Abstract
Background: L-type amino acid transporter 1 (LAT1) is highly expressed in various human neoplasms. Antitumor activity of inhibiting LAT1 was analyzed in non-small cell lung cancer (NSCLC). Materials and Methods: Expression of LAT1 mRNA in 54 lung cancer cell lines was examined by RT-PCR. An inhibitor of LAT1, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), was administered to H1395 cell. LAT1 expression was examined in correlation with clinical features and outcome in 51 NSCLC patients. Results: Inhibition of LAT1 by BCH reduced cell viability in H1395 cells. Furthermore, co-administration of gefitinib with BCH reduced the viability of the cells more than either agent alone. Inhibition of LAT1 reduced the level of phosphorylation of mTOR, p70S6K and 4EBP1. LAT1 protein expression was closely associated with wild type EGFR, and was an independent significant factor to predict a poor prognosis. Conclusion: Inhibition of LAT1 may be a new rationale to the effective therapy of NSCLC without EGFR mutation.
Lung cancer remains the most common cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers (1). Although surgery is the standard treatment, the majority of patients with NSCLC are inoperable at the time of diagnosis. Meta-analysis has demonstrated that platinum-based chemotherapy may have a better potential for prolongation of survival than the best supportive care for these patients (2), however, recent phase III trials demonstrate that a median survival time (MST) of advanced NSCLC is eight to ten months with a one year survival rate of 30-35% (3).
Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is expressed in NSCLC. Approximately 70% of NSCLC cases with EGFR mutations respond to EGFR-tyrosine kinase inhibitors (TKIs) (4). Gefitinib is an effective drug in the treatment of advanced NSCLC with EGFR mutation (5), however, most patients with NSCLC do not benefit from or develop resistance to specific inhibitors of the EGFR TK activity. Nowadays, the use of EGFR-TKI is controversial in NSCLC patients without the EGFR mutation. Therefore, the clinical course of such patients is very poor and a new treatment strategy is needed.
Amino acid transporters are essential for growth and proliferation of normal and transformed cells (6, 7). Amino acid transport system L is a Na+-independent transport agency for large neutral amino acids (6, 8). L-type amino acid transporter 1 (LAT1) is one of the system L amino acid transporters, and transports large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine and histidine (8-10). LAT1 requires covalent association with the heavy chain of 4F2hc cell surface antigen (CD98) for its functional expression on the plasma membrane (8). Previous studies have shown that LAT1 is highly expressed in various human neoplasms (10). Recent studies have demonstrated that the level of expression of LAT1 is a significant factor indicating a poor prognosis in various human cancers including NSCLC (11-15). LAT1 provides cancer cells with the essential amino acids not only for protein synthesis but also for the stimulation of growth of cancer cells via mammalian target of rapamycin (mTOR) (16-18). Recently, Yamauchi et al. reported that an inhibitor of system L amino acid transporter reduced the level of phosphorylation of mTOR and its downstream signaling molecules in a head and neck squamous cell carcinoma cell line (19).
Comparison of mRNA levels of the (A) LAT1 and (B) CD98 genes relative to those of the TBP gene between lung cancer cell lines (N=54) as measured by quantitative real-time RT-PCR analysis. (C) Immunoblots of the membrane fraction of H1395 cells with anti-LAT1 and anti-CD98 antibodies in reducing (DTT+) and non-reducing (DTT-) conditions. (D) The effect of BCH (0.1-20 mM) on [14C] L-leucine (3 μM) uptake in H1395 cells. The leucine uptake is expressed as a percentage of control (NT, no BCH) and plotted against BCH concentration. Each data point represents mean ± SEM (n=3). Abscissa represents BCH concentration in log scale. (E) The effect of BCH on cellular viability. Viability of the H1395 cells in the presence of the different concentrations of BCH for six days was measured by the MTT assay. The data are presented as percentage of control (no BCH) (n=6).
mTOR is a key intracellular kinase integrating proliferation and survival pathways and is associated with the resistance to EGFR inhibitors. Recent studies described that an inhibitor of mTOR causes antitumor activity in EGFR-resistant NSCLC cancer cell lines and xenografts (20, 21). These data suggest that combination of EGFR targeted therapy with mTOR inhibitors potentially benefit a selected group of NSCLC patients. Therefore, EGFR and mTOR are currently under investigation as potential targets for anticancer therapy in various malignant diseases. Since the inhibition of system L amino acid transporter has been associated with down-regulation of mTOR (19), the inhibition of LAT1 is expected to exert similar antitumor effects on lung cancer cell lines. However, to date, it is unknown whether the inhibitor of LAT1 has antitumor activity in lung cancer. Moreover, the antitumor efficacy of combining LAT inhibitor with EGFR TKIs remains to be clarified.
