Introduction

Cancer is presently a highly prevalent pathology and, despite improvements in diagnostic and medical treatment, including the development of new therapies, the mortality rates associated with this condition are still very high. Therefore, it is fundamental to find new anti-tumor drugs with new mechanisms of action to improve cancer prognosis.

Cancer cells are metabolically reprogrammed to support their high proliferative ratio [1, 2]. This characteristic, which is one of the cancer hallmarks, is essential to provide the energy needs and biosynthetic precursors and for maintenance of redox homeostasis of tumor cells [1]. Indeed, metabolic reprogramming is a mechanism of adaptation of cancer cells, allowing them to develop and evolve, competing with other cells for glucose and other nutrients [1, 2]. Of importance, many of the metabolic changes observed in cancer cells are associated with a poor outcome [1, 2]. Thus, interference with metabolic reprogramming mechanisms of cancer cells constitutes a new hotspot in antineoplastic research.

It is known that glucose metabolism plays a very important role in tumor prognosis, but the role of glutamine metabolism in cancer cells is receiving a crescent attention [3]. Glutamine is a nutritionally non-essential amino acid with a very important role in cancer. It is not only a source of energy production and nucleotide/protein biosynthesis, but also possesses other effects in cancer cells (see below) that establish this nutrient as playing an important role in cancer cells, contributing to malignancy [4,5,6]. Thus, knowledge/modulation of glutamine metabolism can be useful, both in the diagnosis, monitoring, and cancer therapy [5].

To meet their high glutamine needs, cancer cells develop mechanisms that allow them to take up high amounts of this amino acid, namely by increasing the expression of membrane transporters that mediate the cellular uptake of glutamine [7]. ASCT2 (Alanine, Serine, Cysteine Transporter 2; encoded by SLC1A5 gene), a Na+-dependent transmembrane transporter involved in the cellular uptake of neutral amino acids such as glutamine, is a primary transporter of glutamine in cancer cells [3]. There is overexpression of ASCT2 in many cancer cells, and ASCT2 appears to have a role in tumorigenesis and in the outcome of malignant neoplasia [3, 8,9,10,11,12,13]. In agreement with this important role of ASCT2 in glutamine uptake by cancer cells, silencing ASCT2 was shown to interfere with cancer cells proliferation and survival, thus improving prognosis [3]. Furthermore, silencing ASCT2 appears to improve response to other antineoplastic therapies [3]. Therefore, ASCT2 is currently attracting a lot of attention in oncology research, and the possibility of using it as a therapeutic target has been investigated [3, 8,9,10,11,12,13].

In this article, we will describe the pharmacological agents that act on ASCT2, which has been attracting a lot of attention in antineoplastic investigation. We will also address the impact of silencing/inhibition of this glutamine transporter on the prognosis of some cancers.

Glutamine metabolism and its functions in cancer cells

Glutamine is a very important amino acid in our body, being the main amino acid flowing in the bloodstream [14]. This nutrient has been receiving a growing attention in cancer research, due to its role in cancer cells and on cancer initiation and progression. Indeed, cancer cells have a high glutamine demand (they use 10–100 times more glutamine than any other amino acid) and become glutamine “addicted”, as glutamine withdrawal can cause cell death [15]. This amino acid performs numerous important functions in cancer cells. It is an important energy source because, in most cancer cells, less pyruvate is oxidized in the mitochondria and an increase in the conversion of glucose to lactate is observed—the Warburg effect—and so other energy sources, including glutamine, branched-chain amino acids, and fatty acids, are used [4, 11, 16, 17]. Additionally, glutamine is also involved in redox homeostasis, cell signaling, apoptosis inhibition, and in autophagy [4,5,6]. In addition, cancer cell biosynthesis is enhanced to support their increased proliferation [18,19,20], and glutamine is a source of carbon and nitrogen, allowing molecules such as amino acids, proteins, nucleotides, and fatty acids, to be synthesized [18, 21, 22], as next described.

Glutaminolysis, which is increased in cancer cells, involves a set of reactions beginning with glutamine (Fig. 1) [21]. After entering the cells, glutamine is hydrolyzed by glutaminases (GLS/GLS2), within the mitochondria, being converted into ammonia and glutamate [18]. Silencing or inhibition of GLS (whose expression correlates with cancer growth and malignancy in rodents) delays tumor growth in both in vitro and animal models [5], including breast cancer and B lymphoma [23] and glioblastoma [24]. Importantly, the GLS inhibitor telaglenastat improved progression-free survival in patients with advanced renal cell carcinoma and is currently in clinical trials for advanced renal cell carcinoma and for non-small cell lung cancer (NSCLC) with mutations that activate the NRF2/KEAP1 pathway [25].

