Amino acid transporter LAT1 (SLC7A5) as a molecular target for cancer
diagnosis and therapeutics
Yoshikatsu Kanai ⁎
Department of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
article info abstract
Available online xxxx
Editor: S.J. Enna
Cancer cells require a massive supply of nutrients, including sugars and amino acids—the upregulation of transporters for each nutrient contributes to meet the demand. Distinct from glucose transporters, amino acid transporters include ones whose expression is specific to cancer cells. For example, LAT1 (SLC7A5) displays protein
expression mostly limited to the plasma membrane of cancer cells. The exceptions are the placental barrier
and the blood-brain barrier, where immunohistochemical and mass spectrometric studies have shown LAT1 expression, although their levels are supposed to be lower than those in cancers. The expression of LAT1 has been
reported in cancers from various tissue origins, where high LAT1 expression is related to the poor prognosis of
patients. LAT1 is essential for cancer cell growth because the pharmacologic inhibition and knockdown/knockout
of LAT1 suppress the proliferation of cancer cells and the growth of xenograft tumors. The inhibition of LAT1 suppresses protein synthesis by downregulating the mTORC1 signaling pathway and mobilizing the general amino
acid control (GAAC) pathway in cancer cells. LAT1 is, thus, a candidate molecular target for the diagnosis and
therapeutics of cancers. 18F-labeled 3-fluoro-L-α-methyl-tyrosine (FAMT) is used as a LAT1-specific PET probe
for cancer detection due to the LAT1 specificity of α-methyl aromatic amino acids. FAMT accumulation is
cancer-specific and avoids non-cancer lesions, including inflammation, confirming the cancer-specific expression
of LAT1 in humans. Due to the cancer-specific nature, LAT1 can also be used for cancer-specific delivery of antitumor agents such as L-para-boronophenylalanine used for boron neutron capture therapy and α-emitting
nuclide-labeled LAT1 substrates developed for nuclear medicine treatment. Based on the importance of LAT1
in cancer progression, high-affinity LAT1-specific inhibitors have been developed for anti-tumor drugs. JPH203
(KYT0353) is such a compound designed based on the structure-activity relationship of LAT1 ligands. It is one
of the highest-affinity inhibitors with less affecting other transporters. It suppresses tumor growth in vivo without significant toxicity in preclinical studies at doses enough to suppress tumor growth. In the phase-I clinical
trial, JPH203 appeared to provide promising activity. Because the mechanisms of action of LAT1 inhibitors are
novel, with or without combination with other anti-tumor drugs, they could contribute to the treatment of cancers that do not respond to current therapy. The LAT1-specific PET probe could also be used as companion diagnostics of the LAT1-targeting therapies to select patients to whom therapeutic benefits could be expected.
Recently, the cryo-EM structure of LAT1 has been solved, which would facilitate the understanding of the mechanisms of the dynamic interaction of ligands and the binding site, and further designing new compounds with
© 2021 Elsevier Inc. All rights reserved.
Amino acid transporters
Amino acid signaling
Pharmacology & Therapeutics xxx (xxxx) xxx
Abbreviations: BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; BNCT, boron neuron capture therapy; L-BPA, L-para-boronophenylalanine; FAMT, 3-fluoro-L-α-methyltyrosine; FDG, 2-fluoro-2-deoxy-D-glucose; FET, O-(2-fluoroethyl)-L-tyrosine; IMT, 3-iodo-L-α-methyl-tyrosine; PET, positron emission tomography; SPECT, single photon emission
computed tomography; T3, triiodothyronine; cryo-EM, cryo-electron microscopy; lysoPC, lysophosphatidylcholine.
⁎ Corresponding author.
E-mail address: [email protected].
JPT-107964; No of Pages 16
0163-7258/© 2021 Elsevier Inc. All rights reserved.
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Please cite this article as: Y. Kanai, Amino acid transporter LAT1 (SLC7A5) as a molecular target for cancer diagnosis and therapeutics, Pharmacology & Therapeutics, https://doi.org/10.1016/j.pharmthera.2021.107964
Transporters are the membrane proteins that mediate the selective
transfer of organic and inorganic solutes through the plasma membrane
and membranes of intracellular organelles. They are essential for the
cellular uptake and epithelial absorption of nutrients such as sugars,
amino acids, lipids, vitamins, and minerals. They also contribute to the
absorption, distribution, metabolism, excretion, and toxicity of drugs
and xenobiotics (Hediger, Clémençon, Burrier, & Bruford, 2013). By
transporting substrates through the plasma membrane, transporters,
in conjunction with metabolic enzymes, strongly influence the distribution of their substrates across the membrane where the transporters are
localized and eventually contribute to the body’s disposition of nutrients and drugs. Therefore, the drugs that target transporters would
alter the distribution of their substrates in the body.
In pathological conditions, the distribution of compounds in the
body is affected by many diseases. For example, glucose, cholesterol,
and urate concentrations in blood elevate in patients with diabetes, hypercholesterolemia, and hyperuricemia, respectively. The drugs
targeting transporters to regulate their functions have, thus, been used
clinically to recover the normal distribution of compounds in the body
and restore the homeostatic states. Antidepressants, such as tricyclic
and tetracyclic antidepressants, selective serotonin reuptake inhibitors
(SSRI), and serotonin noradrenaline reuptake inhibitors (SNRI) inhibit
monoamine transporters in the brain. They are supposed to recover
monoaminergic neurotransmission at the synapses by inhibiting the
neurotransmitter reuptake transporters (Andersen, Kristensen, BangAndersen, & Strømgaard, 2009). Diuretics, furosemide, and thiazides
are the inhibitors of renal Na+ reabsorption transporters, which increase urinary Na+ content and decrease Na+ maintained in the body
(Giménez, 2006; Ko & Hoover, 2009). Ezetimibe, which decreases
blood cholesterol levels by inhibiting cholesterol absorption from the
intestine, has been proved to target the cholesterol transporter in the intestine (Altmann et al., 2004). Uricosuric agents, benzbromarone, probenecid, and losartan, inhibit renal urate reabsorption transporters,
which increase urinary excretion of urate and reduce blood urate level
(Enomoto et al., 2002). Similarly, recently developed anti-diabetic
SGLT2 inhibitors inhibit Na+/glucose cotransporter responsible for
renal reabsorption of glucose and reduce blood glucose level by increasing urinary glucose excretion (Bailey, 2011).
The efficient clinical effects of these transporter inhibitors have
established the importance of transporters as targets of therapeutic
drugs that treat pathological conditions involving the disrupted distribution of compounds in the body. This review article describes
the other aspect of transporter-targeting drugs: to suppress transporters of pathogenic cells to ameliorate the diseases, emphasizing
an amino acid transporter upregulated in cancer cells. Their specific
substrates and inhibitors could be used for cancer diagnosis and
2. Glucose and amino acid transporters in malignant tumors
Transporters do not just play roles in tissue functions such as absorption of nutrients and excretion of metabolites of endogenous and exogenous compounds through the epithelia. However, transporters are, as
described above, also essential for the cellular uptake of nutrients. In
rapidly growing cancer cells, a massive supply of sugars, amino acids,
and other nutrients is critical for continuous growth and proliferation.
The upregulation of transporters for each nutrient contributes to meet
the demand (Christensen, 1990). Tumor cells augment glucose uptake
via the upregulation of facilitative glucose transporters GLUT1
(SLC2A1) and GLUT3 (SLC2A3) (Wright, Loo, & Hirayama, 2011).
Recently it has been reported that some cancer cells express Na+/glucose cotransporters SGLT1 (SLC5A1) and SGLT2 (SLC5A2) to meet the
increased glucose requirement (Scafoglio et al., 2015). The upregulation
of GLUT1 is the basis for the use of its substrate, 2-[18F]fluoro-2-deoxyD-glucose (18F-FDG), as a probe for positron emission tomography (PET)
for the detection of tumors (Adekola, Rosen, & Shanmugam, 2012;
Ganapathy, Thangaraju, & Prasad, 2009; Szablewski, 2013). When
using 18F-FDG as a PET probe for cancer detection, the critical problem
is that malignant tumors and benign lesions such as granulomatous lesions and inflammatory tissues accumulate 18F-FDG, which causes falsepositive findings in the tumor diagnosis (Cook, Maisey, & Fogelman,
1999). The high physiologic background accumulation of 18F-FDG, particularly in the brain, hampers its use to diagnose brain tumors (Cook
et al., 1999). The lack of cancer specificity in the expression of GLUT1,
the primary route for 18F-FDG uptake into cancer cells, causes such
disadvantages of 18F-FDG PET in the diagnosis of malignant tumors.
