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.

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).

Polycystic ovary syndrome 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 polycystic ovary syndrome 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 polycystic ovary syndrome 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.

Breast cancer is the most common cancer in women, affecting 1 in every 8 women, with a combination of radiation, surgery, and chemotherapy as treatment. SLC38A5 is a glutamine/serine/glycine/methionine transporter, which mediates the influx of Na+/amino acid into cells coupled to the efflux of H+. SLC7A11 mediates the cellular uptake of cystine in exchange for glutamate. Both transporters are upregulated in triple-negative breast cancer (TNBC). SLC38A5 supports “glutamine addiction” and the increased need for one-carbon metabolism in cancer cells. SLC7A11 supports cellular synthesis of glutathione by providing cysteine, and thus protects the cancer cells against oxidative stress and iron-induced cell death (ferroptosis). To date, no functional crosstalk has been identified between the two transporters. We hypothesized that (i) SLC38A5 might mediate the cellular uptake of the micronutrient selenium in the form of selenomethionine (Se-Met) like it does Metionine (Met) and (ii) since Se-Met is known to induce the anti-oxidative transcription factor Nrf2 that promotes SLC7A11 expression, there might be a crosstalk between the two transporters via Se-Met. We tested these hypotheses using two TNBC cell lines, MB231 and MB453. The activity of SLC38A5 was monitored by measuring the uptake of serine in the presence of an uptake buffer (pH 8.5) containing LiCl in place of NaCl. Li+ tolerance and higher uptake at alkaline pH are unique features of SLC38A5. The interaction of Met and Se-Met with SLC38A5 was investigated by studying the dose-response effects for the two amino acids in inhibiting serine uptake. Met and Se-Met competed with serine for SLC38A5-mediated uptake in both cell lines with comparable IC50 values (~500 μM). We then investigated the effect of Se-Met on the expression and activity of SLC7A11. Pretreatment of cell lines with 1 mM Se-Met for 16 h increased SLC7A11 mRNA levels and increased the transport activity of SLC7A11. In these studies, monomethylfumarate was used as a positive inducer of Nrf2. Previously work showed shRNA-mediated downregulation of SLC38A5 in TNBC cell lines suppresses growth and proliferation of cancer in cell culture and in mouse xenografts.RNAseq analysis of the control tumors and shRNA-tumors show transcriptomic variance in the profile of the tumors in response to SLC38A5 deficiency. We found upregulation of genes involved in oxidative phosphorylation, TGF-β signaling, and hypoxia signaling and downregulation of KRAS and epithelial-mesenchymal transition pathways in SLC38A5-deficient tumors. We conclude that though both SLC38A5 and SLC7A11 promote TNBC via independent mechanisms, the two transporters are functionally coupled by Se-Met. This study provides valuable insight into how these two amino acid transporters work together to support TNBC growth, thus setting the stage for pharmacologic targeting of these transporters as a novel therapeutic strategy. Citation Format: Marilyn Mathew, Gunadharini Nandagopal Dharmalingam, Sathish Sivaprakasam, Sabarish Ramachandran, Souad R. Sennoune, Vadivel Ganapathy. Functional coupling between the amino acid transporters SLC38A5 and SLC7A11 in TNBC via selenomethionine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 274.