Fig. 1
figure 1

Overview of glutamine metabolism in cancer cells. Glutamine is converted into ammonia and glutamate by glutaminases (GLS/GLS2) [18]. In turn, from glutamate it is possible to produce serine, which, in turn, allows glycine and cysteine to be synthesized. Alternatively, the combination of glutamate, glycine, and cysteine allows the synthesis of glutathione [19, 28], or glutamate can be converted to α-ketoglutarate, an intermediate of the TCA cycle [18, 21, 26], from which malate or citrate can be obtained. Malate can be expelled from the mitochondria, being converted to pyruvate and lactate (NAPDH is produced during these reactions) [20, 27]. Citrate can also be expelled from the mitochondria and it permits both lipid and α-ketoglutarate (in association with NADPH production) syntheses [20, 27]. So, glutamine is a source of nitrogen, allowing amino acids to be synthesized (alanine, serine, proline, aspartate, among others [19]) through its conversion to glutamate, and by transamination reactions [18, 20]. In addition to amino acids, glutamine also allows nucleotides, proteins, and lipids to be synthesized [18, 21, 22]. Taken from [19]. ACC acetyl-CoA carboxylase, AKG α-ketoglutarate, AS asparagine synthetase, ALT alanine transaminase, Ac-CoA acetyl-coenzyme A, AST aspartate transaminase, CS citrate synthase, ACO aconitase, G6P glucose-6-phosphate, FH fumarate hydratase, GCS gamma-glutamylcysteine synthetase, GFAT glutamine:fructose-6-phosphate aminotransferase, F6P fructose-6-phosphate, Gln glutamine, GLS glutaminase isoenzyme, GLS2 glutaminase 2 isoenzyme, Glc glucose, Gln glutamine, GLUD glutamate dehydrogenase, GS glutamine synthetase, IDH-1 cytosolic isocitrate dehydrogenase, GSH glutathione, HIF-1 hypoxia-inducible factor-1, IDH-2 mitochondrial isocitrate dehydrogenase, KGDH ketoglutarate dehydrogenase, LRH-1 nuclear receptor liver receptor homolog 1, LDH lactate dehydrogenase, ME malic enzyme, MDH malate dehydrogenase, NF-κB nuclear factor-kappa B, OAA oxaloacetate, mTOR mammalian target of rapamycin, OXPHOS oxidative phosphorylation, ODC ornithine decarboxylase, PC pyruvate carboxylase, PDH pyruvate dehydrogenase, PKM2 pyruvate kinase, M2 isoform, ROS reactive oxygen species, PPP pentose phosphate pathway, SDH succinate dehydrogenase, SCS succinyl-coenzyme A-synthetase, STAT1 signal transducer and activator of transcription 1

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In turn, from glutamate, it is possible to produce serine, which, in turn, allows glycine and cysteine to be synthesized; glutamate also allows other non-essential amino acids (alanine, proline, aspartate, among others [19]) to be synthesized through transamination reactions [18, 20]. Alternatively, glutamate can be converted to α-ketoglutarate, an intermediate of the Krebs cycle [18, 19, 21, 26], that can be converted into malate and citrate [19, 27, 28]. Malate can be effluxed from the mitochondria, and then converted into pyruvate [20, 27]. From pyruvate, lactate (and NADH) can be produced. Citrate, in turn, can also be removed from the mitochondria, leading to generation of acetyl-CoA (which is essential to form fatty acids and cholesterol) and α-ketoglutarate (in association with NADPH production) in the cytosol [20, 27]. In addition, combination of glutamate, glycine, and cysteine allows the synthesis of glutathione (an antioxidant neutralizing reactive oxygen species, ROS) [19, 28], by the action of glutamate cysteine ligase [18].

So, glutamine metabolism is important for redox balance in cancer cells [18, 21] through glutathione and NADPH synthesis [29]. Consequently, inhibition of glutamine metabolism originates higher ROS levels, increasing cancer cell apoptosis [3]. Moreover, glutamine is also a source of nitrogen, as its γ-nitrogen can be used for the synthesis of aspartate, asparagine, hexosamines, and nucleotides [18]. So, in addition to its role in energy production, glutamine is considered both a carbon source and a nitrogen donor, being involved in the biosynthesis of other amino acids and proteins, nucleotides, fatty acids, cholesterol, and glutathione (Fig. 1) [18, 21, 22]. All of these functions of glutamine are very important for cancer progression. Consequently, interference with its metabolism may constitute a new therapeutic target in cancer.

Glutamine cellular uptake: ASCT2 as a primary transporter of glutamine in cancer cells

Glutamine is a nutrient that has been receiving crescent attention for its role in antineoplastic therapy. As shown in the previous section, glutamine is essential for tumor development, as glutaminolysis is stimulated in cancer cells and cancer cells have an increased demand for this nutrient [4, 7, 30, 31]. This amino acid can be endogenously synthesized by cancer cells [18, 21, 26]. Alternatively, glutamine can be taken up from the extracellular space [4] and, thus, its cellular inlet becomes essential for tumor cells [4]. Glutamine is transported across the plasma membrane using different transporters [30], and there is an overexpression of transporters for this amino acid in cancer cells [7], as shown next.

