (95%CI: 0.897-0.924). The 3-year survival rate of high-LAT1 level group (the percentage of LAT1-positive cells≥45.6%) was 50.00%, which was lower than 78.26% of low-LAT1 level group (P < 0.05). CONCLUSION: The expression level of LAT1 in NHL tissue increases, which affects Ann Arbor stage and extranodal infiltration of patients. LAT1 is a risk factor for death.

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.

Fat mass and obesity-associated protein (FTO) mediated m6A modification of circFAM192A promoted gastric cancer proliferation by suppressing SLC7A5 decay.

Gastric cancer (GC) is a common malignant tumor worldwide, especially in East Asia, with high incidence and mortality rate. Epigenetic modifications have been reported to participate in the progression of gastric cancer, among which m6A is the most abundant and important chemical modification in RNAs. Fat mass and obesity-associated protein (FTO) is the first identified RNA demethylase but little is known about its role in gastric cancer. In our study, data from TCGA and clinical samples showed that FTO was highly expressed in gastric cancer tissues. Kaplan-Meier plotter suggested that patients with the high level of FTO had a poor prognosis. In vitro and in vivo experiments confirmed the role of FTO in promoting gastric cancer cell proliferation. Mechanistically, we found that FTO bound to circFAM192A at the specific site and removed the m6A modification in circFAM192A, protecting it from degradation. CircFAM192A subsequently interacted with the leucine transporter solute carrier family 7 member 5 (SLC7A5) and enhancing its stability. As a result, an increased amount of SLC7A5 was on the membrane, which facilitated leucine uptake and activated the mTOR signaling pathway. Therefore, our study demonstrated that FTO promoted gastric cancer proliferation through the circFAM192A/SLC7A5 axis in the m6A-dependent manner. Our study shed new light on the role of FTO in gastric cancer progression.

Structure-activity characteristics of phenylalanine analogs selectively transported by L-type amino acid transporter 1 (LAT1).

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

A bis-boron boramino acid PET tracer for brain tumor diagnosis.

PURPOSE: Boramino acids are a class of amino acid biomimics that replace the carboxylate group with trifluoroborate and can achieve the 18F-labeled positron emission tomography (PET) and boron neutron capture therapy (BNCT) with identical chemical structure. METHODS: This study reports a trifluoroborate-derived boronophenylalanine (BBPA), a derived boronophenylalanine (BPA) for BNCT, as a promising PET tracer for tumor imaging. RESULTS: Competition inhibition assays in cancer cells suggested the cell accumulation of [18F]BBPA is through large neutral amino acid transporter type-1 (LAT-1). Of note, [18F]BBPA is a pan-cancer probe that shows notable tumor uptake in B16-F10 tumor-bearing mice. In the patients with gliomas and metastatic brain tumors, [18F]BBPA-PET shows good tumor uptake and notable tumor-to-normal brain ratio (T/N ratio, 18.7 ± 5.5, n = 11), higher than common amino acid PET tracers. The [18F]BBPA-PET quantitative parameters exhibited no difference in diverse contrast-enhanced status (P = 0.115-0.687) suggesting the [18F]BBPA uptake was independent from MRI contrast-enhancement. CONCLUSION: This study outlines a clinical trial with [18F]BBPA to achieve higher tumor-specific accumulation for PET, provides a potential technique for brain tumor diagnosis, and might facilitate the BNCT of brain tumors.

FBW7/GSK3ß mediated degradation of IGF2BP2 inhibits IGF2BP2-SLC7A5 positive feedback loop and radioresistance in lung cancer.

