Targeting L-type amino acid transporter 1 for anticancer therapy: clinical impact from diagnostics to therapeutics.

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Review

Targeting L-type amino acid transporter 1 for anticancer therapy: clinical impact from diagnostics to therapeutics

1.

Introduction

2.

Characteristics of LAT1

3.

LAT1 in cancer

Su-Eon Jin, Hyo-Eon Jin & Soon-Sun Hong†

4.

LAT1 for drug development

†

5.

Conclusion

6.

Expert opinion

Department of Drug Development, College of Medicine, Inha University, Incheon, Republic of Korea

Introduction: L-type amino acid transporter 1 (LAT1) is one of the amino acid transporters. It is overexpressed in various types of cancer cells, while it is produced restrictedly in normal tissues. Areas covered: We discuss its characteristics in cancer cells compared with normal cells. We also mention the current applications to target LAT1 for anticancer therapy focusing on prognostic biomarkers, radio-labeled tumor imaging reagents, amino acid-stapled prodrugs, LAT1-mediated enhanced transport of anticancer drugs and LAT1 inhibitors. Expert opinion: LAT1 can be a versatile target to promisingly develop transporter-based drugs with enhanced drug delivery potential for anticancer therapy. Keywords: anticancer drug, cancer, inhibitor, L-type amino acid transporter 1, prodrug, prognostic biomarker, tumor imaging Expert Opin. Ther. Targets [Early Online]

1.

Introduction

L-type amino acid transporter 1 (LAT1, also called 4F2lc, CD98lc and SLC7A5) is an amino acid transporter that belongs to the system L, which is classified as a Na+-independent neutral amino acid transporter [1]. System L is considered as the main uptake route for large neutral amino acids with bulky side chains (Leu, Ile and Phe), which are mostly exchanged with glutamine (SLC7A5, SLC7A8, SLC7A9, SLC7A10 and SLC7A11) except for SLC7A6 and SLC7A7 [2]. LAT1 has been highlighted by the overexpression of various cancer types among amino acid transporters that are essential for nutritional demands in all living cells [3-6]. LAT1 overexpression can represent clinicopathological stages of cancer (e.g., tumor size, metastasis) [3-6]. In this review, we will illustrate the characteristics of LAT1 such as its structure, distribution and genetic control and discuss the clinical impact of LAT1 overexpression based on current approaches of LAT1 for anticancer therapy. 2.

Characteristics of LAT1

Structure LAT1 is a catalytic light chain of the 12 putative membrane-spanning domains (50 -- 60 kDa), which is covalently linked by a disulfide bond to the type II membrane glycoprotein heavy chain (4F2hc, also called CD98hc, and SLC3A2; 85 kDa) (Figure 1) [7,8]. The cysteine residue -- involved in the disulfide bond -- is located between the transmembrane domain III and IV within the structure of LATs. LAT1 and 4F2hc form a heterodimeric complex of transporters (4F2hc/LAT1) 2.1

10.1517/14728222.2015.1044975 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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L-type amino acid transporter 1 (LAT1) is overexpressed in cancer patients representing clinicopathological stages of cancer. LAT1 overexpression is specifically related to coordinately overexpression of ASCT2 and 4F2hc in a wide spectrum of human cancer and metastasis. LAT1 overexpression mediates mTOR and integrin pathways to induce cell survival and metastasis. LAT1 has been exploited in various strategies for anticancer therapy as follows: i) prognostic biomarkers; ii) radio-labeled tumor imaging reagents; iii) amino acidstapled prodrugs; iv) LAT1-mediated enhanced transport of anticancer drugs and v) LAT1 inhibitors. LAT1 of influx transporters would be a new research target for anticancer therapy over the traditional resistance study of anticancer drugs by efflux transporters. We expect that LAT1 can be used to further optimize for targeted therapy and to improve the individualized use of cancer medicines in clinical practice.

This box summarizes key points contained in the article.

for its transporter function that differs from other LAT subtypes, LAT3 and LAT4, which can work without 4F2hc. Distribution LAT1 distribution is restricted to only several organs in normal conditions compared with malignant cancer conditions [9]. Its normal mRNA expression level is in order of placenta, brain, spleen, testes and colon. LAT1 is expressed in the placental membranes supplying hormones and amino acids to developing fetus and placenta [10]. In addition, it is predominantly expressed in the microvessels of the central nervous system (CNS) as well as the inner blood--retinal barrier of the blood--brain barrier (BBB), which plays a key role in the transport maintenance of large neutral amino acids and neurotransmitters [11]. 2.2

Genetic control and disorders LAT1 and 4F2hc are genetically controlled by the solute carrier (SLC) gene (for detailed information about SLC genes, please visit: http://www.bioparadigms.org) [12]. SLCs represent one of the major families in the transporter system together with the ATP-binding cassette (ABC) transporters, which comprise both facilitated and secondary active transporters. LAT1 is one of the SLC7 family members, specifically SLC7A5 (TC 2.A.3.8.1), controlled by human gene locus 16q24.3 (NM_003486). In addition, 4F2hc is a member of the SLC3 family, namely SLC3A2 (TC 8.A.9.2.1) located on the human gene locus 11q13. Due to genetic variants or mutations of genes, transporters generally exhibit abnormal functions in many human diseases. A query of the OMIM database (online Mendelian inheritance in man; http://www.ncbi.nlm.nih.gov/omim) for transporters yielded allelic variants in 198 genes linked to a wide 2.3

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variety of disease phenotypes. Genetic disorder of LAT1, together with 4F2hc, has been reported to be related to cancer [13,14]. The alteration of LAT1 expression, specifically abnormal overexpression of LAT1, is related to cancer promotion in a large number of different types of cancer. In addition, LAT1 can function as a promoter in cancer that is associated with clinicopathological features. In the following section, we will introduce LAT1 function in cancer, focusing on LAT1 overexpression in cancer patients, interaction of other related factors and LAT1-related cancer-inducing signaling pathways. 3.

LAT1 in cancer

LAT1 overexpression in cancer patients Tumor cells have increased demands for essential amino acids and glutamine to maintain tumor cell growth and survival via neoplastic transformation [15]. To sustain nutritional needs of tumor cells, LAT1 is overexpressed in various types of cancer cells by a 4- to 5-fold increase of RNA expression and a 2- to 3-fold increase of protein expression compared with nontumor tissues [16,17]. Figure 2 shows transporters that are upregulated with LAT1 in various cancer tissues: ASCT2 (SLC1A5, ATBo, neutral amino acid transporter 2 for system ASC), ATBo,+ (SLC6A14, neutral and cationic amino acid transporter for system Bo,+), SNAT3 (SLC38A3, neutral amino acid transporters 3 in system N [Na+, co-transport; H+, antiport]), MCT1 (SLC19A1, monocarboxylate transporter 1), ENT1 (SLC29A1, equilibrative nucleoside transporter 1), OATP1B3 (SLCO1B3, OATP8, LST2, organic anion-transporting polypeptide 1B3), GLUT1 (SLC2A1, glucose transporter 1) and PEPT1 (SLC15A1, H+/peptide co-transporter), as well as transporters that are downregulated with LAT1 in cancer tissues: SMCT (SLC5A8, Na+/monocarboxylate transporter). Additionally, Table 1 summarizes LAT1 expression in various types of cancer cell lines and tissues from cancer patients [e.g., non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), gastrointestinal cancer (pancreas, liver, biliary system, colon and rectum), gynecologic cancer (breast, ovary, uterus and cervix), renal cancer, glioma and prostate cancer with an alteration of other associated factors [e.g., 4F2hc, neutral amino acid transporter 2, vascular endothelial growth factor (VEGF), Ki-67, phosphorylated AKT (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), c-Myc and p53] [4,18-21]. The high levels of LAT1 expression are associated with clinicopathological variables used to assess the survival rate of cancer patients such as cancer staging, lymph-node metastasis and lymphatic permeation. Specifically, LAT1 serves as a poor prognostic marker to predict worse outcomes of cancer development and aggressiveness in patients after surgery [4,22,23]. In patients with NSCLC, LAT1 overexpression is consistently detected in combination with an upregulated 4F2hc forming complex [6,18,24] and p-mTOR, which is involved in 3.1

