Acid-base transporters in the context of tumor heterogeneity.

The copious metabolic acid production and -extrusion by cancer cells render poorly vascularized regions of solid tumors highly acidic. A growing list of proton - and bicarbonate transporters has been suggested to contribute to net acid extrusion from cancer cells, and/or been shown to be dysregulated and favor malignant development in various cancers. The great majority of these roles have been studied at the level of the cancer cells. However, recent advances in understanding of the cellular and physicochemical heterogeneity of solid tumors both enable and necessitate a reexamination of the regulation and roles of acid-base transporters in such malignancies. This review will briefly summarize the state-of-the-art, with a focus on the SLC9A and SLC4A families, for which most evidence is available. This is followed by a discussion of key concepts and open questions arising from recent insights and of the challenges that need to be tackled to address them. Finally, opportunities and challenges in therapeutic targeting of the acid-base transportome in cancers will be addressed.

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.

Functional Characterization of a Novel SLC4A4 Variant and Uniparental Isodisomy in Proximal Renal Tubular Acidosis Patient.

SLC4A4 variants are the etiologies of inherited proximal renal tubular acidosis (pRTA), which results in metabolic acidosis, hypokalemia, glaucoma, band keratopathy, and cataract. This study aims to characterize SLC4A4 variant and uniparental isodisomy of chromosome 4 in a patient, and analyse the functional characterization of SLC4A4 variants. This study analyzed renal tubular acidosis disease genes by whole exome sequencing (WES). H3M2 algorithm was used to analyze the run of homozygosity region in chromosomal regions in trio-WES data. The pathogenicity analysis of variants was performed using bioinformatics tools. Additionally, protein stability was analyzed by cycloheximide chase assay. Whole-cell patch clamping was used to examine the electrophysiological properties of NBCe1-A. A novel homozygous SLC4A4 variant was identified in the patient: a missense variant c.496C > T, p. Arg166Trp (NM_003759.4). But the father was heterozygous variant carrier, and the mother did not detect the variant. The H3M2 and UPDio algorithm revealed paternal uniparental isodisomy on chromosome 4 in the patient. SIFT, Poly Phen-2, FATHMM and Mutant Taster showed that the variant might be pathogenic. The tertiary structure analysis showed that the variant could cause structural damage to NBCe1 protein. Foldx results showed that the protein stability of the variant was slightly reduced. Cycloheximide chase assay demonstrated that the variant affects protein stability. The result of electrophysiological studies showed that the variant altered Na+/HCO3- cotransport activity of protein. In conclusion, the study is the first to report a pRTA patient with Arg166Trp variant with UPiD (4) pat and analyze the function of Arg166Trp variant.

Acid-base effects of combined renal deletion of NBCe1-A and NBCe1-B.

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deletion of the A splice variant of Na+-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The purpose of the present study was to determine the effect of combined renal deletion of NBCe1-A and NBCe1-B on systemic and PT ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. As renal NBCe1-A and NBCe1-B expression is limited to the PT, Cre-positive mice had PT NBCe1-A/B deletion [PT-NBCe1-A/B knockout (KO)]. Although on a basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased the expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and increased the expression of glutamine synthetase, an ammonia-recycling enzyme, in PTs in both the cortex and OM. Exogenous acid loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase expression in PTs in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to PT ammonia metabolism in the OM and thereby to systemic acid-base regulation.NEW & NOTEWORTHY The results of the present study show that combined deletion of both A and B splice variants of electrogenic Na+-bicarbonate cotransporter 1 from the proximal tubule impairs acid-base homeostasis and completely blocks changes in ammonia excretion in response to acidosis, indicating that both proteins are critical to acid-base homeostasis.

