Correction: Mathew et al. Metabolic Signature of Warburg Effect in Cancer: An Effective and Obligatory Interplay between Nutrient Transporters and Catabolic/Anabolic Pathways to Promote Tumor Growth. Cancers 2024, 16, 504.

In the original publication [...].

Induction of Oxidative Stress and Ferroptosis in Triple-Negative Breast Cancer Cells by Niclosamide via Blockade of the Function and Expression of SLC38A5 and SLC7A11.

The amino acid transporters SLC38A5 and SLC7A11 are upregulated in triple-negative breast cancer (TNBC). SLC38A5 transports glutamine, methionine, glycine and serine, and therefore activates mTOR signaling and induces epigenetic modifications. SLC7A11 transports cystine and increases the cellular levels of glutathione, which protects against oxidative stress and lipid peroxidation via glutathione peroxidase, a seleno (Se)-enzyme. The primary source of Se is dietary Se-methionine (Se-Met). Since SLC38A5 transports methionine, we examined its role in Se-Met uptake in TNBC cells. We found that SLC38A5 interacts with methionine and Se-Met with comparable affinity. We also examined the influence of Se-Met on Nrf2 in TNBC cells. Se-Met activated Nrf2 and induced the expression of Nrf2-target genes, including SLC7A11. Our previous work discovered niclosamide, an antiparasitic drug, as a potent inhibitor of SLC38A5. Here, we found SLC7A11 to be inhibited by niclosamide with an IC50 value in the range of 0.1-0.2 µM. In addition to the direct inhibition of SLC38A5 and SLC7A11, the pretreatment of TNBC cells with niclosamide reduced the expression of both transporters. Niclosamide decreased the glutathione levels, inhibited proliferation, suppressed GPX4 expression, increased lipid peroxidation, and induced ferroptosis in TNBC cells. It also significantly reduced the growth of the TNBC cell line MB231 in mouse xenografts.

Colonic ketogenesis, a microbiota-regulated process, contributes to blood ketones and protects against colitis in mice.

Ketogenesis is considered to occur primarily in liver to generate ketones as an alternative energy source for non-hepatic tissues when glucose availability/utilization is impaired. 3-Hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2) mediates the rate-limiting step in this mitochondrial pathway. Publicly available databases show marked down-regulation of HMGCS2 in colonic tissues in Crohn's disease and ulcerative colitis. This led us to investigate the expression and function of this pathway in colon and its relevance to colonic inflammation in mice. Hmgcs2 is expressed in cecum and colon. As global deletion of Hmgcs2 showed significant postnatal mortality, we used a conditional knockout mouse with enzyme deletion restricted to intestinal tract. These mice had no postnatal mortality. Fasting blood ketones were lower in these mice, indicating contribution of colonic ketogenesis to circulating ketones. There was also evidence of gut barrier breakdown and increased susceptibility to experimental colitis with associated elevated levels of IL-6, IL-1ß, and TNF-α in circulation. Interestingly, many of these phenomena were mostly evident in male mice. Hmgcs2 expression in colon is controlled by colonic microbiota as evidenced from decreased expression in germ-free mice and antibiotic-treated conventional mice and from increased expression in a human colonic epithelial cell line upon treatment with aqueous extracts of cecal contents. Transcriptomic analysis of colonic epithelia from control mice and Hmgcs2-null mice indicated an essential role for colonic ketogenesis in the maintenance of optimal mitochondrial function, cholesterol homeostasis, and cell-cell tight-junction organization. These findings demonstrate a sex-dependent obligatory role for ketogenesis in protection against colonic inflammation in mice.

Metabolic Signature of Warburg Effect in Cancer: An Effective and Obligatory Interplay between Nutrient Transporters and Catabolic/Anabolic Pathways to Promote Tumor Growth.

