Association of L-type amino acid transporter 1 (LAT1) with the immune system and prognosis in invasive breast cancer.

L-type amino acid transporter 1 (LAT1), also referred to as SLC7A5, is believed to regulate tumor metabolism and be associated with tumor proliferation. In invasive breast cancer, we clinicopathologically investigated the utility of LAT1 expression. LAT1 expression was evaluated via immunohistochemistry analyses in 250 breast cancer patients undergoing long-term follow-up. We assessed the relationships between LAT1 expression and patient outcomes and clinicopathological factors. Breast cancer-specific survival stratified by LAT1 expression was assessed. Human epidermal growth factor receptor 2 (HER2)-positive patients with metastasis received trastuzumab therapy. The density of tumor-infiltrating lymphocytes (TILs) was evaluated according to the International Working Group guidelines. In the current study, high LAT1 expression was significantly correlated with estrogen receptor (ER) negativity, progesterone receptor negativity, high histological grade, increased TILs, and programmed death ligand 1 positivity. Among the ER-positive and HER2-negative patients, high LAT1 was an independent indicator of poor outcomes (hazard ratio (HR) = 2.97; 95% confidence interval (CI), 1.16-7.62; p = 0.023). Moreover, high LAT1 expression was an independent poor prognostic factor in luminal B-like breast cancer with aggressive features (HR = 3.39; 95% CI 1.35-8.52; p = 0.0094). In conclusion, high LAT1 expression could be used to identify a subgroup of invasive breast cancer characterized by aggressive behavior and high tumor immunoreaction. Our findings suggest that LAT1 might be a candidate therapeutic target for breast cancer patients, particularly those with luminal B-like type breast cancer.

Structural and antigenic investigation of Usutu virus envelope protein domain III.

The mosquito-borne flavivirus Usutu virus (USUV) has recently emerged in birds and humans in Europe. Symptoms of a USUV infection resemble those of West Nile virus (WNV); further, the close antigenic relationship of domain III (DIII) of the USUV and WNV envelope (E) proteins has prevented the development of a reliable serological test to distinguish USUV from WNV. To begin to address this deficiency, we identified ten different sequence groups of DIII from 253 complete and 80 partial USUV genome sequences. We solved the DIII structures of four groups, including that of the outlying CAR-1969 strain, which shows an atypical DIII structure. Structural comparisons of the USUV DIII groups and the DIII of WNV bound to the neutralizing antibody E16 revealed why the E16 failed to neutralize all USUV strains tested except for USUV CAR-1969. The analyses allowed predictions to be made to engineer an antibody specific for USUV CAR-1969.

Targeting L-type amino acid transporter 1 in innate and adaptive T cells efficiently controls skin inflammation.

BACKGROUND: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1ß, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored. OBJECTIVE: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis. METHODS: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor-related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin]). RESULTS: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17-secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1ß, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1ß stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23- and IL-1ß-induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling. CONCLUSION: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1ß/IL-17 axis.

Crosstalks between mTORC1 and mTORC2 variagate cytokine signaling to control NK maturation and effector function.

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions.

L-Type Amino Acid Transporter 1 Immunoreactivity as a Possible Diagnostic and Prognostic Marker of Thymic Carcinoma.

L-type amino acid transporter 1 (LAT1) functions to transport large neutral amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine. These amino acids are essential for cell growth and proliferation. Many studies have demonstrated LAT1 expression in various types of cancer, and its high expression level was associated with poor prognosis. However, the significance of LAT1 expression in thymic epithelial tumors is controversial. We conducted this retrospective study to investigate the LAT1 immunoreactivity in thymic epithelial tumors and its impact on prognosis. We analyzed 32 patients with thymoma and 14 patients with thymic carcinoma who underwent surgery at our institute. Immunohistochemical analysis was performed using formalin-fixed paraffin-embedded surgical tissues and an anti-LAT1 polyclonal antibody. We thus found that LAT1 immunoreactivity was undetectable in all of the thymoma specimens, regardless of the subtypes of thymoma. By contrast, LAT1 immunoreactivity was consistently detected in the cytosol of thymic carcinoma cells; namely, all 14 thymic carcinoma specimens demonstrated LAT1 immunoreactivity in the cytosol. Among these 14 thymic carcinoma specimens, four carcinoma specimens also showed LAT1 immunoreactivity in the cell membrane. Survival analysis indicated that the thymic carcinoma with the LAT1 membrane signal was associated with poor prognosis, compared with the specimens with the LAT1 cytosol signal. We therefore propose that LAT1 is expressed in the cytosol of thymic carcinoma cells, which could be a diagnostic marker of thymic carcinoma. Moreover, LAT1 expression in the cell membrane is a prognostic marker of thymic carcinoma.

Single cell analysis of kynurenine and System L amino acid transport in T cells.