SLC4A4 promotes prostate cancer progression in vivo and in vitro via AKT-mediated signalling pathway.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related male deaths worldwide. The purpose of this study was to investigate the effects of homo sapiens solute carrier family 4 member 4 (SLC4A4), which encodes the electrogenic Na+/HCO3- cotransporter isoform 1 (NBCe1), in the development and progression of PCa. METHODS: The expression levels of SLC4A4 in PCa and normal prostate tissues were evaluated by immunohistochemistry. The SLC4A4 knockdown cell model was structured by lentiviral infection, and the knockdown efficiency was validated by RT-qPCR and Western blotting. The effects of SLC4A4 knockdown on cell proliferation, apoptosis and cycle, migration, and invasion were detected by Celigo cell counting assay and CCK-8 assay, flow cytometry analysis, wound-healing, and Transwell assay, respectively. Tumor growth in nude mice was surveyed by in vivo imaging and Ki-67 staining. Furthermore, underlying mechanism of SLC4A4 silence induced inhibition of PCa progression was explored by human phospho-kinase array. RESULTS: Our results revealed that SLC4A4 expression was up-regulated in PCa tissues and human PCa cell lines. High expression of SLC4A4 in tumor specimens was significantly correlated with disease progression. SLC4A4 knockdown inhibited cell proliferation, migration and invasion, while facilitated apoptosis, which was also confirmed in vivo. Moreover, SLC4A4 promoted PCa progression through the AKT-mediated signalling pathway. CONCLUSION: The results of this study indicated that SLC4A4 overexpression was closely associated with the progression of PCa; SLC4A4 knockdown suppressed PCa development in vitro and in vivo. SLC4A4 acts as a tumor promotor in PCa by regulating key components of the AKT pathway and may therefore act as a potential therapeutic target for PCa treatment.

IRBIT activates NBCe1-B by releasing the auto-inhibition module from the transmembrane domain.

KEY POINTS: The electrogenic Na+ /HCO3- cotransporter NBCe1-B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1-B has very low activity under basal condition due to auto-inhibition, but can be fully activated by protein interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). The structural components of the auto-inhibition domain and the IRBIT-binding domain of NBCe1-B are finely characterized based on systematic mutations in the present study and data from previous studies. Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto-inhibition of NBCe1-B. We propose that the auto-inhibition domain functions as a brake module that inactivates NBCe1-B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain via competitive binding to the auto-inhibition domain. ABSTRACT: The electrogenic Na+ /HCO3- cotransporter NBCe1-B is widely expressed in many tissues in the body. NBCe1-B exhibits only basal activity due to the action of the auto-inhibition domain (AID) in its unique amino-terminus. However, NBCe1-B can be activated by interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto-inhibition of NBCe1-B and its activation by IRBIT. The IRBIT-binding domain (IBD) of NBCe1-B spans residues 1-52, essentially consisting of two arms, one negatively charged (residues 1-24) and the other positively charged (residues 40-52). The AID mainly spans residues 40-85, overlapping with the IBD in the positively charged arm. The magnitude of auto-inhibition of NBCe1-B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1-B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1-B; (2) IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain.

Expression of the regulated isoform of the electrogenic Na+/HCO3- cotransporter, NBCe1, is enriched in pacemaker interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility- and electrical slow-wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of Slc4a4-207 and Slc4a4-208 transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICCs from Kittm1Rosay mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and SLC4A4-201 and SLC4A4-204 RNAs were >300-fold enriched relative to SLC4A4-202. In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs.NEW & NOTEWORTHY In this study, we show that the electrogenic Na+/HCO3- cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO3 cotransporter NBCe1-A, expressed in Xenopus oocytes.