This study investigated antitumor activity of the inhibitor of LAT1, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), in NSCLC cell lines. The effect of combining EGFR TKIs with LAT1 inhibitor was also examined. Clinicopathological analyses were performed to determine the association of LAT1 expression and EGFR mutation.
Materials and Methods
Reagents. Dithiothreitol (DTT) was obtained from Wako (Tokyo, Japan). Gefitinib was obtained from LC laboratories (MA, USA). All the other reagents were obtained from Sigma unless otherwise indicated.
Cell culture. Cell lines used in the present study included the following 22 small cell lung cancers (SCLCs) (NCI-H187, -H209, - H345, -H378, -H524, -H526, -H740, -H865, -H889,- H1045, - H1092, -H1184, -H1238, -H1339, -H1607, -H1618,- H1672, - H1963, -H2141, -H2171, -H2227, -HCC33) and 32 NSCLCs (NCI- H23, -H157, -H322, -H358, -H441, -H460, -H520, -H661, -H838, -H1264, -H1299, -H1395, -H1437, -H1648, -H1666, -H1792, - H1819, -H2009, -H2077, -H2087, -H2106, -H2122, -HCC15, - HCC44, -HCC78, -HCC95, -HCC193, -HCC515, -HCC827, - H3255, -A427 and A549). These cell lines were cultured in RPMI 1640 with 5% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a humidified incubator at 37°C with 5% CO2. Cancer cells were kindly provided by Dr. John D. Minna and Adi F. Gazdar of the University of Texas Southwestern Medical Center at Dallas.
Real-time RT-PCR. The expression of LAT1 and CD98 genes was examined by quantitative real-time RT-PCR as previously described (22). Briefly, total RNA was extracted by using the RNeasy mini kit (Qiagen, Valencia, CA, USA), and the cDNA was synthesized by using 2 μg of total RNA with the SuperScript II First-Strand Synthesis with oligo (dT) primer system (Invitrogen) according to the manufacturer's instructions. Primers and probes for LAT1 (SLC7A5) (Assay ID: Hs00185826_m1) and CD98 (SLC3A2) (Assay ID: Hs00374243_m1) were obtained from Applied Biosystems (Tokyo, Japan). For the quantitative analysis, the TBP gene was used as an internal reference gene to normalize input cDNA as described previously (22). PCR was performed in a reaction volume of 20 μl, including 2 μl of cDNA by using the Gene Amp 7700 Sequence Detection System and software (Applied Biosystems). The comparative Ct method was used to compute relative expression values.
Western blot analysis. Cells were seeded at 1×106 cells in 9 cm plates, cultured for two days, then washed twice with ice-cold PBS and harvested by scraping in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1% TritonX-100) with protease inhibitor cocktail (Roche, Switzerland). After homogenization and centrifugation for 30 min at 13,000g, the supernatants (30 μg of protein) in the presence or absence of DTT were subjected to SDS–PAGE, followed by transferring to nitrocellulose membranes (BIORAD, Hercules, CA, USA). Immunoblotting was performed using either anti-LAT1 antibody (1:500) (9) or anti-CD98 antibody (1:2000) (Santa Cruz Biotechnology, CA, USA) and horseradish peroxidase-conjugated anti-rabbit IgG as a secondary antibody (GE Healthcare, UK). The images were obtained with high performance chemiluminescence film (GE Healthcare, UK). The levels of phosphorylation were analyzed as follows: cells were seeded at 1×105 cells/well in 6-well plates, cultured for two days, then treated with or without 20 mM BCH. After six days of treatment, the cells were washed twice with ice-cold PBS and harvested by scraping in cell lysis buffer (see above) with PhosSTOP (Roche, Switzerland) and protease inhibitor cocktail (Roche, Switzerland). After homogenization and centrifugation for 30 min at 13,000g, the supernatants (15 or 30 μg of protein) were subjected to SDS–PAGE, followed by transferring to nitrocellulose membranes (BIORAD, Hercules, CA, USA). For immunodetection, following antibodies were used: antiphospho-mTOR (1:5000), anti-phospho-p70S6K (1:3000), anti-phospho-4EBP1 (1:1000) (all obtained from Cell Signaling Technology, USA) and anti-β-actin (1:5000). The images were obtained with high performance chemiluminescence film (GE Healthcare, UK).