The tryptophan metabolite kynurenine has critical immunomodulatory properties and can function as an aryl hydrocarbon receptor (AHR) ligand. Here we show that the ability of T cells to transport kynurenine is restricted to cells activated by the T-cell antigen receptor or proinflammatory cytokines. Kynurenine is transported across the T-cell membrane by the System L transporter SLC7A5. Accordingly, the ability of kynurenine to activate the AHR is restricted to T cells that express SLC7A5. We use the fluorescence spectral properties of kynurenine to develop a flow cytometry-based assay for rapid, sensitive and quantitative measurement of the kynurenine transport capacity in a single cell. Our findings provide a method to assess the susceptibility of T cells to kynurenine, and a sensitive single cell assay to monitor System L amino acid transport.

Role of LAT1 in the promotion of amino acid incorporation in activated T cells.

Intake of nutrients from the environment is fundamental to cellular activity. The requirement of nutrients depends on the situation in which cells are placed. Activation of T cells changes the structure and scale of cellular metabolism, which requires a large amount of nutrients. Hydrophilic nutrients such as glucose and amino acids cannot diffuse beyond the cellular membrane; thus transporters are required to assist the incorporation of these nutrients into cells. Based on this observation, metabolic changes accompanying activation of T cells must occur simultaneously with the reorganization of transporters that are capable of providing sufficient nutrients to the cell. This review describes the functional advantages of using special nutrient transporters in activated T cells and discusses the mechanisms of responses to nutrient starvation in T cells.

Imaging the L-type amino acid transporter-1 (LAT1) with Zr-89 immunoPET.

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [(89)Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [(18)F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [(89)Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.

High expression of L-type amino-acid transporter 1 (LAT1) in gastric carcinomas: comparison with non-cancerous lesions.

Amino acid transporters are essential for maintenance and proliferation of both normal and transformed cells. In the present study, L-type amino-acid transporter 1 (LAT1) immunoreactive expression was investigated in gastric carcinomas, in comparison with gastric adenomas and non-neoplastic lesions, using our recently developed novel monoclonal antibody. In a total of 87 cases of advanced gastric cancer, high LAT1 expression was observed in carcinoma cells, predominantly at plasma membranes with greater intensity in non-scirrhous than scirrhous carcinomas. Gastric carcinoma cases with lymph node metastasis showed significantly higher LAT1 expression than cases without lymph node metastasis. A positive correlation with Ki-67 LI was observed and the highly expressing non-scirrhous carcinomas showed a significantly poorer prognosis than the low LAT1 group. Cox hazard test revealed that TNM stage and LAT1 expression were independent prognostic factors in non-scirrhous carcinoma group. Further, a significant poor prognosis was confirmed in high LAT1 expression group, when limited to undifferentiated carcinoma cases excluding scirrhous carcinoma. Lower levels were found in adenomas. In conclusion, LAT 1 expression may be linked with cell proliferation and prognosis of gastric carcinomas, and offers a potential target for future anticancer therapy by inhibitors.

Properties of L-type amino acid transporter 1 in epidermal ovarian cancer.

HYPOTHESIS: To investigate the expression and the functional properties of L-type amino acid transporter 1 (LAT1) in human epithelial ovarian cancer to provide a basis for potential new therapies to control the growth and the metastasis of ovarian cancer. METHODS: The material used comprised 63 surgically resected specimens obtained from female patients undergoing gynecologic surgery at Kyorin University School of Medicine (Tokyo, Japan). The expression of LAT1 in 53 cases of ovarian cancers was determined by Western blot and immunohistochemical staining, and results were compared with those of normal ovarian tissues (5 cases) and benign ovarian tumors (5 cases). Furthermore, we examined the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), the classic inhibitor of system L on the survival, the migration, and the uptake of l-leucine by human epithelial ovarian cancer cell line (OVCAR-3). RESULTS: The LAT1 was significantly up-regulated in various human epithelial ovarian cancers that was localized predominantly on their plasma membrane and in the plasma membrane of the ovarian cancer cell line in conjunction with 4F2hc via disulfide bonds. The BCH inhibited the proliferation and the migration of the OVCAR-3 cells and the uptake of [14C]l-leucine by these cells in a dose-dependent manner. The OVCAR-3 cells did not express LAT2, and the uptake of [14C]l-leucine by these cells was Na-independent and almost completely inhibited by BCH. Thus, our findings indicated that most l-leucine uptake in OVCAR-3 cells was mediated by LAT1. CONCLUSIONS: The LAT1 plays significant roles in nutrition, proliferation, and migration of ovarian cancer. Then, LAT1 inhibition would be useful for anticancer therapy in suppressing tumor growth without affecting normal tissues.

Chemosensitivity and stratification by a five monoclonal antibody immunohistochemistry test in the NSABP B14 and B20 trials.

