RESEARCH ARTICLE

The impact of L-type amino acid transporter 1 (LAT1)

in human hepatocellular carcinoma

Juan Li & Juan Qiang & Shu-Fen Chen & Xin Wang &

Jing Fu & Yao Chen

Received: 28 April 2013 / Accepted: 10 May 2013

International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Upregulation of L-type amino acid transporter 1

(LAT1) has been reported to be associated with a poor

prognosis in a variety of malignant tumors. However, the

impact of LAT1 in hepatocellular carcinoma (HCC) remains

unclear. The objective of this study was to investigate

whether the expression of LAT1 in HCC was associated

with established clinicopathological features. Quantitative

reverse transcription polymerase chain reaction was used

to detect LAT1 mRNA expression in 23 pairs of fresh-

frozen HCC tissues and corresponding noncancerous tissues.

Results showed that LAT1 mRNA expression level in

HCC tissues was significantly higher than that in

corresponding noncancerous tissues. To investigate the

association between LAT1 protein expression and clinico-

pathological characteristics of HCC, immunohistochemistry

was performed in 148 archived paraffin-embedded HCC

samples. High LAT1 expression in HCC was associated

significantly with tumor size (P=0.032), histological differen-

tiation (P=0.003), and tumor stage (P=0.01). Kaplan–Meier

curves demonstrated that patients with a high expression

of LAT1 have a significantly increased risk of shortened

survival time. Moreover, stepwise Cox regression showed

that LAT1 expression may be an independent prognostic

factor. Collectively, our study demonstrated that LAT1was

overexpressed in HCC and could be served as a potential

prognostic marker.

Keywords LAT1

Hepatocellular carcinoma

Biomarker

Prognosis

Introduction

Hepatocellular carcinoma (HCC) is a highly aggressive

solid tumor associated with poor prognosis [1]. Cura tive

therapies of surgical treatment, including hepatic resection

and liver transplantation, improve the chances of survival of

patients with HCC [2–4]. However, a limited number of

patients can be treated with surgery because of the damage

to liver function. The prognosis for most patients remains

poor after surgery for multicentric recurr ence and intrahe-

patic metastasis [5, 6]. Therefor e, the development of a

systemic therapy that targets a new molecule involved in

HCC is needed.

L-type amino acid transporter 1 (LAT1) is one of the

system L amino acid transporters, transporting large neutral

amino acids such as leucine, isoleucine, valine, phenylala-

nine, tyrosine, tryptophan, methionine, and histidine [7–9].

LAT1 is mainly expressed in human brain, spleen, thymus,

testis, placenta, skeletal muscle, and carcinoma cells such as

prostatic, esophageal, and lung carcinoma [ 8 , 10, 11].

Previous studies have shown that LAT1 is highly expressed

in various human neoplasms [10, 12– 19]. Over expression

of LAT1 protein has now been strongly linked to poor

prognosis in cancer, such as non-small cell lung cancer

[13], transitional cell carcinoma [14], prostate cancer

[10], and pancreatic cancer [15, 16]. However, no infor-

mation is available regarding LAT1 expression in human

HCC. To explore the vita l role of LAT1 in the tumori-

genesis and progression of HCC, we examined expres-

sion patterns of LAT1 in HCC tissues and analyzed the

relationship between LAT1 expression and clinicopatho-

logical factors of HCC.

J. Li (*)

J. Qiang

S.<F. Chen

X. Wang

Department of Clinical Nutrition, Changhai Hospital,

Second Military Medical University, No. 168, Changhai Road,

Shanghai 200433, China

e-mail: [email protected]

J. Fu

Y. Chen

International Cooperation Laboratory on Signal Transduction,

Eastern Hepatobiliary Surgery Institute, Second Military

Medical University, No. 225, Changhai Road,

Shanghai 200438, China

Tumor Biol.

DOI 10.1007/s13277-013-0861-5

Materials and methods

Patients and tissue samples

A total of 148 HCC specimens were obtained as paraffin-

embedded samples from Changhai Hospital, Second

Military Medical University, Shanghai, China. The 148

HCC cases comprised 79 males and 69 females with an

age range of 35 to 76 years (median age, 55.2 years). The

diagnoses were confirmed histologically in all cases, based

mainly on the examination of sections stained with hema-

toxylin and eosin. Table 1 shows the clinicopathological

features of these patients. In addition, 23 self-pairs of HCC

tissue samples and adjacent nonneoplastic tissue samples

were snap frozen in liquid nitrogen and stored at −80 °C

following surgery for real- time quantitative RT-PCR assay.