[14C]L-leucine uptake assay. H1395 cells were seeded on 24-well plates at 1×105/ well in MEM at 37°C in 5% CO2 and cultured for two days. After removing the medium, the cells were washed three times with fresh MEM and then incubated in MEM containing 3 μM [14C] L-leucine for 1 min in the absence or presence of BCH (0.1 mM, 1 mM, 10 mM, 20 mM) (23). After washing twice with ice-cold uptake solution, the cells were solubilized with 0.1N NaOH and the radioactivity was measured by a liquid scintillation counter.
Cell viability assay. Cell viability was measured by using the 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) Cell Growth Assay Kit (Chemicon International, Temecula, CA, USA), according to the manufacturer's protocol. Trypan blue-negative viable cells were plated and cultured in 96-well plates in replicates of eight. The cells were maintained for six days under the Indicated treatments and then incubated with 0.5 mg/ml MTT for 4 hr at 37°C. After MTT withdrawal, the resulting blue formazan cristae were solubilized, and the absorbance was read at 570/630 nm with a microtiter plate reader.
Patients and tissue sample collections. Tumor specimens for real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunohistochemical staining were obtained from 51 patients with a histologically confirmed NSCLC who underwent resection lobectomy with mediastinal lymph node dissection at Gunma University Hospital between April 2003 and May 2007. None of the patients had been treated before surgery. Institutional review board permission and informed consent were obtained for all cases.
The age of the patients ranged from 40 to 80 years, and the mean age at the time of surgery was 69 years. The tumor specimens were histologically classified according to the criteria of the World Health Organization. Postsurgical pathologic stage determined by the current tumor-node-metastasis classification was used for the selection of patients in this study (24). The stage of the disease was postoperative pathologic stage. Therefore, the stage III and IV diseases included preoperative clinical stage I or II diseases. The tumors were frozen and stored at −80°C until DNA extraction. Genomic DNA was prepared by a previously described method (25) or by using a QIAamp DNA Mini Kit (Qiagen, Tokyo, Japan). As postoperative adjuvant therapies, platinum-based chemotherapy and oral administration of tegafur (a fluorouracil derivative drug) were administered to one and three patients, respectively. Intra-operative therapy was not performed on any of the patients. The postoperative clinical course was assessed by analyzing outpatient medical records and by making telephone inquiries. The day of surgery was considered the starting day for counting postoperative survival. The follow-up duration ranged from 7 to 69 months (median, 47 months).
(A) Treatment schedule; H1395 cells were seeded in 96-well plates at 5×103/well in their growth medium and cultured for 2 days. Indicated treatment was started at this point and continued for 6 days followed by the MTT assay. (B) The effect of BCH treatment on gefitinib action; Viability of the H1395 cells was measured at various concentration of gefitinib by the MTT assay and expressed as percentage of control (no BCH or gefitinib). Each data point represents mean ± SEM (n=8). **p<0.01
Immunohistochemical staining
LAT1 and CD98: LAT1 expression was determined by immunohistochemical staining with an affinity-purified rabbit polyclonal anti-human LAT1 antibody (1.2 mg/ml; 1:3200) (10). An oligopeptide corresponding to amino acid residues 497-507 of human LAT1 (CQKLMQVVPQET) was synthesized. The N-terminal cysteine residue was introduced for conjugation with keyhole limpet hemocyanine. Antipeptide antibody was produced as described elsewhere (26). For immunohistochemical analysis, antiserum was affinity-purified as described previously (26). CD98 (Santa Cruz Biotechnology, Inc. 1:200 dilution) is an affinity purified goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of CD98 of human origin. The detailed protocol for immunostaining has been published elsewhere (27-29). The expression of LAT1 and CD98 was considered positive only if distinct membrane staining was present. Staining intensity was scored as follows: 1, ≤10% of tumor area stained; 2, 11-25% stained; 3, 26-50% stained; and 4, ≥51% stained. The tumors in which stained tumor cells made up more than 10% of the tumor were graded as positive.