KEY POINTS: According to the HCO3- metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO3 cotransporter NBCe1-A speeds transport by regenerating/consuming HCO3- . The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1-A, heterologously expressed in Xenopus oocytes. We confirm the essential elements of the previous experimental observations, taken as support for the HCO3- metabolon hypothesis. However, using our own experimental protocols or those of others, we find that NBCe1-A function is unaffected by cytosolic CAs. Previous conclusions that cytosolic CAs do stimulate NBCe1-A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na-H exchanger. Theoretical analyses show that, although CAs could stimulate non- HCO3- transporters (e.g. Na-H exchangers) by accelerating CO2 / HCO3- -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying HCO3- , CO3= , or NaCO3- ion pairs. ABSTRACT: The HCO3- metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes HCO3- replenishment/consumption, and enhances transport. Using a short step-duration current-voltage (I-V) protocol with Xenopus oocytes expressing eGFP-tagged NBCe1-A, our group reported that neither injecting human CA II (hCA II) nor fusing hCA II to the NBCe1-A carboxy terminus affects background-subtracted NBCe1 slope conductance (GNBC ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (Vh ) far from NBCe1-A's reversal potential (Erev ) for prolonged periods - found that bCA increases total membrane current (ΔIm ), which apparently supports the metabolon hypothesis. We systematically investigated differences in the two protocols. In oocytes expressing untagged NBCe1-A, injected with bCA and clamped to -40 mV, CO2 / HCO3- exposures markedly decrease Erev , producing large transient outward currents persisting for >10 min and rapid increases in [Na+ ]i . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔIm and d[Na+ ]i /dt, it does not reduce GNBC . In oocytes not expressing NBCe1-A, CO2 / HCO3- triggers rapid increases in [Na+ ]i that both hCA II and bCA enhance in concentration-dependent manners. These d[Na+ ]i /dt increases are inhibited by EZA and blocked by EIPA, a Na-H exchanger (NHE) inhibitor. In oocytes expressing untagged NBCe1-A and injected with bCA, EIPA abolishes the EZA-dependent decreases in ΔIm and d[Na+ ]i /dt. Thus, CAs/EZA produce their ΔIm and d[Na+ ]i /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of HCO3- transport, supporting the conclusion that an NBCe1-A- HCO3- metabolon does not exist in oocytes.

Exploring the autoinhibitory domain of the electrogenic Na+ /HCO3- transporter NBCe1-B, from residues 28 to 62.

KEY POINTS: Slc4a4 (mouse) encodes at least five variants of the electrogenic sodium/bicarbonate transporter NBCe1. The initial 41 cytosolic amino acids of NBCe1-A and -D are unique; NBCe1-A has high activity. The initial 85 amino acids of NBCe1-B, -C and -E are unique; NBCe1-B and -C have low activity. Previous work showed that deleting residues 1-85 or 40-62 of NBCe1-B, or 1-87 of NBCe1-C, eliminates autoinhibition. These regions also include binding determinants for IRBIT (inositol trisphosphate (IP3 )-receptor binding protein released with IP3 ), which relieves autoinhibition. Here, systematically replacing/deleting residues 28-62, we find that only the nine amino acid cationic cluster (residues 40-48) of NBCe1-B is essential for autoinhibition. IRBIT stimulates all but one low-activity construct. We suggest that electrostatic interactions - which IRBIT presumably interrupts - between the cationic cluster and the membrane or other domains of NBCe1 play a central role in tempering the activity of NBCe1-B in the pancreas, brain and other organs. ABSTRACT: Variant B of the electrogenic Na+ /HCO3- cotransporter (NBCe1-B) contributes to the vectorial transport of HCO3- in epithelia (e.g. pancreatic ducts) and to the maintenance of intracellular pH in the central nervous systems (e.g. astrocytes). NBCe1-B has very low basal activity due to an autoinhibitory domain (AID) located, at least in part, in the unique portion (residues 1-85) of the cytosolic NH2 -terminus. Previous work has shown that removing 23 amino acids (residues 40-62) stimulates NBCe1-B. Here, we test the hypothesis that a cationic cluster of nine consecutive positively charged amino acids (residues 40-48) is a necessary part of the AID. Using two-electrode voltage clamping of Xenopus oocytes, we assess the activity of human NBCe1-B constructs in which we systematically replace or delete residues 28-62, which includes the cationic cluster. We find that replacing or deleting all residues within the cationic cluster markedly increases NBCe1-B activity (i.e. eliminates autoinhibition). On the background of a cationic clusterless construct, systematically restoring Arg residues restores autoinhibition in two distinct quanta, with one to three Arg residues restoring ∼50%, and four or more Arg residues restoring virtually all autoinhibition. Systematically deleting residues before the cluster reduces autoinhibition by, at most, a small amount. Replacing or deleting residues after the cluster has no effect. For constructs with low NBCe1 activity (but good surface expression, as assessed by biotinylation), co-expression with super-IRBIT (lacking PP1-binding site) restores full activity (i.e. relieves autoinhibition). In summary, the cationic cluster is a necessary component of the AID of NBCe1-B.

Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney.

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study's purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

SLC4A4 compound heterozygous mutations in exon-intron boundary regions presenting with severe proximal renal tubular acidosis and extrarenal symptoms coexisting with Turner's syndrome: a case report.

BACKGROUND: Congenital NBCe1A deficiency with the SLC4A4 mutation causes severe proximal renal tubular acidosis, which often comprises extrarenal symptoms, such as intellectual disability and developmental delay, glaucoma, cataract and band keratopathy. To date, almost all mutations have been found to be homozygous mutations located in exons. CASE PRESENTATION: We performed direct nucleotide sequencing analysis of exons and exon-intron boundary regions of the SLC4A4 in a patient presenting with severe renal proximal tubule acidosis, glaucoma and intellectual disability and her parents without these signs. The examination revealed compound heterozygous mutations in exon-intron boundary regions, c.1076 + 3A > C and c.1772 - 2A > T, neither of which have been reported previously. While the former mutation was found in the mother, the latter was found in the father. The transcript of the SLC4A4 gene was almost undetectable, and the patient was also diagnosed with Turner's syndrome. CONCLUSIONS: We identified two novel SLC4A4 mutations, c.1076 + 3A > C and c.1772 - 2A > T. When presented in a compound heterozygous state, these mutations caused a phenotype of severe renal proximal tubular acidosis along with glaucoma and mental retardation. This is the first report of congenital proximal renal tubular acidosis carrying compound heterozygous SLC4A4 mutations in exon-intron boundary regions. We suggest that an mRNA surveillance mechanism, nonsense-mediated RNA decay, following aberrant splicing was the reason that the SLC4A4 transcript was almost undetectable in the proband.

Effect of heat stress on the gene expression of ion transporters/channels in the uterus of laying hens during eggshell formation.

Heat stress is a problem in laying hens as it decreases egg quality by decreasing eggshell mineralization. Heat stress alters gene expression, hence our aim was to investigate effects of heat stress on gene expression of ion transport elements involving in uterine mineralization (TRPV6, CALB1, ITPR3, SCNN1G, SLC4A4, KCNJ15, SLC4A9, and CLCN2) by real time quantitative PCR. Forty 23-week-old White Leghorn laying hens were housed in two rooms. The control group (n = 20) was maintained at 21-23 °C, and the heat stress group (n = 20) was exposed to 36-38 °C for 8 weeks. All parameters of egg quality including egg weight, surface area, volume, and eggshell weight, thickness, ash weight, and calcium content were decreased in the heat stress group compared to the control group (by 26.9%, 32.7%, 44.1%, 38.4%, 31.7%, 39.4%, and 11.1%, respectively). Total plasma calcium was decreased by 13.4%. Levels of ITPR3, SLC4A4, and SLC4A9 transcripts in the uterine lining were decreased in the heat stress group compared to the control group (by 61.4%, 66.1%, and 66.1%, respectively). CALB1 transcript level was increased (by 34.2 fold) in the heat stress group of hens compared to controls. TRPV6, SCNN1G, KCNJ15, and CLCN2 transcript levels did not significantly differ between control and heat stress groups of laying hens. It is concluded that the down-expression of ITPR3, SLC4A4, and SLC4A9 genes may impair transportation of Cl-, HCO3-, and Na+ in eggshell mineralization during heat stress. Increased CALB1 gene expression may increase resistance of uterine cells to detrimental effects of heat stress.

SLC4A4 is a novel driver of enzalutamide resistance in prostate cancer.