Aerobic glycolysis in cancer cells, originally observed by Warburg 100 years ago, which involves the production of lactate as the end product of glucose breakdown even in the presence of adequate oxygen, is the foundation for the current interest in the cancer-cell-specific reprograming of metabolic pathways. The renewed interest in cancer cell metabolism has now gone well beyond the original Warburg effect related to glycolysis to other metabolic pathways that include amino acid metabolism, one-carbon metabolism, the pentose phosphate pathway, nucleotide synthesis, antioxidant machinery, etc. Since glucose and amino acids constitute the primary nutrients that fuel the altered metabolic pathways in cancer cells, the transporters that mediate the transfer of these nutrients and their metabolites not only across the plasma membrane but also across the mitochondrial and lysosomal membranes have become an integral component of the expansion of the Warburg effect. In this review, we focus on the interplay between these transporters and metabolic pathways that facilitates metabolic reprogramming, which has become a hallmark of cancer cells. The beneficial outcome of this recent understanding of the unique metabolic signature surrounding the Warburg effect is the identification of novel drug targets for the development of a new generation of therapeutics to treat cancer.

Amino acid transporter SLC38A5 is a tumor promoter and a novel therapeutic target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) cells have a great demand for nutrients in the form of sugars, amino acids, and lipids. Particularly, amino acids are critical for cancer growth and, as intermediates, connect glucose, lipid and nucleotide metabolism. PDAC cells meet these requirements by upregulating selective amino acid transporters. Here we show that SLC38A5 (SN2/SNAT5), a neutral amino acid transporter is highly upregulated and functional in PDAC cells. Using CRISPR/Cas9-mediated knockout of SLC38A5, we show its tumor promoting role in an in vitro cell line model as well as in a subcutaneous xenograft mouse model. Using metabolomics and RNA sequencing, we show significant reduction in many amino acid substrates of SLC38A5 as well as OXPHOS inactivation in response to SLC38A5 deletion. Experimental validation demonstrates inhibition of mTORC1, glycolysis and mitochondrial respiration in KO cells, suggesting a serious metabolic crisis associated with SLC38A5 deletion. Since many SLC38A5 substrates are activators of mTORC1 as well as TCA cycle intermediates/precursors, we speculate amino acid insufficiency as a possible link between SLC38A5 deletion and inactivation of mTORC1, glycolysis and mitochondrial respiration, and the underlying mechanism for PDAC attenuation. Overall, we show that SLC38A5 promotes PDAC, thereby identifying a novel, hitherto unknown, therapeutic target for PDAC.

Sigma Receptors: Novel Regulators of Iron/Heme Homeostasis and Ferroptosis.

Sigma receptors are non-opiate/non-phencyclidine receptors that bind progesterone and/or heme and also several unrelated xenobiotics/chemicals. They reside in the plasma membrane and in the membranes of the endoplasmic reticulum, mitochondria, and nucleus. Until recently, the biology/pharmacology of these proteins focused primarily on their role in neuronal functions in the brain/retina. However, there have been recent developments in the field with the discovery of unexpected roles for these proteins in iron/heme homeostasis. Sigma receptor 1 (S1R) regulates the oxidative stress-related transcription factor NRF2 and protects against ferroptosis, an iron-induced cell death process. Sigma receptor 2 (S2R), which is structurally unrelated to S1R, complexes with progesterone receptor membrane components PGRMC1 and PGRMC2. S2R, PGRMC1, and PGRMC2, either independently or as protein-protein complexes, elicit a multitude of effects with a profound influence on iron/heme homeostasis. This includes the regulation of the secretion of the iron-regulatory hormone hepcidin, the modulation of the activity of mitochondrial ferrochelatase, which catalyzes iron incorporation into protoporphyrin IX to form heme, chaperoning heme to specific hemoproteins thereby influencing their biological activity and stability, and protection against ferroptosis. Consequently, S1R, S2R, PGRMC1, and PGRMC2 potentiate disease progression in hemochromatosis and cancer. These new discoveries usher this intriguing group of non-traditional progesterone receptors into an unchartered territory in biology and medicine.

Potent Inhibition of Macropinocytosis by Niclosamide in Cancer Cells: A Novel Mechanism for the Anticancer Efficacy for the Antihelminthic.