Tumor stage was determined according to the 2002

International Union Against Cancer TNM classification system

[20]. Tumor differentiation was graded by the Edmondson

grading system. The study was approved by the Research

Ethics Committee of Changhai Hospital, Second Military

Medical University, Shanghai, China. Informed consent

was obtained from all of the patients. All specimens were

handled and made anonymous according to accepted ethical

and legal standards.

Quantitative reverse-transcriptase polymerase chain reaction

Total RNA was extracted using Trizol reagent (Invitrogen Life

Technologies, Ontario, Canada). The qPCR primers to amplify

LAT1 were designed using the qPrimerDepot website

(http://primerdepot.nci.nih.gov/). LAT1 primer patterns are

as follows: 5′-TTGAATTCCGGAACTGACCTTCCAACC

ACC-3′ (forward) and 5′-TTAACCTTAACCTAGGCATA

TTA-3′ (reverse), and glyceraldehyde 3-phosphate dehydro-

genase (GAPDH) primers included: 5′-ATTCCACCCATG

HCCAAATTC-3′, (forward) and 5′-ATTCCACCCATGHC

CAAATTC-3′ (reverse). Quantitative reverse transcription

polymerase chain reaction (qRT-PCR) was carried out using

the FastStart Universal SYBR Green Master (ROX; Roche,

Toronto, Ontario, Canada) on the Bio-Rad CFX96 qRT-PCR

detection system (Applied Biosystems Inc., Foster City, CA,

USA). The CFX Manager software was used to calculate a

threshold cycle (Ct) value for GAPDH and LAT1 during the

log phase of each cycle. Expression data were normalized to

the geometric mean of the housekeeping gene GAPDH to

control the variability in expression levels and then analyzed

using the 2

−ΔΔct

method, where ΔΔCt=ΔCt

LAT1

−

ΔCt

GAPDH

. T o minimize experimental variability, each sam-

ple was tested in triplicate and the mean femtogram expres-

sion level was calculated.

Immunohistochemistry

We performed immunohistochemistry (IHC) assays to

evaluate the expression of LAT1 in HCC according to

standard protocols [21, 22]. Specimens were fixed in

10 % formalin for 12 h, and then paraffin-embedded.

The paraffin-embedded tissues were stored at room tem-

perature. All these collection methods were standardized.

The paraffin sections were deparaffinized by sequential

washing with xylene, graded ethanol, and phosphate-

buffered saline (PBS). After quenching the endogenous

peroxidase activ ity with 3 % hydrogen peroxide for

5 min at room temperature, the sections were treated for 1 h

with 5 % bovine serum albumin to block nonspecific staining.

LAT1 goat-antihuman polyclonal antibody was then added

and incubated at 4 °C overnight. After washing with PBS,

the secondary horseradish peroxidase-conjugated antibodies

were incubated for 30 min at 37 °C. Antibody binding was

visualized by incubating with fresh 3,3′-diaminobenzidine

buffer. The sections were then washed in running water and

counterstained with hematoxylin, followed by dehydration

and mounting. The extent and intensity of IHC were assessed

Table 1 Correlations of LAT1 expression with the clinicopathological

features of HCC

Variable No. of cases LAT1 expression P value

Low High

Age (years)

<60 66 32 34 0.481

≥60 82 35 47

Sex

Male 79 38 41 0.459

Female 69 29 40

Tumor size (cm)

≤5 61 34 27 0.032

>5 87 33 54

Histological differentiation

Well/moderate 64 38 26 0.003

Poor 84 29 55

Liver cirrhosis

With 115 44 71 0.104

Without 33 13 10

HBsAg

Positive 107 48 59 0.871

Negative 41 19 22

AFP (ng/ml)

≤400 25 15 10 0.105

>400 123 52 71

Tumor stage

I–II 52 31 21 0.01

III–IV 96 36 60

Tumor Biol.

to determine the expression level of LAT1. The intensity of

the immunostaining was categorized as follows: no brown

particle staining: 0; light brown particle: 1; moderate brown

particle: 2; and dark brown particle: 3. The extent of

immunostaining was quantified by counting the percentage

of positive cells and classified into four groups: 0, less than

25 % positive cells; 1, 25–50 % positive cells; 2, 51–75 %

positive cells; and 3, more than 75 % positive cells. The

sum of the extent and intensity scores was defined as

staining index (SI). SI less than 3 was considered as low

expression, while SI of 4 or more was considered as high

expression. To avoid interindividual bias of IHC staining

differentiations, all slides were determined by two experi-

enced pathologists.

