1. SLC6A14 and SLC38A5 and Their Relevance to Cancer

Cancer cells exhibit increased demands for all amino acids, essential as well as non-essential. Since the essential amino acids cannot be generated de novo, these have to be obtained solely from extracellular sources. In contrast, the non-essential amino acids can be produced endogenously in cells from other precursors; nonetheless, cancer cells rely on extracellular sources to a significant extent even for these amino acids because their synthetic capacity does not meet the highly increased demands. Therefore, cancer cells upregulate specific amino acid transporters in their plasma membrane to mediate the entry of essential and non-essential amino acids from blood. To date, four distinct amino acid transporters have received increasing attention for their role in the promotion of cancer: SLC1A5 (also known as ASCT2), SLC7A5 (also known as LAT1), SLC7A11 (also known as x_c⁻), and SLC6A14 (also known as ATB^0,+) [46,47,48,49,50]. SLC38A5 (also known as SN2) [51,52] should also be added to this elite list prompted by the findings that it is a target for the c-Myc oncogene [53], but relatively much less is known on this transporter in cancer compared to the other four transporters. Among these five amino acid transporters that have received attention with regard to their biological relevance to cancer, SLC6A14 and SLC38A5 stand out in terms of their functional features that are ideal for the promotion of cancer (Table 1).

SLC6A14 and SLC38A5 mediate the transfer of their amino acid substrates in one direction, mostly into the cells in the influx mode (Figure 2). SLC6A14 is also known as ATB^0,+, referring to its identity with the amino acid transport system B^0,+ (B stands for “broad-specific”; uppercase letter stands for Na⁺-dependence; 0,+ in the superscript stands for the ability of the transporter to accept neutral and cationic amino acids as substrates). It is energized by three driving forces: a Na⁺ gradient, a Cl⁻ gradient, and membrane potential [54,55,56]. Therefore, this transporter primarily mediates the influx of its substrates into cells because of the high magnitude of the combined driving forces. SLC38A5 is also known as SN2 (i.e., the second isoform of the amino acid transport system N that is capable of accepting the amino acids glutamine, asparagine, and histidine, which contain nitrogen in the side chain; N stands for nitrogen in the side chain) and SNAT5 (sodium-coupled neutral amino acid transporter 5). It is also driven by a Na⁺ gradient, but interestingly it is an electroneutral transporter with no involvement of membrane potential. This is because of the coupling of the transport process with a transmembrane H⁺ gradient in which the transfer of Na⁺ and the amino acid substrate in one direction is functionally coupled to the transfer of H⁺ in the opposite direction [51,52,57]. As SLC38A5 accepts only neutral amino acids as its substrates, the transport process is electroneutral. Based on this operational mechanism, the transfer of amino acids and Na⁺ into cells via SLC38A5 leads to the removal of H⁺ from the cells, thus resulting in intracellular alkalinization [52]. Stated differently, SLC38A5 functions as an amino acid-dependent Na⁺/H⁺ exchanger. Since the transmembrane Na⁺ gradient is quantitatively the principal driving force for SLC38A5 under normal physiologic conditions, this transporter could also mediate the efflux of glutamine from the cells if the cells express high levels of glutamine synthetase, thus causing a concentration gradient for the amino acid across the plasma membrane in the outward direction. This is the case for the function of this transporter in astrocytes in the brain and Muller cells in the retina where SLC38A5 mediates the release of glutamine from the cells with subsequent uptake of glutamine by neurons; this is a part of the glutamine–glutamate cycle that operates between astrocytes/Muller cells and neurons [58,59]. However, the functioning of this transporter in the glutamine efflux mode is unique to astrocytes/Muller cells. This transporter is expected to function solely in the influx mode in cancer cells because of the avid utilization of glutamine by these cells, thus creating a concentration gradient for the amino acid across the plasma membrane that facilitates influx rather than efflux. In contrast to SLC6A14 and SLC38A5, the other three transporters related to cancer are all amino acid exchangers, meaning that the influx of one amino acid substrate into cells is coupled to the efflux of another amino acid substrate out of the cells [48]. Consequently, these three transporters are not ideal to meet the increased demands for amino acids in cancer cells because when the cells acquire one given amino acid via these transporters, they lose some other amino acid.