Amino acid transporters are grouped into transport systems (e.g., system A, ASC, N, and L), depending on the substrates for which they have greater specificity and on their Na+ dependence or Na+ independence, among other differentiating characteristics [32].

In normal (non-cancerous) cells, glutamine transporter expression varies depending on the tissue [33,34,35,36,37,38]. For instance, SNAT1 (SLC38A1) is a glutamine transporter expressed in the neocortex, hippocampus, and neuroepithelium, and it provides glutamine to neurons, contributing to neuronal glutamate synthesis [38]. On the other hand, SNAT3 (SLC38A3) is expressed in astrocytes (which produce glutamine), allowing them to release glutamine [37]. Thus, both SNAT1 and SNAT3 are involved in brain glutamine transport and in glutamine–glutamate cycling: SNAT3 allows the release of glutamine by astrocytes, and SNAT1 allows neurons to take up glutamine [37, 38]. SNAT3 is also expressed in hepatic and renal tissues, allowing periportal cells and proximal renal tubule cells to obtain glutamine, and liver perivenous cells to release glutamine [33, 35, 36]. Additionally, SNAT2 (SLC38A2) is widely expressed in our body, playing very important roles [34]. It is associated with glutamine uptake and mTOR pathway signaling (contributing to cell growth and differentiation) [34, 39, 40].

As previously mentioned, although glutamine transporters exist in normal cells [33,34,35,36,37,38], their expression is increased in some cancers [7]. ASCT2 (Alanine, Serine, Cysteine Transporter 2) is a Na+-dependent transmembrane transporter, transporting glutamine and other neutral amino acids across the plasma membrane [41]. These other neutral amino acids include serine, cysteine, valine, threonine, and alanine [11, 41, 42]. ASCT2 is considered an obligatory exchanger of neutral amino acids and it is associated with both glutamine uptake and glutamine efflux [8, 43]. ASCT2 belongs to the SLC1A family (solute carrier 1A) [8], being encoded by the SLC1A5 gene (solute-linked carrier family A1 member 5), and it is considered a primary transporter for glutamine uptake in cancer cells [41], including breast cancer cells [44]. The SLC1A family of transporters includes ASCT1, ASCT2 (neutral amino acid transporters), and the EAAT1-5 transporters (excitatory amino acid transporters, which are associated with glutamate transport) [8].

ASCT2 (which is overexpressed in cancer cells) accounts for the inlet of glutamine into the cell, favoring tumorigenesis and tumor progression with consequent metastasis [45], and is associated with a poor prognosis [3]. Consequently, inhibition of ASCT2 may prevent tumor proliferation by interfering with glutamine influx [45,46,47,48,49]. So, we will focus on ASCT2 (SLC1A5) as a possible target for the development of new antineoplastic therapies, due to its notable role in glutamine uptake by cancer cells.

However, other transporters are also involved in glutamine transport by cancer cells. One of these transporters is LAT1 [50]. LAT1 (SLC7A5) is a transporter responsible for leucine uptake into the cells [3]. LAT1 allows an exchange of glutamine with leucine, allowing leucine to enter the intracellular space and glutamine, in turn, to pass into the extracellular space (Fig. 1) [3, 26, 50]. LAT1-mediated leucine uptake into the intracellular space allows activation of mTOR, being associated with tumor proliferation and, so, indirectly, glutamine allows mTOR activation [3, 6, 51]. LAT1 and CD98 constitute a heterodimeric complex, but whether CD98 is necessary for the transport function of this complex, or is only responsible for LAT1 membrane insertion, remains controversial [50, 52,53,54], although the activity of mTORC1 appears to be dependent on LAT1, but not CD98 [54]. Since LAT1 action depends on the intracellular concentration of glutamine (because it acts as an exchanger), there is an association between LAT1 and ASCT2 activities [3]. Both ASCT2 and LAT1 expression are independent prognostic factors in different cancers, and according to El Ansari et al., the combined expression of ASCT2 and LAT1 seems to influence breast cancer outcome [55].

Besides ASCT2 and LAT1, glutamine uptake may also involve other transporters. In relation to breast cancer cells, for instance, the following transporters are also able to transport glutamine across the plasma membrane: (a) SLC6A14 (also known as ATB0,+), known to be upregulated in ER-positive breast cancer cell lines [56]; (b) SLC3A1 (also known as rBAT), a Na+-independent transporter of cystine and neutral and dibasic amino acids, reported to be associated with breast cancer tumorigenesis [57]; (c) SLC7A7 (also known as y + LAT1), which mediates the influx of dibasic amino acids in a Na+-independent manner and is highly overexpressed in triple-negative breast cancer cells (MDA-MB-231 cells) [58] and in HER2 positive breast cancer cell lines [59]; (d) SLC7A8, a Na+-independent, large neutral amino acid transporter 2 (LAT2) that was reported as a predictive biomarker of good response to endocrine therapy in ER-positive breast cancer [60], and (e) SLC38A1, also known as Na+-coupled neutral amino acid transporter 1 (SNAT1), which is upregulated in breast cancer cell lines and human breast cancer tissues [61], and which overexpression is related to tumor size, nodal metastasis, and advanced tumor stage [62].

