Evidence for a relationship between genetic polymorphisms of the L-DOPA transporter LAT2/4F2hc and risk of hypertension in the context of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) and hypertension are chronic diseases affecting a large portion of the population frequently coexistent and interdependent. The inability to produce/use adequate renal dopamine may contribute to the development of hypertension and renal dysfunction. The heterodimeric amino acid transporter LAT2/4F2hc (SLC7A8/SLC3A2 genes) promotes the uptake of L-DOPA, the natural precursor of dopamine. We examined the plausibility that SLC7A8/SLC3A2 gene polymorphisms may contribute to hypertensive CKD by affecting the L-DOPA uptake. METHODS: 421 subjects (203 men and 218 women, mean age of 78.9 ± 9.6 years) were recruited and divided in four groups according to presence/absence of CKD, defined as reduced estimated glomerular filtration rate (eGFR < 60 ml/min/m2) calculated using the creatinine-based Berlin Initiative Study-1 (BIS1) equation, and to presence/absence of hypertension (systolic blood pressure ≥ 140 and/or diastolic blood pressure ≥ 90 mmHg). Subjects were analysed for selected SNPs spanning the SLC7A8 and SLC3A2 loci by Sequenom MassARRAY iPLEX platform. RESULTS: The most significant SNP at the SLC3A2 (4F2hc) locus was rs2282477-T/C, with carriers of the C-allele having a lower chance to develop hypertension among CKD affected individuals [OR = 0.33 (CI 0.14-0.82); p = 0.016]. A similar association with hypertensive CKD was found for the SLC7A8 (LAT2) rs3783436-T/C, whose C-allele resulted associated with decreased risk of hypertension among subjects affected by CKD [OR = 0.56 (95% CI 0.35-0.90; p = 0.017]. The two variants were predicted to be potentially functional. CONCLUSIONS: The association between SLC3A2 and SLC7A8 variants to hypertension development in patients with renal failure could be linked to changes in L-DOPA uptake and consequently dopamine synthesis. Although the associations do not survive correction for Bonferroni multiple testing, and additional research is needed, our study opens new avenues for future basic and translational research in the field of hypertensive CKD.

SLC34A2 promotes cell proliferation by activating STX17-mediated autophagy in esophageal squamous cell carcinoma.

BACKGROUND: Solute carrier family 34 member 2 (SLC34A2) has been implicated in the development of various malignancies. However, the clinical significance and underlying molecular mechanisms of SLC34A2 in esophageal squamous cell carcinoma (ESCC) remain elusive. METHODS: Western blotting, quantitative real-time PCR and immunohistochemistry were utilized to evaluate the expression levels of SLC34A2 mRNA/protein in ESCC cell lines or tissues. Kaplan-Meier curves were employed for survival analysis. CCK-8, colony formation, EdU and xenograft tumor model assays were conducted to determine the impact of SLC34A2 on ESCC cell proliferation. Cell cycle was examined using flow cytometry. RNA-sequencing and enrichment analysis were carried out to explore the potential signaling pathways. The autophagic flux was evaluated by western blotting, mRFP-GFP-LC3 reporter system and transmission electron microscopy. Immunoprecipitation and mass spectrometry were utilized for identification of potential SLC34A2-interacting proteins. Cycloheximide (CHX) chase and ubiquitination assays were conducted to test the protein stability. RESULTS: The expression of SLC34A2 was significantly upregulated in ESCC and correlated with unfavorable clinicopathologic characteristics particularly the Ki-67 labeling index and poor prognosis of ESCC patients. Overexpression of SLC34A2 promoted ESCC cell proliferation, while silencing SLC34A2 had the opposite effect. Moreover, SLC34A2 induced autophagy to promote ESCC cell proliferation, whereas inhibition of autophagy suppressed the proliferation of ESCC cells. Further studies showed that SLC34A2 interacted with an autophagy-related protein STX17 to promote autophagy and proliferation of ESCC cells by inhibiting the ubiquitination and degradation of STX17. CONCLUSIONS: These findings indicate that SLC34A2 may serve as a prognostic biomarker for ESCC.

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.

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

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.

Acidosis activates breast cancer ferroptosis through ZFAND5/SLC3A2 signaling axis and elicits M1 macrophage polarization.