In contrast to glucose transporters, amino acid transporters include
one that exhibits high cancer-specificity in its expression. That is, LAT1
(SLC7A5)1 upregulates in cancer cells, as described later in detail. Except
for LAT1, cancer cells upregulate various amino acid transporters (Bhutia,
Babu, Ramachandran, & Ganapathy, 2015; Bhutia & Ganapathy, 2016;
Ganapathy et al., 2009). They include ASCT2 (SLC1A5) and SNAT2
(SLC38A2) that mainly transport small neutral amino acids as well as glutamine; neutral and cationic amino acid transporter ATB0,+ (SLC6A14);
LAT3 (SLC43A1) transporting branched-chain amino acids and phenylalanine; acidic amino acid transporter EAAC1 (SLC1A1); cystine/glutamate
exchanger xCT (SLC7A11) (Babu et al., 2003; Ganapathy et al., 2009;
Wang et al., 2011; Hu et al., 2014; Bröer, Rahimi, & Bröer, 2016). xCT
that provides cancer cells with cystine has been considered important
from the therapeutic point of view. Cystine taken up by cancer cells via
xCT is converted to cysteine and used for glutathione synthesis essential
for cancer cells to survive against oxidative stress caused by radiation
therapies and chemotherapies (Lo, Wang, & Gout, 2008; Nagano,
1 Properties of amino acid transporters described in this article are briefly summarized
in Table 1.
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
Okazaki, & Saya, 2013). xCT binds with a variant isoform (CD44v) of the
cancer stem cell marker CD44 (Ishimoto et al., 2011). The biding of xCT
with CD44v stabilizes xCT on the plasma membrane of cancer stem
cells and maintains intracellular glutathione level, contributing to the
therapeutic resistance of cancer stem cells. Sulfasalazine that inhibits
xCT suppressed the growth of xenograft tumors and increased the sensitivity to anti-tumor drugs, which confirms the importance of xCT for the
survival of cancer cells against oxidative stress (Ishimoto et al., 2011). In
the clinical trial of sulfasalazine targeting CD44v-positive cancer stem
cells in advanced gastric cancers, some patients treated with sulfasalazine
exhibited reduced CD44v-positive cells and glutathione levels (Shitara
et al., 2017).
3. LAT1 as a cancer-type amino acid transporter
Among amino acid transporters upregulated in cancer cells, LAT1 is
conspicuous because of its cancer-specific expression, which lets us
consider it as a candidate molecular target for cancer treatment. Before
identifying LAT1 as an amino acid transporter, a partial nucleotide sequence of LAT1 designated TA1 (tumor-associated gene 1) was recognized for its cancer-associated expression (Sang, Lim, Panzica, Finch, &
Thompson, 1995). The embryo and tumors expressed TA1. Its expression was closely associated with tumor progression in rat hepatoma
models (Sang et al., 1995). The full-length cDNA encoding LAT1, the
functional protein, was initially identified by functional expression cloning from rat C6 glioma cells in search of the transporter covalently
linked with a single-membrane-spanning protein 4F2hc (4F2 heavy
chain, also known as CD98 or SLC3A2) (Kanai et al., 1998). 4F2hc was
once proposed as an activator or a regulator of unknown amino acid
transporters (Bröer, Bröer, & Hamprecht, 1997). Therefore, the functional expression cloning was performed by co-expressing 4F2hc and
cDNA library from C6 glioma cells, which revealed LAT1 as the transporter that becomes functional by forming a heterodimeric complex
with 4F2hc via a disulfide bond (Kanai et al., 1998). The heterodimer
formation proved to be essential for the proper or stable plasma membrane localization. LAT1 has, thus, become the first example of heterodimeric amino acid transporters (Fotiadis, Kanai, & Palacín, 2013).
Following LAT1, multiple amino acid transporters corresponding to
various amino acid transport systems were identified in this new category of transporters, which contributed a lot to reveal the molecular nature of classic amino acid transport systems. Six transporters (system L
transporters LAT1 and LAT2 (SLC7A8), system asc transporter Asc-1
(SLC7A10), system y+L transporters y+LAT1 (SLC7A7) and y+LAT2
(SLC7A6) and system x-C transporter xCT) have been identified to link
with 4F2hc, whereas system b0,+ transporter b0,+AT (SLC7A9) and glutamate/aspartate/cystine transporter AGT1 (SLC7A13) were found to
link with other single-membrane-spanning protein rBAT (SLC3A1)
structurally similar to 4F2hc (Fotiadis et al., 2013; Nagamori et al.,
2016a; Verrey et al., 2004). LAT1 transports leucine, isoleucine, valine,
phenylalanine, tyrosine, tryptophan, methionine, and histidine with
high affinity in a Na+-independent manner, corresponding to the properties of classically defined amino acid transport system L (Kanai et al.,
1998; Prasad et al., 1999; Yanagida et al., 2001). Most of its substrates
are essential amino acids.
The tissue expression of LAT1 was initially studied by northern blot
or PCR to measure the mRNA expression and by western blot to estimate the protein expression in the tissues (Kanai et al., 1998; Prasad
et al., 1999; Yanagida et al., 2001). As for transporters, however, they
do not provide sufficient information to estimate the functions in vivo.
It is essential to evaluate the protein localization on the plasma membrane by using, for example, specific antibodies in immunohistochemical analyses. In normal tissues of adult organs, LAT1 protein on the
plasma membrane has been reported in brain capillary endothelial
cells and in syncytiotrophoblasts forming a blood-brain barrier and placental barrier, respectively, although mRNA expression and nonplasma-membrane cytoplasmic staining were reported in some other
tissues (Gaccioli et al., 2015; Kageyama et al., 2000; Matsuo et al.,
2000; Ohgaki et al., 2017; Ritchie & Taylor, 2001). In liver and skeletal
muscle, LAT1 staining was reported, but its localization was predominantly in the cytoplasm (Elorza et al., 2012; Hodson et al., 2017). The
role of LAT1 in lysosomal membranes has been proposed to play an essential role in mTOR activation on the lysosomal membranes for the activation of mTORC1 recruited onto the lysosomal membrane (Milkereit
et al., 2015). However, LAT1 is at least not functional for cellular amino
acid uptake as long as it is not present on the plasma membrane. The expression of transporters should be carefully evaluated in terms of precise membrane localization.
In the developing placenta, a unique role of LAT1 was reported in the
cell fusion of trophoblasts (Ohgaki et al., 2017). LAT1, as a covalently
bound partner forming the heterodimeric complex, maintains 4F2hc,
which possesses a fusogenic function in trophoblastic cells, on the
plasma membrane and promotes syncytiotrophoblast formation by
contributing to cell fusion in the developing placenta (Ohgaki et al.,
2017). After all, LAT1 homo-knockout mice are embryonic lethal due
to the importance of LAT1 in placental formation and its essential role
in the growth and development of LAT1-expressing fetuses (Ohgaki
et al., 2017).
Many clinicopathological studies have shown that LAT1 is highly
expressed in cancer. Precise plasma membrane localization of LAT1 protein has been reported in primary cancers of various tissue origins such
as lung, pancreas, biliary tract, liver, breast, prostate, ovary, brain,
esophagus, stomach, oral cavity, uterus, skin, bone, and their metastatic
legions (Furuya, Horiguchi, Nakajima, Kanai, & Oyama, 2012; Honjo
et al., 2016; Ichinoe et al., 2011; Isoda et al., 2014; Kaira et al., 2007a,
2008a, 2008b, 2009a, 2012, 2013a, 2015; Kobayashi et al., 2008;
Namikawa et al., 2015; Nawashiro et al., 2006; Nikkuni et al., 2015;
Nobusawa et al., 2013; Sakata et al., 2009; Shimizu et al., 2015;
Toyoda et al., 2014; Watanabe et al., 2014). Interestingly, the rate of
LAT1 positive tumors is, in general, higher in squamous cell carcinoma
than adenocarcinoma. For example, in surgically-resected lung nonsmall cell carcinoma, high LAT1 expression was detected in 91% of squamous cell carcinoma and 29% of adenocarcinoma (Kaira et al., 2008a).
LAT1 expression is in general associated with tumor proliferation (Ki-
67 labeling index), angiogenesis, and poor prognosis (Honjo et al.,
2016; Ichinoe et al., 2011; Isoda et al., 2014; Kaira et al., 2008a, 2008b,
2012, 2015; Kaira, Oriuchi, Imai, et al., 2009a; Kaira, Sunose, et al.,
2013a; Kobayashi et al., 2008; Namikawa et al., 2015; Nawashiro et al.,
2006; Nikkuni et al., 2015; Nobusawa et al., 2013; Sakata et al., 2009;
Shimizu et al., 2015; Toyoda et al., 2014; Watanabe et al., 2014). In the
clinicopathological relevance study conducted on completely resected
pathologic stage I–III non-small cell lung carcinoma, the 5-year survival
rate of LAT1-positive patients (51.8%) was significantly worse than that
of LAT1-negative patients (87.8%; P < 0.001) (Kaira et al., 2008a).
Positive expression of LAT1 was an independent factor for predicting a
poor prognosis in the multivariate analysis (Imai et al., 2010; Kaira
et al., 2008a). Similarly, in the multivariate analysis, it has been shown
that high-LAT1 expression is a significant prognostic factor in stage I
pulmonary adenocarcinoma and stage I non-small cell lung cancer. It
is also a significant prognostic factor in biliary tract cancer, adenoid cystic carcinoma, tongue cancer, multiple myeloma, cutaneous melanoma,
hepatocellular carcinoma, and ovarian tumors (Imai et al., 2009; Isoda
et al., 2014; Kaira et al., 2013b, 2015; Kaira, Oriuchi, Imai, et al., 2009a;
Kaira, Sunose, et al., 2013a; Namikawa et al., 2015; Shimizu et al.,
2015; Toyoda et al., 2014).