The effect of BCH on the phosphorylation of mTOR, p70S6K, 4EBP1 in H1395 cells. Cells were incubated in the normal growth medium for 6 days under the presence or absence of 20 mM BCH. Whole cell lysates (15 or 30 μg of protein) were subjected to Western blot analysis. Phospho-mTOR, phospho-p70S6K, phospho-4EBP1 and β-actin were probed. β-actin expression was used to assess for protein extract quality and loading. Two independent samples were examined for each condition.
Ki-67: The detailed protocol for immunostaining has been published elsewhere (27-29). For immunostaining, the murine monoclonal antibody MIB-1 (Dako, Denmark), specific for human nuclear antigen Ki-67, was used in a 1:40 dilution. All epithelial cells with nuclear staining of any intensity were defined as positive. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of MIB-1-stained nuclei (Ki-67 labeling index) in the sample.
VEGF, CD31, and CD34: The detailed protocol for immunostaining has been published elsewhere (27-29). The antibodies used were: a monoclonal antibody against VEGF (Immuno-Biological Laboratories Co.,Ltd., Japan, 1:100 dilution); a mouse monoclonal antibody against CD31 (Dako,1:50 dilution); and a mouse monoclonal antibody against CD34 (Nichirei, Tokyo, Japan,1:200 dilution). The expression of VEGF was assessed according to the percentage of immunoreactive cells in a total of 1000 neoplastic cells (quantitative analysis). MVD was assessed using the criteria of Weidner et al. (30). The areas of highest neovascularization were identified as regions of invasive carcinoma with the highest numbers of discrete microvessels stained for CD31 and CD34. The number of CD31- and CD34-positive vessels was counted in four selected hot spots in a × 400 field (0.26 mm2 field area). The mean value of the two independent readings of the same specimen was calculated, and MVD was defined as the mean count of microvessels per 0.26 mm2 field area (31).
Expression of these immunohistochemical markers (LAT1, CD98, Ki-67, VEGF, CD31 and CD34) was evaluated by two investigators without knowledge of patient outcome.
Mutation analysis of the EGFR, KRAS, P53 genes. Genomic DNA was extracted from an approximately 3-5mm cube of tumor tissue using a DNA Mini Kit (Qiagen; Tokyo, Japan) and serially diluted to 20 ng/ul. Exon 19 and 21 EGFR mutations were detected using a non-radioactive single-strand conformation polymorphism (Non-RI-SSCP) (32) and the Smart Amplification Process version 2 (SmartAmp2) (33, 34). The SmartAmp2 method is a unique genotyping technology that can detect a mutation in a single step within 30 min under isothermal conditions. It is based on strand-displacing DNA polymerase activity and can amplify and detect mutations directly from simple lung cancer sample preparations (33, 34).
(A) Kaplan-Meier estimates of overall survival in 51 patients with surgically resected non-small cell lung cancer according to the expression of LAT1 mRNA. LAT1 mRNA levels were measured by real-time quantitative reverse transcriptase-polymerase chain reaction in primary tumor specimens. The relative amount of tissue LAT1 mRNA was standardized against the geometric mean of TBP and glyceraldehydes 3-phosphate dehydrogenase mRNAs. The median LAT1 value was used to divide patients into high LAT1- and low LAT1-expressing groups. P value was determined using a two-sided log-rank test. High LAT1 mRNA expression was not associated with significantly decreased overall survival (p=0.1533). (B) Kaplan-Meier estimates of overall survival in 51 patients with surgically resected non-small cell lung cancer according to the expression of LAT1 protein. LAT1 protein expression was examined by immunohistochemical analysis of primary tumor specimens. P value was determined using a two-sided log-rank test. Positive LAT1 staining of tumor cells was associated with poor overall survival in patients with non-small cell lung cancer (p=0.0002).
The KRAS mutation was also detected by the SmartAmp2 assay using DNA extracted from tumor tissue (35). The EGFR and KRAS Mutation Detection Kit for SmartAmp2 assay was obtained from DNAFORM K.K. (Yokohama, Japan).