Expert Opin. Ther. Targets (2015) 19(10)

Targeting LAT1 for anticancer therapy: clinical impact from diagnostics to therapeutics 4F2hc (CD98hc; SLC3A2)

SS

Cell membrane


Integrin N

LAT1 (4F2lc; CD98lc; SLC7A5) C

4F2hc/LAT1 complex (CD98)

Figure 1. Schematic diagram of LAT1 structure. CD98 is composed of a heavy chain (4F2hc) and a light chain (LAT1). LAT1 is a 12 putative membrane spanning domain associated with a type II membrane glycoprotein, 4F2hc, via a disulfide bond. Adapted from [1].

tumorigenesis induction [25,26]. Its positive expression is also correlated with Ki-76 [23,24] and VEGF [20,26] used to predict cancer stages in patients with resected NSCLC. The frequency of LAT1 expression in the plasma membrane is associated with tumor histology, differentiation grade, pathologic stage, T classification, pleural invasion, lymph-vessel invasion and overall survival rate [27]. LAT1 is also reported as an independent significant prognostic factor in HCC [3] and gastrointestinal cancer such as gastric cancer [28], pancreatic cancer [4,21,29,30], biliary tract cancer [5] and colorectal cancer [31,32]. In pancreatic cancer [4] and biliary tract cancer [5], LAT1 overexpression was significantly associated with cancer stages with lymphatic metastasis and tumor size and strongly correlated with 4F2hc expression in patients with pancreatic cancer [4] and in DLD-1 colorectal cancer cells [31]. ASCT2 was also associated with LAT1 overexpression in DLD-1 cells [31]. However, VEGF was independently overexpressed in pancreatic cancer patients [4]. Hayashi et al. described the mechanism of genetic regulation of LAT1 expression in pancreatic cancer using a c-Myc small interfering RNA (siRNA) in MIA Paca-2 cells [21]. LAT1 is also overexpressed in gynecologic cancer including ovarian cancer [33,34], breast cancer [35], triple negative breast cancer (TNBC) [36] and uterine cervical cancer [37] in association with 4F2hc, ASCT2 and p-mTOR. In patients with clear cell renal cell carcinoma (RCC), Betsunoh et al. described that LAT1 mRNA was also related to invasive and progressive potential of clear cell RCC by quantifying LAT1, LAT2, LAT3, LAT4 and 4F2hc mRNA expression [16].

In glioma [38-42] and prostate cancer [22,43,44], LAT1 is also a significant factor that serves as a prognostic marker associated with 4F2hc, ASCT2 and mTOR. Specifically, LAT1 corresponds to tumor-associated gene 1(TA-1), an oncofetal antigen in glioma cells [39,40], which provided information on malignancy of glioma [38,41] and prostate cancer [22]. LAT1-related factors and signaling in cancer LAT1 is overexpressed in a wide spectrum of human cancer and metastasis as we described in previous section, which is specifically related to ASCT2 and 4F2hc [45-47]. LAT1 overexpression mediates cancer-related signaling pathways, namely mTOR and integrin pathways to induce cell survival and metastasis [14]. Figure 3 shows a schematic diagram of the association between LAT1 and signaling pathways of mTOR and integrin in cancer. In the next section, we will focus on the interaction of LAT1 overexpression and other SLC families that induce signaling pathway involved in tumorigenesis. 3.2

4F2hc/LAT1 and ASCT2 As mentioned above, LAT1 expression increases in various types of cancer collaborated with 4F2hc and ASCT2. The interaction between 4F2hc/LAT1 and ASCT2 was analyzed by Cytoscape (Figure 4A). LAT1, 4F2hc and ASCT2 interacted in various ways generating a protein interactome based on BioGrid, an online database. This network contained 77 nodes, and 154 edges filtering for LAT1, 4F2hc, ASCT2, human and cancer as keywords. From a statistical analysis of topology, the plot of topological coefficients versus number of neighbors provided the equation of 3.2.1


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Na+

LAT1

Na

H+

SMCT

MCT1

Breast cancer

+

Na Na+ ASCT2

LAT1

ATBo+

LAT1

Cl–

H+

Na+ ATB

Ovarian o+

ENT1

Pancreatic cancer

cancer

OATP1B3

Liver cancer PEPT1

LAT1

LAT1

Cl– Cl–

Na a+ ATBo+


LAT1

SNAT3

Glioma

+

Lung cancer

Na+

ASCT2

GLUT1 Prostate cancer

Na LAT1

+

SMCT

Colon cancer

OCTs MCT1

Figure 2. LAT1 overexpression in a wide spectrum of cancer. Orange and green represent upregulated and downregulated LAT1 and other transporters, respectively, in cancer compared with normal organs. Adapted from [13]. ASCT2 (SLC1A5, ATBo): Neutral amino acid transporter 2 for system ASC; ATBo,+ (SLC6A14): Neutral and cationic amino acid transporter for system Bo,+; ENT1 (SLC29A1): Equilibrative nucleoside transporter 1; GLUT1 (SLC2A1): Glucose transporter; MCT1 (SLC19A1): Monocarboxylate transporter 1; OATP1B3 (SLCO1B3, OATP8, LST2): Organic anion-transporting polypeptide 1B3; PEPT1 (SLC15A1): H+/peptide co-transporter; SMCT (SLC5A8): Na+/monocarboxylate transporter; SNAT3 (SLC38A3): Neutral amino acid transporters 3 in system N (Na+, co-transport; H+, antiport).

y = 0.880x--0.753 (R-squared, 0.963) with a correlation of 0.991. All these factors formed network hubs in cancer extended to pp36 of immunity, which suggested that cancer could be related to immunological disorders together with metabolic disorders of amino acid transports [14]. Interaction among 4F2hc, LAT1 and ASCT2 could match for their overexpression in cancer based on the 4F2hc/LAT1 complex formation and the transport of shared substrates (Gln) by LAT1 and ASCT2. In cancer cells, LAT1 and ASCT2 are coordinately overexpressed. LAT1 uses intracellular ASCT2 substrates to adjust the concentration of essential amino acids, while ASCT2 drives the cell growth and 4

survival processes mediating glutamine net uptake. Inhibition of LAT1 as well as ASCT2 downregulates the cancer cell proliferation suggesting a potential linkage between LAT1 and ASCT2 via a cancer-mediated signaling, mTOR [45]. The expression of unidirectional transporter has been proposed to overlap substrate selectivity (e.g., SNATs, SLC38) and to provide efflux substrates which are necessary for LAT1 and ASCT2 [48]. In the tumor microenvironment, amino acid transporters support metabolism, triggering cell growth and survival. As such, 4F2hc/ LAT1 affects the downstream trafficking of mTOR in cancer, and 4F2hc mediates integrin signaling [14].