4. Cancer specificity of LAT1 demonstrated by LAT1-specific PET
The cancer specificity of the expression of LAT1 has been proved not
only by immunohistochemistry but also by PET using a LAT1-specific
probe (Kaira, Oriuchi, Otani, Shimizu, et al., 2007a; Kaira et al., 2009b:
Wei et al., 2016b; Wiriyasermkul et al., 2012; Jin et al., 2020). The
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
LAT1-specific PET probe 3-fluoro-L-α-methyl-tyrosine (FAMT)2 was
generated by replacing iodine of 3-iodo-L-α-methyl-tyrosine (IMT)
with fluorine (Fig. 1). IMT has been used for single-photon emission
computed tomography (SPECT) and exhibited high cancer specificity
(Jager et al., 2001). FAMT and IMT are selective to LAT1 because of
their α-methyl moieties (Wiriyasermkul et al., 2012) (Fig. 2).
α-Methyl aromatic amino acids have been suggested to be
LAT1-selective, because α-methyl phenylalanine, α-methyl tyrosine,
and α-methyl DOPA inhibited LAT1 but less affected LAT2, a system L
transporter expressed in non-cancer cells (Khunweeraphong et al.,
2012; Morimoto et al., 2008). Similarly, FAMT and IMT selectively
inhibited LAT1 but not LAT2, whereas other amino acid probes developed for cancer imaging such as O-(2-fluoroethyl)-L-tyrosine (FET)
(Fig. 1), 3-fluoro-L-tyrosine, 2-fluoro-L-tyrosine, L-tyrosine, and
L-methionine were not LAT1-selective. They are transported by both
LAT1 and LAT2 (Wiriyasermkul et al., 2012). The LAT1-selectivity of
FAMT was further confirmed by uptake measurements using 14C-labeled FAMT. 14C-FAMT was transported only by LAT1 among
neutral amino acid transporters (Wei et al., 2016b). It was shown that
α-methyltryptophan is a non-transportable blocker of the broad scope
Na+-dependent amino acid transporter ATB0,+ (Karunakaran et al.,
2008). Although it has not been investigated whether FAMT, which is
also an α-methyl amino acid, inhibits ATB0,+, FAMT is at least not a
transportable substrate of ATB0,+ and any other amino acid transporters
excluding LAT1 (Wei et al., 2016b). Such a striking LAT1-selectivity
makes FAMT a LAT1-specific probe to detect LAT1 in PET imaging.
Clinical PET studies using LAT1-specific probe FAMT have been conducted on patients with cancers. In 18F-FAMT-PET, FAMT is specifically
accumulated in malignant tumors with low physiologic background
(Inoue et al., 2001; Kaira, Oriuchi, Otani, Shimizu, et al., 2007a). In the
brain showing high accumulation in 18F-FDG-PET, FAMT accumulation
is pretty low in contrast to tumors, although LAT1 is present in the
blood-brain barrier (Inoue et al., 1999). Consistent with this, the
amount of LAT1 protein in the isolated mouse brain capillaries is
much lower than that of GLUT1 (2.19 fmol/μg protein for LAT1 versus
90.0 fmol/μg protein for GLUT1) (Kamiie et al., 2008). So, 18F-FAMTPET can image brain tumors (Inoue et al., 1999). The level of FAMT accumulation in PET images is well correlated with the level of LAT1 protein
expression on the plasma membrane of cancer cells evaluated by immunohistochemistry (Kaira, Oriuchi, Otani, Shimizu, et al., 2007a). FMAT
does not significantly accumulate in non-cancer legions such as granulomatous lesions and inflammatory legions (Kaira et al., 2007b;
Nobusawa et al., 2013). In sarcoidosis, a granulomatous lesion, FDG is
accumulated at a high level, whereas FAMT accumulation in sarcoidosis
is similar to the background level (Kaira, Oriuchi, Otani, Yanagitani,
et al., 2007b). Remarkably, FAMT can differentiate cancers from inflammatory lesions. In oral cancer cases, it has been shown that the lymph
nodes with cancer metastasis are positive for both FDG-PET and
FAMT-PET, whereas the lymph nodes with inflammation are positive
for FDG but negative for FAMT (Nobusawa et al., 2013). These observations demonstrate the usefulness of LAT1-specific probes for cancer diagnosis with high specificity that distinguishes cancers from
granulomatous and inflammatory legions. In addition, they ultimately
confirm cancer-specific expression of LAT1 in humans.
Besides FAMT, other amino acid probes also exhibit low physiologic
background compared with FDG (Jager et al., 2001). For example, 11Cmethionine showing low brain background has been developed as a
PET probe to diagnose brain tumors. However, the accumulation of
11C-methionine is detected in the pancreas, liver, and inflammatory lesions because multiple amino acid transporters present in non-tumor
tissues and inflammatory lesions transport L-methionine (Deloar et al.,
1998; Rau et al., 2002; Salber et al., 2007; Wei et al., 2016b). Compared
with 11C-methionine, 18F-FET, similar to 18F-FAMT, shows lower
physiologic backgrounds and accumulates more selectively to malignant tumors because more limited transporters transport aromatic
amino acids compared with L-methionine (Jager et al., 2001). 18F-FET
still suffers from various levels of physiologic background (Deloar
et al., 1998; Pauleit et al., 2003). It accumulates in reactive astrocytes
around brain abscesses and shows physiologic uptake in skeletal muscle
(Pauleit et al., 2003; Salber et al., 2007; Stober et al., 2006). FET uptake in
skeletal muscle is supposed to be due to the uptake via LAT2 expressed
in the muscle (Pauleit et al., 2003, 2004). These observations further
confirm the importance of LAT1 selectivity for the cancer specificity of
5. Validation of LAT1 as a target of cancer therapy
Because of the cancer-specificity of expression, LAT1 has been proposed to be a molecular target for cancer diagnosis and therapeutics.
In order to validate LAT1 as the molecular target, it is crucial to know
how much LAT1 contributes to the cellular uptake of amino acids. In
T24 bladder cancer cells, the uptake of leucine was almost exclusively
mediated by LAT1 (Kim et al., 2002). Similarly, LAT1 is the primary
transporter responsible for leucine uptake in the other cancer cell
lines (Nagamori et al., 2016b). Because leucine triggers mTORC1-
mediated signaling, LAT1 is a proposed upstream of mTORC1 (Li, Yin,
Tan, Kong, & Wu, 2011; Shigemitsu et al., 1999; Xu et al., 1998). The inhibitors of LAT1 and knockdown of LAT1 reduced the phosphorylation
of p70 S6 kinase and 4E-BP1, downstream targets of mTORC1
(Nagamori, Wiriyasermkul, Okuda, et al., 2016b; Nicklin et al., 2009;
Yamauchi et al., 2009).
The importance of LAT1 in cell growth has been examined by gene
knockout and knockdown experiments. Homozygous LAT1-disrupted
cells generated by targeted gene disruption in chicken B cell-derived
DT40 cell line exhibited pretty slow growth and decreased colonyformation capacity in soft agar, indicating the essential roles of LAT1 in
the cell proliferation and occurrence of malignant phenotypes
(Ohkawa et al., 2011). Furthermore, the knockdown of LAT1 by siRNAs
resulted in a marked reduction of cell growth in human cancer cells
such as HCT116 cells (colon cancer), HeLa cells (cervical cancer), and
T24 cells (bladder cancer) (Ohkawa et al., 2011). Antisense oligonucleotides designed against LAT1 as well as system L inhibitors suppressed
the proliferation of tumor cells and the growth of xenograft tumors
(Cormerais et al., 2016; Kaira, Sunose, et al., 2013a; Marshall et al.,
2016; Najumudeen et al., 2021; Oda et al., 2010; Ohshima et al., 2016;
Quan et al., 2020; Rosilio et al., 2015), and prolonged the survival of
tumor-bearing mice (Nawashiro et al., 2006). Importantly, LAT1 inhibitors did not exhibit remarkable toxicity in vivo at the doses effective for
anti-tumor activity, supporting the usefulness of LAT1 as the target of
cancer therapeutics. Neurological symptoms were also not detected
probably because the other amino acid transporters could compensate
for the inhibition of LAT1 at the blood-brain barrier in the brain capillary
endothelial cells. However, a potential caution would be required to use
LAT1 inhibitors for cancer therapy because a recent study reported that
LAT1 in the blood-brain barrier might be compromised in children with
autism (Tărlungeanu et al., 2016).