BACKGROUND: The development of radioresistance seriously hinders the efficacy of radiotherapy in lung cancer. However, the underlying mechanisms by which radioresistance occurs are still incompletely understood. The N6-Methyladenosine (m6A) modification of RNA is involved in cancer progression, but its role in lung cancer radioresistance remains elusive. This study aimed to identify m6A regulators involved in lung cancer radiosensitivity and further explore the underlying mechanisms to identify therapeutic targets to overcome lung cancer radioresistance. METHODS: Bioinformatic mining was used to identify the m6A regulator IGF2BP2 involved in lung cancer radiosensitivity. Transcriptome sequencing was used to explore the downstream factors. Clonogenic survival assays, neutral comet assays, Rad51 foci formation assays, and Annexin V/propidium iodide assays were used to determine the significance of FBW7/IGF2BP2/SLC7A5 axis in lung cancer radioresistance. Chromatin immunoprecipitation (ChIP)-qPCR analyses, RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (MeRIP)-qPCR analyses, RNA pull-down analyses, co-immunoprecipitation analyses, and ubiquitination assays were used to determine the feedback loop between IGF2BP2 and SLC7A5 and the regulatory effect of FBW7/GSK3ß on IGF2BP2. Mice models and tissue microarrays were used to verify the effects in vivo. RESULTS: We identified IGF2BP2, an m6A "reader", that is overexpressed in lung cancer and facilitates radioresistance. We showed that inhibition of IGF2BP2 impairs radioresistance in lung cancer both in vitro and in vivo. Furthermore, we found that IGF2BP2 enhances the stability and translation of SLC7A5 mRNA through m6A modification, resulting in enhanced SLC7A5-mediated transport of methionine to produce S-adenosylmethionine. This feeds back upon the IGF2BP2 promoter region by further increasing the trimethyl modification at lysine 4 of histone H3 (H3K4me3) level to upregulate IGF2BP2 expression. We demonstrated that this positive feedback loop between IGF2BP2 and SLC7A5 promotes lung cancer radioresistance through the AKT/mTOR pathway. Moreover, we found that the ubiquitin ligase FBW7 functions with GSK3ß kinase to recognize and degrade IGF2BP2. CONCLUSIONS: Collectively, our study revealed that the m6A "reader" IGF2BP2 promotes lung cancer radioresistance by forming a positive feedback loop with SLC7A5, suggesting that IGF2BP2 may be a potential therapeutic target to control radioresistance in lung cancer.

m6A modification mediates SLC3A2/SLC7A5 translation in 3-methylcholanthrene-induced uroepithelial transformation.

3-Methylcholanthracene (3-MC) is one of the most carcinogenic polycyclic aromatic hydrocarbons (PAHs). Long-term exposure to PAHs has been thought of as an important factor in urothelial tumorigenesis. N6-methyladenosine (m6A) exists widely in eukaryotic organisms and regulates the expression level of specific genes by regulating mRNA stability, translation efficiency, and nuclear export efficiency. Currently, the potential molecular mechanisms that regulate m6A modification for 3-MC carcinogenesis remain unclear. Here, we profiled mRNA, m6A, translation and protein level using "-omics" methodologies, including transcriptomes, m6A profile, translatomes, and proteomics in 3-MC-transformed urothelial cells and control cells. The key molecules SLC3A2/SLC7A5 were screened and identified in 3-MC-induced uroepithelial transformation. Moreover, SLC7A5/SLC3A2 promoted uroepithelial cells malignant phenotype in vitro and in vivo. Mechanically, METTL3 and ALKBH5 mediated m6A modification of SLC3A2/SLC7A5 mRNA in 3-MC-induced uroepithelial transformation by upregulating the translation of SLC3A2/SLC7A5. Furthermore, programmable m6A modification of SLC3A2/SLC7A5 mRNA affected the expression of its proteins. Taken together, our results revealed that the m6A modification-mediated SLC3A2/SLC7A5 translation promoted 3-MC-induced uroepithelial transformation, suggesting that targeting m6A modification of SLC3A2/SLC7A5 may be a potential therapeutic strategy for bladder cancer related to PAHs.

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.

CircSCUBE3 Reduces the Anti-gastric Cancer Activity of Anti-PD-L1.

The progression of gastric cancer (GC) is closely related to tumor immune escape. The research, therefore, studied the impact of possible circRNAs on the immune escape of GC tumors and the underlying mechanisms. Here, to explore circRNAs that may affect GC, the differential circRNAs in six normal gastric mucosal tissues and six GC samples (GSM2005868-GSM2005879) were analyzed through the bioinformatics website circmine, and hsa_circ_0076092 (circSCUBE3) was identified as the research object. In vitro assays revealed the functions of circSCUBE3 and its downstream miRNA/mRNA axis in GC cells. The effect of circSCUBE3 against PD-1 anti-tumor activity was evaluated in vivo. The relationship between circSCUBE3 and miR-744-5p, miR-744-5p, and SLC7A5 was identified by RNA immunoprecipitation and dual-luciferase reporter experiments. The effect of SLC7A5 on GC immune escape by regulating PD-L1 expression was assessed by co-culture system and flow cytometry. CircSCUBE3 was up-regulated in human GC tissues and GC cell lines. circSCUBE3 was associated with poor prognosis in GC patients. Functional experiments reported that circSCUBE3 knockdown could suppress GC immune escape. Mechanistically, circSCUBE3 bound to miR-744-5p, which further targeted SLC7A5, and SLC7A5 can affect GC immune escape by regulating PD-L1. Furthermore, in vivo assay manifested that circSCUBE3 attenuated the anti-tumor effect of PD-L1. Our study revealed the importance of the circSCUBE3/miR-744-5p/SLC7A5 axis in GC immune escape and anti-PD-1 resistance.