Targeting LAT1 for anticancer therapy: clinical impact from diagnostics to therapeutics

Table 1. LAT1 expression in various types of cancer. Types of cancer


NSCLC

HCC

Target (cells/stage)

Other related factors

282 patients (stage I)

Ki-67 labeling index (Ki-67)

84 patients (resected pathologic stage I SCC of the lung)

4F2hc

93 patients (primary site and a concordant pulmonary metastatic site, undergone thoracotomy) 321 patients (resectable stage I -- III)

CD98, Ki-67, VEGF, CD31 and CD34

220 patients (resected NSCLC with lymph node metastases)

4F2hc and Ki-67

160 patients (resected)

HIF-1a and mTOR

56 patients (platinumbased chemotherapy with postoperative recurrence)

CD98, VEGF, Ki-67, p-AKT, p-mTOR and p53

237 cases 40 normal lung tissues

-

H1395 51 patients

Wild-type EGFR

23 pairs (fresh-frozen HCC tissues)

-

Ki-67

Observation

Ref.

Positive LAT1 expression in 114 patients (40%) (19% of adenocarcinoma [36 of 186 patients], 83% of SCC [73 of 88 patients] and 63% of large cell carcinoma [five of eight patients]) associated with gender, disease stage and pathological features High expression of LAT1 in SCC associated with lymph node metastasis and disease stage in NSCLC; co-expression of LAT1 and CD98 predicted the poor prognosis in patients LAT1 expression correlated with CD98 expression, angiogenesis and cell proliferation, stronger in the primary and metastatic sites

[6]

LAT1 expression as a promising pathological factor to predict the prognosis of patients with resectable stage I -- III NSCLC significantly correlated with Ki-67 High LAT1 expression in SCC (91%; 65/71) and large cell carcinoma (82%; 9/11); LAT1 expression correlated with CD98 and Ki-67 predicted prognosis in resectable adenocarcinoma patients with N2 disease A significant association of LAT1 expression observed with CD98, hypoxic markers (GLUT1, HIF-1a, hexokinase I, VEGF and CD34) and mTOR pathway (EGFR, a loss of PTEN, p-mTOR and p-S6K), especially in lung adenocarcinoma LAT1, CD98, VEGF, Ki-67 and p53 to predict poor outcome; LAT1 expression associated with chemoresistance; positive expression of LAT1 and VEGF as an independent factor to predict poor prognosis after chemotherapy Positive staining indicating a plasma membrane expression of LAT1 made up > 10% of the tumor; The frequency of LAT1 membrane expression associated with tumor histology, differentiation grade, pathologic stage, T classification, pleural invasion, lymph-vessel invasion and overall survival rate in informing the prognosis in NSCLC cases Inhibition of LAT1 by BCH or co-treatment of gefitinib and BCH to predict a poor prognosis for the effective therapy of NSCLC without EGFR mutation High LAT1 expression in HCC specimens associated with tumor size (p = 0.032), histological differentiation (p = 0.003) and tumor stage (p = 0.01); LAT1 overexpression in HCC served as a potential independent prognostic marker

[23]

[18]

[20]

[24]

[25]

[26]

[27]

[46]

[3]

BCH: 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; 18F-DOPA: 6-18F-Fluoro-L-3,4-dihydroxy-phenylalanine; 5-FU: 5-Fluorouracil; GBM: Glioblastoma; HCC: Hepatocellular carcinoma; HIF-1a: Hypoxia inducible factor-1a; LAT1: L-type amino acid transporter 1; mTORC1: Mammalian target of rapamycin complex 1; p-AKT: Phosphorylated AKT; PDAC: Pancreatic ductal adenocarcinomas; PET: Positron-emission tomography; p-mTOR: Phosphorylated mammalian target of rapamycin; SCC: Squamous cell carcinoma; TA1: Tumor-associated gene 1; TNBC: Triple negative breast cancer.


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Table 1. LAT1 expression in various types of cancer (continued).


Types of cancer



Gastric cancer

87 cases (advanced gastric cancer)

Ki-67

Pancreatic cancer

97 patients (surgically resected pathological stage I -- IV PDAC)

CD98, Ki-67 and VEGF

MIA Paca-2

c-Myc

66 patients (resected PDAC) Colo357

Ki-67 -

Biliary tract cancer

139 patients (resected pathologic stage I -- IV biliary tract adenocarcinoma)

Ki-67

Colorectal cancer

DLD-1

System L (4F2hc, and LAT2), system A (ATA1 and ATA2) and system ASC (ASCT1) -

44 patients (advanced rectal cancer)

Ovarian cancer

SKOV3, IGROV1, A2780 and OVCAR3

ASCT2, SN2 and p70S6K (a downstream effector of mTOR)

63 specimens (surgically resected human epithelial ovarian cancer) OVCAR-3

LAT2 and 4F2hc

Observation

Ref.

High LAT1 expression observed in carcinoma cells, predominantly on the plasma membranes with greater intensity in non-scirrhous than scirrhous carcinomas; a significantly higher LAT1 expression in gastric carcinoma cases with lymph node metastasis than in cases without lymph node metastasis LAT1 and CD98 highly expressed in 52.6% (51/ 97) and 56.7% (55/97) of cases, respectively (p = 0.568); LAT1 expression within pancreatic cancer cells significantly associated with disease stage, tumor size, Ki-67, VEGF, CD34, p53 and CD98 LAT1 expression as a promising pathological marker for the prediction of outcome in patients with pancreatic cancer c-Myc overexpression increased LAT1 promoter activity, whereas mutation of c-Myc binding site diminished this effect; Biological significance of LAT1 in tumor growth and molecular signaling that could explain why the preferential expression of LAT1 in cancer cells LAT1 aberrant overexpression in PDAC predicted poor prognosis, which was independent of Ki-67 Increased LAT1 expression in transplanted Colo357 cells of pancreatic cancer compared to non-malignant control cells LAT1 expression closely correlated with lymphatic metastases, cell proliferation and angiogenesis as a significant indicator predicting poor outcome after surgery; BCH significantly suppressed tumor growth and yielded an additive effect to gemcitabine and 5-FU treatments Mostly LAT1 and its substrates’ (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1; b-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1 A positive LAT1 expression recognized in 50.0% (22/44) of patients; LAT1 expression in marginally significant association with response to hyperthermochemoradiotherapy (p = 0.05) Increased LAT1 expression observed in human ovarian cancer cell lines; combination therapy of LAT1 inhibitor (BCH) with antiproliferative aminopeptidase inhibitors (bestatin) caused 10.5and 4.3-fold decrease in the IC50 value of bestatin in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy has a synergistic effect Significantly upregulated LAT1 expression predominantly localized on plasma membrane in various human epithelial ovarian cancer; significant participation of LAT1 in nutrition, proliferation and migration of ovarian cancer

[28]

[4]

[21]

[29] [30]

[5]

[31]

[32]

[33]

[34]

BCH: 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; 18F-DOPA: 6-18F-Fluoro-L-3,4-dihydroxy-phenylalanine; 5-FU: 5-Fluorouracil; GBM: Glioblastoma; HCC: Hepatocellular carcinoma; HIF-1a: Hypoxia inducible factor-1a; LAT1: L-type amino acid transporter 1; mTORC1: Mammalian target of rapamycin complex 1; p-AKT: Phosphorylated AKT; PDAC: Pancreatic ductal adenocarcinomas; PET: Positron-emission tomography; p-mTOR: Phosphorylated mammalian target of rapamycin; SCC: Squamous cell carcinoma; TA1: Tumor-associated gene 1; TNBC: Triple negative breast cancer.

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Table 1. LAT1 expression in various types of cancer (continued).