In the LAT1-homo knockout cells and shRNA-induced knockdown
cells, the amount of 4F2hc, forming a heterodimeric complex with
LAT1, was markedly reduced on the plasma membrane (Cormerais
et al., 2016). Consistently, as mentioned above, LAT1 maintains 4F2hc
on the plasma membrane of the developing placenta via a LAT1′s
chaperone-like function maintaining 4F2hc protein level (Ohgaki
et al., 2017). Because 4F2hc engages β-integrin signaling and promotes
tumorigenesis (Fenczik, Sethi, Ramos, Hughes, & Ginsberg, 1997; Feral
et al., 2005; Hara, Kudoh, Enomoto, Hashimoto, & Masuko, 1999;
Poettler et al., 2013), it is essential to know whether the effects of
LAT1-knockout/knockdown were due to the decrease of 4F2hc or the
role of LAT1 itself in cell growth. It was, thus, examined which subunit
of the heterodimer (LAT1 or 4F2hc) carries the most prevalent
2 Available IC50, Ki, or Km values of the compounds described in this article are listed in
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
protumoral action (Cormerais et al., 2016). The knockout of LAT1, but
not 4F2hc, in human cancer cell lines, resulted in prominent in vitro
and in vivo tumor growth arrest. Therefore, LAT1 transport activity
was proposed as the crucial growth-limiting step of the heterodimeric
complex (Cormerais et al., 2016). These observations support the role
of LAT1 in cancer cell growth and advocate LAT1 to be a molecular target
for anti-cancer therapeutics.
6. Delivery of anti-tumor agents through LAT1
Because the expression of LAT1 is highly cancer-specific, LAT1 could
be used for the cancer-specific delivery of anti-tumor agents. For example, anti-tumor L-phenylalanine mustard melphalan (Fig. 1) was designed to improve the cellular uptake of nitrogen mustard by
connecting the nitrogen mustard to the para-position of the aromatic
ring of L-phenylalanine (Hosoya et al., 2008). Melphalan was
transported by LAT1, although other transporters, probably organic
anion transporters, were also proposed to contribute to cellular uptake
of melphalan (Kim et al., 2002; Lin et al., 2004; Shnitsar et al., 2009).
However, the velocity of melphalan transport was pretty low compared
with natural amino acid substrates (Kim et al., 2002; Uchino et al.,
2002). It was found that phenylglycine-mustard (Fig. 1), one-carbonshorter than melphalan, was transported at a much higher rate by
LAT1 than melphalan (Hosoya et al., 2008). Phenylglycine-mustard
was, thus, a better substrate of LAT1 and proposed to be more appropriate as a therapeutic drug. Because LAT1 contributes to the blood-brain
barrier permeation of aromatic amino acid drugs, although the level of
expression is supposed to be much less than that of GLUT1 as described
above (Kamiie et al., 2008), the adverse effects caused by the bloodbrain barrier permeation are necessary to be taken into consideration,
when the amino acid mustards become more permeable via LAT1. Melphalan is selective to LAT1 because of the bulky hydrophobic moiety
added at the para-position of the aromatic ring, as discussed later
(Morimoto et al., 2008) (Fig. 2). It is interesting to know whether
Fig. 1. Substrates of LAT1.
Except for high-affinity natural amino acid substrates (leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, histidine), the compounds shown in this figure are
transported by LAT1 as its substrates. FAMT, IMT and α-methyl-tyrosine with an α-methyl group are LAT1-specific substrates. Triiodothyronine is also LAT1-selective due to the bulky
hydrophobic moiety added at position 4 (para-position) of the aromatic ring (see Fig. 2). Gabapentin, a γ-amino acid, is also LAT1-selective. FET and L-BPA are not specific to LAT1.
LAT1 transports phenylglycine-mustard at a higher rate compared with melphalan.
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
phenylglycine-mustard is also LAT1-selective, not just from the therapeutic aspect but also from the structure-activity relationship point of
Another example of anti-tumor agent delivered to cancer cells via
LAT1 is L-para-boronophenylalanine (L-BPA) (Fig. 1), used for boron
neutron capture therapy (BNCT) (Barth et al., 2012). In BNCT, boron
10B) compounds such as L-BPA are loaded into cancer cells and, then,
the thermal neutron irradiation causes, in the cancer cells, a nuclear reaction that emits α-ray and lithium nuclei to destroy cancer cells. This
treatment is quite effective even for advanced cancer cases. Because
the range of travel of such particles is less than the size of cancer cells,
the influence of the treatment is not extended to the surrounding
non-cancer cells as long as non-cancer cells do not take up the boron
compounds (Barth et al., 2012). L-BPA is, however, not specific to LAT1
(Wongthai et al., 2015). LAT2 expressed in non-cancer cells transports
L-BPA. L-BPA is also a substrate of ATB0,+ expressed in the normal tissues
and detected in some cancer cells (Wongthai et al., 2015). The uptake of
L-BPA into cancer cells is mainly due to the high-affinity transport mediated by LAT1. In the cancer cells expressing a high level of ATB0,+, however, the contribution of lower affinity uptake by ATB0,+ becomes
significant when the concentration of L-BPA is increased (Wongthai
et al., 2015). The therapeutic dose of L-BPA is high enough that plasma
L-BPA concentration reaches the level at which ATB0,+ could contribute
to the uptake of L-BPA into cancer cells in addition to LAT1 when cancer
cells express a high level of ATB0,+ (Wongthai et al., 2015). Because
LAT2 and ATB0,+ are present in non-cancer cells, the LAT1-specific
boron compounds would be required to avoid the effects on normal
Recently, substrates of LAT1 labeled with α-emitting nuclides have
been developed, and attempts have begun to utilize the high cancer
specificity of LAT1 to deliver α-emitting nuclides in a cancer-specific
manner. Astatine-labeled phenylalanine 211At-Phe and 211At-AAMT in
which 18F of the LAT1-specific PET probe FAMT was replaced with 211At were generated (Kaneda-Nakashima et al., 2021; Watabe et al.,
2020). In the xenograft tumor model, they exerted a strong antitumor effect with a single intravenous injection, although the safety of
these compounds needs to be evaluated in the future. Since amino
acid derivatives may also be taken up by transporters other than
amino acid transporters, it is crucial to evaluate the systemic distribution. For example, FAMT, which has a structure similar to 211At-AAMT,
is highly specific for LAT1 among amino acid transporters but is recognized as an organic anion by the organic anion transporter OAT1 (Jin
et al., 2020; Wei et al., 2016a). It is, thus, taken up by the renal proximal
tubule where OAT1 is located in the kidney. From the structure-activity
relationship analysis of FAMT-related compounds, a compound design
that avoids interaction with OAT1 has also been proposed, contributing
to the production of compounds that are not distributed in normal organs, including kidneys, in the future (Jin et al., 2020).
7. Anti-tumor agents suppressing LAT1
As indicated above, the essential roles of LAT1 in cancer cell growth
have been established from various aspects. Furthermore, the expression of LAT1 exhibits high-specificity to cancer cells. Therefore, it is expected that the inhibitors of LAT1 could be developed as anti-tumor
drugs clinically used for cancer therapeutics. As an inhibitor of LAT1,
BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid) (Fig. 3) has
been used in in vitro and in vivo experiments to address the roles of
LAT1 in cancer cells (Christensen, 1990; He, Zhang, & Zhao, 2016; Imai
et al., 2010; Kaira, Sunose, et al., 2013a; Kanai et al., 1998; Lin et al.,
2004; Marshall et al., 2016; Ohshima et al., 2016; Wang et al., 2013).
BCH inhibits the cancer cell growth in vitro and suppresses the growth
of xenograft tumors in nude mice. BCH was originally established as
an inhibitor of cellular amino acid uptake in the 1960s (Christensen,
Handlogten, Lam, Tager, & Zand, 1969). Because of its bulky side chain,
BCH was supposed to inhibit specifically system L, a classically defined
amino acid transport system that prefers large neutral amino acids, including branched-chain and aromatic amino acids with bulky side
chains (Christensen, 1990). Now system L is known to be composed of
4 isoforms: LAT1 and LAT2 corresponding to system L1 and LAT3
(SLC43A1) and LAT4 (SLC43A2) corresponding to system L2 (Babu
et al., 2003; Bodoy et al., 2005; Bodoy, Fotiadis, Stoeger, Kanai, &
Palacín, 2013; Fotiadis et al., 2013; Kanai et al., 1998; Segawa et al.,
Amino acid transporters a).
Names Amino acid
Substrates c) Na+-dependence Involvement
LAT1 (SLC7A5) L (L1) Leu d), Ile, Val, Phe, Tyr, Trp, Met, His, DOPA, BCH, BPA, FAMT, Gabapentin No Yes
LAT2 (SLC7A8) L (L1) neutral amino acids, BCH No Yes
LAT3 (SLC43A1) L (L2) Leu, Ile, Val, Phe, amino alcohols No No
LAT4 (SLC43A2) L (L2) Leu, Ile, Val, Phe, amino alcohols No No
(SLC6A14) B0,+ Neutral and cationic amino acids Yes No
(SLC6A19) B0 Neutral amino acids Yes No
ASCT2 (SLC1A5) ASC Ala, Ser, Cys, Thr, Gln, Asn Yes No
(SLC38A2) A/N Ala, Asn, Cys, Gln, Gly, His, Met, Pro, Ser Yes No
(SLC7A10) asc Ala, Gly, Ser, Thr, Cys, α-aminoisobutyric acid, D-Ser No Yes
(SLC7A7) y+L cationic amino acids (Na+-independent), large neutral amino acids (partially
See “Substrate” in the left
(SLC7A6) y+L cationic amino acids (Na+-independent), large neutral amino acids (partially
See “Substrate” in the left
EAAC1 (SLC1A1) X−
A,G Glu, Asp, D-Asp Yes No
xCT (SLC7A11) x−
C cystine, Glu No Yes
(SLC7A9) b0,+ cationic amino acids, neutral amino acids, cystine No rBAT e)
AGT1 (SLC7A13) Asp, Glu, cystine No rBAT e)
a) The amino acid transporters described in this article are included in the table. This table is constructed based on the “SLC TABLES” (http://slc.bioparadigms.org).
b) Classically characterized amino acid transport systems (Christensen, 1990).
c) Representative substrates are listed.
d) Amino acids listed in the table are L-forms unless otherwise indicated.
e) b0,+AT and AGT-1 form heterodimers with rBAT instead of 4F2hc.