Niclosamide, a drug used to treat tapeworm infection, possesses anticancer effects by interfering with multiple signaling pathways. Niclosamide also causes intracellular acidification. We have recently discovered that the amino acid transporter SLC38A5, an amino acid-dependent Na+/H+ exchanger, activates macropinocytosis in cancer cells via amino acid-induced intracellular alkalinization. Therefore, we asked whether niclosamide will block basal and SLC38A5-mediated macropinocytosis via intracellular acidification. We monitored macropinocytosis in pancreatic and breast cancer cells using TMR-dextran and the function of SLC38A5 by measuring Li+-stimulated serine uptake. The peptide transporter activity was measured by the uptake of glycylsarcosine. Treatment of the cancer cells with niclosamide caused intracellular acidification. The drug blocked basal and serine-induced macropinocytosis with differential potency, with an EC50 of ~5 µM for the former and ~0.4 µM for the latter. The increased potency for amino acid-mediated macropinocytosis is due to direct inhibition of SLC38A5 by niclosamide in addition to the ability of the drug to cause intracellular acidification. The drug also inhibited the activity of the H+-coupled peptide transporter. We conclude that niclosamide induces nutrient starvation in cancer cells by blocking macropinocytosis, SLC38A5 and the peptide transporter. These studies uncover novel, hitherto unknown, mechanisms for the anticancer efficacy of this antihelminthic.

Expression and Function of StAR in Cancerous and Non-Cancerous Human and Mouse Breast Tissues: New Insights into Diagnosis and Treatment of Hormone-Sensitive Breast Cancer.

Breast cancer (BC) is primarily triggered by estrogens, especially 17ß-estradiol (E2), which are synthesized by the aromatase enzyme. While all steroid hormones are derived from cholesterol, the rate-limiting step in steroid biosynthesis is mediated by the steroidogenic acute regulatory (StAR) protein. Herein, we demonstrate that StAR mRNA expression was aberrantly high in human hormone-dependent BC (MCF7, MDA-MB-361, and T-47D), modest in hormone-independent triple negative BC (TNBC; MDA-MB-468, BT-549, and MDA-MB-231), and had little to none in non-cancerous mammary epithelial (HMEC, MCF10A, and MCF12F) cells. In contrast, these cell lines showed abundant expression of aromatase (CYP19A1) mRNA. Immunofluorescence displayed qualitatively similar patterns of both StAR and aromatase expression in various breast cells. Additionally, three different transgenic (Tg) mouse models of spontaneous breast tumors, i.e., MMTV-Neu, MMTV-HRAS, and MMTV-PyMT, demonstrated markedly higher expression of StAR mRNA/protein in breast tumors than in normal mammary tissue. While breast tumors in these mouse models exhibited higher expression of ERα, ERß, and PR mRNAs, their levels were undetected in TNBC tumors. Accumulation of E2 in plasma and breast tissues, from MMTV-PyMT and non-cancerous Tg mice, correlated with StAR, but not with aromatase, signifying the importance of StAR in governing E2 biosynthesis in mammary tissue. Treatment with a variety of histone deacetylase inhibitors (HDACIs) in primary cultures of enriched breast tumor epithelial cells, from MMTV-PyMT mice, resulted in suppression of StAR and E2 levels. Importantly, inhibition of StAR, concomitant with E2 synthesis, by various HDACIs, at clinical and preclinical doses, in MCF7 cells, indicated therapeutic relevance of StAR in hormone-dependent BCs. These findings provide insights into the molecular events underlying the differential expression of StAR in human and mouse cancerous and non-cancerous breast cells/tissues, highlighting StAR could serve not only as a novel diagnostic maker but also as a therapeutic target for the most prevalent hormone-sensitive BCs.

Polycystic ovary syndrome and iron overload: biochemical link and underlying mechanisms with potential novel therapeutic avenues.