Types of cancer



Breast cancer

MCF-7, ZR-75-1 and MDA-MB-231

4F2hc

TNBC

129 patients

Uterine cervical cancer

Cervical intraepithelial neoplasia

CD98, HER2, TN, Ki-67, ER and PgR Ki-67 and p16

Renal cancer

82 Japanese patients (clear cell renal cell carcinoma)

LAT2, LAT3, LAT4 and 4F2hc

Glioma

Human malignant glioma

4F2hc

60 patients (human primary astrocytic tumor tissue) C6 68 patients (human primary glioma tissues)

CD98, TA1 (an oncofetal antigen) and 4F2hc

33 patients (newly diagnosed human glioma)

4F2hc, CD34 and Ki-67

Established GBM cell lines (T98, GBM28) Primary GBM xenografts

-

114 patients

Ki-67

LNCaP, PC-3, DU145, C4-2B and MDAPCa-2b

LAT3, mTORC1 and ATF4

Human prostate cancer tissue PC-3 xenograft mouse model

LAT3, ASCT1, ASCT2 and 4F2hc (regulated by androgen receptor); UBE2C, CDC20 and CDK1

Prostate cancer

TA1

Observation

Ref.

System L required to maintain MCF-7, ZR-75-1 and MDA-MB-231 cell growth; 4F2hc/ LAT1 as a suitable target to inhibit breast cancer progression CD98 considered as a risk factor for relapse in TNBC and as a prognostic factor LAT1 expression decreased because of human papillomavirus infection as reflected by p16 overexpression in cervical intraepithelial neoplasia; LAT1 expression associated with acquired malignant potential in invasive carcinoma Significant increase of LAT1 mRNA expression in tumor tissue compared with non-tumor tissue which showed reduced expression of LAT2 and LAT3 mRNAs; no difference in the expression of LAT4 and 4F2hc mRNAs between tumor and non-tumor tissues LAT1, the major transporter of system L, frequently expressed at higher levels in highgrade gliomas than in low-grade gliomas and brain tissues Inhibited the growth of C6 glioma cells in vitro and in vivo in a dose-dependent manner by BCH

[35]

Higher LAT1 expression in infiltrating glioma cells than in cells located in the center of the tumor Higher expression of LAT1 and 4F2hc in highgrade gliomas than in low-grade gliomas; LAT1, mainly expressed in the tumor cytoplasm and vascular endothelium; 4F2hc, mainly expressed in the tumor cytoplasm and plasma membrane LAT1 as a key determinant of amino acid tracer, 18 F-DOPA accumulation in GBM during PET imaging, which may provide better spatial and functional information in human gliomas than CT or MRI alone Elevated LAT1 expression in prostate cancers as a novel independent biomarker of high-grade malignancy, which can be utilized together with the Gleason score Inhibiting LAT function to decrease cell growth and mTORC1 signaling in prostate cancer cells, which maintained levels of amino acid influx through androgen receptor-mediated regulation of LAT3 expression and ATF4 regulation after amino acid deprivation; increased levels of LAT1 after hormone ablation and in metastatic lesions Inhibition of LAT transporters to provide novel therapeutic targets in metastatic castrationresistant prostate cancer, via suppression of mTORC1 activity and M-phase cell cycle genes

[40]

[36] [37]

[16]

[38]

[39]

[41]

[42]

[22]

[43]

[44]

BCH: 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; 18F-DOPA: 6-18F-Fluoro-L-3,4-dihydroxy-phenylalanine; 5-FU: 5-Fluorouracil; GBM: Glioblastoma; HCC: Hepatocellular carcinoma; HIF-1a: Hypoxia inducible factor-1a; LAT1: L-type amino acid transporter 1; mTORC1: Mammalian target of rapamycin complex 1; p-AKT: Phosphorylated AKT; PDAC: Pancreatic ductal adenocarcinomas; PET: Positron-emission tomography; p-mTOR: Phosphorylated mammalian target of rapamycin; SCC: Squamous cell carcinoma; TA1: Tumor-associated gene 1; TNBC: Triple negative breast cancer. Expert Opin. Ther. Targets (2015) 19(10)

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Essential amino acids Glutamine CD98 Cell membrane

LAT1

4F2hc

Unidirectional transporter

4F2hc region for integrin signaling

Glutamine


Leucine AKT p130CAS FAK

mTOR activation Autophagy

Survival Anchorage-Independence Metastasis

Survival and growth

Figure 3. Schematic diagram of LAT1 function in cancer. CD98 functions as an amino acid transporter through its light chain (LAT1) and as a mediator of integrin signaling through its heavy chain (4F2hc). Overexpression of CD98 in cancer cells induces tumorigenesis via the activation of mTOR as well as integrin. Adapted from [14].

3.2.2

mTOR signaling

LAT1 overexpression leads to an increase of amino acid influx (especially Leu) and activation of mTOR pathway in tumor cells (Figure 4B). The mTOR is a serine/threonine kinase that regulates cell growth, proliferation, motility and survival dysregulated in cancer [49,50]. The mTOR pathway is activated by a nutritional uptake of essential amino acids via LAT1 transport [25,51,52]. In brief, LAT1 heterodimerizes with 4F2hc and the 4F2hc/LAT1 complex then imports essential amino acids (e.g., Leu, Ile, Arg) with an exchange of intracellular small neutral amino acids (e.g., Gln) in a Na+-independent manner. After the amino acid uptake, mTOR activation is mediated by vacuolar protein sorting 34. In Figure 4B, mTORC1 -- an active form of mTOR -- is constituted by rapamycin-sensitive adaptor protein of mTOR (raptor), G protein-b-subunit-like protein and proline-rich AKT substrate of 40 kDa [52]. It interacts with downstream substrates such as S6K1 and 4E-BP1. Specifically, S6K1 is sensitive to intracellular ATP levels and it is also regulated by a negative feedback loop to insulin receptor substrate 1 (IRS1). Upregulation and activation of S6K1 against 4E-BP1 drive cell growth and metabolism in cancer, which is based on increased intracellular nutrients as fuel. Integrin signaling 4F2hc also plays a key role in integrin signaling of cancer cells due to its plasma membrane domain that interacts with 3.2.3

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integrins [14,53]. The overexpression of 4F2hc/LAT1 leads to the integrin signaling pathway activation, which is cascaded by phosphatidylinositide 3-kinase/AKT (PI3K/AKT), p130CAS and focal adhesion kinase (FAK) [51,53-55]. Although the mechanism of 4F2hc-dependent integrin signaling is unknown, it seems to be independent of 4F2hc-associated transporter activity that requires an interaction with integrins. The association of 4F2hc heterodimer at the cell surface is necessary to trigger integrin signaling and promote tumorigenesis in cancer cells [53,56]. Poettler et al. reported that, in order to possess tumorigenic potential in vivo, 4F2hc/integrin interaction was required for adhesion-induced FAK phosphorylation and activation of major downstream signaling partners, PI3K/AKT and MEK/ERK, in renal cancer cell lines [56]. 4.

LAT1 for drug development

Clinical significance In cancer cells, several types of influx transporters are overexpressed by metabolic transition (e.g., LAT1, GLUTs, MCT1) based on a massive increase in amino acid demands (e.g., essential amino acids, Gln) [57]. Thus, transporters are selected as chemotherapeutic targets since their function and transporter-based drugs are proposed to increase clinical efficacy and reduce side effects [58]. LAT1 can be a promising therapeutic target with clinical significance between its role and clinical outcome to predict a prognosis in cancer 4.1




A.

B.