PURPOSE: To test the association between risk stratification and outcome in a prospectively designed, blinded retrospective study using tissue arrays of available paraffin blocks from the estrogen receptor-expressing, node-negative samples from the National Surgical Adjuvant Breast and Bowel Project B14 and B20 tamoxifen and chemotherapy trials. EXPERIMENTAL DESIGN: Tissue arrays were stained by immunohistochemistry targeting p53, NDRG1, SLC7A5, CEACAM5, and HTF9C. Risk stratification was done using predefined scoring rules, algorithm for combining scores, and cutoff points for low-risk, moderate-risk, and high-risk patient strata. RESULTS: In a univariate Cox model, this test was significantly associated with recurrence-free interval [HR, 1.3 (95% confidence interval, 1.1-1.6); P = 0.006]. In a multivariate model it contributed information independent of age, tumor size, and menopausal status (P = 0.007). The Kaplan-Meier estimates of the proportion of recurrence-free after 10 years were 73%, 86%, and 85% for the high-risk, moderate-risk, and low-risk groups (P = 0.001). The Kaplan-Meier estimates of the breast-cancer-specific-death rate were 23%, 10%, and 9% (P < 0.0001). Exploratory analysis in patients >/=60 years old showed Kaplan-Meier estimates of the proportion of recurrence-free of 78%, 89%, and 92%. Both high-risk and low-risk groups showed significant improvement on treatment with cytotoxic chemotherapy. CONCLUSIONS: Immunohistochemistry using five monoclonal antibodies assigns breast cancer patients to a risk index that was significantly associated with clinical outcome among the estrogen receptor-expressing, node-negative tamoxifen-treated patients. It seems that the test may be able to identify patients who have greater absolute benefit from adjuvant chemotherapy compared with unstratified patient populations. Exploratory analysis suggests that this test will be most useful in clinical decision making for postmenopausal patients.

Production and characterization of highly tumor-specific rat monoclonal antibodies recognizing the extracellular domain of human L-type amino-acid transporter 1.

L-type large amino acid transporter (LAT) 1, the first light chain (lc) of cluster of differentiation 98 (CD98) to be identified, is associated with the heavy chain (hc) of CD98 and expressed on the surface of various tumor cells irrespective of their origin. Because LAT1 is a 12-pass membrane protein and its possible immunogenic extracellular region is very small, specific monoclonal antibodies (mAb) had not been developed. We report the successful preparation and characterization of mAb recognizing the extracellular domain of human LAT1 protein. Two mAb were selected from hybridoma clones established by fusing mouse myeloma cells and spleen cells from rats immunized against RH7777 rat hepatoma cells expressing recombinant green fluorescent protein fused to human LAT1 protein. Designated SOL22 and SOL69, these mAb specifically reacted with the extracellular domain of LAT1 on cells transfected with cDNA of LAT1, but not with cells transfected with cDNA of other CD98 lc, namely, LAT2, y(+)LAT1, y(+)LAT2, and xCT amino acid transporters. These mAb immunoprecipitated 35- and 90-kDa proteins under reducing conditions in extracts prepared from human HeLa tumor cells, indicating the existence of intermolecular disulfide bonds between cysteine residues in the 90-kDa hc and 35-kDa lc (LAT1). SOL22 and SOL69 mAb reacted with a wide variety of living unfixed human tumor cell lines, but were only weakly reactive with HEK293F human embryonic kidney cells and human peripheral blood cells. Comparative immunohistochemical analyses of normal human tissues with anti-CD98 hc and anti-LAT1 revealed LAT1 to be an excellent molecular target for antibody therapy, possibly even superior to CD98 hc.

Expression of L-type amino acid transporter 1 (LAT1) in esophageal carcinoma.

BACKGROUND AND OBJECTIVES: It has been reported that amino acid transport systems play an important role in cell proliferation. Their activity is increased in malignant cells compared to benign cells. In this study, we investigated whether L-type amino acid transporter 1 (LAT1) is expressed in human non-cancerous esophageal mucosa and esophageal squamous cell carcinoma. We also examined whether LAT1 expression is correlated with histopathological features. METHODS: From January 1999 to December 2001, sections of formalin-fixed, paraffin-embedded tissue from 11 cases of early esophageal carcinoma (T1) and 19 cases of advanced esophageal carcinoma (T2, T3) were entered in the study. Histopathologically, all 30 cases were squamous cell carcinoma. Immunohistochemical staining was performed using rabbit anti-LAT1 IgG, with the standard avidin-streptavidin immuno-peroxidase method. Measurement was performed by means of computer-assisted image analysis. The ratio of cells with LAT1 expression in esophageal squamous cell carcinoma and non-cancerous esophageal mucosa was used for analysis in this study. RESULTS: Non-cancerous esophageal mucosa expressed LAT1 only in the basal layer of the esophageal wall. Esophageal squamous cell carcinoma expressed LAT1 throughout the tumor. LAT1 expression in esophageal squamous cell carcinoma was significantly higher than that in non-cancerous esophageal mucosa. LAT1 expression in esophageal squamous cell carcinoma increased as the depth of invasion progressed (T1 < T2 (P = 0.0477), T2 < T3 (P = 0.0415), T1 < T3 (P = 0.0044)), and as the tumor size increased. Also, high LAT1 expression was significantly associated with well-differentiated carcinoma. CONCLUSION: These results suggest that LAT1 plays a significant role in cell proliferation, differentiation, and invasion in esophageal squamous cell carcinoma.