Genomic DNA fragments covering the entire coding region of the p53 gene, between exon 5 and exon 8, were amplified by PCR with p53-specific oligonucleotide primers as follows; exon 5 (forward primer 5′-TGTTCACTTGTGCCCTGACT-3′ and reverse primer 5′-AGCAATCAGTGAGGAATCAG-3′), exon 6 (forward primer 5′-CCCAGGCCTCTGATTCCTCA-3′ and reverse primer 5′-CAACCACCCTTAACCCCTCC-3′), exon 7 (forward primer 5′-CTTGCCACAGGTCTCCCCAA-3′ and reverse primer 5′-AGGGGTCAGCGGCAAGCAGA-3′), and exon 8 (forward primer 5′-TCCTTACTGCCTCTTGCTTC-3′ and reverse primer 5′-TCTCCTCCACCGCTTCTTGT-3′). PCR was carried out by 35 amplification cycles of 94°C (4 min) for denaturation, 55°C (exon 5), 62°C (exon 6,7) or 60°C (exon 8) (30 s) for annealing, 72°C (30 s) for extension followed by 10 min extension at 72°C. The PCR products were then purified using the QIAquick PCR Purification kit (Qiagen Inc., CA, USA). DNA sequencing was performed with the ABI PRISM 3100 DNA Analyzer (Applied Biosystems; CA, USA) using the ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems) with the p53ex5-8F primer.
Statistical analysis. Probability values of <0.05 were taken to indicate statistically significance. Fisher's exact test was used to examine the association of two categorical variables. Correlation analysis was performed using the nonparametric Spearman's rank test. Survival time was determined as the time from tumor resection to death from any cause. For survivors, survival times were censored on the last date that patients were known to be alive. The Kaplan-Meier method was used to estimate survival as a function of time, and survival difference were analyzed by the log-rank test. Multivariate analyses were performed using stepwise Cox proportional hazards model to identify independent prognostic factors. Statistical analysis was performed using JMP 8 (SAS, Institute Inc., Cary, NC, USA) for Windows.
Results
H1395 cells expressed LAT1 and CD98. The expression of LAT1 and CD98 mRNA was examined by quantitative real-time RT-PCR in 54 lung cancer cell lines (22 SCLCs and 32 NSCLCs). The levels of expression of LAT1 (Figure 1A) and CD98 (Figure 1B) were the highest in H1395. The expression of LAT1 mRNA was significantly correlated with that of CD98 mRNA. Next, a Western blot analysis was performed to confirm the existence of LAT1 and CD98 proteins in H1395 cells. Both antibodies against LAT1 and CD98 recognized the 125-kDa-protein under the non-reducing condition (DTT-), whereas a 37-kDa-protein for LAT1 and an 85-kDa-protein for CD98 were detected under the reducing condition (DTT+), confirming that H1395 expresses both LAT1 and CD98 and that they form a heterodimer via a disulfide bond between them (Figure 1C). H1395 is a cell line of adenocarcinoma characterized as EGFR wild type and KRAS wild type.
BCH inhibited [14C] L-leucine uptake by H1395 cells and cellular proliferation. BCH is a well-documented inhibitor for system L amino acid transporters (36). To confirm its inhibitory activity on system L-mediated amino acid transport in H1395 cells, the uptake of radiolabeled leucine, a substrate of system L amino acid transporter, in the presence or absence of BCH was measured. BCH inhibited the uptake of [14C] L-leucine in a concentration-dependent manner (Figure 1D).
Clinicopathological characteristics according to LAT1 expression.
The effect of BCH on cellular proliferation of H1395 cells was then examined by the MTT assay. In the presence of different concentrations of BCH, the total number of viable H1395 cells decreased as compared with control (no BCH) (Figure 1E). These results suggest that the effect of BCH seems to be mediated through the inhibition of amino acid uptake but not through non-specific toxic effect of the drug.
Combination of gefitinib with BCH enhanced its anti-tumor activity. Whether the combination of gefitinib with BCH enhances the anti-tumor activity of gefitinib was examined. The treatment schedule is shown in Figure 2A. Cells were treated with various concentration of gefitinib. Incubation with gefitinib alone caused cytotoxicity in a concentration-dependent manner. Addition of 5 mM BCH to gefitinib further reduced the number of viable cells (Figure 2B). The result indicated the effects of gefitinib and BCH are additive.
BCH inhibited mTOR pathway in H1395 cells. The effect of BCH on the activity of mTOR signaling pathway was investigated to elucidate the mechanism of additive effect of BCH to gefitinib. Treatment with 20 mM BCH for six days reduced the level of phosphorylation of mTOR and its downstream signaling molecules, p70S6K and 4EBP1 (Figure 3).