Glucose

Amino acids Insulin / IGF Leu Glutamine Tyrosine kinase receptor 4F2hc/ LAT1

Cell membrane

IRS1 P

ASCT2

Cytoplasm

Glutamine

ATP

VPS34

Lipid synthesis

mTORC1 P P

S6K1

4E-BP1

Mitochondrial Autophagy Microtubule organization metabolism

Cell growth and metabolism (Protein synthesis)

Figure 4. LAT1-mediated network and signaling in cancer. (A) Network analysis of LAT1-related proteins. Protein-protein interactions of LAT1, 4F2hc and ASGT2 were generated based on cellular location and function using Cytoscape v3.1.1 with BioGrid database. This network contains interactions of 77 nodes and 154 edges filtering between transporters using LAT1, 4F2hc, ASGT2, human and cancer as keywords. Nodes of LAT1, 4F2hc and ASGT2 are in pink corresponding to major transporters overexpressed in cancer. Edge path analysis resulted in the association of interaction types to some of the edges (dash dot lines, ‘colocalization’). (B) LAT1-mediated mTOR pathway in tumorigenesis. Adapted from [52].

patients [4,5,23]. The alteration of LAT1 expression is an adaptive response to utilize the host nutrition in cancer patients. Investigating levels of LAT1 expression can be clinically and pharmacologically relevant to determine the exact status of cancer patients. Since LAT1 is a gatekeeper that regulates the entry of anticancer drugs into cancer cells, it can also be a target for the enhanced drug delivery in anticancer therapy.

ii) imaging with amino acid-stapled positron-emission tomography (PET) tracers as LAT1 substrates; iii) developing prodrugs for enhancing delivery potential; iv) transporting anticancer drugs through LAT1 and v) inhibiting LAT1 for developing anticancer drugs (Figure 5). We will highlight these applications of LAT1 for translational research in the upcoming sections. Cancer biomarker LAT1 can be used as a prognostic cancer biomarker because it is overexpressed in various types of cancer and provides information regarding the state of cancer disease in an untreated individual [59]. Nowadays, prognostic studies usually include patients who received systemic anticancer treatment, including 4.2.1

Recent approaches with LAT1 for anticancer therapy

4.2

LAT1 has been exploited in various strategies for anticancer therapy, including: i) predicting prognosis in cancer patients;


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LAT1 inhibitors

LAT1-mediated transport Altered transport of anticancer drugs as LAT1 substrates

Small molecules Combinations Genetic materials

Prodrug development

LAT1 as a target for anticancer therapy

Cancer biomarker Prognostic marker


Amino acid-stapled anticancer drugs Conjugation of anticancer drugs with amino acid derivatives or peptides Tumor imaging Diagnosis PET Amino acid-stapled PET tracer

Figure 5. Potential clinical applications of LAT1 for anticancer therapy. LAT1 can be applied for: i) a cancer biomarker for predicting prognosis in cancer patients; ii) a tumor imaging with PET tracers by conjugating amino acids as LAT1 substrates; iii) a prodrug development for enhanced uptake of anticancer drugs; iv) a LAT1-mediated transport of anticancer drugs for an altered response of chemotherapeutics via drug--drug or drug--transporter interaction and v) a development of LAT1 inhibitors as chemotherapeutic drugs.

post-surgical treatment. However, it may influence the natural course of cancer. Patients with low risk -- in early cancer stages -- do not receive adjuvant treatment such as preventive chemotherapy despite some of these patients experiencing cancer recurrence. In these cases, evidence-based cancer biomarkers -- prognostic markers -- can be helpful in selecting patients for adjuvant treatment. Clinical significance of LAT1 has been demonstrated in patients with pancreatic cancer [4,29], breast cancer [17], prostate cancer [22] and NSCLC [6,24]. In these studies, LAT1 was monitored as a prognostic marker, based on the correlation between its elevated expression and cancer progression. LAT1 can be used as a molecular biomarker to predict cancer prognosis and to detect and manage cancer recurrence. It can also offer assistance to anticancer therapy in other aspects of personalized medicine. Tumor imaging with PET Radio-labeled amino acid analogues with bulky side chains that are transported by LAT1 are used as PET probes for cancer diagnosis. These include O-(2-18F-fluoroethyl)-Ltyrosine (18F-FET) [60], L-3-18F-a-methyl tyrosine (18F-FAMT) [61-65], 6-18F-fluoro-L-3,4-dihydroxy-phenylalanine (18F-DOPA) [42,66], S-(3-18F-fluoropropyl)-D-homocysteine (18F-d-FPHCys) [67] and 89Zirconium-labeled antibody (89ZrDFO-Ab) [68]. The majority of the radio-labeled amino acid tracers targets system L transporters, which includes derivatives of natural amino acids, L-phenylalanine and L-tyrosine [69]. The upregulation of LAT1 in malignant tumor 4.2.2

10

tissues should contribute to an intratumoral accumulation of the PET probes for the detection of tumors as well as noninvasive evaluation and monitoring of tumor growth. Table 2 summarizes the radio-labeled amino acid analogues used as PET tracers for tumor imaging. 18 F-FET is a substrate for system L amino acid transporters including LAT1. It has advantages over natural amino acids with incorporation of prolonged remaining radionuclides (18F), using simplified radiosynthetic methods and without formation of radio-labeled metabolites. The use of 18F-FET can avoid potential confounding accumulation of the activity in non-target tissues and simplify the kinetic analysis based on the absence of confounding radio-labeled metabolites [70]. However, additional data are still needed to better define the accuracy of these tracers for their uses in the clinic [70]. Its use also showed a limited success for oncologic imaging outside the brain. Thus, new strategies are being studied for imaging LAT1 in systemic cancer and several PET tracers have currently been developed such as 18F-DOPA, 18FFAMT, 18F-d-FPHCys and 89ZrDFO-Ab. 18 F-DOPA is a widely used PET probe in neuroendocrine tumors. Youland et al. reported the use of 18F-DOPA for tumor imaging via PET in a glioblastoma model [42]. It is metabolized by aromatic amino acid decarboxylase, which contributes to its favorable imaging properties with an enhanced substrate activity to system L compared with other substrates [66,71]. 18F-FAMT, a selective LAT1 substrate, has been shown to be useful in PET tumor imaging due to its



Table 2. Radio-labeled amino acids for PET tracers transported via LAT1.


Tracers 18

F-FET

18

F-DOPA

18

F-FAMT

Types of cancer LGG of WHO grade II, early detection of progression to WHO grade III or IV Human glioma

Target (cells/stage) Patients (27)

T98 and GBM28

Suspected malignant brain tumors

Patients (10)

NSCLC

Patients (98)

NSCLC

Patients (37)

Multiple myeloma

Patients (11)

OSCC

Consecutive patients (68)

-

Mouse renal proximal tubule cell line S2 stably expressing human LAT1 (S2-LAT1) A431 PC3 Colo 205 and HT-29

18

F-D-FPHCys

Squamous cell carcinoma Prostate cancer Colorectal cancer

89

ZrDFO-Ab

Human colorectal cancer cell line

HCT-116 and HCT116 xenograft model

Observation

Ref.