Although other glutamine transporters may be therapeutic targets for some cancers [34], ASCT2 is crucial for its primary and essential role in glutamine transport in several different cancers, and the therapeutic role of ASCT2 inhibition in some cancers has been well documented, as shown below [3, 9,10,11,12,13, 63, 64].

ASCT2 regulation in cancer cells

Tumor cells overexpress ASCT2. However, the mechanisms that regulate this increased expression are yet not completely understood, and more research is needed. Nevertheless, the following mechanisms have been described to regulate ASCT2.

Glutamine

Glutamine regulates the expression of ASCT2 [8]. According to Bungard et al., glutamine deprivation declined both ASCT2 expression, as well as tumor growth in a hepatoma (HepG2) cell line [65]. On the other hand, adding glutamine and, consequently, increasing glutamine availability, increased ASCT2 promoter activity and ASCT2 expression, favoring tumor growth [65]. Similarly, Dolinska et al. verified that in C6 glioma cells, glutamine deprivation was associated with lower ASCT2-mediated glutamine uptake [66]. Glutamine appears to regulate ASCT2 expression in cancer cells either by interfering with its transcription or at post-transcriptional level [8].

C-MYC

The MYC family of transcription factors is associated, in cancer, with nutrient uptake, metabolism, and proliferation. MYC activates the expression of some genes related to the acquisition and metabolism of glutamine, namely ASCT2 [67, 68].

Rb tumor suppressor

Retinoblastoma (Rb) tumor suppressor family can also regulate ASCT2 [68]. The entry of glutamine into the cell can be negatively regulated by Rb tumor suppressor, whose deletion enhances ASCT2-mediated glutamine uptake and upregulates GLS1 (via an E2F-dependent manner) [68].

microRNA-137 (miR-137)

This microRNA acts on ASCT2 mRNA, promoting its degradation or inhibiting its translation, and consequently decreasing ASCT2 levels [69, 70], glutamine uptake, and metabolism. Interestingly, MYC is associated with microRNA downregulation [67].

RNF5

Another regulator of ASCT2 is RNF5 [71]. Paclitaxel is a stress factor for breast cancer, promoting ubiquitination, RNF5 association, and a reduction of ASCT2 levels [71]. Accordingly, RNF5 deletion is associated with greater expression of ASCT2, which is related to a worse prognosis and less response to paclitaxel in breast cancer [71].

Leptin

An in vitro study using a human colon adenocarcinoma cell line (Caco-2 cells) evaluated the impact of leptin on the uptake of glucose and amino acids by these cells [72]. In relation to glutamine, this study demonstrated that, after the addition of leptin, uptake of glutamine rapidly decreased, together with a decrease in membrane ASCT2 protein expression [72]. Thus, leptin appears to inhibit glutamine uptake by downregulating ASCT2 [72]. Similarly, leptin was also found to decrease ASCT2-mediated glutamine uptake by both ER-positive and triple-negative breast cancer cell lines (Silva et al. unpublished).

Insulin

A study using rat adipocytes showed that both insulin and cell swelling stimulate ASCT2-mediated glutamine uptake [73]. In addition, insulin increased glutamine uptake not only directly, but also indirectly, through increased cell swelling [73]. The mechanism by which insulin increased ASCT2-mediated glutamine transport involves activation of the ERK/MAPK cascade pathway, suggesting that activation of the ERK pathway contributes to the regulation of ASCT2 by insulin [73]. In contrast, insulin appears to decrease ASCT2-mediated glutamine uptake by breast cancer cell lines (Silva et al. unpublished).

Interferon-γ and oxidative stress

In breast cancer cell lines, ASCT2-mediated uptake of glutamine was increased by INF-γ and by the oxidative stress inducer tert-butyl hydroperoxide (Silva et al. unpublished). Because higher levels of these parameters are associated with obesity/type 2 diabetes, this effect may contribute to the known higher prevalence of breast cancer in obese/type 2 diabetic women [74].

ASCT2 silencing/inhibition strategies

Currently, glutamine metabolism is seen as a target for cancer therapy development [3]. Glutamine has an essential role, both in the development and progression of the tumor [1, 2]. This amino acid is particularly important for cancer cells, compared to normal cells [4]. It is associated with metabolic reprogramming, allowing cells to adapt to their demands [3]. By interfering with glutamine metabolism, it is possible to inhibit one of the mechanisms of tumor adaptation. Therefore, inhibiting glutamine metabolism seems to be a good therapeutic strategy [3, 9].