Statistical analysis

Statistical analyses were performed using the SPSS 16.0

software (SPSS, Chicago, IL, USA). The Student s’ t test

was used for comparison between groups. The χ

test

was performed to analyze the correlation between LAT1

expression and clinicopathological parameters. The

Kaplan–Meier method (the log-rank test) was used for

survival curves. Cox regression model with stepwise

manner (forward, likelihood ratio) was utilized to perform

a multivariate analysis. A P value <0.05 was considered

to be statistically significant.

Results

Expression of LAT1 mRNA in HCC tissues by qRT-PCR

We examined LAT1 mRNA expression in 23 pairs of HCC

and adjacent noncancerous tissues by qRT-PCR. As shown

in Fig. 1, the increased LAT1 mRNA expression in HCC

was observed in 16 of the 23 cases, suggesting that LAT1

mRNA level was significantly higher in HCC tissues com-

pared to that in adjacent noncancerous tissues (1.56±0.31 vs

0.82±0.18, P <0.001; Fig. 1).

Correlation of LAT1 protein expression

with clinicopathological characteristics

The correlation between the clinicopathological characteris-

tics and LAT1 expression is shown in Table 1.Accordingto

the immunohistochemical results, LAT1 was highly and

lowly expressed in 81 (54.7 %) and 67 (45.3 %) of the

148 HCC patients, respectively (Fig. 2). LAT1 expression

was positively correlated with tumor size (P=0.032), histo-

logical differentiation (P=0.003), and tumor stage (P=0.01).

However, it was not correlated with patients’ age, gender, liver

cirrhosis, HBsAg, and serum AFP (Table 1).

Association of LAT1 expression with overall survival

in patients with HCC

Kaplan–Meier analysis using the log-rank test was performed

to determine the association of LAT1 expression with clinical

outcome of HCC patients (Fig. 3). The results showed that

high LAT1 expression was markedly associated with a

shorter overall survival (P<0.001). Univariate Cox regres-

sion analysis also identified that clinical variables includ-

ing tumor size, histological differentiation, tumor stage,

and LAT1 expression were significantly associated with

overall survival (Table 2). Furthermore, multivariate Cox

regression analysis was performed to evaluate the potential

of LAT1 expression as an independent predictor for overall

survival of HCC patients. While other factors failed t o demon-

strate independence, tumor size, histological dif ferentiation,

Fig. 1 LAT1 mRNA expression in HCC tissues. LAT1 mRNA level in

HCC tissues was significantly higher compared to that in adjacent

noncancerous tissues (1.56±0.31 vs 0.82±0.18, P<0.001)

Fig. 2 Immunohistochemical

analysis of LAT1 in HCC. a

Positive LAT1 expression in

HCC tissues. b Negative LAT1

expression in HCC tissues

Tumor Biol.

tumor stage, and LAT1 expression may play a role in predicting

overall survival in HCC (Table 2).

Discussion

Tumor invasion and metastasis are an important issue for

understanding tumor biology and further improving the

prognosis of patients with carcinomas, including HCC.

This is a very complex process with multiple promoters or

suppressor genes invol ved. Understa nding the genes respon-

sible for either enhancing or suppressing this process would

enable novel diagnostic, therapeutic, and prognostic appli-

cations to evolve and thus improve the clinical outcome of

HCC patients.

LAT1 is one of the system L amino acid transporters,

transporting large neutral amino acids such as leucine,

isoleucine, valine, phenylalanine, tyrosine, tryptophan, methi-

onine, and histidine [7–9]. LAT1 is mainly expressed in human

brain, spleen, thymus, testis, placenta, skeletal muscle, and

carcinoma cells such as prostatic, esophageal, and lung carci-

noma [8, 10, 11]. Previous studies have shown that LAT1 is

highly expressed in various human neoplasms [10, 12–19].