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder in women with components of significant genetic predisposition and possibly multiple, but not yet clearly defined, triggers. This disorder shares several clinical features with hemochromatosis, a genetically defined inheritable disorder of iron overload, which includes insulin resistance, increased adiposity, diabetes, fatty liver, infertility, and hyperandrogenism. A notable difference between the two disorders, however, is that the clinical symptoms in PCOS appear at much younger age whereas they become evident in hemochromatosis at a much later age. Nonetheless, noticeable accumulation of excess iron in the body is a common finding in both disorders even at adolescence. Hepcidin, the iron-regulatory hormone secreted by the liver, is reduced in both disorders and consequently increases intestinal iron absorption. Recent studies have shown that gut bacteria play a critical role in the control of iron absorption in the intestine. As dysbiosis is a common finding between PCOS and hemochromatosis, changes in bacterial composition in the gut may represent another cause for iron overload in both diseases via increased iron absorption. This raises the possibility that strategies to prevent accumulation of excess iron with iron chelators and/or probiotics may have therapeutic potential in the management of polycystic ovary syndrome.

Carbidopa suppresses estrogen receptor-positive breast cancer via AhR-mediated proteasomal degradation of ERα.

Estrogen receptor-α (ERα) promotes breast cancer, and ER-positive cancer accounts for ~ 80% of breast cancers. This subtype responds positively to hormone/endocrine therapies involving either inhibition of estrogen synthesis or blockade of estrogen action. Carbidopa, a drug used to potentiate the therapeutic efficacy of L-DOPA in Parkinson's disease, is an agonist for aryl hydrocarbon receptor (AhR). Pharmacotherapy in Parkinson's disease decreases the risk for cancers, including breast cancer. The effects of carbidopa on ER-positive breast cancer were evaluated in cell culture and in mouse xenografts. The assays included cell proliferation, apoptosis, cell migration/invasion, subcellular localization of AhR, proteasomal degradation, and tumor growth in xenografts. Carbidopa decreased proliferation and migration of ER-positive human breast cancer cells in vitro with no significant effect on ER-negative breast cancer cells. Treatment of ER-positive cells with carbidopa promoted nuclear localization of AhR and expression of AhR target genes; it also decreased cellular levels of ERα via proteasomal degradation in an AhR-dependent manner. In vivo, carbidopa suppressed the growth of ER-positive breast cancer cells in mouse xenografts; this was associated with increased apoptosis and decreased cell proliferation. Carbidopa has therapeutic potential for ER-positive breast cancer either as a single agent or in combination with other standard chemotherapies.

Binding of Citrate-Fe3+ to Plastic Culture Dishes, an Artefact Useful as a Simple Technique to Screen for New Iron Chelators.

NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe3+), citrate uptake/binding as monitored by the association of [14C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe3+-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe3+ chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe3+ chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe3+ and prevent the formation and adsorption of citrate-Fe3+ to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.

Peptide Tags and Domains for Expression and Detection of Mammalian Membrane Proteins at the Cell Surface.

Normal functions of cell-surface proteins are dependent on their proper trafficking from the site of synthesis to the cell surface. Transport proteins mediating solute transfer across the plasma membrane constitute an important group of cell-surface proteins. There are several diseases resulting from mutations in these proteins that interfere with their transport function or trafficking, depending on the impact of the mutations on protein folding and structure. Recent advances in successful treatment of some of these diseases with small molecules which correct the mutations-induced folding and structural changes underline the need for detailed structural and biophysical characterization of membrane proteins. This requires methods to express and purify these proteins using heterologous expression systems. Here, using the solute carrier (SLC) transporter NaCT (Na+-coupled citrate transporter) as an example, we describe experimental strategies for this approach. We chose this example because several mutations in NaCT, distributed throughout the protein, cause a severe neurologic disease known as early infantile epileptic encephalopathy-25 (EIEE-25). NaCT was modified with various peptide tags, including a RGS-His10, a Twin-Strep, the SUMOstar domain, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. When transiently expressed in HEK293 cells, recombinant NaCT proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited citrate transport activity similar to the nontagged protein. Surface NaCT expression was enhanced by the presence of SUMOstar on the N-terminus. The dual-purpose peptide epitopes RGS-His10 and Twin-Strep facilitated detection of NaCT by immunohistochemistry and western blot and may serve useful tags for affinity purification. This approach sets the stage for future analyses of mutant NaCT proteins that may alter protein folding and trafficking. It also demonstrates the capability of a transient mammalian cell expression system to produce human NaCT of sufficient quality and quantity to augment future biophysical and structural studies and drug discovery efforts.