Several strategies to inhibit glutamine metabolism have been investigated. One of the strategies consists of inhibiting transmembrane transporters involved in glutamine cellular uptake. In this context, ASCT2 is considered as a primary glutamine transporter and ASCT2 inhibitors have been developed [8], using distinct strategies: neutral amino acid analogs, which act as competitive inhibitors [8]; monoclonal antibodies targeting ASCT2 [9, 12, 13]; non-competitive ASCT2 inhibitors [75]; and strategies causing ASCT2 downregulation [10, 64] (Fig. 2). However, only a few ASCT2 inhibitors exist presently, and those are described below.

Fig. 2
figure 2

Mechanisms to inhibit ASCT2. Several ASCT2 inhibitors have been developed. Nevertheless, most of the existing inhibitory drugs are not sufficiently potent or selective [8]. GPNA inhibits several transporters related to glutamine metabolism, such as ASCT2, LAT1, SNAT1, and SNAT [79, 80]; V-9302 does not act by inhibiting ASCT2, but rather SNAT2, another glutamine transporter [81]; monoclonal antibodies recognizing ASCT2 appear to have a therapeutic role in cancer, slowing tumor growth and proliferation [9, 13]; δ-tocotrienol inhibits not only ASCT2, but also LAT1 and the mTOR pathway [63]; miRNA-137 acts on ASCT2 mRNA, promoting its degradation and inhibiting its translation [69, 70]

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Benzyl-serine/benzyl-cysteine derivatives

Benzyl-serine/benzyl-cysteine derivatives are competitive inhibitors of ASCT2 [76]. They bind to ASCT2, competing for the binding site with neutral amino acids, that are transported [76]. Consequently, these drugs prevent the transport of neutral amino acids through ASCT2 [76]. However, benzyl-serine reduces, significantly, both glutamine and leucine uptake in melanoma cells [48] and breast cancer cells (MDA-MB-231, MCF-7, and HCC1806 cells) in vitro [77]. Therefore, benzyl-serine is not a specific ASCT2 inhibitor, as it also blocks the leucine transporter LAT1 [77].

Phenyl-glycine analogs

These compounds are not ASCT2-specific inhibitors, inhibiting also other amino acid transporters such as ASCT1 [78]. They were used for targeting both ASCT1 and ASCT2 in rat visual cortex slices [78].

L-γ-glutamyl-p-nitroanilide (GPNA)

It is an analog of glutamine [8, 79]. GPNA inhibits several transporters related to glutamine metabolism, such as ASCT2, LAT1, SNAT1, and SNAT2, in several human cancer cell lines (Fig. 2) [79, 80]. So, it is not specific for ASCT2.

Monoclonal antibodies (mAb)

Monoclonal antibodies are being created to recognize ASCT2, interfering with its function or promoting its internalization [9, 13]. Hara et al. developed a monoclonal antibody that recognizes ASCT2, trying to understand if resorting to this therapeutic strategy would improve KRAS-mutated cancer prognosis [9]. They verified that it would be a good strategy for KRAS-mutated cancer treatment, improving its outcome and being promising, because cancers with this mutation have few effective pharmacological treatments [9]. Ab 3-8 mAb binds to ASCT2, promoting its internalization in SW1116 and HCT116 colon cancer cell lines harboring KRAS mutation [9]. Consequently, Ab 3-8 mAb reduces glutamine uptake, and inhibits in vivo tumor growth of these cells in athymic mice, by promoting ASCT2 internalization (Fig. 2) [9]. This monoclonal antibody was also found to bind ASCT2 in colon cancer cells without a KRAS mutation (HT29 cells) [9]. However, contrary to what happens in mutated cancer cells, Ab3-8 mAb does not cause ASCT2 internalization in cells not harboring KRAS mutation (HT29 cells) [9]. The reason for non-internalization of ASCT2 in these non-mutated cancer cells is unknown [9]. Additionally, Suzuki et al. also developed mAbs recognizing ASCT2 (Fig. 2) [13]. They isolated KM4008, KM4012, and KM4018 mAb, and found that they bind to cells expressing ASCT2, mainly recognizing EL2 (an extracellular domain of ASCT2) [13]. KM8094 mAb was found to suppress gastric cancer cell growth in vitro [12]. KM8094 appears to decrease tumor growth by inhibiting glutamine uptake, increasing oxidative stress and inducing antibody-dependent cellular cytotoxicity in gastric tumor cells [12]. So, monoclonal antibodies recognizing glutamine transporters appear to have a therapeutic role in cancer, slowing tumor growth and proliferation [9, 13]. Monoclonal antibodies have some advantages, being selective and causing other effects such as antibody-dependent cellular cytotoxicity [12, 13].

microRNA-137 (miR-137)

This compound suppresses tumor development by interfering with ASCT2 [69]. More specifically, decreasing miR-137 transcription increases ASCT2 expression and, consequently, glutamine uptake and metabolism and cancer cell proliferation in vitro and in vivo [69]. Thus, there is an inverse association between miR-137 and ASCT2 [69].

1, 2, 3-Dithiazoles

According to a study carried out on proteoliposomes, these compounds appear to inhibit ASCT2 [75]. It is a non-competitive inhibition with a covalent interaction between these compounds and cysteine thiol groups [75]. This non-competitive inhibition is advantageous because it is associated with a longer duration of inhibition [75].