Overexpression of LAT1 protein has now been strongly linked

to poor prognosis in cancer , such as non-small cell lung cancer

[13], transitional cell carcinoma [14], prostate cancer [10],

and pancreatic cancer [15, 16]. However, no information is

available regarding LAT1 expression in human HCC. To

explore the vital role of LAT1 in the tumorigenesis and

progression of HCC, we examined expression patterns of

LAT1 in HCC tissues and analyzed the relationship between

LAT1 expression and clinicopathological factors of HCC.

In this study, we demonstrated that LAT1 mRNA level in

HCC tissues was significantly higher compared to that in

adjacent noncancerous tissues. It suggested that LAT1 might

play a role in the tumorigenesis of HCC. To investigate

whether LAT1 can accurately predict the outcome in

patients with HCC, IHC was performed in 148 archived

paraffin-embedded HCC samples. Interestingly, the expres-

sion of LAT1 in HCC was closely correlated with tumor size

(P=0.032), histological differentiation (P=0.003), and tumor

stage (P=0.01).

Kaplan–Meier analysis was used to evaluate the survival

of patients with HCC. Patients with high LAT1 expression

were likely to be with significantly shorter overall survival.

We next evaluated the LAT1 expression and other clinico-

pathological factors on prognosis of HCC, using univariate

analyses. Results indicated that tumor size (hazard ratio

(HR), 1.314; 95 % CI, 0.786–2.016; P=0.021), histological

grade (HR, 1.197; 95 % CI, 0.535–2.193; P=0.028), tumor

stage (HR, 1.297; 95 % CI, 0.674–2.188; P=0.019), and LAT1

expression (HR, 1.375; 95 % CI, 0.693–2.184; P<0.001)

were significant predictors of cancer-specific survival. Age,

gender, liver cirrhosis, HBsAg, and serum AFP did not

significantly affect cancer-specific survival (Table 2).

Furthermore, LAT1 expression and those clinicopathological

variables significant in univariate analysis were further eval-

uated in multivariate analysis. Results indicated that tumor

size (HR, 1.296; 95 % CI, 0.719–2.087;

P=0.017), histo-

logical grade (HR, 1.215; 95 % CI, 0.635–2.017; P=0.011),

tumor stage (HR, 1.246; 95 % CI, 0.708–2.056; P=0.015),

and LAT1 expression (HR, 1.425; 95 % CI, 0.832–2.293;

Fig. 3 Kaplan–Meier analyses of overall survival periods among 148

curatively resected HCC patients are shown stratified according to

LAT1 expression

Table 2 Univariate analysis and

multivariate analyses showing

the overall survival rate for HCC

patients

Variables Univariate analysis Multivariate analysis

HR 95 % CI P value HR 95 % CI P value

LAT1 1.375 0.693–2.184 <0.001 1.425 0.832–2.293 <0.001

Age 1.234 0.673–1.983 0.623 1.288 0.687–1.978 0.586

Gender 1.293 0.619–1.904 0.575 1.176 0.782–2.013 0.498

Tumor size 1.314 0.786–2.016 0.021 1.296 0.719–2.087 0.017

Histological grade 1.197 0.535–2.193 0.028 1.215 0.635–2.017 0.011

Liver cirrhosis 1.297 0.674–2.006 0.585 1.043 0.586–1.988 0.624

HBsAg 1.318 0.649–2.248 0.679 1.108 0.586–2.198 0.576

AFP (ng/ml) 1.193 0.694–1.983 0.283 1.294 0.619–2.095 0.382

Tumor stage 1.297 0.674–2.188 0.019 1.246 0.708–2.056 0.015

Tumor Biol.

P<0.001) were also independent predictor for overall survival

of HCC patients (Table 2).

In conclusion, we showed that LAT1 is overexpressed in

HCC tissues. Moreover, our study provides the clinical

evidence that LAT1 is independently prognostic for outcome

in HCC. Independent validation of these clinical findings,

examination of LAT1 expression in other kinds of cancers,

and further investigation of the cell biology of LAT1 and its

potential as a therapeutic target are clearly warranted.

Acknowledgments This work was supported by the National

Natural Science Foundation of China (no. 31200887).

Conflicts of interest None

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