Expression and roles of system L amino acid transporters in human embryonal carcinoma cells.

BACKGROUND: Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive. MATERIALS AND METHODS: Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated. RESULTS: Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation. CONCLUSION: Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs.

Expression of amino acid transporter (LAT1 and 4F2hc) in pulmonary pleomorphic carcinoma.

Amino acid transporters are necessary for tumor growth, metastasis, and survival of various neoplasms; however, the clinicopathological significance of L-type amino acid transporter 1 (LAT1) and 4F2 cell surface antigen (4F2hc) in patients with pulmonary pleomorphic carcinoma (PPC) remainsunknown. The aim of this study is to clarify the prognostic impact of these amino acid transporters in PPC. One hundred five patients with surgically resected PPC were assessed by immunohistochemistry. The expression of LAT1 and 4F2hc, and Ki-67 labeling index were investigated using specimens of the resected tumors. LAT1 and 4F2hc were highly expressed in 35% and 53% of all patients (n = 105, P < .01), 25% and 48% of patients with an adenocarcinoma component (n = 48, P = .02), and 44% and 58% of patients with a nonadenocarcinoma component (n = 57, P = .18), respectively. A high LAT1 expression was significantly related to advanced disease, lymphatic permeation, tumor cell proliferation, and 4F2hc expression. By multivariate analysis, LAT1 and 4F2hc were identified as significant independent markers for predicting a worse prognosis. LAT1 is highly expressed in PPC, and high LAT1 expression can serve as a significant predictor linked to a worse prognosis in patients with PPC.

L-Leucine and L-isoleucine enhance growth of BBN-induced urothelial tumors in the rat bladder by modulating expression of amino acid transporters and tumorigenesis-associated genes.

We investigated the underlying mechanisms of L-leucine and L-isoleucine mediated promotion of bladder carcinogenesis using an initiation-promotion model. Rats were administered N-butyl-N-(4-hydroxybutyl) nitrosamine for 4 weeks and then fed AIN-93G basal diet or diet supplemented with L-leucine or L-isoleucine for 8 weeks followed by the basal diet for another 8 weeks. At the end of the experiment, week 20, there was a significant elevation of papillary and nodular (PN) hyperplasia multiplicity in the amino acid groups. L-Leucine and L-isoleucine transporters were up-regulated in PN hyperplasias and/or bladder tumors compared with concomitant normal-appearing bladder urothelium at weeks 12 and/or 20 in all groups. In addition, in normal-appearing bladder urothelium, significantly increased mRNA levels of y+LAT1, LAT2, LAT4, and 4F2hc were observed in the amino acid groups compared with the BBN control group at both weeks 12 and 20, and increased mRNA levels of LAT1 were observed at week 20. Furthermore, up-regulation of TNF-α, c-fos, ß-catenin, p53, p21(Cip1/WAF1), cdk4, cyclin D1 and caspase 3 in the amino acid groups was detected in normal-appearing bladder urothelium. Overall, our results indicate that supplementation with l-leucine or l-isoleucine enhanced growth of bladder urothelial tumors by triggering expression of amino acid transporters and tumorigenesis-associated genes.

4F2hc-silencing impairs tumorigenicity of HeLa cells via modulation of galectin-3 and ß-catenin signaling, and MMP-2 expression.

4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with ß-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with ß1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/ß1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of ß-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial-mesenchymal transition by inhibition of ß-catenin proteasomal degradation through Akt/GSK-3ß signaling and enabling cell motility.

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris.

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.

Expression of 4F2hc (CD98) in pulmonary neuroendocrine tumors.

4F2hc (CD98) has been associated with tumor growth, and is highly expressed in various tumors. The aim of this study was to evaluate the clinicopathological significance of 4F2hc expression in pulmonary neuroendocrine (NE) tumors. Surgically-resected patient tumors including 16 large cell neuroendocrine carcinoma (LCNEC), 12 small cell lung cancer (SCLC), 1 atypical carcinoid (AC) and 5 typical carcinoid (TC) samples were included in this study. Tumor sections were immunohistochemically stained for 4F2hc (CD98), glucose transporter 1 (Glut1) and 3 (Glut3), hypoxia-inducible factor-1α (HIF-1α), hexokinase I, vascular endothelial growth factor (VEGF), microvessel density (CD34), epidermal growth factor receptor (EGFR), Akt/mammalian target of rapamycin (mTOR) signaling pathway (p-Akt, p-mTOR and p-S6K) and for a cell cycle regulator (p53). 4F2hc was overexpressed in 0% of the pulmonary carcinoids (TCs and ACs), 62.5% of the LCNECs and 50.0% of the SCLCs. A positive 4F2hc expression was significantly associated with age, histology and Glut1 expression. Moreover, a significant correlation was found between 4F2hc expression, and Glut1, HIF-1α, p-Akt, p-mTOR and p-S6K. The expression of 4F2hc was also significantly associated with poor overall survival. The expression of 4F2hc expression tended to increase from low-grade to high-grade pulmonary NE tumors. Our results suggest that 4F2hc may play a significant role in tumor progression, hypoxic conditions and poor outcome in patients with pulmonary NE tumors.

Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in oral squamous cell carcinoma and its precusor lesions.

BACKGROUND: Amino acid transporters play an important role in supplying organic nutrients to cells. The expression of L-type amino acid transporter 1 (LAT1) and its subunit 4F2 heavy chain (4F2hc) was evaluated to determine the alterations of these transporters in oral normal mucosa (ONM), oral precancerous lesion (OPL) and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Sections from formalin-fixed, paraffin-embedded samples of ONM, OPL or OSCC were examined using immunohistochemical staining to detect L4T1 and 4F2hc proteins. RESULTS: The LAT1 and 4F2hc expression increased progressively from ONM to hyperplastic and to dysplastic lesions and OSCC. In particular, LAT1 may be a more specific indicator of tumor progression than 4F2hc. CONCLUSION: LAT1 and 4F2hc may have an important role in the early stages of multistep oral carcinogenesis. In addition, the specific inhibition of LAT1 and 4F2hc might be a new rationale to suppress oral cancer progression.

Lysophosphatidylcholine enhances cytokine production of endothelial cells via induction of L-type amino acid transporter 1 and cell surface antigen 4F2.

OBJECTIVE: A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. We performed DNA microarray experiments to explore novel responses of human umbilical vein endothelial cells (HUVECs) to oxLDL and its components. METHODS AND RESULTS: cDNA microarray analysis showed that oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2-nonenal, and oxysterols altered gene expression specifically, but some genes were commonly induced in HUVECs. Solute carrier family 3 member 2 and family 7 member 5, encoding the heavy chain of the cell surface antigen 4F2 (4F2hc) and the L-type amino acid transporter 1 (LAT1), respectively, were induced by oxLDL and many oxidation products. LAT1 requires 4F2hc to form a heterodimeric functional complex to transport neutral amino acids into the cell. LysoPC increased membrane protein levels of LAT1 confirmed by Western blot analysis and also uptake of L-[(14)C]leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor. CONCLUSIONS: These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells.

Sequential increases in 4F2hc expression during DMBA-induced hamster buccal pouch carcinogenesis.

BACKGROUND: Although it is known that amino acid transporters are up-regulated for continuous growth in transformed cells, alterations in the 4F2hc expression levels between normal and cancer cells are unclear. The purpose of this study, therefore, was to examine the alteration of the inducible 4F2hc expression level in hamster buccal cancers. MATERIALS AND METHODS: The expression profile of 4F2hc on normal buccal mucosa and squamous cell carcinoma (SCC) from different differentiation stages in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal carcinogenesis was examined using immunohistochemical analysis. RESULTS: Immunoreactivity for 4F2hc was detected in almost all the layers of the normal mucosa as well as in the tumor cells and keratin layer of SCC. In particular, the 4F2hc expression level increased progressively via hamster buccal pouch malignant progression. CONCLUSION: The results suggest that 4F2hc expression is associated with the development of DMBA-induced oral carcinomas. In addition, 4F2hc overexpression in SCC indicates that 4F2hc may play an important role in the development and progression of oral SCC.

Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesions of rat models.

BACKGROUND AND OBJECTIVES: It has been said that amino acid transporters play an important role in supplying nutrition to cells and for cell proliferation. In this study, we examined whether LAT1 and 4F2hc are closely related to tumor growth. METHODS: Rat colon cancer cells (RCN-9) were injected into the spleen of 12 male rats (inbred F344/DuCrj). In each rat, liver samples including tumor lesions were immunostained with anti-LAT1 and anti-4F2hc antibodies. The staining area of LAT1 and 4F2hc tumor lesions was calculated by computer analysis. RESULTS: Sixty-eight tumor nodules were observed in 12 livers. Out of the 68 tumor nodules, 36 nodules (52.9%) indicated a positive staining of LAT1 and 32 (47.1%) had a negative staining of LAT1. However, the LAT1 expression was scarcely detected in non-tumor areas. In terms of the 4F2hc expression, there were 56 nodules (82.4%) with 4F2hc positive and 12 (17.6) with 4F2hc negative. In addition, the expression of 4F2hc in non-tumor areas was almost the same as the expression of 4F2hc in tumor lesions. The average tumor size of the group with LAT1 positive and 4F2hc positive (n = 31) was 0.845 +/- 0.232 mm(2), which was significantly larger than that of the group with LAT1 negative and 4F2hc negative group (n = 7) (0.090 +/- 0.028 mm(2)) or the group with LAT1 positive and 4F2hc negative (n = 5) (0.097 +/- 0.025 mm(2)), respectively (P = 0.0017, P = 0.007). CONCLUSION: LAT1 was related to tumor growth. We think that LAT1 can possibly enhance its ability to promote tumor growth in cooperation with 4F2hc.