Both tumor-to-brain ratio and kinetic parameters of 18 F-FET PET uptake provided valuable diagnostic information for the noninvasive detection of malignant progression of LGG LAT1 expression was positively correlated with 18FDOPA PET uptake (p = 0.04) in clinical samples and strongly associated with 3H-L-DOPA uptake in vitro and 18F-DOPA uptake in patient biopsy samples 18 F-DOPA PET SUVmax may more accurately identify regions of higher-grade/higher-density disease in patients with astrocytomas and will have utility in guiding stereotactic biopsy selection 18 F-FAMT uptake correlated with LAT1 expression and both LAT1 expression and 18F-FMT uptake were significantly higher in nonadenocarcinomatous disease than in adenocarcinoma The metabolic activity of primary tumors as evaluated by PET with 18F-FAMT and 18F-FDG is related to tumor angiogenesis and proliferative activity in NSCLC 18 F-FAMT PET provides a useful imaging modality for detecting active myelomatous lesions 18 F-FAMT accumulation correlated with OSCC tumor proliferation. 18F-FAMT PET may be a suitable replacement for 18F-FDG PET in assessing malignant lymph nodes of oral cancer, especially non-tongue cancer. The uptake of 18F-FAMT was significantly correlated with LAT1 expression, cell proliferation and advanced stage FAMT is selective to LAT1 and not transported by LAT2 based on its a-methyl moiety, which is proposed to contribute to highly tumor-specific accumulation of 18F-FAMT in PET 18 F-labeled methionine derivative. 18F-d -FPHCys showed in vitro and in vivo uptake as follows; A431 cells > Colo 205 > PC3 > HT-29 (similarly observed with 14C-MET). 18F-d-FPHCys retention was strongly correlated with LAT1 expression both in vitro and in vivo, demonstrating a clear dependence on this transporter for tumor uptake and cellular proliferation 89 ZrDFO-Ab targeted the extracellular domain of LAT1 with in vitro and in vivo specificity, showing excellent tumor imaging properties in xenograft tumor models of colorectal cancer

[60]

[42]

[66]

[61]

[62]

[63] [64]

[65]

[67]

[68]

F-DOPA: 6-18F-Fluoro-L-3,4-dihydroxy-phenylalanine; 18F-D-FPHCys: S-(3-18F-fluoropropyl)-D-homocysteine; 18F-FAMT: L-3-18F-a-methyl tyrosine; 18F-FET: O-(2-18FFluoroethyl)-L-tyrosine; LAT1: L-type amino acid transporter 1; LGG: Low-grade glioma; OSCC : Oral squamous cell carcinoma; PET: Positron-emission tomography; WHO: World Health Organization; 89ZrDFO-Ab: 89Zirconium-labeled antibody. 18

stable expression and a high tumor-specific accumulation in the renal cells overexpressing human LAT1 [65]. In addition, it provides a useful imaging modality for detecting active myelomatous lesions in multiple myeloma patients [63] and its accumulation correlates with tumor proliferation and advanced stage in patients with oral squamous cell carcinoma [64] and NSCLC [18,24]. 18F-d-FPHCys is a new 18F-labeled methionine derivative developed as a PET tracer. It shows high in vitro and

in vivo uptake potentials in A431, Colo 205, PC3 and HT-29 correlated with LAT1 expression [67]. Although 18F has a long half-life (110-min), making batch production and remote distribution feasible, it substantially alters the biological properties of amino acids [70]. Overcoming these disadvantages of 18F, 89ZrDFO-Ab is a novel zirconium89-labeled antibody that targets LAT1 extracellular domain in a preclinical colorectal cancer model [68]. This tracer


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S. -E. Jin et al.

demonstrated a specificity for LAT1 with excellent tumor imaging properties in cancer xenograft mice model [68]. Prodrug development for enhanced delivery Amino acid prodrugs have a large structural diversity with derivatization of 20 common natural amino acids and synthetic amino acids via linkage chemistry (e.g., ester, amide, carbonate, carbamate) [72]. They can target carrier-mediated transporters to improve drug delivery across the cell membrane by overcoming resistance and enhancing permeability [73]. Many amino acid prodrugs that target transporters have been developed and some of them are already commercially available for clinical use (e.g., BMS-582664, CAM-4562, CEP-7055, QC12). Using these approaches, LAT1 can be a useful target to deliver amino acid-stapled cytostatic agents. For example, quinidine was conjugated to the carboxylic group of L-isoleucine to prepare L-isoleucine-quinidine prodrug [74]. The L-isoleucine-quinidine prodrug was selectively transported to the site of action via LAT1 for the treatment of human prostate cancer. The uptake of L-isoleucine-quinidine prodrug showed a 2-fold increase in PC-3 cells compared with that of quinidine. LAT1 has been used to transport nutrients and drugs across BBB [75,76]. Thus, LAT1 prodrug can also be used for the delivery of drugs such as phenylalanine derivative of dopamine [77], phenylalanine derivatives of valproic acid [78], L-tyrosine [76] and L-lysine analogues of ketoprofen [79] to the brain by overcoming BBB. In these studies, prodrugs showed a brain uptake potential, which was inhibited by a LAT1 inhibitor, suggesting that these amino acid prodrugs had entered the brain compartment through a mechanism of LAT1-mediated transport. Chemotherapeutic drugs showed a differential response in cancer cells due to LAT1 expression, which dictated their differential transport into cells. Specifically, LAT1 allows anticancer drugs to overcome BBB without disruption of its integrity. For example, melphalan, a chemotherapeutic drug, has a moderately low affinity to LAT1 as a L-phenylalanine derivative (Km = 90 ~ 150 mM), showing poor brain penetration [80]. However, Matharu et al. reported the enhanced anticancer effect of the melphalan analog, phenylglycine-mustard, due to the improvement of their affinity for LAT1 [80]. In addition, Li et al. reported O,N-carboxymethyl chitosanGly-Gly-melphalan conjugates as prodrugs for melphalan presented good cathepsin X-sensitivity, lower toxicity and improved drug release pattern [81]. Based on the current knowledge, we prepared the amino acid--gemcitabine conjugates showing the enhanced delivery potential by 20 -- 30% in pancreatic cancer cells (unpublished).


4.2.3

LAT1-mediated transport of anticancer drugs Several anticancer drugs are transported by LAT1 as substrates such as melphalan [82-84], acivicin [85-87] and fenclonine (Table 3) [86,88]. Their anticancer activity can be altered via 4.2.4

12

drug--drug or drug--transporter interaction due to their LAT1-mediated transport in cancer cells. For example, melphalan, which behaves more like a LAT1 inhibitor, is a widely used anticancer drug for the treatment of multiple myeloma [82]. Giglia et al. reported that the differential LAT1 transport based on SLC7A5 gene polymorphism caused an altered response to melphalan for the treatment of malignant multiple myeloma [83]. They recommended an individual dosage for patients for effective therapy without gastrointestinal toxicity. In addition, Tsubaki et al. described the enhanced cytotoxic effects of melphalan after a combination with rapamycin -- an mTOR inhibitor -- in multiple myeloma cells, which blocks the LAT1-mediated mTOR pathway in tumorigenesis [84]. Acivicin has a potential anticancer activity for melanoma [85], glioblastoma [86] and recurrent high-grade astrocytoma [87]. Specifically, Geier et al. reported that acivicin anticancer activity is based on the inhibition of LAT1-dependent cancer cell proliferation in T98G cells [86]. In cholangiocarcinoma, fenclonine also has a therapeutic potential to inhibit the increased local serotonin release as a serotonin inhibitor via LAT1-mediated BBB transport [86,88]. Alpini et al. confirmed that the inhibition of serotonin release decreased in vitro and in vivo tumor cell growth in cholangiocarcinoma cell lines, tissues and bile samples from patients [88]. LAT1 inhibitors Specific inhibitors of LAT1 have a pharmacological potential as anticancer drugs to slowdown the cell growth rate in several types of cancer [13,57,89]. Various small molecules (e.g., 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid [BCH], KYT-0353 and JPH203) and genetic materials (e.g., LAT1 siRNA) have been reported as potential anticancer drugs. Table 4 shows the currently used LAT1 inhibitors with specific details regarding their application in various types of cancer. BCH is a widely used LAT1 inhibitor. It is a nonmetabolizable analogue of leucine. In human breast cancer cells such as MCF-7, ZR-75-1 and MDA-MB-231 cells, BCH markedly inhibited the metabolism of water-soluble tetrazolium salt-1 in a dose-dependent manner suggesting that LAT1 was required to maintain cell growth [35]. In KB human oral epidermoid carcinoma cells, Saos2 human osteogenic sarcoma cells and C6 rat glioma cells treated with BCH, LAT1 was inhibited in a time- and dose-dependent manner, resulting in the suppression of cancer cell growth [90]. Specifically, in KB human oral cancer cells, BCH induced cell cycle arrest at G1 phase by inhibiting the cyclin D3--CDK6 complex [91]. A potent inhibitor of LAT1, KYT-0353, was recently reported to have a 50% inhibitory concentration (IC50) value of 4.1 µM for cell growth, which was comparable to that of cisplatin and 5-fluorouracil in HT-29 cells [92]. KYT-0353 also inhibited 14C-leucine uptake in HT-29 cells with an IC50 of 0.06 µM which was about 70-fold lower than its IC50 value for cell growth, suggesting a difference in the susceptibility between cell growth and leucine uptake. 4.2.5