Acidosis is involved in multiple pathways in tumor cells and immune cells among the tumor microenvironment (TME). Ferroptosis is a nonapoptotic and iron-dependent form of cell death characterized by accumulation of lipid peroxidation involved in various cancers. The role of ferroptosis in the breast cancer (BC) acidic microenvironment remains unrevealed. Here, we reported that short-term acidosis induced ferroptosis of BC cells in the zinc finger AN1-type domain 5 (ZFAND5)/solute carrier family 3 member 2 (SLC3A2) dependent manner to suppress tumor growth using in silico and multiple biological methods. Mechanistically, we demonstrated that short-term acidosis increased total/lipid reactive oxygen species (ROS) level, decreased glutathione (GSH) level and induced the morphological changes of mitochondria. Specifically, acidosis restrained the protein stability of SLC3A2 by promoting its ubiquitination process. The prognostic analysis showed that higher expression of ZFAND5 and lower expression of SLC3A2 were correlated with longer overall survival of BC patients, respectively. Furthermore, in combination with ferroptosis agonist metformin, short-term acidosis could synergistically inhibit viability and enhance the ferroptosis of BC cells. Meanwhile, by the exploration of immune cells, short-term acidosis also induced M1 macrophage polarization, triggering processes of phagocytosis and ferroptosis in BC cells. This study demonstrated that short-term acidosis induced BC cell ferroptosis through ZFAND5/SLC3A2 signaling axis and promoted phagocytosis and ferroptosis of BC cells with M1 macrophage polarization, which might be a new mechanism for BC therapy.

Effect of disulfidptosis-related genes SLC3A2, SLC7A11 and FLNB polymorphisms on risk of autoimmune thyroiditis in a Chinese population.

PURPOSE: This study aimed to evaluate the associations between disulfidptosis related genes-SLC3A2, SLC7A11 and FLNB polymorphisms and risk of autoimmune thyroiditis (AIT). METHODS: Six SNPs in the SLC3A2, SLC7A11 and FLNB were genotyped in 650 AIT cases and 650 controls using a MassARRAY platform. RESULTS: Minor alleles of SLC3A2-rs12794763, rs1059292 and FLNB-rs839240 might lead to a higher risk of AIT (p < 0.001), while SLC7A11-rs969319-C allele tends to decrease the risk of the disease (p = 0.006). Genetic model analysis showed that SLC3A2-rs12794763, SLC3A2-rs1059292 and FLNB-rs839240 polymorphisms were risk factors for AIT (p < 0.001); while SLC7A11-rs969319 showed a protective role for the disease in all genetic models (p < 0.005). Stratification analysis showed that SLC3A2-rs1059292 and rs12794763 were correlated with higher risk of AIT regardless of sex (p < 0.05). Moreover, FLNB-rs839240 exhibited higher risk of disease only in females (p < 0.05). By contrast, SLC7A11-rs969319 showed a protective role only in females (p < 0.05). CONCLUSION: Our results shed new light on the association between disulfidptosis-related genes and AIT risk.

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.

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.

SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 has distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect)-driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2∗SLC7A5 heterodimer with amino-acid/Na+ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na+/K+ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.

An amino acid transporter subunit as an antibody-drug conjugate target in colorectal cancer.

BACKGROUND: Advanced colorectal cancer (CRC) is difficult to treat. For that reason, the development of novel therapeutics is necessary. Here we describe a potentially actionable plasma membrane target, the amino acid transporter protein subunit CD98hc. METHODS: Western blot and immunohistochemical analyses of CD98hc protein expression were carried out on paired normal and tumoral tissues from patients with CRC. Immunofluorescence and western studies were used to characterize the action of a DM1-based CD98hc-directed antibody-drug conjugate (ADC). MTT and Annexin V studies were performed to evaluate the effect of the anti-CD98hc-ADC on cell proliferation and apoptosis. CRISPR/Cas9 and shRNA were used to explore the specificity of the ADC. In vitro analyses of the antitumoral activity of the anti-CD98hc-ADC on 3D patient-derived normal as well as tumoral organoids were also carried out. Xenografted CRC cells and a PDX were used to analyze the antitumoral properties of the anti-CD98hc-ADC. RESULTS: Genomic as well proteomic analyses of paired normal and tumoral samples showed that CD98hc expression was significantly higher in tumoral tissues as compared to levels of CD98hc present in the normal colonic tissue. In human CRC cell lines, an ADC that recognized the CD98hc ectodomain, reached the lysosomes and exerted potent antitumoral activity. The specificity of the CD98hc-directed ADC was demonstrated using CRC cells in which CD98hc was decreased by shRNA or deleted using CRISPR/Cas9. Studies in patient-derived organoids verified the antitumoral action of the anti-CD98hc-ADC, which largely spared normal tissue-derived colon organoids. In vivo studies using xenografted CRC cells or patient-derived xenografts confirmed the antitumoral activity of the anti-CD98hc-ADC. CONCLUSIONS: The studies herewith reported indicate that CD98hc may represent a novel ADC target that, upon well-designed clinical trials, could be used to increase the therapeutic armamentarium against CRC.