Deletion of Slc6a14 reduces cancer growth and metastatic spread and improves survival in KPC mouse model of spontaneous pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal. There is a dire need for better therapeutic targets. Cancer cells have increased demand for sugars, amino acids, and lipids and therefore up-regulate various nutrient transporters to meet this demand. In PDAC, SLC6A14 (an amino acid transporter (AAT)) is up-regulated, affecting overall patient survival. Previously we have shown using in vitro cell culture models and in vivo xenograft mouse models that pharmacological inhibition of SLC6A14 with α-methyl-l-tryptophan (α-MLT) attenuates PDAC growth. Mechanistically, blockade of SLC6A14-mediated amino acid transport with α-MLT leads to amino acid deprivation, eventually inhibiting mTORC1 signaling pathway, in tumor cells. Here, we report on the effect of Slc6a14 deletion on various parameters of PDAC in KPC mice, a model for spontaneous PDAC. Pancreatic tumors in KPC mice show evidence of Slc6a14 up-regulation. Deletion of Slc6a14 in this mouse attenuates PDAC growth, decreases the metastatic spread of the tumor, reduces ascites fluid accumulation, and improves overall survival. At the molecular level, we show lower proliferation index and reduced desmoplastic reaction following Slc6a14 deletion. Furthermore, we find that deletion of Slc6a14 does not lead to compensatory up-regulation in any of the other amino transporters. In fact, some of the AATs are actually down-regulated in response to Slc6a14 deletion, most likely related to altered mTORC1 signaling. Taken together, these results underscore the positive role SLC6A14 plays in PDAC growth and metastasis. Therefore, SLC6A14 is a viable drug target for the treatment of PDAC and also for any other cancer that overexpresses this transporter.

Unconventional Functions of Amino Acid Transporters: Role in Macropinocytosis (SLC38A5/SLC38A3) and Diet-Induced Obesity/Metabolic Syndrome (SLC6A19/SLC6A14/SLC6A6).

Amino acid transporters are expressed in mammalian cells not only in the plasma membrane but also in intracellular membranes. The conventional function of these transporters is to transfer their amino acid substrates across the lipid bilayer; the direction of the transfer is dictated by the combined gradients for the amino acid substrates and the co-transported ions (Na+, H+, K+ or Cl-) across the membrane. In cases of electrogenic transporters, the membrane potential also contributes to the direction of the amino acid transfer. In addition to this expected traditional function, several unconventional functions are known for some of these amino acid transporters. This includes their role in intracellular signaling, regulation of acid-base balance, and entry of viruses into cells. Such functions expand the biological roles of these transporters beyond the logical amino acid homeostasis. In recent years, two additional unconventional biochemical/metabolic processes regulated by certain amino acid transporters have come to be recognized: macropinocytosis and obesity. This adds to the repertoire of biological processes that are controlled and regulated by amino acid transporters in health and disease. In the present review, we highlight the unusual involvement of selective amino acid transporters in macropinocytosis (SLC38A5/SLC38A3) and diet-induced obesity/metabolic syndrome (SLC6A19/SLC6A14/SLC6A6).

Resetting amino acid metabolism of cancer cells by ATB0,+-targeted nanoparticles for enhanced anticancer therapy.

Reprogramed cellular metabolism is one of the most significant hallmarks of cancer. All cancer cells exhibit increased demand for specific amino acids, and become dependent on either an exogenous supply or upregulated de novo synthesis. The resultant enhanced availability of amino acids supports the reprogramed metabolic pathways and fuels the malignant growth and metastasis of cancers by providing energy and critical metabolic intermediates, facilitating anabolism, and activating signaling networks related to cell proliferation and growth. Therefore, pharmacologic blockade of amino acid entry into cancer cells is likely to have a detrimental effect on cancer cell growth. Here we developed a nanoplatform (LJ@Trp-NPs) to therapeutically target two transporters, SLC6A14 (ATB0,+) and SLC7A5 (LAT1), that are known to be essential for the sustenance of amino acid metabolism in most cancers. The LJ@Trp-NPs uses tryptophan to guide SLC6A14-targeted delivery of JPH203, a high-affinity inhibitor of SLC7A5. In the process, SLC6A14 is also down-regulated. We tested the ability of this strategy to synergize with the anticancer efficacy of lapatinib, an inhibitor of EGFR/HER1/HER2-assocated kinase. These studies show that blockade of amino acid entry amplifies the anticancer effect of lapatinib via interference with mTOR signaling, promotion of apoptosis, and suppression of cell proliferation and metastasis. This represents the first study to evaluate the impact of amino acid starvation on the anticancer efficacy of widely used kinase inhibitor.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of ß-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