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
1999). LAT1 and LAT2 are functional as the heterodimeric proteins
forming a complex with 4F2hc as described above, whereas LAT3 and
LAT4 belonging to the distinct transporter family (SLC43) are functional
by themselves (Bodoy et al., 2013; Fotiadis et al., 2013). BCH inhibits all
these system L isoforms (Babu et al., 2003; Bodoy et al., 2005; Kanai
et al., 1998; Segawa et al., 1999). Furthermore, it has been known that
BCH is not a selective system L inhibitor. It also inhibits ATB0,+ from system B0,+ and B0
AT1 from system B0 (Bröer et al., 2004; Sloan & Mager,
1999). The disadvantage of using BCH as a system L inhibitor is not
just because of its less selective nature, but BCH is low affinity and requires high concentration to exert its inhibitor actions
(Khunweeraphong et al., 2012; Kim et al., 2002).
In order to develop anti-tumor agents targeting LAT1, it is required
to generate the compounds with high affinity and high selectivity to
LAT1. The compounds should not inhibit other transporters expressed
in non-cancer cells to avoid unexpected side effects. As indicated
above, α-methylated aromatic amino acids showing high-selectivity
to LAT1 could be the candidates of LAT1-specific inhibitor
(Khunweeraphong et al., 2012; Morimoto et al., 2008; Wei et al.,
2016b; Wiriyasermkul et al., 2012). α-Methylation, however, in general,
decreases the affinity to LAT1 (Khunweeraphong et al., 2012; Kim et al.,
2002; Uchino et al., 2002). The Ki value of α-methyl-tyrosine for LAT1
measured in T24 human bladder carcinoma cells was 153 μM, whereas
that of tyrosine measured under the same condition was 60.4 μM (Kim
et al., 2002). It was also found that gabapentin (Fig. 1), a γ-amino acid, is
transported by LAT1 but not by LAT2, suggesting that γ-amino acids
might be LAT1-selective (Kim et al., 2002; Morimoto et al., 2008)
(Fig. 2). However, the affinity of gabapentin to inhibit LAT1 was also
low (Table 2) (Kim et al., 2002; Su, Feng, & Weber, 2005; Uchino et al.,
2002). Therefore, other strategies to obtain LAT1-specific compounds
should be required to generate LAT1 inhibitors appropriate for clinical
The structure-activity relationship study was performed on phenylalanine derivatives and the related compounds to design high-affinity
compounds (Uchino et al., 2002). It was found in the study that, for
the compounds to interact with LAT1 as high-affinity inhibitors, they
Fig. 2. LAT1-selective compounds.
α-Methyl aromatic amino acids such as FAMT and γ-amino acid gabapentin are LAT1-selective substrates transported by LAT1, although the affinity is lower than natural amino acid
substrates. When O-linked bulky hydrophobic moieties are added at position 4 of the aromatic ring, the compounds become LAT1-selective non-transportable high-affinity blockers
such as JPH203). Triiodothyronine is also more like a blocker than a substrate because the transport rate is low (see text). Melphalan in which nitrogen mustard is added directly (without
intervening oxygen atom) at para-position is also selective to LAT1, although the affinity and the transport rate are low.
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
should possess a free carboxyl and an amino group, and the carbonyl oxygen of the carboxyl group must not participate in hydrogen bonding.
Furthermore, the hydrophobic interaction between the substrate side
chain and the substrate-binding site of LAT1 is crucial for substrate
binding. When Connolly accessible area of the compound becomes
large, or the compound has a highly hydrophobic side chain (with
high calculated logP value), the compound becomes a blocker rather
than a substrate of LAT1 (Uchino et al., 2002) (Fig. 2). Such structural requirements for the recognition of the compounds by LAT1 was
confirmed and further refined in the structure-activity relationship
study conducted on leucine derivatives and the related compounds as
follows: the compound to be a substrate of LAT1 should have carbonyl
oxygen and alkoxy oxygen of carboxyl group, intact amino group and
hydrophobic side chain, whereas carboxyl esters of amino acids could
be transported with decreased affinity (Nagamori, Wiriyasermkul,
Okuda, et al., 2016b) (Fig. 4). Therefore, to obtain high-affinity LAT1 inhibitors, the compounds should be designed to have an α-amino acid
structure with free carboxyl and amino groups and a hydrophobic
Fig. 3. LAT1 inhibitors.
JPH203 is a high-affinity LAT1-specific inhibitor. SKN101 and SKN102 are also LAT1-selective inhibitors: SKN101 with stronger inhibition on LAT1 than SKN102. KYT-0284 is a high-affinity
inhibitor, but it inhibits both LAT1 and LAT2. JX-009 is also supposed to inhibit both LAT1 and LAT2. JX-075, JX-078, and JX-119 are high-affinity LAT1 inhibitors. BCH is a less-selective lowaffinity inhibitor that inhibits all the system L isoforms and system B0,+, and system B0 transporters. 3,5-Diiodotyrosine, 3-iodo-L-tyrosine, fenclonine, and acivicin were identified through
comparative modeling, in silico virtual screening, and experimental validation (Geier et al., 2013). Because trans-stimulation effects were observed for fenclonine and acivicin, it was
suggested that they could have some properties as the substrates of LAT1 (Geier et al., 2013).
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
bulky side chain, where the carbonyl oxygen of the carboxyl group
should not be involved in intramolecular hydrogen bonding. Triiodothyronine (T3) (Fig. 1), a thyroid hormone, is the one that meets such
requirements and, in fact, exhibits the highest affinity to inhibit LAT1
among naturally occurring compounds (Table 2) (Kim et al., 2002;
Uchino et al., 2002). Fortunately, T3 was not just a high-affinity inhibitor
of LAT1, but it was also selective to LAT1 with less affecting LAT2-
mediated transport (Khunweeraphong et al., 2012; Morimoto et al.,
2008) (Fig. 2).
Based on the structure of T3, structural development was conducted
to increase the affinity to inhibit LAT1 (Endou, Kanai, Tsujihara, & Saito,
2008; Wempe et al., 2019). Among the compounds synthesized, JPH203
(KYT-0353) and KYT-0284 (Fig. 3) were representative ones with distinctive properties (Oda et al., 2010). Both compounds possess high af-
finity (more than a thousand times higher than BCH; several tens times
higher than T3) to inhibit LAT1. JPH203 is highly selective to LAT1 with
less inhibiting LAT2 (more than 500 times selective to LAT1 based on
IC50 values), whereas KYT-0284 equally inhibited both LAT1 and LAT2
(Table 2) (Oda et al., 2010). Although extensive studies are required
to assess compound selectivity among transporters, JPH203 can distinguish at least structurally and functionally similar transporters such as
LAT1 and LAT2. Based on the structure-activity relationship of the series
of compounds, it was suggested that the addition of O-linked hydrophobic bulky moiety at para-position (position 4) of the aromatic ring (see
JPH203) makes the compound LAT1-specific (Fig. 2). SKN101, whose
side chain was extended by adding O-linked hydrophobic bulky moiety
to the para position (Fig. 3), also showed LAT1-specific inhibition without acting on LAT2. Interestingly, SKN102, with a terminal benzene ring
added at different position on the naphthalene ring (Fig. 3), showed a
significantly reduced inhibition on LAT1 compared with SKN101
(Kongpracha et al., 2017). It is noted that melphalan, in which nitrogen
mustard is added directly (without intervening oxygen atom) at paraposition, is also selective to LAT1, although the affinity of melphalan to
inhibit LAT1 is lower (Table 2; Fig. 2). In contrast, the addition of Olinked hydrophobic bulky moiety at meta-position (position 3) of the
aromatic ring (for example, KYT-0284 in Fig. 3) makes the compound
high-affinity to both LAT1 and LAT2 (Table 2). Consistently, JX-009
(Fig. 3), whose O-linked side chain is extended at the meta-position of
the benzene ring, was supposed to inhibit both LAT1 and LAT2 with
high affinity (Zaugg et al., 2020). New series of compounds, bicyclic
meta-tyrosine derivatives JX-075, JX-078, and JX-119 (Fig. 3) were developed as LAT1 inhibitors. These compounds inhibited LAT1 at a pretty
high affinity, whereas selectivity to LAT1 was not reported yet (Table 2).
JPH203 effectively inhibited tumor cell growth in vitro and suppressed
the tumor growth at lower doses in vivo than BCH, which establishes
the concept of LAT1-specific inhibitors as anti-tumor agents (Oda
et al., 2010).