The androgen receptor signaling inhibitor (ARSI) enzalutamide (Enz) has shown critical efficacy in the treatment of advanced prostate cancer (PCa). However, the development of drug resistance is a significant factor contributing to mortality in PCa patients. We aimed to explore the key mechanisms of Enz-resistance. Through analysis of GEO databases, we identified SLC4A4 as a novel driver in Enz resistance. Long-term Enz treatment leads to the up-regulation of SLC4A4, which in turn mediates P53 lactylation via the NF-κB/STAT3/SLC4A4 axis, ultimately leading to the development of Enz resistance and progression of PCa. SLC4A4 knockdown overcomes Enz resistance both in vitro and in vivo. Hence, our results suggest that targeting SLC4A4 could be a promising therapeutic strategy for Enz resistance. STATEMENT OF SIGNIFICANCE: SLC4A4 is a novel driver of enzalutamide resistance.

SLC4A4 moulds the inflammatory tumor microenvironment and predicts therapeutic expectations in colorectal cancer.

AIM: In this report, we performed a comprehensive analysis of data in colorectal cancer (CRC), to elucidate the association among Solute Carrier Family 4 Member 4 (SLC4A4) and the abundance of immunological features and immune cell infiltration in CRC, and to explore the impact of SLC4A4 on the CRC tumor microenvironment. BACKGROUND: Colorectal cancer (CRC) cases with advanced or distal metastases experience a survival rate of less than 20%, with the lack of spectral therapeutic targets and prognostic markers posing a significant challenge for CRC treatment. SLC4A4 may be a CRC-targeted therapy for which there is currently inadequate evidence Objective: To deeply and systematically reveal the characteristics of the tumor microenvironment created by SLC4A4. METHODS: We downloaded RNA sequencing files (TCGA-COADREAD), clinical data for Colon Cancer (COAD) and Rectal Cancer (READ) from the Cancer Genome Atlas. We evaluated the spearman correlation of SLC4A4 with immune features, Tracking Tumor Immunophenotype (TIP) score, and immune checkpoint gene expression. SLC4A4/immunity-related differentially expressed genes (DEGs) were identified in SLC4A4 expression groups and immune groups, and an assessment system for predicting CRC prognosis was constructed based on univariate COX and multivariate COX analyses. Based on the prognostic factors in CRC, we also constructed a nomogram to assess the survival risk status of CRC. Besides, we evaluated the potential association of SLC4A4 to immunotherapy. RESULTS: We found that SLC4A4 expression trended positively with immune checkpoint expression (PD-L1, CTLA4) and promoted infiltration of 27 immune cells. SLC4A4 promoted the infiltration of CD8 T cells, Dendritic cells, Macrophage, NK cells, and Th1 cells in CRC, shaping the inflammatory tumor microenvironment. Up-regulation of SLC4A4 expression might promote drug response to Anti-FGFR3_therapy, Anti-PPARG_therapy, Nivolumab, Ipilimumab in CRC patients, and down-regulation of SLC4A4 expression might promote drug response to Anti-EGFR_therapy, Aflibercept drug response. Based on the SLC4A4/immunization-related DEGs, we constructed RiskScore to assess the prognosis of CRC, which showed excellent predictive effect and robustness. RiskScore showed a trend of negative correlation with SLC4A4, which was consistent with the trend of the effect of SLC4A4 on CRC survival. Besides, RiskScore could also be useful for predicting patient prognosis. Finally, we constructed a nomogram for predicting CRC survival based on metrics with independent prognostic value (Age, M stage, Stage, RiskScore), which showed potential clinical value. CONCLUSION: Overall, upregulation of SLC4A4 expression promoted an inflammatory tumor microenvironment in CRC, and RiskScore predicted therapeutic expectancy. SLC4A4 could be a potentially clinically valuable target for CRC therapy.

Identification of Polymorphisms in EAAT1 Glutamate Transporter Gene SLC1A3 Associated with Reduced Migraine Risk.

Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a role in susceptibility to associated neurological disorders, including pathogenesis of a migraine. Rare pathogenic variants in specific ion channels have been implicated in monogenic migraine subtypes. In this study, we further examined the channelopathic nature of a migraine through the analysis of common genetic variants in three selected ion channel or transporter genes: SLC4A4, SLC1A3, and CHRNA4. Using the Agena MassARRAY platform, 28 single-nucleotide polymorphisms (SNPs) across the three candidate genes were genotyped in a case-control cohort comprised of 182 migraine cases and 179 matched controls. Initial results identified significant associations between migraine and rs3776578 (p = 0.04) and rs16903247 (p = 0.05) genotypes within the SLC1A3 gene, which encodes the EAAT1 glutamate transporter. These SNPs were subsequently genotyped in an independent cohort of 258 migraine cases and 290 controls using a high-resolution melt assay, and association testing supported the replication of initial findings-rs3776578 (p = 0.0041) and rs16903247 (p = 0.0127). The polymorphisms are in linkage disequilibrium and localise within a putative intronic enhancer region of SLC1A3. The minor alleles of both SNPs show a protective effect on migraine risk, which may be conferred via influencing the expression of SLC1A3.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests that ion homeostasis is a cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption, which was rescued by pharmacological or genetic inhibition of the CCL2-CCR2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-CCL2 and endothelial CCR2 axis as a mechanism controlling BBB integrity and repair, while providing insights for a therapeutic approach against BBB-related CNS disorders.

Nanoparticles loaded with circ_0086375 for suppressing the tumorigenesis of pancreatic cancer by targeting the miR-646/SLC4A4 axis.

Nanoparticles possess the ability to adsorb and load other compounds. This study aimed to synthesize a gene carrier with polyethyleneimine (PEI), hyaluronic acid (HA) and mesoporous silica nanoparticles (MSNs) for circ_0086375 delivery to investigate the role and mechanism of circ_0086375 in pancreatic cancer (PC) progression. The expression of genes and proteins was detected by quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were performed by cell counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, transwell assay, and wound healing assay, respectively. Dual-luciferase activity assay was used to investigate the target relationship between miR-646 and circ_0086375 or SLC4A4 (solute carrier family 4 member 4). Circ_0086375 loaded PEI/HA-based mesoporous silica nanoparticles (MSNs) were prepared, and in vivo assay was performed by using xenograft tumor model. Circ_0086375 expression was decreased in PC tissues and cells. Restoration of circ_0086375 suppressed PC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, circ_0086375 acted as a sponge for miR-646 to elevate SLC4A4 expression, which was confirmed to be a target of miR-646. The prepared circ_0086375/MSN/PEI/HA nanocomplexes showed excellent fluorescent properties and a higher cellular uptake of circ_0086375 in PC cells. Moreover, circ_0086375/MSN/PEI/HA showed relatively more anticancer effects in PC than that of circ_0086375 alone in vitro and in vivo. Delivery of circ_0086375 by nanoparticles suppresses the tumorigenicity of pancreatic cancer by miR-646/SLC4A4 axis, suggesting a new potential target for future pancreatic cancer treatment.

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

Objective: To explore new biomarkers related to microsatellite instability in order to better predict prognosis and guide medication. Methods: 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. Results: 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. Conclusion: 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.

Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a NO-CCL2-CCR2 pathway.

Astrocytes play vital roles in blood-brain barrier (BBB) maintenance, yet how they support BBB integrity under normal or pathological conditions remains poorly defined. Recent evidence suggests pH homeostasis is a new cellular mechanism important for BBB integrity. In the current study, we investigated the function of an astrocyte-specific pH regulator, Slc4a4, in BBB maintenance and repair. We show that astrocytic Slc4a4 is required for normal astrocyte morphological complexity and BBB function. Multi-omics analyses identified increased astrocytic secretion of CCL2 coupled with dysregulated arginine-NO metabolism after Slc4a4 deletion. Using a model of ischemic stroke, we found that loss of Slc4a4 exacerbates BBB disruption and reactive gliosis, which were both rescued by pharmacological or genetic inhibition of the NO-CCL2 pathway in vivo. Together, our study identifies the astrocytic Slc4a4-NO-CCL2 axis as a pivotal mechanism controlling BBB integrity and repair, while providing insights for a novel therapeutic approach against BBB-related CNS disorders.