Integrative Multi-omics Analysis Reveals Different Metabolic Phenotypes Based on Molecular Characteristics in Thyroid Cancer.

PURPOSE: Thyroid cancer metabolic characteristics vary depending on the molecular subtype determined by mutational status. We aimed to investigate the molecular subtype-specific metabolic characteristics of thyroid cancers. EXPERIMENTAL DESIGN: An integrative multi-omics analysis was conducted, incorporating transcriptomics, metabolomics, and proteomics data obtained from human tissues representing distinct molecular characteristics of thyroid cancers: BRAF-like (papillary thyroid cancer with BRAFV600E mutation; PTC-B), RAS-like (follicular thyroid cancer with RAS mutation; FTC-R), and ATC-like (anaplastic thyroid cancer with BRAFV600E or RAS mutation; ATC-B or ATC-R). To validate our findings, we employed tissue microarray of human thyroid cancer tissues and performed in vitro analyses of cancer cell phenotypes and metabolomic assays after inducing genetic knockdown. RESULTS: Metabolic properties differed between differentiated thyroid cancers of PTC-B and FTC-R, but were similar in dedifferentiated thyroid cancers of ATC-B/R, regardless of their mutational status. Tricarboxylic acid (TCA) intermediates and branched-chain amino acids (BCAA) were enriched with the activation of TCA cycle only in FTC-R, whereas one-carbon metabolism and pyrimidine metabolism increased in both PTC-B and FTC-R and to a great extent in ATC-B/R. However, the protein expression levels of the BCAA transporter (SLC7A5) and a key enzyme in one-carbon metabolism (SHMT2) increased in all thyroid cancers and were particularly high in ATC-B/R. Knockdown of SLC7A5 or SHMT2 inhibited the migration and proliferation of thyroid cancer cell lines differently, depending on the mutational status. CONCLUSIONS: These findings define the metabolic properties of each molecular subtype of thyroid cancers and identify metabolic vulnerabilities, providing a rationale for therapies targeting its altered metabolic pathways in advanced thyroid cancer.

Identification of autophagy and angiogenesis modulators in colorectal cancer based on bioinformatics analysis.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related death worldwide. The purpose of this study was to discover novel molecular pathways and potential prognosis biomarkers. To achieve this, we acquired five microarray datasets from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes between CRC and adjacent normal tissue samples and further validated them using The Cancer Genome Atlas (TCGA) database. Using various analytical approaches, including the construction of a competing endogenous RNA (ceRNA) network, Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analyses, as well as survival analysis, we identified key genes and pathways associated with the diagnosis and prognosis of CRC. We obtained a total of 185 differentially expressed genes, comprising 17 lncRNAs, 30 miRNAs, and 138 mRNAs. The ceRNA network consisted of 17 lncRNAs, 25 miRNAs, and 7 mRNAs. Among the 7 mRNAs involved in the ceRNA network, SLC7A5 and KRT80 were found to be upregulated, while ADIPOQ, CCBE1, KCNB1, CADM2, and CHRDL1 were downregulated in CRC. Further analysis revealed that ADIPOQ and SLC7A5 are involved in the AMPK and mTOR signaling pathway, respectively. In addition, survival analysis demonstrated a statistically significant relationship between ADIPOQ, SLC7A5, and overall survival rates in CRC patients. In conclusion, our findings suggest that downregulation of ADIPOQ and upregulation of SLC7A5 in tumor cells lead to increased mTORC1 activity, reduced autophagy, enhanced angiogenesis, and ultimately contribute to cancer progression and decreased survival in CRC patients.

Circular RNA circ-SLC7A5 Functions as a Competing Endogenous RNA to Impact Cell Biological Behaviors in Esophageal Squamous Cell Carcinoma (ESCC).