8. The inhibitors and synthetic substrates of LAT1
Besides the aforementioned LAT1 ligands, other compounds that interact with LAT1 have also been reported. Employing comparative
modeling, in silico virtual screening, and experimental validation, 3,5-
L-diiodotyrosine, 3-iodo-L-tyrosine, fenclonine, and acivicin (Fig. 3)
were identified as new LAT1-ligands that inhibit LAT1 (Geier et al.,
2013). Among them, acivicin and 3-iodo-L-tyrosine suppressed tumor
cell growth. Because all of these compounds possess α-amino acid
structures, they were supposed to inhibit LAT1 by binding to the
substrate-biding site of LAT1. In another series of approaches to find
the compounds with potent and prolonged inhibition of the transporter,
the compounds based on dithiazole and dithiazine scaffold were
screened in the proteoliposome experimental model (Napolitano
et al., 2017). In the screening, two compounds indicated in Fig. 5 were
identified to inhibit LAT1 potently (IC50 < 1 μM) with mixed type
inhibition in their inhibition kinetics due to the disulfide bond
formation between the compounds and LAT1. Molecular docking and
Kinetic parameters of compounds.
Compounds Kinetic parameters References
Leucine Km, 80.3 μM Km, 318 μM Morimoto et al., 2008
FAMT Km, 72.7 μM No transport Wei et al., 2016a
α-Methyl-tyrosine Ki, 90.7 μM Ki, 839.9 μM Khunweeraphong et al., 2012
L-BPA Km, 20.3 μM Km, 88.3 μM Wongthai et al., 2015
Gabapentin Ki, 191 μM No inhibition Kim et al., 2002
Morimoto et al., 2008
Triiodothyronine Ki, 7.3 μM No inhibition Kim et al., 2002
Khunweeraphong et al., 2012
Melphalan Ki, 101 μM No inhibition Hosoya et al., 2008
Morimoto et al., 2008
Phenylglycine-mustard Ki, 231 μM N.D.a) Hosoya et al., 2008
JPH203 (KYT-0353) IC50, 0.14 μM No inhibition Oda et al., 2010
KYT-0284 IC50, 2.0 μM IC50, 0.45 μM Oda et al., 2010
JX-075 IC50, 0.165 μM N.D. Yan et al., 2021
JX-078 IC50, 0.121 μM N.D. Yan et al., 2021
JX-119 IC50, 0.234 μM N.D. Yan et al., 2021
BCH Ki, 78.3 μM Ki, 150.5 μM Khunweeraphong et al., 2012
3,5-Diiodotyrosine IC50, 7.9 μM N.D. Geier et al., 2013
acivicin IC50, 340 μM N.D. Geier et al., 2013
a) N.D. indicates “not determined”.
Fig. 4. Proposed important moieties for the recognition by LAT1.
The important moieties for the recognition by LAT1 are proposed on the structure of
leucine. The compounds recognized by the substrate-binding site of LAT1 should have
carbonyl oxygen and alkoxy oxygen of carboxyl group (blue), amino group (purple),
and hydrophobic side chain (green). Alkoxy oxygen is preferably closer to carbonyl
oxygen (shown in orange), although hydroxamic acid could be accepted with lower
affinity (Nagamori, Wiriyasermkul, Okuda, et al., 2016b; Zur et al., 2016).
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
site-directed mutagenesis study of LAT1 revealed that the compounds
form a covalent bond with the residue C407 in the proposed
substrate-binding site of LAT1. Such compounds exhibited prolonged
inhibition of LAT1-mediated transport and potent suppression of
tumor cell growth (Napolitano et al., 2017). For the use of these compounds as anti-tumor agents, selectivity to LAT1 should be established
to avoid unexpected side effects.
Another approach to obtain LAT1 ligands is to produce amino acid
prodrugs transported by LAT1, which can be possible by taking advantage of the broad substrate selectivity of LAT1 (Nagamori,
Wiriyasermkul, Okuda, et al., 2016b; Uchino et al., 2002). Several
amino acid prodrugs have been proposed to use LAT1 for efficient
blood-brain barrier permeation. Because LAT1 can accept various neutral side chains and carboxyl esters of amino acids (Nagamori,
Wiriyasermkul, Okuda, et al., 2016b; Uchino et al., 2002), many strategies are possible to design LAT1 ligands that could be used for prodrugs,
depending on their affinity and transport velocity. For example,
ketoprofen was conjugated to L-lysine via a terminal amino group of
side-chain to mimic a neutral amino acid with a bulky side chain to become a LAT1 substrate (Gynther et al., 2010) (Fig. 6). Similarly,
ketoprofen conjugated to L-phenylalanine at meta-position was
transported by LAT1 (Kärkkäinen et al., 2018). Valproic acid was conjugated to the aromatic ring of L-phenylalanine at meta-position with a
methylene group between the amide bond and aromatic ring of Lphenylalanine to make it transported by LAT1 (Gynther et al., 2016;
Kärkkäinen et al., 2018) (Fig. 6). Interestingly, the meta-conjugation of
L-phenylalanine was shown to increase its affinity for LAT1 evaluated
by IC50 compared with para-conjugation or the conjugation to an aliphatic amino acid moiety (Kärkkäinen et al., 2018). Although carboxyl
ester of isoleucine and quinidine was once shown to be transported by
LAT1 (Patel et al., 2013), it is now proved to have no affinity for LAT1
(Rautio, Kärkkäinen, Huttunen, & Gynther, 2015). Furthermore,
hydroxamic acid compounds can be the substrates of LAT1, although
the affinity to LAT1 seems less than corresponding amino acid esters
(Zur et al., 2016).
9. Substrate binding site of LAT1 identified in the cryo-EM structure
The heterodimer structure of LAT1 and 4F2hc has recently been
solved by cryo-electron microscopy (cryo-EM) analysis. Based on the
substrate-bound structures, the substrate-binding site of LAT1 and the
binding-related interactions have been revealed (Fig. 7) (Lee et al.,
2019; Newstead, 2019; Yan et al., 2021; Yan, Zhao, Lei, & Zhou, 2019).
From the crystal structure of bacterial homologs, it has been speculated
that LAT1 has a so-called LeuT fold structure, one of the typical structural folds of transporters (Newstead, 2019). The cryo-EM analysis of
LAT1 has confirmed this. In the LeuT fold structure, transmembrane helices 1 and 6 unwind in the middle portion of the plasma membrane,
which is involved in substrate binding (Fig. 7) (Newstead, 2019).
Based on the crystal structure of the substrate-bound bacterial homolog
and the BCH-bound LAT1 cryo-EM structure, it has been proposed that
the carboxyl group of the substrate amino acid binds to the backbone
of the unwound portion of the transmembrane helix 1 primarily
through hydrogen bonds. On the other hand, the amino group of the
substrate amino acid binds to the backbone of the unwound portion of
the transmembrane helix 6 (Lee et al., 2019; Newstead, 2019; Yan
et al., 2019). Gly255, contained in the unwound portion of the helix 6
to which the amino group of the substrate amino acid binds in its backbone, corresponds to a residue having a bulky side chain in bacterial homologs with narrow substrate selectivity (Lee et al., 2019). Therefore, it
was suggested that the space created by the small side chains of Gly255
(H atom only) could contribute to the pockets where LAT1 could accommodate the bulky side chains of the substrates. To test the involvement
of Gly255 in substrate recognition, a site-directed mutant that converts
Gly255 to Ala was analyzed (Lee et al., 2019). The G255A mutation introduced a methyl group into the space that should accept the side
chain of substrate amino acids, narrowing that space and reducing the
Fig. 5. LAT1 inhibitors covalently bound to LAT1.
The compounds shown are covalently bound to LAT1 via a disulfide bond and exhibit potent and prolonged inhibition (Napolitano et al., 2017). IC50 values were reported as 0.98 μM and
0.89 μM for compounds in the left and right, respectively (Napolitano et al., 2017).
Fig. 6. Amino acid-conjugate prodrugs as substrates of LAT1.
Lysine-ketoprofen prodrug (Gynther et al., 2010), phenylalanine-ketoprofen prodrug
(Kärkkäinen et al., 2018), and valproic acid-phenylalanine prodrug (Gynther et al.,
2016) have been designed as substrates of LAT1. Km and Vmax of the lysine-ketoprofen
prodrug of rat brain uptake in rat brain perfusion study were reported to be 231.6 μM
and 1.50 pmol/mg/min, respectively (Gynther et al., 2010).
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
uptake of amino acids with large side chains such as tryptophan. Thus,
this portion adjacent to Gly255 was confirmed to be involved in the
structure that accepts the large side chains of substrate amino acids
(Lee et al., 2019). As expected, the G255A mutation increased the uptake of amino acids with small side chains like alanine, initially not a
high-affinity substrate for LAT1 (Lee et al., 2019). The G255A mutation
was supposed to narrow the side chain binding space to accept amino
acids with small side chains (Lee et al., 2019). This observation further
verified that the relatively wide pocket around Gly255 contributed to
accepting the side chains of substrate amino acids.