Others

In addition to these strategies, some drugs already known for other functions also seem to interfere with ASCT2-mediated glutamine transport. These include (a) topotecan, an inhibitor of DNA topoisomerase I (Topo I) which prevents both the replication of DNA and RNA synthesis, causing the death of malignant cells. It is used to treat non-small cell lung cancer and ovarian cancer [64]. Despite its anti-tumor effect acting as a Topo I inhibitor, according to a study that evaluated the impact of this compound on gastric cancer cell metabolism, topotecan also downregulates ASCT2, reducing the entry of glutamine into the cell, decreasing proliferation and increasing oxidative stress and apoptosis in a gastric cancer cell line in vitro [64]; (b) resveratrol, which downregulates ASCT2 in human hepatocellular carcinoma cell lines [10]; and (c) δ-tocotrienol, which inhibits not only ASCT2, but also LAT1 and the mTOR pathway in non-small cell lung cancer cell lines [63] (Fig. 2).

V-9302

This compound was initially presented as an ASCT2 inhibitor, decreasing glutamine uptake with a higher potency than GPNA, both in vitro and in mice [47]. Currently, it is known that this molecule does not act by inhibiting ASCT2, but rather SNAT2, another glutamine transporter, and LAT1 (Fig. 2) [81]. Combining V-0302 with an ASCT2 inhibitor more drastically decreases glutamine uptake in some cancer cell lines, such as head and neck squamous carcinoma cell lines [3]. Nevertheless, caution is needed when combining an ASCT2 inhibitor with V-9302 because non-malignant cells also use glutamine, triggering adverse effects [3].

So, it is necessary to develop new strategies to inhibit ASCT2 since most of the existing inhibitory drugs are not sufficiently potent or selective [8].

Impact of ASCT2 silencing/inhibition in different cancers

As next described, ASCT2 is associated with growth/proliferation, survival, autophagy, and apoptosis of several cancer cell lines in vitro (Table 1), and with tumor development and disease progression and outcome of several cancers (namely gastric cancer, colorectal carcinoma, breast cancer, prostate cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, and liver cancer) in animal models (Table 2) [3, 42, 82,83,84,85,86]. So, ASCT2 inhibition can be a therapeutic strategy, mainly if associated with other therapies, by improving the response to them [3].

Table 1 In vitro effects of ASCT2 silencing/inhibition on different tumor cell lines
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Table 2 In vivo effects of ASCT2 silencing/inhibition on several tumor types
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Head and neck squamous cell carcinoma (HNSCC)

Several studies have demonstrated an association between glutamine and the development and progression of HNSCC [3, 87, 88].

According to Zhang et al., ASCT2 inhibition reduced intracellular glutamine levels and significantly decreased the survival and growth of HNSCC tumor cells (SCC15 and FaDu cell lines), both in vitro and in vivo (human xenograft that was transfected to mice) (Tables 1 and 2) [3]. Moreover, tumor cells in which ASCT2 was silenced responded more effectively to cetuximab [3]. Cetuximab (an anti-EGFR monoclonal antibody approved for HNSCC treatment) acts on EGFR, which is expressed at high levels in HNSCC [3, 89, 90]. Interestingly, ASCT2 forms a complex with EGFR and AP1G1 [3, 89, 90]. Thus, cetuximab, by acting on EGFR (causing EGFR endocytosis), also targets ASCT2, decreasing glutamine transport into the cells [3, 89, 90]. Zhang et al. used three different strategies to inhibit ASCT2: ASCT2 silencing with shRNA, miR-137, and shRNA combined with V-9302. All strategies decreased, significantly, glutamine uptake. However, combining ASCT2 silencing with V-9302 (a SNAT2 inhibitor) further reduced the entry of glutamine into the cells (Table 1) [3]. It was suggested that ASCT2 is not the unique transporter accounting for glutamine uptake in HNSCC and that SNAT2 (which is inhibited by V-9302) is also involved in this process and, probably, it is induced when ASCT2 is silenced to compensate the entry of glutamine into the cancer cell [3, 30].

Thus, based on the actual knowledge, glutamine cellular uptake and its metabolism appear to be essential for the development and progression of HNSCC [3]. Moreover, inhibiting ASCT2 and combining ASCT2 inhibitors and cetuximab may constitute an effective strategy for HNSCC treatment [3].

Colorectal carcinoma (CRC)

Suzuki et al. developed monoclonal antibodies recognizing ASCT2 (KM4008, KM4012, and KM4018 mAbs). They found that these antibodies inhibited the proliferation of colorectal cancer cells (WiDr cells), in vitro, by blocking glutamine uptake (Table 1) [13]. Therefore, ASCT2 inhibition could have an anti-tumor effect on colorectal carcinoma [13].