Integrated analysis of FKBP1A/SLC3A2 axis in everolimus inducing ferroptosis of breast cancer and anti-proliferation of T lymphocyte.

Background: Solute Carrier Family 3 Member 2 (SLC3A2) is a member of the solute carrier family that plays pivotal roles in regulation of intracellular calcium levels and transports L-type amino acids. However, there are insufficient scientific researches on the prognostic and immunological roles of SLC3A2 in breast cancer (BC) and whether everolimus regulates novel SLC3A2 related molecular mechanism in the immuno-oncology context of the tumor microenvironment (TME), therefore, we see a necessity to conduct the current in silico and biological experimental study. Methods: Using diverse online databases, we investigated the role of SLC3A2 in therapy response, clinicopathological characteristics, tumor immune infiltration, genetic alteration, methylation and single cell sequencing in BC. WB, Co-IP, cell proliferation assay, Edu staining, ROS and GSH assay and in vivo tumor xenograft assays were performed to verify FKBP1A/SLC3A2 axis in everolimus inducing ferroptosis of breast cancer. Co-cultures and IL-9 ELISA were performed to demonstrate the T lymphocyte function. Results: We demonstrated that SLC3A2 was aberrantly expressed among various BC cohorts. Our results also suggested that SLC3A2 expression was associated with chemotherapeutic outcome in BC patients. Our results further indicated that SLC3A2 was associated with tumor infiltration of cytotoxic T cell but not other immune cells among BC TME. The alterations in SLC3A2 gene had a significant correlation to relapse free survival and contributed a significant impact on BC tumor mutational burden. Finally, SLC3A2 was illustrated to be expressed in diverse BC cellular populations at single cell level, and negatively linked to angiogenesis, inflammation and quiescence, but positively correlated with other functional phenotypes. Noteworthily, everolimus (a targeted therapy drug for BC) related protein, FK506-binding protein 1A (FKBP1A) was found to bind with SLC3A2, and negatively regulated SLC3A2 expression during the processes of everolimus inducing ferroptosis of BC cells and promoting anti-proliferation of Th9 lymphocytes. Conclusions: Altogether, our study strongly implies that SLC3A2 is an immuno-oncogenic factor and FKBP1A/SLC3A2 axis would provide insights for a novel immunotherapy approach for the treatment of BC in the context of TME.

Potent nanoreactor-mediated ferroptosis-based strategy for the reversal of cancer chemoresistance to Sorafenib.

The drug resistance of cancer cells is related to a variety of mechanisms, among which the destruction of redox homeostasis is one of the key factors. Ferroptosis, an intracellular iron-dependent form of cell death, is related to the production of oxidative stress. The accumulation of lipid peroxidation (LPO) during ferroptosis disrupts intracellular redox homeostasis, thereby affecting the sensitivity of tumor cells to drugs. In this work, we proposed a ferroptosis strategy based on LPO accumulation, reduced glutathione generation via inhibition of SLC3A2 protein and inactivated glutathione peroxidase 4 (GPX4) to reverse the chemoresistance of cancer cells. The Fenton reaction based on the ferroptosis-inducing nanoreactors (Au/Fe-GA/Sorafenib@PEG) not only generated hydroxyl radicals (·OH) under laser irradiation to realize the accumulation of LPO, but also depleted GSH to increase the accumulation of LPO. Meanwhile, the cystine uptake of cells was inhibited by Sorafenib, resulting in reduced GSH synthesis and inactivated GPX4. In vitro and in vivo experiments demonstrated AFG/SFB@PEG + Laser group could inactivate GPX4 and the enhanced ferroptosis can reverse chemo-resistance caused by continuous upregulation of GPX4 levels in cells through 'self-rescue'. The study proposed the mechanism and feasibility of ferroptosis to reverse drug resistance, providing a promising strategy for chemo-resistant cancer treatment. STATEMENT OF SIGNIFICANCE: Herein, we proposed a ferroptosis strategy based on LPO accumulation, reduced glutathione generation via inhibition of SLC3A2 protein, and inactivated glutathione peroxidase 4 (GPX4) to reverse chemoresistance of cancer cells. The Fenton reaction based on the ferroptosis-inducing nanoreactors (Au/Fe-GA/Sorafenib@PEG) not only generated hydroxyl radicals (·OH) under laser irradiation to realize the accumulation of LPO but also depleted GSH to increase the accumulation of LPO. Meanwhile, the cystine uptake of cells was inhibited by Sorafenib, resulting in reduced GSH synthesis and inactivated GPX4. In vitro and in vivo experiments demonstrated AFG/SFB@PEG + Laser group could inactivate GPX4 and the enhanced ferroptosis can reverse chemo-resistance caused by continuous upregulation of GPX4 levels in cells through 'self-rescue'.