As already mentioned, it is assumed that hydrophobic interaction
between the side chain of the LAT1 ligand, which is an amino acid derivative, and the substrate-binding site is essential. Therefore, the inhibitors have been designed to make the side chains bulky and
hydrophobic. When the cryo-EM structure of LAT1 was unraveled, it
was confirmed that there was a large hydrophobic pocket containing
the space around Gly255 involved in side-chain recognition (Fig. 7)
(Lee et al., 2019). It was assumed that this pocket would accept the
large hydrophobic side chains of high-affinity inhibitors. In the recently
reported LAT1 cryo-EM structure, high-affinity LAT1 inhibitors JX-075,
JX-078, and JX-119 bind, and the proximal part of their side chains
close to the amino and carboxyl groups located at the proposed
substrate-binding site with the carboxyl and amino groups binding to
unwound portions of the helix 1 and helix 6, respectively (Yan et al.,
2021). However, unexpectedly, distal parts of the side chains were
bent and bound to a site other than the assumed hydrophobic pocket
(Fig. 8). Unlike regular α-amino acids, these inhibitors have a bicyclic
meta-tyrosine structure in the basic skeleton. Therefore, further structural analysis is awaited on how high-affinity inhibitors with α-amino
acid structures such as JPH203 bind to LAT1.
10. Use of LAT1 as a molecular target for cancer diagnosis and
As mentioned above, the expression of LAT1 protein in the plasma
membrane shows high specificity for cancer cells, except for the
blood-brain barrier and placental barrier. Such high cancer specificity
makes LAT1 unique among cancer-upregulated amino acid transporters. Furthermore, LAT1 is responsible for supplying cancer cells
with many essential amino acids, including leucine that activates
mTORC1 to regulate cancer cell growth. In colon cancer, it was shown
that LAT1 upregulates under the strong drive by Myc oncogene product
and is responsible for the global metabolic reprogramming associated
with cancer development (Satoh et al., 2017). Therefore, LAT1 has
been proposed as a molecular target of cancer diagnosis and therapeutics. LAT1-specific PET probe FAMT has been demonstrated to be
cancer-specific to discriminate cancers from non-cancer lesions, including inflammation. LAT1-specific PET probes, thus, have, potentials to be
developed as a “post-FDG” PET probe that could solve the issues accompanied with the use of conventional FDG-PET (Jager et al., 2001). The
cancer-specific accumulation of LAT1-specific probe FAMT finally supports the cancer-specific expression of LAT1 in humans. The LAT1-
specific PET probe is not only useful as a cancer diagnosis with fewer
false-positive findings, but it could also be used as companion diagnostics of the LAT1-targeting therapies for the selection of patients to
whom therapeutic benefits could be expected. In FDG, its advantage as
a PET probe is the high accumulation of radioactivity in cancer cells
due to the high expression of GLUT1 in cancer cells and the trapping
and accumulation of FDG metabolite (FDG-6-phosphate) into the cancer cells (Szablewski, 2013). The LAT1-specific PET probe FAMT is an
α-methyl amino acid and was developed to assume that it is not metabolized intracellularly and exhibits high accumulation (Inoue et al.,
Fig. 7. The cryo-EM structure of substrate binding site of LAT1.
The structural model of substrate binding site of LAT1 (modified from Lee et al., 2019) was
constructed from PDB data 6JMQ. Transmembrane helices 1 and 6 unwind in the middle so
that they are divided into helices 1a/1b and 6a/6b, respectively. The carboxyl group of the
substrate amino acid binds to the backbone of the unwound portion of the
transmembrane helix 1 involving G65, S66, and G67 primarily through hydrogen bonds.
The amino group of the substrate amino acid binds to the backbone of the unwound
portion of the transmembrane helix 6 involving G255. There is a large hydrophobic
pocket surrounded by hydrophobic side chains of F400, W405, V408, and W257,
containing the space around G255 involved in side-chain recognition (see text).
Fig. 8. LAT1-bound structures of JX-075, JX-078, and JX-119.
The structures of JX-075, JX-078, and JX-119 were constructed from PDB data of
compound-bound LAT1 structures (7DSK, 7DSL, and 7DSN, respectively). The structures
of the three compounds are overlaid and shown at the top. The proximal part of their
side chains, close to the amino and carboxyl groups (Fig. 3), is located at the proposed
substrate-binding site. Their carboxyl and amino groups are bound to unwound portions
of helix 1 and helix 6, respectively (see Fig. 7). The distal parts of the side chains are,
however, bent and bound to a site other than the large hydrophobic pocket (indicated
with a dotted circle; also see Fig. 7) adjacent to the substrate-binding site.
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
1999). However, its metabolism has not been fully studied. Tumor accumulation of 18F-FAMT compared in the same patient has been reported
to be lower than that of 18F-FDG (Kaira, Oriuchi, Otani, Shimizu, et al.,
2007a; Kaira, Oriuchi, Shimizu, et al., 2009b). Comparing LAT1 and
GLUT1 expression in tumors and the analysis of the metabolism of the
probe, affecting PET probe accumulation in tumor cells, are required
for further study.
Taking advantage of cancer-specific expression and essential roles in
cancer cell growth, LAT1 could be a therapeutic target. Because LAT1 is a
transporter, it would be used as a route for delivering anti-tumor agents
into cancer cells as discussed above; melphalan, L-BPA used for BNCT,
and α-ray emitting 211At-labeled LAT1 substrates for nuclear medicine
treatment are examples of using LAT1 for drug delivery to cancer cells.
Finally, the biggest challenge would be developing LAT1 inhibitors
that can be used clinically as anti-tumor drugs. Based on the
structure-activity relationship analysis of LAT1 ligands, a high-affinity
LAT1-specific blocker JPH203 was generated. It is among the compounds with the highest affinity to LAT1 developed so far and highly selective to LAT1, supposed to avoid acting on other transporters. The
first-in-human phase I clinical trial of JPH203 was recently conducted
in patients with advanced solid tumors. JPH203 appeared to be welltolerated and to provide promising activity against biliary tract cancer
(Okano et al., 2020).
LAT1-specific high-affinity inhibitors are expected to be beneficial in
cancer treatment, given the critical role of LAT1 in cancer cell proliferation and cancer progression demonstrated in preclinical studies. In combination with other anti-tumor agents, synergic effects would
furthermore be expected because the mechanisms of action of LAT1 inhibitors are novel and distinct from other anti-tumor drugs. The synergic effect of LAT1 inhibitors (or knockdown/knockout) and anti-tumor
drugs such as cisplatin, gemcitabine, 5-FU, gefitinib, and bicalutamide
has been reported (Imai et al., 2010; Kaira, Sunose, et al., 2013a;
Ohshima et al., 2016; Xu et al., 2016; Yamauchi et al., 2009). Recently,
a significant reduction in cell proliferation was observed in the combination of JPH203 with cell cycle-related kinase inhibitors such as
dinadiclib, whose targets were inactivated by LAT1 inhibition in cholangiocarcinoma cells (Okanishi, Ohgaki, Okuda, Endou, & Kanai, 2021).
The combination would also be beneficial to reduce the dose of combined anti-tumor drugs to decrease their side effects. Another aspect
of combined treatment is the potential use of LAT1 inhibitors with the
inhibitors of other amino acid transporters such ASCT2. It has been demonstrated that ASCT2 is one of the proteins in the macromolecular assembly involving LAT1-4F2hc heterodimeric complex, where LAT1
and ASCT2 cooperate efficiently in the “transportsome” (Fuchs & Bode,
2005; Xu & Hemler, 2005). It has been proposed that ASCT2 transports
glutamine into the cells to provide glutamine to the intracellular
substrate-binding site of LAT1 and promotes obligatory exchange transport in which LAT1 takes up essential amino acids into the cell in exchange for releasing glutamine, thereby supporting the function of
amino acid exchanger LAT1. (Fuchs & Bode, 2005; Nicklin et al., 2009;
Yanagida et al., 2001). ASCT2 inhibition itself also suppressed cancer
cell growth by the mechanisms distinct from LAT1 (Cormerais et al.,
2018). The combination of inhibitors of LAT1 and ASCT2 is, thus, expected to enhance their anti-tumor actions in a synergic manner. In
this combination, the caner specificity of LAT1 is also the basis of the
use of the inhibitors to reduce side effects on normal tissues because
other transporters, including ASCT2, are less cancer-specific.
11. Anti-tumor effects of LAT1 inhibitors
Many studies have shown that LAT1 regulates mTORC1 activity upstream of mTORC1 by being responsible for leucine uptake into tumor
cells (Li et al., 2011; Nagamori, Wiriyasermkul, Okuda, et al., 2016b;
Nicklin et al., 2009). Therefore, LAT1 inhibitors suppress the downstream effectors of mTORC1, including ribosomal protein S6 kinase
p70S6K, to inhibit translation initiation (Fig. 9A) (Liu & Sabatini,
2020). At the same time, LAT1 inhibitors are also supposed to cause
the accumulation of uncharged tRNAs due to intracellular amino acid
deprivation and activate the general amino acid control (GAAC) pathway (Fig. 9A). The uncharged tRNAs activate general control
nonderepressible 2 (GCN-2) kinase and induce phosphorylation of
eIF2α, causing a global down-regulation of translation (Fig. 9A) (Baird
& Wek, 2012; Kilberg, Shan, & Su, 2009). In this way, LAT1 inhibitors
suppress protein synthesis and inhibit tumor cell growth. It was
shown that LAT1 inhibitors cause the accumulation of G0/G1 phase
cells (Okanishi et al., 2021). Combined proteomics and
phosphoproteomics performed on cultured tumor cells (cholangiocarcinoma cells) treated with JPH203 revealed many changes in the signaling
Fig. 9. The effects of LAT1 inhibitors on signaling pathways regulating translation
A, Downstream signaling affected by LAT1 inhibitors. LAT1 inhibitors suppress the
downstream effectors of mTORC1, including p70S6K, to inhibit translation initiation. At
the same time, LAT1 inhibitors cause the accumulation of uncharged tRNAs and activate
the general amino acid control (GAAC) pathway. Uncharged tRNAs activate GCN-2 and
induce phosphorylation of eIF2α, causing a global down-regulation of translation.