CRC is associated with several mutations, including KRAS mutation [9]. KRAS mutation occurs in several cancers (CRC, non-small-cell lung cancer, and pancreatic ductal adenocarcinoma) [91,92,93,94]. KRAS (codified by an oncogene and, when mutated, it contributes to cancer development) is a GTPase, which interferes with MAPK signaling pathway, contributing to cancer development, by propagating the signal from the extracellular environment to the nucleus and increasing the expression of proteins involved in tumorigenesis and cell proliferation [95]. It is necessary to create new treatments for tumors with this mutation since there are a limited number of effective drugs [9]. A very recent work investigated whether ASCT2 inhibition could interfere with the outcome of KRAS-mutated tumors [9]. It was verified that, in CRC cells with KRAS mutation, Ab3-8 mAb (a monoclonal antibody that targets human ASCT2) reduced the entry of glutamine into the cells in vitro (Table 1) and tumor development in vivo (Table 2) [9]. On the other hand, in CRC cells without KRAS mutation, Ab3-8 mAb was not effective in decreasing cell growth (Table 1) [9]. So, interference with the entry of glutamine into the intracellular space seems to be a good strategy to treat KRAS-mutated CRC [9].

Leukemia

The role of ASCT2 in hematopoiesis and in the development of leukemia is being investigated [11]. According to Ni et al., silencing of ASCT2 has a slight impact on hematopoiesis, in the absence of a stress factor, but, in contrast, it decreases the development and progression of leukemia [11]. According to these authors, the role of ASCT2 in tumorigenesis is not entirely justified for its association with glutamine transport, and other amino acids that are transported by ASCT2 are also important [11]. Moreover, although ASCT2 inhibition had a different impact on malignant and non-malignant cells, mainly interfering with the malignant ones, it was concluded that caution is necessary, when combining ASCT2 inhibition with chemotherapy that damages DNA [11]. This is because in the presence of the myelotoxic agent fluorouracil (5-FU), both in in vitro and in vivo models, glutamine starts to play an important role in hematopoiesis, even in non-malignant cells, and, consequently, combining these two therapies can be associated with adverse consequences (Tables 1 and 2) [11].

Gastric cancer

A new monoclonal antibody recognizing ASCT2—KM8094—appears to improve gastric cancer prognostic, causing cytotoxicity and suppressing tumor growth both in vitro (Table 1) and in vivo (using human xenograft-transfected mice) (Table 2) [12]. Moreover, combination of M8094 with docetaxel (an antineoplastic therapy used in gastric cancer) improves tumor outcome in vivo (Table 2) [12]. KM8094 was found to decrease the cellular entry of glutamine and intracellular glutathione levels, resulting in an increase in oxidative stress levels in vitro (Table 1) [12].

Similar results were obtained with the ASCT2 inhibitor GPNA or ASCT2 knockdown [64]. These treatments reduced glutamine uptake, decreasing glutathione production, increasing ROS level and significantly suppressing tumor cell growth, both in vitro (Table 1) and in vivo (Table 2) [64]. ASCT2 knockdown also induced apoptosis via the mitochondrial pathway [64].

Topotecan is a Topo I inhibitor, preventing DNA replication and RNA synthesis and being indicated to treat some cancers [64]. However, topotecan appears to perform anti-tumor effects on gastric cancer cells both by acting as a Topo I inhibitor and by interfering with cancer metabolism [64]. More specifically, it was found to cause ASCT2 downregulation and to reduce glutamine metabolism in vitro (Table 1), and a reduction of gastric cancer growth in vivo (Table 2) [64].

Hepatocellular carcinoma (HCC)

HCC is associated with a poor outcome [96]. Consequently, developing new therapeutic strategies is necessary. ASCT2 expression is increased in HCC cells and ASCT2 upregulation is directly related to tumor size and outcome in humans [96]. So, ASCT2-based therapies may be of interest for this type of cancer.

One of the most effective chemotherapeutic agents for HCC treatment is cisplatin [10]. However, some people with HCC relapse due to resistance to cisplatin [10]. Interestingly, the dietary polyphenol resveratrol was found to decrease ASCT2 expression and cell proliferation, potentiating cisplatin-induced cytotoxicity by causing apoptosis (Table 1) [10].

Non-small cell lung cancer (NSCLC)

ASCT2 expression is increased and directly related to tumor size, disease stage, lymphatic and vascular invasion, metastasis, and prognosis in patients with pulmonary adenocarcinoma [86]. So, ASCT2-based therapies may be also interesting for this type of cancer. In support of this, δ-tocotrienol was recently found to inhibit the glutamine transporters ASCT2 and LAT1, which prevents glutamine uptake into cancer cells, inhibiting the in vitro proliferation of malignant cells and increasing apoptosis, associated with mTOR pathway downregulation (Table 1) [63].

Clear cell renal cell carcinoma (ccRCC)

An increased expression of ASCT2 (SLC1A5) is related to a more advanced TNM (tumor, node, metastases) stage, higher Fuhrman degree and worst outcomes, in patients with ccRCC [85]. So, ASCT2 is an independent prognostic factor in ccRCC [85].