Clinicopathological analysis. To explore the prognostic significance of LAT1 and CD98 expression in human NSCLC, real-time quantitative RT-PCR was performed to examine LAT1 mRNA expression in 51 patients with NSCLC. The relative amount of LAT1 and CD98 mRNA in tumor tissues was standardized against the geometric mean TATA box binding protein (TBP) and glyceraldehydes 3-phosphate dehydrogenase mRNAs, and individual samples were categorized as low or high LAT1 and CD98 expression by the median expression level. High LAT1 mRNA expression was not significantly associated with the overall survival (Figure 4A; p=0.1533).
Immunohistochemical analysis was performed to examine the expression of LAT1, CD98, Ki-67, VEGF, CD31, and CD34 protein in tumor specimens from the 51 patients as indicated above. Expression of LAT1 and CD98 was localized predominantly on the plasma membrane of carcinoma cells in tumor tissues and the cytoplasmic staining was rarely evident as described previously (27-29). Positive LAT1 staining of tumor cells was associated with the poor overall survival (Figure 4B; p=0.0002).
Result of the univariate analyses (log-rank test) (n=51).
Clinicopathological analyses were performed by real-time qRT-PCR and immunohistochemistry to correlate with LAT1 expression (Table I). LAT1 protein expression was significantly associated with gender, disease staging, CD98, Ki-67, VEGF, CD31, CD34, EGFR wild-type, lymphatic permeation and vascular invasion. LAT1 mRNA expression was significantly associated with gender, CD98, Ki-67, VEGF and EGFR wild-type. Expression of LAT1 protein was closely associated with that of LAT1 mRNA. Expression of LAT1 protein and LAT1 mRNA was also significantly associated with CD98 expression, cell proliferation, angiogenesis and EGFR wild-type.
A univariate analysis of prognostic factors was performed in 51 patients with NSCLC (Table II). According to the results of log-rank test, the following ten factors were screened with a cut-off of p=0.10; disease staging, LAT1 protein, Ki-67, VEGF, CD31, CD34, KRAS, pleural involvement, lymphatic permeation and vascular invasion. Multivariate analysis confirmed that LAT1 protein expression, disease staging, MVD, and pleural involvement were independent significant factors to predict a poor prognosis.
Discussion
The present study confirmed antitumor activity of the inhibition of LAT1 in NSCLC cell lines and the antitumor effects of combining LAT1 inhibitor with EGFR TKIs. The results demonstrated that a lung cancer cell line, H1395, expressed LAT1 and CD98 and the inhibition of LAT1 reduced the cellular proliferation in lung cancer cells. The effect of BCH and that of gefitinib was additive in antitumor actions. Moreover, the inhibitor of LAT1 reduced the level of phosphorylation of mTOR and its downstream signaling molecules, p70S6K and 4EBP1. A previous study showed that the inhibitor of LAT1 decreased tumor cell viability and BCH synergistically enhanced cytotoxic effect of cisplatin (19). However, the association between EGFR-targeted therapy and LAT inhibitor was not been examined in that study. In the present study, antitumor activity of gefitinib was enhanced by the additive administration of BCH. System L amino acid transporters regulated multiple cellular activities including apoptosis through mTOR pathway (37), and the current results indicated that phosphorylation of mTOR was decreased by the inhibition of LAT.
The inhibition of mTOR signaling pathway is believed to be a promising therapeutic option in various neoplasms. Everolimus (RAD001), an orally bioavailable derivative of rapamycin, is one of the inhibitors of mTOR currently under investigation. RAD001 has shown antitumor activity both as a single agent and in combination with other anticancer agents in both in vitro and in vivo tumor model, including NSCLC (38, 39). Recent studies showed that everolimus was effective in not only tumor cells with acquired resistance to anti-EGFR drugs, but was also in tumors sensitive to anti-EGFR drugs (20, 21). These preclinical data suggested either additive or synergistic interactions by a combination of EGFR and mTOR inhibitors in NSCLC. The present study indicated an additive effect by the combination of gefitinib and LAT1 inhibitor in NSCLC cell line. The possible mechanism of the additive effect is thought to be an inactivation of mTOR by the treatment with BCH. This is similar to the effect of mTOR inhibitor. However, it is unknown whether an inhibitor of LAT1 would be able to restore gefitinib sensitivity in the resistant NSCLC cell lines. Further study is warranted to investigate how LAT1 expression is associated with two major intracellular signaling pathways activated by EGFR, the PI3K/AKT/mTOR and the RAS/RAF/MARK cascades.