Table 3. LAT1-mediated transport of anticancer drugs. Drugs

Types of cancer

Target (cells/stage) Mechanism of Observation study

Melphalan

-

Measurement by LAT1 transport in Xenopus laevis oocyte

Transporter assay (substrate)

Multiple myeloma

Patients (135)

Multiple myeloma

ARH-77 and IM9

Melanoma

B16F10

GBM

T98G

Recurrent highgrade astrocytoma

Patients

-

HEK-LAT1

Phenotypic assessment of gastrointestinal toxicity and DNA genotyping of SLC7A5 Cell growth Combination treatment with a TNF-ainhibition assay neutralizing antibody and melphalan inhibited NF-kB p65 nuclear translocation and mTOR activation enhancing the cytotoxic effect of melphalan on multiple myeloma cells. NF-kB inhibitor (dimethyl fumarate) or the mTOR inhibitor (rapamycin) suppressed NF-kB p65 nuclear translocation and enhanced the cytotoxic effect of melphalan Cell growth Acivicin had a synergistic action of glutaminase inhibition assay and potentiation of cisplatin cytotoxicity in metastatic melanoma, based on the growth, angiogenic activity and invasiveness of B16F10 cells in vitro and in B16F10 allografts of C57BL/6 mice Cell growth Acivicin presented an antineoplastic activity via inhibition assay inhibition of LAT1-dependent cancer cell (substrate) proliferation in T98G Clinical trial In Phase II study of acivicin in patients, the dose (Phase II) was 12 mg/m2/day intravenously over 30 min, daily for 5 days to be repeated every 3 weeks. There were no objective responders, two patients remained stable, two were not evaluable for response and the other 12 progressed on treatment. The median survival of the patients was 128 days Modeling assay Fenclonine is a serotonin inhibitor, which pass (substrate) blood-brain barrier via LAT1-mediated transport as a LAT1 substrate Cell growth In cholangiocarcinoma, increased local serotonin inhibition assay release may have implications on cholangiocarcinoma cell growth. Inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. Fenclonine is a potential therapeutic agent in cholangiocarcinoma

Acivicin

Fenclonine

Cholangiocarcinoma Cholangiocarcinoma cell line, tissue and bile from patients

Ref.

[82] Melphalan is not transported by LAT1 at high rates. Thus, it was not regarded as a good substrate, and behaves more like a blocker based on relatively large Connolly accessible areas (> 500 A˚2) of chemical structure [83] Individual therapeutic dose of melphalan for multiple myeloma was recommended to be effective in malignant multiple myeloma because of differential LAT1 transport, based on SLC7A5 gene polymorphism [84]

[85]

[86]

[87]

[86]

[88]

GBM: Glioblastoma; LAT1: L-type amino acid transporter 1; mTOR: Mammalian target of rapamycin; NF-kB: Nuclear factor kB.

The activity of JPH203 was reported as a potent LAT1 selective inhibitor in vitro and in vivo [93,94]. It suppressed the growth of YD-38 human oral cancer cells expressing LAT1 and 4F2hc [93]. In this case, JPH203 completely inhibited L-leucine uptake in YD-38 cells, showing a superior activity than BCH. JPH203 can mediate the apoptosis of YD-38 cells through the activation of apoptotic factors, including caspases and

poly(ADP-ribose) polymerase. In addition, it significantly inhibited tumor growth in nude mice bearing HT029 tumors after intravenous administration [94]. LAT1 inhibitors can also be used for combination therapy with other anticancer drugs [33,46,47,95]. Depending on the anticancer drug combined with LAT1 inhibitor, a synergistic or an additive effect is observed on cancer [96]. BCH and bestatin -- antiproliferative aminopeptidase inhibitors -- showed


13

S. -E. Jin et al.


Table 4. LAT1 inhibitors for anticancer therapy. Inhibitors

Types of cancer

Target (cells)

Observation

Small molecules BCH

Human breast cancer

MCF-7, ZR-75-1 and MDA-MB-231

KB Saos2 C6

KYT-0353

Human nasopharyngeal epidermoid carcinoma Human osteogenic sarcoma Rat glioma Human nasopharyngeal epidermoid carcinoma Human colon cancer

Treating cells with BCH markedly inhibited the metabolism [35] of WST-1 in a dose-dependent manner. The system L is required to maintain MCF-7, ZR-75-1 and MDAMB-231 cell growth L-Leucine transport was inhibited in a concentration[90] dependent manner resulting in cell growth inhibition in a time-dependent manner

JPH203 (LAT1 selective inhibitor) Co-treatment BCH + bestatin

KB

The cell cycle arrest at G1 phase caused by inhibiting cyclin D3-CDK6 complex while increasing the expression of a CDK inhibitor, p27 KYT-0353 inhibited 14C-leucine uptake and cell growth in human colon cancer-derived HT-29 cells (IC50, 0.06 and 4.1 µM, respectively) JPH203 significantly inhibited a tumor growth in HT-29 xenograft nude mice model after intravenous administration

[91]

The IC50 value of bestatin, an antiproliferative aminopeptidase inhibitor, was reduced by co-treatment with BCH (10.5- and 4.3-fold decrease in IGROV1 and A2780 cells, respectively), suggesting that the combined therapy has a synergistic effect Cell viability was reduced in H1395 cells. Combination of gefitinib and BCH reduced cell viability more than either agent alone Leucine uptake was inhibited by BCH decreasing Hep-2 cell viability. Co-treatment with cisplatin and BCH reduced cell viability more than either agent alone. An additive effect was observed when cells were first treated with BCH then with cisplatin, while a synergistic effect was observed when cells were first treated with cisplatin then with BCH The combination of melphalan and LAT1 inhibitors showed a synergistic or an additive effect in cancer cells. LAT-1 is highly expressed in Barrett’s adenocarcinoma cell lines which are sensitive to melphalan

[33]

KB

The uptake of [14C]L-leucine was inhibited by LAT1 siRNA

[97]

U266, SK-MM-2, RPMI-8226, OPM-2, NCIH929, LP-1 and L363 HL-60

Downregulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with the accumulation or cytotoxicity of melphalan. Tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affected the intracellular accumulation of melphalan, and thus, determined its cytotoxicity

[98]

HT-29

Human colon cancer

HT-29 xenograft model

Human ovarian cancer

SKOV3, IGROV1, A2780 and OVCAR3

BCH + gefitinib

NSCLC

H1395

BCH + cisplatin

Human head and neck cancer

Hep-2

BCH + melphalan Barrett’s adenocarcinoma Genetic materials LAT1 siRNA Human nasopharyngeal epidermoid carcinoma Human multiple myeloma Human acute myeloid leukemia

Ref.

-

[92]

[94]

[46]

[47]

[95]

BCH: 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; LAT1: L-type amino acid transporter 1; WST-1: Water-soluble tetrazolium salt-1.

synergistic anticancer effects in human ovarian cancer cells blocking the recycling of amino acids [33]. Imai et al. reported that the combination of gefitinib and BCH reduced the viability of NSCLC cells more than either agent alone suggesting that LAT1 overexpression is associated with the wild-type epidermal 14

growth factor receptor (EGFR) as an independent factor [46]. In Hep-2, head and neck squamous cell carcinoma cell line, Yamauchi et al. reported that BCH enhanced the antitumor action of cisplatin via an inhibition of the mTOR pathway decreasing the level of mTOR, p70S6K and 4E-BP1 [47].