BACKGROUND: Circular RNAs (circRNAs) have profound effects on establishment and pathogenesis of esophageal squamous cell carcinoma (ESCC). Here, we defined whether circRNA solute carrier family 7 member 5 (circ-SLC7A5, also called hsa_circ_0040796) is causally involved in the pathogenesis of ESCC. METHODS: Circ-SLC7A5, microRNA (miR)-874-3p and coronin-1C (CORO1C) expression levels were gauged by qRT-PCR or immunoblotting. Cell functional phenotypes were tested by colony formation, EdU, flow cytometry, transwell and wound-healing assays. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were applied to ascertained circ-SLC7A5/miR-874-3p and miR-874-3p/CORO1C relationships. RESULTS: Circ-SLC7A5 was highly expressed in human ESCC. Circ-SLC7A5 depletion impaired cell growth, migration, invasiveness, and promoted apoptosis. Circ-SLC7A5 knockdown diminished ESCC cell tumorigenicity. Mechanistically, circ-SLC7A5 contained a binding site for miR-874-3p. Also, miR-874-3p was responsible for circ-SLC7A5's function in ESCC cells. CORO1C was a direct miR-874-3p target. Circ-SLC7A5 functioned as a competing endogenous RNA (ceRNA) to control CORO1C by competing for shared miR-874-3p. Furthermore, CORO1C knockdown phenocopied miR-874-3p overexpression in impacting the biological behaviors of ESCC cells. CONCLUSION: These findings identify circ-SLC7A5 as a crucial modulator of ESCC cells and establish a novel circ-SLC7A5/miR-874-3p/CORO1C ceRNA network in ESCC.

Targeted delivery of liposomal Ribociclib to SLC7A5 transporters in breast cancer cells.

This study aimed to prepare SLC7A5 transporters targeted liposomes of Ribociclib (RB) by stear(o)yl conjugation of Phe, Asp, Glu amino acids to liposomes as targeting moieties. The liposomes were optimized for their formulations. Cell analysis on two cell lines of MCF-7 and NIH-3T3 were done including; cell viability test by MTT assay, cellular uptake, and cell cycle arrest by flow cytometry. The optimal liposomes showed the particle size of 123.6 ± 1.3 nm, drug loading efficiency and release efficiency of 83.87% ± 1.33% and 60.55% ± 0.46%, respectively. The RB loaded liposomes showed no hemolysis activity. Targeted liposomes increased cytotoxicity on MCF-7 cells more significantly than NIH-3T3 cells. Cell flow cytometry indicated that targeted liposomes uptake was superior to plain (non-targted) liposomes and free drug. Free drug and RB-loaded liposomes interrupted cell cycle in G1. However, amino acid-targeted liposomes arrested cells more than the free drug at this stage. Targeted liposomes reduced cell cycle with more interruption in the G2/M phase compared to the negative control.

Hypoxia-mimetic by CoCl2 increases SLC7A5 expression in breast cancer cells in vitro.

OBJECTIVE: Increased expression of the amino acid transporter solute Carrier Family 7 Member 5 (SLC7A5) has been observed in neoplastic cells during hypoxic conditions in vitro, indicating an adaptation for cell survival. To further explore this, we evaluated hypoxia-mimetic by CoCl2 as a model for hypoxia in breast cancer cell lines and the effect on SLC257A5 expression. Four different breast cancer cell lines (MCF7, T-47D, BT-474 and ZR-75-1) were exposed to 100 µM CoCl2 for 48 h. Subsequently, cell viability, gene- and protein expression analyses were performed. RESULTS: The gene expression of VEGF, a marker of hypoxia, was significantly elevated in all four cell lines compared to the control (p < 0.0001), indicating that CoCl2 exposure generates a hypoxic response. Moreover, CoCl2 exposure significantly upregulated SLC7A5 gene expression in T-47D (p < 0.001), BT-474 (p < 0.0001) and ZR-75-1 (p < 0.0001) cells, as compared to vehicle control. Immunofluorescence staining showed increased SLC7A5 protein expression in MCF7, T-47D and BT-474 cells compared to vehicle control. This report suggests that hypoxia-mimetic by CoCl2 can be used as a simple model for inducing hypoxia in breast cancer cell lines and in fact influence SLC7A5 gene and protein expression in vitro.

Increased sodium fluorescein transport by corticosteroids is inhibited by a LAT-1 specific inhibitor in retinal pigment epithelial cells in vitro.