In the present study, the association with LAT1 expression and EGFR mutation was also evaluated in tumor specimens. The results indicated that LAT1 protein expression was significantly higher in NSCLC samples without EGFR mutation than those with EGFR mutation. A recent study demonstrated that NSCLC with EGFR mutation was sensitive to gefitinib, whereas that without EGFR mutation was refractory to gefitinib (5). Because LAT1 expression was significantly associated with EGFR wild type, patients with NSCLC with LAT1 expression may be refractory to gefitinib treatment. Previous reports demonstrated that LAT1 expression was significantly higher in patients with squamous cell carcinoma (positive rate: 91%) than those with adenocarcinoma (29%), and that LAT1 expression was closely associated with lymph node or distant metastases (12, 29). Moreover, LAT1 expression was closely correlated with CD98 expression, and a cooperative expression of LAT1 and CD98 was a stronger prognostic factor to predict poor outcome as compared with LAT1 expression alone (40, 41). Although LAT1 expression has been seen frequently in squamous cell carcinoma, LAT1 expression as a poor prognostic marker has been more closely associated with adenocarcinoma than with squamous cell carcinoma (12, 40, 41). The results of present study support the previous findings that LAT1 protein expression is a poor prognostic factor in NSCLC (12). On the other hand, EGFR mutation has been observed in approximately 40% of pulmonary adenocarcinoma, but not in pulmonary squamous cell carcinoma (4). EGFR mutation was known as a prognostic factor to predict good outcome in pulmonary adenocarcinoma (42). These data indicate that the combination of EGFR wild type and LAT1 expression may be a biological marker for the prediction of poor outcome in pulmonary adenocarcinoma. As LAT1 inhibitors have not been tested in patients with NSCLC, the additive effect of combination of gefitinib and LAT1 inhibitor is unknown in the clinical setting. The specific inhibitor of LAT1 is yet to be developed and investigated in clinical trials.
The expression of LAT1 mRNA was investigated in 54 lung cancer cell lines in the present study. The results indicated that LAT1 expression was not higher in squamous cell carcinoma compared to adenocarcinoma. This result was inconsistent with that of LAT1 protein expression, and the profile of LAT1 expression was different between the in vitro and in vivo levels. Although the exact cause of the above discrepancy is uncertain, the mechanism and function of LAT1 expression may be different between cancer cell lines and in vivo human tumors. Whereas, pathological analysis of the tumor specimen revealed that the expression of LAT1 protein was closely associated with that of LAT1 mRNA, and LAT1 mRNA was significantly associated with EGFR wild-type. The expression of both mRNA and protein was closely associated with cell proliferation and angiogenesis. Recently, Takeuchi et al. also reported the expression of LAT1 mRNA in 237 NSCLCs and LAT1 protein in 295 NSCLCs (43). In their study, LAT1 mRNA levels were closely correlated with the immunoreactivity of LAT1 protein in NSCLC, but showed no association with the prognosis of patients. However, their study revealed that LAT1 protein expression was significantly associated with poor outcome. The results of the present study were consistent with their results. LAT1 mRNA level was not closely associated with clinicopathological features or prognosis of patients as compared with LAT1 protein.
The limitations of this study must be addressed. As LAT1-specific inhibitor has not been developed, the inhibitor of system L amino acid transporter, BCH was used. Because BCH is not specific to LAT1, there may be a discrepancy between the treatment with BCH and the LAT1 inhibition therapy. it was not possible to investigate the phosphorylation of mTOR and its downstream signaling molecules, p70S6K and 4EBP1 using the tissue specimens. Moreover, the sample size of the present study was small. Further studies should be performed to elucidate the association between LAT1 expression and mTOR signaling pathway.
Conclusion
Inhibition of LAT1 showed antitumor activity and the combination of gefitinib and LAT1 inhibitor showed additive effect on NSCLC cell line. The level of phosphorylation of mTOR was decreased by the BCH treatment, suggesting that the activation of mTOR signaling pathway is associated with LAT1 expression. Moreover, LAT1 protein expression was significantly associated with EGFR wild type. Inhibiting of LAT1 may be a possible therapeutic rationale to treat NSCLC without EGFR mutation.
Acknowledgements
This work was supported in part by Grant 21790793 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and National Hospital Organization Policy Based Medical Services. We thank Toshiaki Ohara (Department of Gastroenterological surgery, transplant, and Surgical Oncology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University) for technical assistance.
Footnotes
-
↵* Both authors contributed equally to this article.
- Received October 25, 2010.
- Revision received November 2, 2010.
- Accepted November 3, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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