The substrate selectivity of SLC6A14 and SLC38A5 bears a key connection to glutaminolysis and serine–glycine–one-carbon pathway. Both transporters transport glutamine, serine, glycine, and methionine (Figure 2; Table 1). Some functional features do distinguish the two transporters. SLC6A14 transports 18 of the 20 proteinogenic amino acids, including all essential amino acids; as leucine is a potent activator of oncogenic mTOR signaling, the functional activity of this transporter is coupled to mTOR activation. Both of these features are essential for cancer cells to support their growth and proliferation. In contrast, the substrate selectivity of SLC38A5 is relatively restricted. This transporter can mediate the uptake of only glutamine, asparagine, histidine, serine, glycine, and methionine; this list of substrates contains all the four amino acids related to glutaminolysis and one-carbon metabolism. However, SLC38A5 exhibits a functional feature that is not seen with SLC6A14. The transport function of SLC38A5 is coupled to H⁺ efflux. As cancer cells generate massive amounts of lactic acid, there is an absolute need for these cells to prevent intracellular acidification, and SLC38A5 contributes to this process. The resultant intracellular alkalinization promotes the entry of the cells into the DNA-synthesis phase of the cell cycle and consequently accelerates cell proliferation [60,61]. It has been documented that SLC38A5 causes an increase in pH in the immediate vicinity of the plasma membrane on the cytoplasmic side of the cell in the presence of its amino acid substrates [52]. This effect is expected to promote another nutrient delivery system, known as macropinocytosis, in cancer cells. The NHE1 isoform (SLC9A1) of the Na⁺/H⁺ exchanger is known for its mitogenic potential with its role in the prevention of intracellular acidification as well as in the promotion of macropinocytosis [62]; inhibitors of NHE1 are widely used to block macropinocytosis [63]. The alkaline pH on the cytoplasmic surface of the plasma membrane influences the phosphorylation and activity of cytoskeletal scaffold proteins to initiate and promote macropinocytosis, a non-selective mode of uptake of nutrients from the extracellular milieu by “drinking” minute droplets of extracellular fluid [64,65,66,67]. As mentioned earlier, SLC38A5 functions as an amino acid-dependent Na⁺/H⁺ exchanger. Therefore, this transporter is expected to promote macropinocytosis in the presence of its amino acid substrates, such as glutamine, serine, glycine, asparagine, histidine, and methionine. It has already been documented that increased activity of macropinocytosis is a hallmark of certain types of solid tumors, particularly tumors associated with KRAS mutations [66,67].

4. SLC6A14 and SLC38A5 as Actionable Drug Targets for Cancer Therapy

SLC6A14 and SLC38A5 are cell-surface proteins that support amino acid nutrition in cancer cells. The amino acids supplied by these transporters serve not only as the building blocks for protein synthesis but also function as signaling molecules (e.g., mTOR activation) and provide substrates for the cancer-cell-specific metabolic pathways, such as glutaminolysis and serine–glycine–one-carbon pathway. As such, SLC6A14 and SLC38A5 drive cancer cell proliferation and tumor growth in specific types of solid tumors. In theory, if small molecules can be developed that have high affinity and high selectivity to inhibit the transport function of these transporters, such inhibitors could potentially have efficacy as anticancer drugs. This idea has already been validated with α-methyl-L-tryptophan as a fairly selective inhibitor of SLC6A14 [72]. It is important to note that α-methyl-L-tryptophan is not a transportable substrate for SLC6A14; it binds to the substrate-binding site of the transporter and blocks the transport function without itself being transported into the cells. As such, α-methyl-L-tryptophan is a blocker of the transporter rather than a competitive inhibitor. The IC₅₀ value for the L-enantiomer to block the transport function of SLC6A14 is ~10 μM. With this small molecule, published reports have demonstrated the therapeutic utility of SLC6A14 blockade in the treatment of multiple tumor types that are associated with upregulation of this transporter: breast cancer [72,73,74], pancreatic cancer [76,95], and colon cancer [69].

There have been some studies describing selective inhibitors of SLC38A5 [96]; the best inhibitor in terms of selectivity is glutamate-γ-hydroxamate. It is a competitive inhibitor of the transporter, but it is not known whether the compound is actually transported into cells via the transporter. Furthermore, the inhibitor shows low affinity with an IC₅₀ value of ~0.6 mM. Even though selective high-affinity inhibitors/blockers for SLC38A5 are not yet available, the experience with α-methyl-L-tryptophan and SLC6A14 indicates that a similar approach would also work for SLC38A5-positive tumors as well. Another feasible approach is to generate monoclonals specific to extracellular epitopes of these transporters that could be used to block the function of these cell-surface proteins. Even though it is widely acknowledged that amino acid metabolism comprising of numerous biochemical pathways is obligatory for cancer cell survival and that amino acid transporters play an essential role in supplying the amino acid substrates to feed into these pathways, the possibility of exploiting these transporter proteins as actionable drug targets for cancer therapy is just beginning to be recognized. If successful, this would represent a novel, hitherto unexplored, therapeutic approach for the treatment of cancer.