Interestingly, if BCH and cisplatin treatment are provided in different orders, a combination effect was differentially obtained. BCH treatment after cisplatin was observed to have a synergistic effect on reducing the cell viability while BCH treatment prior to cisplatin was shown to have an additive effect on reducing Hep-2 cell viability. In Barrett’s adenocarcinoma cells, Lin et al. reported a synergistic or an additive effect of the BCH and melphalan combination [95]. The siRNAs are also used to inhibit LAT1 function as a potential anticancer drug in various types of cancer cells such as KB human oral squamous cell carcinoma [97], and multiple myeloma [98]. The siRNA-mediated LAT1 knockdown suppressed cell proliferation and indicated a biological significance of LAT1 in tumor growth. Regarding melphalan, LAT1 siRNA affected the intracellular accumulation of melphalan, determining its cytotoxicity [98]. 5.

Conclusion

In this review, we focused on the clinical significance of LAT1 as a target for anticancer therapy. LAT1 can be a prognostic biomarker based on its overexpression to reflect cancer stages in patients. In addition, targeting LAT1 with amino acid prodrugs and anticancer drugs transported by LAT1 may offer enhanced drug delivery potentials via transporter--drug interaction. Moreover, radio-labeled LAT1 substrates can be used as PET tracers to diagnose tumor with metastasis. Furthermore, the pivotal role of LAT1 in cancer has been highlighted to suggest the pharmacological benefits of LAT1 inhibitors as anticancer drugs. Although the function of LAT1 still needs to be confirmed by mechanistic studies with regard to evaluating the downstream signaling molecules, it can be a versatile and successful chemotherapeutic target for anticancer therapy. 6.

Expert opinion

Transporters can be attractive targets for drug discovery and development based on the transporter function in disease. Efflux transporters (e.g., P-glycoprotein encoded by ABCB1, MRP1 encoded by ABCC1, etc.) have been extensively studied to prevent the multidrug resistance of chemotherapeutics so far. In spite of researches to control efflux transporters, the modulators for efflux transporters still have many limitations. The modulators may change the pharmacological characteristics of drugs and affect drug metabolism particularly through cytochrome P450. There are multiple resistance mechanisms via several efflux transporters. Therefore, in order to overcome them for the successful chemotherapy, we need to explore with novel approaches. As cutting-edge strategies for anticancer therapy, the influx transporters would be a new research target for anticancer therapy beyond efflux transporters. Of the influx transporters, LAT1 is overexpressed in cancer cell lines and tissues of cancer patients and is associated with

the activation of signaling pathways involved in tumorigenesis. Based on these properties, LAT1 has been studied in various strategies for anticancer therapy such as prognostic biomarkers, radio-labeled tumor imaging reagents, amino acid-stapled prodrugs, LAT1-mediated enhanced transportation of anticancer drugs and LAT1 inhibitors. Using the characteristics of LAT1, we can improve drug delivery potentials avoiding the efflux of anticancer drugs, which will be the pharmacological and pharmaceutical intervention via the interaction between transporters and anticancer drugs. However, several challenges still exist to precisely characterize the function of LAT1 with many signaling pathway, to develop LAT1 specific anticancer drugs and to apply them as a biomarker in pathology, because influx transporters still need to investigate the expression levels in many patients for the prediction of cancer stages and the enhanced delivery of anticancer drugs. The mechanistic study for calculating the uptake potentials and drugabilities of various anticancer drugs transported by LAT1 would also be necessary to obtain the precise therapeutic indexes in clinics [99]. Moreover, the study for the network between LAT1 and other transporters, which were upregulated or downregulated, would be needed to achieve the development of LAT1-based transporter drugs in anticancer therapy. Nevertheless, we expect that novel therapeutic or diagnostic agents might be developed via LAT1 or other influx transporters. LAT1 can be used to further optimize enhanced bioavailability and to improve the individualized use of cancer medicines in clinical practice [100]. Several substrates are well known for the transport through LAT1. By the conjugation of LAT1 substrate on the anticancer drugs in market, their affinity for LAT1 could be improved [80]. LAT1 targeted drugs are constructed in a relatively simple way compared to new drug development processes. One of the recent trends in drug development is the drug repositioning to reduce the timelines of pharmaceutical research and development, which are often associated with increasing risk [101]. If we can modify the existing drugs as candidates for LAT1 targeting, shorter drug development periods and routes to clinic can be possible based on many accumulated data for the existing drugs (i.e., in vitro and in vivo screening, chemical optimization, toxicology, bulk manufacturing, formulation and clinical development data) [101]. Therefore, LAT1 can be a versatile target to be promisingly developed for transporter-based drugs with enhanced drug delivery potential for anticancer therapy.

Declaration of interest The authors were supported by the Inha University Grant and Medical Research Center (No. 2014009392) funded by MSIP, Korea. HE Jin was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2011-357-E00083). The authors have


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no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.


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Affiliation Su-Eon Jin1, Hyo-Eon Jin2 & Soon-Sun Hong†3 PhD † Author for correspondence 1 Yonsei University, College of Pharmacy, Incheon, 406-840, Republic of Korea 2 University of California, Lawrence Berkeley National Laboratory, Department of Bioengineering, Berkeley and Physical Biosciences Division, Berkeley, CA 94720, USA 3 Professor, Inha University, College of Medicine, Department of Drug Development, Incheon, 400-712, Republic of Korea Tel: +82 32 890 3683; Fax: +82 32 890 2462; E-mail: [email protected]

19

Targeting Lipid Metabolic Reprogramming as Anticancer Therapeutics.

Targeting apoptosis for anticancer therapy.

Epigenetic Biomarkers for Parkinson's Disease: From Diagnostics to Therapeutics.

Designing Novel Nanoformulations Targeting Glutamate Transporter Excitatory Amino Acid Transporter 2: Implications in Treating Drug Addiction.

Clinical significance of coexpression of L-type amino acid transporter 1 (LAT1) and ASC amino acid transporter 2 (ASCT2) in lung adenocarcinoma.

Structure and allosteric inhibition of excitatory amino acid transporter 1.

Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

Amino acid ester prodrugs conjugated to the α-carboxylic acid group do not display affinity for the L-type amino acid transporter 1 (LAT1).

Novel clinical therapeutics targeting the epithelial to mesenchymal transition.

Molecular diagnostics and therapeutics for ectopic pregnancy.

Quinone-amino acid conjugates targeting Leishmania amino acid transporters.

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics.

Small molecule-facilitated degradation of ANO1 protein: a new targeting approach for anticancer therapeutics.

Foreword. Respiratory diagnostics and therapeutics.

Targeting glutamate transporter-1 in neurological diseases.

Targeting NK Cells for Anticancer Immunotherapy: Clinical and Preclinical Approaches.

Development of nanoscale approaches for ovarian cancer therapeutics and diagnostics.

Novel diagnostics and therapeutics for drug-resistant tuberculosis.

Conjugation of vitamin E analog α-TOS to Pt(IV) complexes for dual-targeting anticancer therapy.

Telomere biology: Rationale for diagnostics and therapeutics in cancer.

Targeting Neddylation pathways to inactivate cullin-RING ligases for anticancer therapy.

L-type amino acid transporter-1 and CD98 expression in bone and soft tissue tumors.

The role of L-type amino acid transporter 1 in human tumors.

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter.