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Cancer Sci
2016 Mar 01;1073:347-52. doi: 10.1111/cas.12878.
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Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging.
Wei L
,
Tominaga H
,
Ohgaki R
,
Wiriyasermkul P
,
Hagiwara K
,
Okuda S
,
Kaira K
,
Oriuchi N
,
Nagamori S
,
Kanai Y
.
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3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 μM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.
Figure 1. [14C]FAMT transport by system L transporters. The uptakes of 50 μM [14C]FAMT (FAMT) and l‐[14C]methionine (Met) as well as a typical substrate l‐[14C]leucine (Leu) (50 μM) were measured for 15 min for LAT1 (a), LAT2 (b) and LAT3 (c), and for 30 min for LAT4 (d). The uptakes were measured in Na+‐free uptake buffer on the control oocytes (“−”) and the oocytes expressing each transporter (“+”). The uptake rates were expressed as mean ± SEM (n = 8–12). *P < 0.05; n.s., not significant.
Figure 2. [14C]FAMT transport by neutral amino acid transporters other than system L that transport aromatic amino acids. The uptakes of 50 μM l‐[14C]tyrosine (Tyr), [14C]FAMT (FAMT) and l‐[14C]methionine (Met) by TAT1 (a), the uptakes of 50 μM l‐[14C]leucine (Leu), [14C]FAMT (FAMT) and l‐[14C]methionine (Met) by B0
AT1 (b), ATB
0,+ (c), y+
LAT1 (e) and y+
LAT2 (f), and the uptakes of 50 μM l‐[14C]cystine (Cyst), [14C]FAMT (FAMT) and l‐[14C]methionine (Met) by b0,+ (d) were measured on the control oocytes (“−”) and the oocytes expressing each transporter (“+”). For the functional expression of b0,+ in (d), rBAT, an auxiliary subunit of system b0,+ transporter strongly inducing system b0,+ activity in Xenopus oocytes, was expressed in Xenopus oocytes, therefore, the induced system b0,+ activity was not that of human but of Xenopus although human rBAT was expressed.16
l‐[14C]Tyrosine and l‐[14C]cystine were used as typical substrates of TAT1 and b0,+, respectively. l‐[14C]Leucine was used as a typical substrate of B0
AT1, ATB
0,+, y+
LAT1 and y+
LAT2. Na+‐free uptake buffer was used for TAT1 and b0,+, whereas ND96 solution was used for the others. Uptakes were measured for 30 min for B0
AT1 and for 15 min for the others. Uptake rates were expressed as mean ± SEM (n = 5–10). *P < 0.05; n.s., not significant.
Figure 3. Concentration dependence of [14C]FAMT transport. Concentration dependence of [14C]FAMT transport mediated by LAT1 was determined. LAT1‐mediated transport of [14C]FAMT at each concentration was measured for 15 min in Na+‐free uptake buffer. Transport rates were expressed as means ± SEM (n = 7–9) and fit to Michaelis–Menten curve.
Figure 4. Effect of LAT1 knockdown on the uptake of [14C]FAMT and l‐[14C]methionine in HeLa S3 cells. HeLa S3 cells were transfected with LAT1 siRNA (LAT1 siRNA#1, #2, #3) or non‐targeting control siRNA (Ctrl siRNA#1, #2) and compared with non‐treated control (“(−)”). Knockdown of LAT1 with siRNA highly reduced LAT1 protein level in HeLa S3 cells in western blot (a). Accordingly, [14C]FAMT uptake was decreased by the treatment with LAT1 siRNA (b). The uptake of l‐[14C]methionine (Met) was also reduced, although the reduction of [14C]methoinine uptake by LAT1 knockdown was less than that of [14C]FAMT uptake (c). Control siRNA did not affect the levels of LAT1 protein and the uptake of [14C]FAMT and l‐[14C]methionine (a–c).
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