B, Crosstalk between growth factor signaling and amino acid signaling. The LAT1-
mediated amino acid signaling is independent of the PI3K-Akt axis downstream of
growth factor receptors and is supposed to recruit mTORC1 onto the lysosomal
membrane in order for mTORC1 to be activated by the input from the growth factor
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
pathways due to secondary and tertiary effects following the suppression of the mTOR system. Cell cycle regulators such as the cyclindependent kinases CDK1 ~ 6 and AURKA were identified as the most
downregulated upstream kinases, explaining the suppression of cell
proliferation by JPH203 (Okanishi et al., 2021).
As already mentioned, LAT1 is expressed in the brain capillary endothelial cells that make up the blood-brain barrier, whereas the endothelial cells of other normal tissues do not express LAT1. However, certain
stimuli can induce LAT1 expression in endothelial cells separate from
the blood-brain barrier. Previously, it was shown that vascular endothelial cells HUVEC upregulate LAT1 upon the stimulation with
lysophosphatidylcholine (lysoPC), a component of oxidized LDL
(Takabe et al., 2004). Inhibition of LAT1 by BCH suppressed the release
of lysoPC-induced inflammatory cytokines such as IL-6 and IL-8 from
HUVEC, probably because LAT1 inhibition suppressed protein synthesis.
It was suggested that upregulation of LAT1 by lysoPC contributes to the
early process of arteriosclerosis. LAT1 was actually expressed in the endothelial cells of LDL receptor-deficient mice fed a high-fat diet but not
in a standard diet (Takabe et al., 2004).
Since it was suggested that the expression of LAT1 is induced by
stimulating vascular endothelial cells, the expression of LAT1 in the endothelial cells of tumor blood vessels was then examined. Vascular endothelial cells of pancreatic cancer tissues highly expressed LAT1,
whereas those of normal human pancreatic tissue did not (Quan et al.,
2020). This is because angiogenic growth factors VEGF-A and FGF2 released in the cancer microenvironment induce LAT1 expression in vascular endothelial cells. Pharmacologic inhibition and genetic disruption
of LAT1 in vascular endothelial cells suppressed migration, invasion, and
tube formation of endothelial cells and suppressed angiogenesis. In addition, inhibition and knockout of LAT1 suppressed tumor angiogenesis
in xenograft and allogeneic transplant models. Furthermore, LAT1
knockout specific for vascular endothelial cells showed that LAT1
expressed in vascular endothelial cells contributes to tumor growth
(Quan et al., 2020). In analyzing the effects of amino acid transporter inhibitors on vascular endothelial cells, important crosstalk between
growth factor signaling and amino acid signaling was revealed
(Fig. 9B). In HUVEC, VEGF-A activated mTORC1, but this VEGF
receptor-mediated activation of mTORC1 did not occur in the presence
of LAT1 inhibitors, which suggests that LAT1-mediated amino acid signals regulate growth factor signal-mediated mTORC1 activation in an
“on/off” manner (Quan et al., 2020). It is presumed to be a regulatory
mechanism that monitors amino acid availability, allows input from
growth factor signals to pass through mTORC1, and prevents excessive
protein synthesis without sufficient amino acids. Suppression of
amino acid signals by LAT1 inhibitors is suggested to predominate
growth factor signals in mTORC1 activation, which may be behind the
antitumor effect of LAT1 inhibitors. The fact that amino acid signals predominate the growth factor signals in the activation of mTORC1 is understandable because the amino acid signals must recruit mTORC1
onto the lysosomal membrane for mTORC1 to be activated by the
input from the growth factor signals (Fig. 9B). Even if signals from a
growth factor activate Rheb, Rheb cannot activate mTORC1 unless
mTORC1 is recruited onto the lysosomal membrane by amino acid
The effect of long-term treatment with LAT1 inhibitors was investigated concerning the acquisition of resistance by treatment with LAT1
inhibitors. Incubation of the medulloblastoma cell lines HDMB03 and
DAOY with JPH203 for 120 days increased the expression levels of
LAT1 and 4F2hc (Cormerais et al., 2019). This increase of LAT1 and
4F2hc expression is thought to be due to a JPH203-stimulated increase
in the transcription factor ATF4, which induces upregulation of LAT1
and 4F2hc (Cormerais et al., 2016, 2018, 2019). Nevertheless, JPH203
still exhibited a strong anti-proliferative effect, suggesting that treatment for at least four months does not affect the efficacy of LAT1 inhibitors in these cell lines. However, the potential for upregulation of other
transporters or adaptation of cellular signaling pathways could hamper
efficient LAT1 inhibitor treatment, depending on the cell line, which
should be addressed in future studies.
12. LAT1 in immune cells
4F2hc, which forms a heterodimer with LAT1, was first recognized as
a heavy chain of the 4F2 antigen upregulated by lymphocyte activation.
After molecular identification of LAT1, it was confirmed that lymphocyte activation also upregulates LAT1 expression (Nii et al., 2001). In
activated primary human T cells, induction of LAT1 expression was mediated by NF-kB and AP-1 (Hayashi, Jutabha, Endou, Sagara, & Anzai,
2013). Inhibition of LAT1 resulted in the reduction of cytokine production via inhibition of NF-kB and NFAT activities. Furthermore, LAT1
knockout T cells could not reprogram their metabolism responding to
the antigen and did not undergo clonal expansion or effector differentiation (Sinclair et al., 2013). The T cell activation required uptake of leucine to activate mTORC1 and the expression of c-Myc (Sinclair et al.,
2013). It is of note that regulatory T (Treg) cells are not dependent on
LAT1 for their active replication, although they require isoleucine via
4F2hc-dependent metabolic reprogramming (Ikeda et al., 2017). Some
other transporters of 4F2hc partners would be responsible for it. In the
skin of atopic dermatitis accompanied by T-cell activation, high LAT1
expression was detected in CD4+ T cells. LAT1 inhibition suppressed allergic skin inflammation in mouse atopic dermatitis model induced by
ovalbumin-specific Th2 cells, suggesting the importance of LAT1 in
Th2 cell-mediated dermal inflammation (Hayashi et al., 2020). These reports indicate the involvement of LAT1 in T cell-mediated immunity. Although preclinical and clinical trials using LAT1 inhibitors as anti-tumor
agents have not reported apparent signs of immunosuppression, the effects of LAT1 inhibitors on the immune system need to be carefully considered (Oda et al., 2010; Okano et al., 2020; Quan et al., 2020).
High expression of LAT1 has been confirmed in many cancers. LAT1-
specific PET probes have shown cancer-specific expression of LAT1. Taking advantage of highly cancer-specific properties, LAT1 can be used as a
tool for cancer-selective compound delivery, making it a target for BNCT
and nuclear medicine treatment. LAT1 regulates cell proliferation upstream of mTORC1. In addition to inhibiting mTORC1, LAT1 inhibitors
activate the GAAC pathway to suppress translation initiation. LAT1 is induced to be expressed in response to lysoPC and angiogenic growth factors in vascular endothelial cells, contributing to arteriosclerosis and
tumor angiogenesis, respectively. In addition to suppressing the growth
of cancer cells themselves, suppression of tumor angiogenesis also contributes to the in vivo anti-tumor effect of LAT1 inhibitors. LAT1-
mediated amino acid signals converge on mTOCR1 with growth signals
from growth factor receptors. In mTOCR1 activation, amino acid signals
dominate growth factor signals, which explains why LAT1 inhibitors are
effective in the presence of growth factor stimulation. Strategies have
been proposed to improve LAT1 selectivity and affinities, such as α-
methylation and O-linked extension at position 4 of the benzene ring
on the side chain of aromatic amino acids. The LAT1 cryo-EM structure
has recently been solved, contributing to designing more active compounds based on structural information. Since LAT1 is a highly cancerspecific transporter that provides essential nutrients for cancer cells, it
is a target for PET diagnosis and delivery of anti-tumor agents, and its inhibitors have anti-tumor effects. It is an ideal target for integrated diagnosis and therapeutics through compound design that maximizes the
interaction between compounds and transporters.
Declaration of Competing Interest
The author declares there are no conflicts of interest.
Y. Kanai Pharmacology & Therapeutics xxx (xxxx) xxx
This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and by the
Project for Development of Innovative Research on Cancer Therapeutics
from Japan Agency for Medical Research and Development. The author
thanks Chunhuan Jin for critical discussion on structural insights of
transporter ligands, and Ryuichi Ohgaki, Chunhuan Jin, and Lili Quan
for helping with preparing figures.
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