TBX2 Drives Neuroendocrine Prostate Cancer through Exosome-Mediated Repression of miR-200c-3p.

Deciphering the mechanisms that drive transdifferentiation to neuroendocrine prostate cancer (NEPC) is crucial to identifying novel therapeutic strategies against this lethal and aggressive subtype of advanced prostate cancer (PCa). Further, the role played by exosomal microRNAs (miRs) in mediating signaling mechanisms that propagate the NEPC phenotype remains largely elusive. The unbiased differential miR expression profiling of human PCa cells genetically modulated for TBX2 expression led to the identification of miR-200c-3p. Our findings have unraveled the TBX2/miR-200c-3p/SOX2/N-MYC signaling axis in NEPC transdifferentiation. Mechanistically, we found that: (1) TBX2 binds to the promoter and represses the expression of miR-200c-3p, a miR reported to be lost in castrate resistant prostate cancer (CRPC), and (2) the repression of miR-200c-3p results in the increased expression of its targets SOX2 and N-MYC. In addition, the rescue of mir-200c-3p in the context of TBX2 blockade revealed that miR-200c-3p is the critical intermediary effector in TBX2 regulation of SOX2 and N-MYC. Further, our studies show that in addition to the intracellular mode, TBX2/miR-200c-3p/SOX2/N-MYC signaling can promote NEPC transdifferentiation via exosome-mediated intercellular mechanism, an increasingly recognized and key mode of propagation of the NEPC phenotype.

Drosophila INDY and Mammalian INDY: Major Differences in Transport Mechanism and Structural Features despite Mostly Similar Biological Functions.

INDY (I'm Not Dead Yet) is a plasma membrane transporter for citrate, first identified in Drosophila. Partial deficiency of INDY extends lifespan in this organism in a manner similar to that of caloric restriction. The mammalian counterpart (NaCT/SLC13A5) also transports citrate. In mice, it is the total, not partial, absence of the transporter that leads to a metabolic phenotype similar to that caloric restriction; however, there is evidence for subtle neurological dysfunction. Loss-of-function mutations in SLC13A5 (solute carrier gene family 13, member A5) occur in humans, causing a recessive disease, with severe clinical symptoms manifested by neonatal seizures and marked disruption in neurological development. Though both Drosophila INDY and mammalian INDY transport citrate, the translocation mechanism differs, the former being a dicarboxylate exchanger for the influx of citrate2- in exchange for other dicarboxylates, and the latter being a Na+-coupled uniporter for citrate2-. Their structures also differ as evident from only ~35% identity in amino acid sequence and from theoretically modeled 3D structures. The varied biological consequences of INDY deficiency across species, with the beneficial effects predominating in lower organisms and detrimental effects overwhelming in higher organisms, are probably reflective of species-specific differences in tissue expression and also in relative contribution of extracellular citrate to metabolic pathways in different tissues.

Expression and function of SLC38A5, an amino acid-coupled Na+/H+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis.

Metabolic reprogramming in cancer necessitates increased amino acid uptake, which is accomplished by up-regulation of specific amino acid transporters. However, not all tumors rely on any single amino acid transporter for this purpose. Here, we report on the differential up-regulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The up-regulation is evident in TNBC tumors, conventional and patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous TNBC mammary tumor. The up-regulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since cell-surface Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to the induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger. Down-regulation of SLC38A5 in TNBC cells attenuates serine-induced macropinocytosis and reduces cell proliferation significantly as assessed by multiple methods, but does not induce cell death. The Cancer Genome Atlas database corroborates SLC38A5 up-regulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition and proliferation.