SLC3A2 promotes tumor-associated macrophage polarization through metabolic reprogramming in lung cancer.

Tumor-associated macrophages (TAMs) are one of the most abundant immunosuppressive cells in the tumor microenvironment and possess crucial functions in facilitating tumor progression. Emerging evidence indicates that altered metabolic properties in cancer cells support the tumorigenic functions of TAMs. However, the mechanisms and mediators the underly the cross-talk between cancer cells and TAMs remain largely unknown. In the present study, we revealed that high solute carrier family 3 member 2 (SLC3A2) expression in lung cancer patients was associated with TAMs and poor prognosis. Knockdown of SLC3A2 in lung adenocarcinoma cells impaired M2 polarization of macrophages in a coculture system. Using metabolome analysis, we identified that SLC3A2 knockdown altered the metabolism of lung cancer cells and changed multiple metabolites, including arachidonic acid, in the tumor microenvironment. More importantly, we showed that arachidonic acid was responsible for SLC3A2-mediated macrophage polarization in the tumor microenvironment to differentiate into M2 type both in vitro and in vivo. Our data illustrate previously undescribed mechanisms responsible for TAM polarization and suggest that SLC3A2 acts as a metabolic switch on lung adenocarcinoma cells to induce macrophage phenotypic reprogramming through arachidonic acid.

The human LAT1-4F2hc (SLC7A5-SLC3A2) transporter complex: Physiological and pathophysiological implications.

LAT1 and 4F2hc form a heterodimeric membrane protein complex, which functions as one of the best characterized amino acid transporters. Since LAT1-4F2hc is required for the efficient uptake of essential amino acids and hormones, it promotes cellular growth, in part, by stimulating mTORC1 (mechanistic target of rapamycin complex 1) signalling and by repressing the integrated stress response (ISR). Gain or loss of LAT1-4F2hc function is associated with cancer, diabetes, and immunological and neurological diseases. Hence, LAT1-4F2hc represents an attractive drug target for disease treatment. Specific targeting of LAT1-4F2hc will be facilitated by the increasingly detailed understanding of its molecular architecture, which provides important concepts for its function and regulation. Here, we summarize (i) structural insights that help to explain how LAT1 and 4F2hc assemble to transport amino acids across membranes, (ii) the role of LAT1-4F2hc in key metabolic signalling pathways, and (iii) how derailing these processes could contribute to diseases.

Identify metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 as potential prognostic markers and correlate with immune infiltrates in head and neck squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of head and neck squamous cell carcinoma (HNSCC) with poor prognosis. Metabolic reprogramming may regulate the tumor microenvironment (TME) by adapting quickly to cellular stress and regulating immune response, but its role in HSCC has not been reported. We used the nCounter® Metabolic Pathways Panel to investigate metabolic reprogramming, cellular stress, and their relationship in HSCC tissues and adjacent normal tissues. Metabolism-related pathways nucleotide synthesis and glycolysis pathways were significantly upregulated, while amino acid synthesis and fatty acid oxidation pathways were significantly downregulated in HSCC tissues compared to adjacent normal tissues. There is a significant correlation between metabolism-related pathways and cellular stress pathways. Enrichment of immune cell and tumor infiltrating lymphocyte (TIL) analysis showed changes in immune responses between HSCC tissues and adjacent normal tissues. Overall survival analysis showed that upregulated genes CD276, LDHB, SLC3A2, EGFR, SLC7A5, and HPRT1 are potential unfavorable prognostic markers in HNSCC, while downregulated genes EEA1, IDO1, NCOA2, REST, CCL19, and ALDH2 are potential favorable prognostic markers in HNSCC. Moreover, metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 are correlated with immune infiltrates in HNSCC. These results suggest that metabolic reprogramming occurs and correlates with cellular stress and immune response in HSCC, which may help researchers understand mechanisms of metabolic reprogramming and develop effective immunotherapeutic strategies in HNSCC.

N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98).

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of inflammatory processes, host-pathogen interactions to association with membrane transporters of the SLC7 family. The recent resolution of CD98 structure in complex with LAT1 showed that four Asn residues, N365, N381, N424, N506, harbour N-glycosylation moieties. Then, the role of N-glycosylation on CD98 trafficking and stability was investigated by combining bioinformatics, site-directed mutagenesis and cell biology approach. Single, double, triple and quadruple mutants of the four Asn exhibited altered electrophoretic mobility, with apparent molecular masses from 95 to 70 kDa. The quadruple mutant displayed a single band of 70 kDa corresponding to the unglycosylated protein. The presence in the membrane and the trafficking of CD98 were evaluated by a biotinylation assay and a brefeldin assay, respectively. Taken together, the results highlighted that the quadruple mutation severely impaired both the stability and the trafficking of CD98 to the plasma membrane. The decreased presence of CD98 at the plasma membrane, correlated with a lower presence of LAT1 (SLC7A5) and its transport activity. This finding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafficking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.

CD98hc in host-pathogen interactions: roles of the multifunctional host protein during infections.

The eukaryotic protein CD98hc (also known as 4F2, FRP-1, or SLC3A2) is a membrane glycoprotein and one of the heavy chains of the family of heterodimeric amino acids transporters. It can associate with any of 6 different light chains to form distinct amino acid transporters. CD98hc is also involved in mediation of intracellular integrin signaling. Besides its physiological roles in the development of the placenta and the immune system, CD98hc is important during pathological processes such as tumorigenesis and host-pathogen interaction. Since its first identification as Fusion Regulatory Protein 1 regulating cell fusion in cells infected by the Newcastle disease virus, CD98hc has been reported to be mediating many viral, apicomplexan, and bacterial infectious processes. In this review we describe the role of CD98hc and its associated light chains in bacterial, apicomplexan, and viral pathogenesis. We also discuss the consequences of infection on the expression and localization of these proteins. The identification of the cellular processes in which CD98hc is involved during pathogenesis highlights the key role of this host protein in infectious diseases.

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.

Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer.

BACKGROUND: Despite the incorporation of novel therapeutics, advanced triple negative breast cancer (TNBC) still represents a relevant clinical problem. Considering this, as well as the clinical efficacy of antibody-drug conjugates (ADCs), we aimed at identifying novel ADC targets that could be used to treat TNBC. METHODS: Transcriptomic analyses were performed on TNBC and normal samples from three different studies. Plasma membrane proteins of three cell lines representative of the TNBC subtype were identified by cell surface biotinylation or plasma membrane isolation, followed by analyses of cell surface proteins using the Surfaceome online tool. Immunofluorescence and western studies were used to characterize the action of a CD98hc-directed ADC, which was prepared by in house coupling of emtansine to an antibody that recognized the ectodomain of CD98hc. Xenografted TNBC cells were used to analyze the antitumoral properties of the anti-CD98hc ADC. RESULTS: Comparative genomic studies between normal breast and TNBC tissues, together with proteomic and bioinformatic analyses resulted in the elaboration of a catalog of potential ADC targets. One of them, the CD98hc transmembrane protein, was validated as an ADC target. An antibody recognizing the ectodomain of CD98hc efficiently internalized and reached the lysosomal compartment. An emtansine-based ADC derived from such antibody was prepared and showed antitumoral properties in TNBC in vitro and in vivo models. Mechanistically, the anti-CD98hc ADC blocked cell cycle progression, that was followed by cell death caused by mitotic catastrophe. CONCLUSIONS: This work describes a list of potential ADC targets in TNBC and validates one of them, the transmembrane protein CD98hc. The studies presented here also demonstrate the robustness of the multiomic approach herewith described to identify novel potential ADC targets.