Breast cancer

The expression of glutamine metabolism-related proteins was found to differ according to human breast cancer phenotype, with ASCT2 expression being the highest in HER2 type (hormone-receptor negative (ER- and PR-negative) and HER2 positive) and lowest in luminal A type (ER- and/or PR-positive) and HER2 negative) human breast cancers [83]. However, our group recently verified that GPNA reduced glutamine uptake in both MCF-7 (ER + , PR + , and HER2−) and MDA-MB-231 (ER−, PR−, and HER2−) cells to a similar degree (by ± 70%) (Silva et al. submitted) (Table 1). Nevertheless, ASCT2 inhibition appears a promising strategy also in this type of cancer.

Other cancer types

Besides the cancer types mentioned above, targeting glutamine metabolism improves the prognosis of other cancers, including epithelial ovarian cancer and tongue cancer [97, 98].

Discussion and conclusions

Metabolic reprogramming is one of the hallmarks of cancer and favors tumor progression, being a mechanism for malignant cells adaptation [1, 2]. It is fundamental to provide the energy needs and biosynthetic precursors and to decrease oxidative stress levels in tumor cells [1]. To compensate for these greatest demands, an increase in the expression of some transmembrane transporters, including transporters for the nutrient glutamine, is observed in most cancer cells [3]. ASCT2 is a primary transporter of glutamine, being upregulated in malignant cells [3, 7]. So, the possibility of inhibiting this transporter, and consequently decreasing the entry of glutamine into the cell and its subsequent metabolism, has been investigated, and strategies have been developed targeting ASCT2 [3, 13, 47, 79,80,81].

As reviewed here, inhibition of glutamine cellular uptake, by inhibition/silencing of ASCT2, appears to reduce cancer cell proliferation and survival in vitro and tumor development, progression, disease progression and outcome in vivo. This association was verified for several distinct cancers, namely gastric cancer, colorectal carcinoma, breast cancer, prostate cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, liver cancer, among others [3, 42, 82,83,84,85,86]. Thus, inhibiting glutamine uptake and its metabolism, through ASCT2 inhibition/silencing, appears to be a promising strategy in antineoplastic therapy [3, 9,10,11,12,13, 63, 64].

ASCT2 inhibitors have been developed via different strategies: neutral amino acid analogs, acting as competitive inhibitors [8]; monoclonal antibodies targeting ASCT2 [9, 12, 13]; non-competitive ASCT2 inhibitors [75]; and other compounds causing ASCT2 downregulation [10, 64]. However, several limitations are presently known for research in this area, and these must be overcome in order that the full impact of ASCT2 in cancer therapy may be ascertained. First, the ASCT2 inhibitors currently known are not sufficiently potent or selective for this transporter, blocking not only ASCT2 but also other amino acid transporters [8]. These characteristics embarrass assessment of ASCT2 inhibition effects on cancer because the outcomes obtained do not result from an effective or selective inhibition of this transporter [8]. So, developing ASCT2-targeting therapeutic strategies to treat cancer has been hampered by the lack of effective and selective inhibitors [8]. Second, the development of this therapeutic strategy is troubled by the fact that different membrane transporters carry the same amino acids (in the specific case of glutamine, it is transported not only by ASCT2, but also by other transporters such as LAT1 and ASCT1 [8]). Moreover, and related to this fact, some reports concluded that ablation of ASCT2 resulted in an increased expression of other glutamine transporters in some cancers, namely SNAT1 and SNAT2, which are also able to mediate glutamine cellular uptake [99], and that this can be sufficient to supply the tumor needs, compensating for the silencing of ASCT2 [30]. So, cancer cells which have the ability to increase the expression of other glutamine transporters are less vulnerable to ASCT2 inhibition [99]. Third, in addition to its role in cancer, ASCT2 is also expressed in non-malignant tissues in our body: brain, lung, intestine, kidney, and skeletal muscle [8, 100, 101]. So, inhibition of ASCT2 might result in unwanted negative effects in non-cancer cells. Fourth, the role attributed to glutamine and, consequently, to ASCT2, appears to be different, depending on the tumor type [4, 100]. This may be due to not only the different origins of the tissues undergoing malignancy but also the tumor microenvironment [4, 100].

Despite these limitations, silencing/inhibition of ASCT2 appears to improve the outcome of different cancers by itself [3, 9, 11,12,13]. So, we believe that targeting ASCT2 may have a hopeful effect on the prognosis of different cancers, by interfering with the uptake and consequent metabolism of glutamine [1, 4, 7, 8, 29, 81]. Additionally, the combination of other anti-tumor therapies with ASCT2 inhibitors may be a very effective strategy to treat some cancers, improving cancer cell response to treatment, and this should be further investigated [3, 12]. So, more research is needed in this area, and of particular interest, the development of more effective and selective drugs to inhibit ASCT2 is a crucial topic [8].