To investigate whether aldosterone (ALD) and hydrocortisone (HC) change the gene expression of SLC7A5, which encodes the large neutral amino acid transporter small subunit 1 (LAT1), and the transport activity of LAT1 in the retinal pigment epithelium (RPE) in vitro. ARPE-19 cells were grown to confluence. After withdrawing the serum, ALD or HC was added with several doses and incubated, and SLC7A5 gene expression was measured. The influx and efflux transport of sodium fluorescein (Na-F) were evaluated using the Transwell culture system. SLC7A5 gene expression was upregulated by ALD and downregulated by HC in a dose-dependent manner. Both ALD and HC significantly increased the influx and efflux Na-F transport of RPE cells at a dose that did not change the expression of SLC7A5. JPH203, a specific inhibitor of LAT1, significantly reduced accelerated Na-F transport. Both ALD and HC increased the gene expression of zonula occludin-1 (ZO-1) although they did not change the immunoreactivity of ZO-1 in RPE cells. LAT1 may play an important role in increasing Na-F transport associated with ALD and HC administration. A specific LAT1 inhibitor may effectively regulate the increased material transport of RPE induced by ALD and HC.

MicroRNA-626 inhibits mTOR pathways activity of retinal pigment epithelial cells by targeting SLC7A5 in human ARPE-19 Cells.

Recent studies have shown that miRNAs are associated with the pathological process involved in age-related macular degeneration (AMD). However, the microRNA-mediated post-transcriptional regulation in human retinal pigment epithelium (RPE) cells has not been adequately investigated. We investigated how miR-626 inhibits mTOR activity pathways and pathway-related genes in retinal pigment epithelial cells by targeting the solute carrier family seven-member 5 (SLC7A5) in ARPE19 cells.    We transfected mir-626 mimic, mir-626 inhibitör and siRNA in human retinal pigment epithelial cell line was examined using RT-PCR and western blot, respectively. We knocked down mir-626 levels and overexpression by mir-626-siRNA transfection of human RPE cell lines, and using an MTT assay, we assessed the role of SLC7A5 on RPE cell proliferation. We additionally measured the expression of mTOR, Akt1, caspase 3, Bax, SLC17A7, SLC17A8, Creb1, Pten, HIF1A, HIFI. The findings demonstrate that mir-626 inhibits SLC7A5 gene expression and proliferation of ARPE-19 cells. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-626, has the same effect on ARPE-19 cells. We identified how miR-626 causes apoptosis and macula degeneration in RPE cells by targeting SLC7A5 through the mTOR signaling pathway. miR-626 was an essential regulator of the expression of the Slc7a5 gene. Importantly, we determined that miR-626 is essential to play a role in AMD. This research project shows that SLC7A5 is a direct target of mir-626 in ARPE-19 cells for the first time.

Gene purging and the evolution of Neoave metabolism and longevity.

Maintenance of the proteasome requires oxidative phosphorylation (ATP) and mitigation of oxidative damage, in an increasingly dysfunctional relationship with aging. SLC3A2 plays a role on both sides of this dichotomy as an adaptor to SLC7A5, a transporter of branched-chain amino acids (BCAA: Leu, Ile, Val), and to SLC7A11, a cystine importer supplying cysteine to the synthesis of the antioxidant glutathione. Endurance in mammalian muscle depends in part on oxidation of BCAA; however, elevated serum levels are associated with insulin resistance and shortened lifespans. Intriguingly, the evolution of modern birds (Neoaves) has entailed the purging of genes including SLC3A2, SLC7A5, -7, -8, -10, and SLC1A4, -5, largely removing BCAA exchangers and their interacting Na+/Gln symporters in pursuit of improved energetics. Additional gene purging included mitochondrial BCAA aminotransferase (BCAT2), pointing to reduced oxidation of BCAA and increased hepatic conversion to triglycerides and glucose. Fat deposits are anhydrous and highly reduced, maximizing the fuel/weight ratio for prolonged flight, but fat accumulation in muscle cells of aging humans contributes to inflammation and senescence. Duplications of the bidirectional α-ketoacid transporters SLC16A3, SLC16A7, the cystine transporters SLC7A9, SLC7A11, and N-glycan branching enzymes MGAT4B, MGAT4C in Neoaves suggests a shift to the transport of deaminated essential amino acid, and stronger mitigation of oxidative stress supported by the galectin lattice. We suggest that Alfred Lotka's theory of natural selection as a maximum power organizer (PNAS 8:151,1922) made an unusually large contribution to Neoave evolution. Further molecular analysis of Neoaves may reveal novel rewiring with applications for human health and longevity.