Correlation of L-type amino acid transporter 1 and CD98 expression with triple negative breast cancer prognosis

Triple negative breast cancer (TNBC) is a heterogeneous, aggressive cancer for which there is no effective chemotherapy or targeted therapy. We aimed to evaluate L-type amino acid transporter (LAT) 1 and CD98 expression immunohistochemically in patients with breast cancer, especially TNBC. Out of 129 patients, LAT1 was positive in 56 patients (43.4%), and CD98 was positive in 41 patients (31.8%). The positive ratio of LAT1 expression in luminal A cases was 7.9%, 30.0% in luminal B cases, 71.4% in HER2 cases and 64.0% in TN cases. HER2 and TN subtypes expressed LAT1 and CD98 at higher levels than luminal A and B subtypes (both P < 0.001). LAT1 and CD98 expression correlated with tumor size (LAT1, P = 0.010; CD98, P = 0.007), nuclear grade (LAT1, P < 0.001; CD98, P < 0.001) and Ki67 labeling index (LAT1, P < 0.001; CD98, P = 0.001). LAT1 and CD98 expression was negatively associated with ER and PgR (both P < 0.001). In TNBC, the 5-year disease-free rate of CD98+ (63.6%) or LAT1+/CD98+ (61.9%) patients was significantly worse than that of CD98- (89.3%) patients or those with no co-expression of LAT1 and CD98 (89.7%), respectively (P = 0.014, P = 0.009). The 5-year survival rates of CD98 positive/negative patients were 77.3% and 100% (P = 0.050), respectively, whereas that of patients with LAT1+/CD98+ (76.2%) was significantly worse (100%) (P = 0.040). Multivariate analysis confirmed that CD98+ or LAT1+/CD98+ expression were risk factors for relapse in TNBC (P = 0.023, P = 0.019). Thus, in the present study we show that LAT1 and CD98 expression are prognostic factors. Inhibition of these proteins might provide a new therapeutic strategy in TNBC.     

Anti‐tumor effects of mAb against l‐type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes

l ‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and K_A values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.     

Expression of L‐type amino acid transporter 1 (LAT1) as a prognostic and therapeutic indicator in multiple myeloma

L‐type amino‐acid transporter 1 (LAT1) plays a key role in cell growth and survival. To determine the prognostic significance of LAT1 in multiple myeloma (MM), we investigated the expression of LAT1 and its functional subunit, 4Fc heavy chain (CD98), on myeloma cells by immunohistochemistry in 100 newly diagnosed MM patients. High expression (moderate or strong staining intensity) of LAT1 and CD98 was detected in 56% and 45% of patients, respectively. The LAT1 expression score was positively correlated with Ki‐67 index (r = 0.631, P < 0.001), and there was a statistically significant difference in Durie–Salmon stage between patients with high and low LAT1 expression (P = 0.03). In 43 patients treated with melphalan and prednisolone, the overall response rate was significantly higher in the high LAT1 expression group (60.0%) than in the low LAT1 expression group (17.6%) (P = 0.03). Multivariate analysis confirmed that high expression of LAT1 was a significant prognostic factor for predicting poor overall survival independently from the International Staging System (both P = 0.01). Here, we show that the overexpression of LAT1 is significantly associated with high proliferation and poor prognosis in newly diagnosed MM patients. Thus, LAT1 may be a promising pathological marker for identifying high‐risk MM.     

High expression of L-type amino acid transporter 1 in infiltrating glioma cells

L-type amino acid transporter 1 (LAT1), a neutral amino acid transport agent, is essential for the transport of large neutral amino acids. LAT1 also corresponds to tumor-associated gene-1 (TA1), an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells such as glioma cells. We have investigated the expression of the transporter in the human primary glioma tissue from 68 patients. Among these patients, we could see the border zone between tumors and normal bain tissues in 10 patients. By WHO criteria, two of the specimens were diagnosed as grade 2, three as grade 3, and five as grade 4 [glioblastoma multiforme (GBM)]. In 9 of 10 cases, we could identify the infiltrating glioma cells associated with stronger immunoreactivity for LAT1. These tumor cells aggregated around the neurons in the border zone and were often found in the perivascular space. In one GBM case, the tumors seemed to develop expansively and separated from the normal brain with a border of arachnoid membrane. The expression of LAT1 was always higher in infiltrating glioma cells than in cells located in the center of the tumor. These findings suggest that LAT1 is one of the molecular targets for glioma therapy.     

Immunohistochemical and soluble expression of CD44 in primary and metastatic human prostate cancers

Immunohistochemical expression of standard and v6 isoforms of CD44 was performed on specimens from three groups of prostate cancer patients: Group I, primary prostate cancers (N=31); Group II? lymph node metastases (N=18); and Group III, bone metastases (N=15). In addition, serum from all Group I patients was analyzed for soluble CD44 expression. Benign glands exhibited strong CD44s and CD44v6 expression in basal cells. Weak basolateral staining was identified in superficial luminal cells. Malignant glands and metastatic tumors revealed diminished or absent expression of both CD44s and CD44vG with a heterogeneous pattern. Pretreatment with chondroitinase did not significantly alter CD44 expression. Soluble CD44 was present in all serum samples, however, expression was variable. There was no statistically significant correlation between immunohistochemical CD44 expression, soluble CD44 expression, and clinical progression.     

Prognostic significance of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (CD98) expression in early stage squamous cell carcinoma of the lung

The purpose of this study was to evaluate the prognostic value of L-type amino acid transporter 1 (LAT 1) and 4F2 heavy chain (CD98) in patients with stage I squamous cell carcinoma of the lung. A total of 84 consecutive patients with completely resected pathologic stage I squamous cell carcinoma of the lung were retrospectively reviewed. All patients underwent resection of the tumor and the immunohistochemical analysis was done to determine the expression of LAT 1, CD98, Ki-67 labeling index, vascular endothelial growth factor, and microvessel density. These pathological parameters were correlated with the prognosis of patients after complete resection of the tumor. A positive rate of LAT 1 expression (87%; 73/84) was significantly higher than that of CD98 expression (65%; 55/84) ( P =  0.0018). Cooperative expression of LAT 1 and CD98 was recognized in 62% (52/84). LAT 1 expression was significantly correlated with CD98, Ki-67 labeling index, vascular endothelial growth factor, and microvessel density. The 5-year survival rates of the LAT 1-positive and LAT1-negative patients were 59% and 88%, respectively ( P =  0.2186). Tumor cell proliferation and angiogenesis were not also (a) prognostic factor. However, the 5-year survival rate of patients with both LAT 1 and CD98-positivity (57%) was significantly worse than that of other patients (88%; P  = 0.0136). Multivariate analysis confirmed that positive cooperative expression of LAT 1 and CD98 was an independent factor for predicting a poor prognosis. A cooperative expression of LAT 1 and CD98 is a significant pathological factor for predicting the poor prognosis in patients with resectable stage I squamous cell carcinoma of the lung. ( Cancer Sci 2009; 100: 249–254)     

L-type amino acid transporter 1 (LAT1) expression in malignant pleural mesothelioma

L-Type amino acid transporter 1 (LAT1) is known to be highly expressed in various human neoplasms. However, little is known about how LAT1 is expressed in malignant pleural mesothelioma (MPM). Twenty-one patients were included in this study. Tumor sections were stained by immunohistochemistry for LAT1, glucose transporter 1 (GLUT1), GLUT3, hypoxia inducible factor-1α (HIF-1α), hexokinase I, vascular endothelial growth factor (VEGF), microvessel density (MVD) by determination of CD34, epidermal growth factor receptor (EGFR), phosphatase and tensin analog (PTEN), p-AKT, p-manmalian target of rapamycin (mTOR), p-S6K, p53 and BCL-2. LAT1 was overexpressed in approximately 50% of the patients with MPM. LAT1 expression was closely correlated with CD98, hypoxic markers, the mTOR pathway, Ki-67 and p53. The overexpression of LAT1 was closely associated with poor outcome in patients with MPM. LAT1 is closely associated with tumor development and progression in patients with MPM.     

Glypican‐3 protein expression in primary and metastatic melanoma

BACKGROUND:

The incidence of melanoma is increasing. Fine‐needle aspiration (FNA) is critical in documenting recurrent/metastatic disease in established cases. The potential of metastatic melanoma (MM) to mimic epithelial tumors presents a diagnostic dilemma. In liver FNA, the distinction between hepatocellular carcinoma (HCC) and MM is a frequent challenge. Glypican‐3 (GPC3), a heparan sulfate proteoglycan, is a highly sensitive and specific marker for HCC. Serum GPC3 was shown to be expressed in 40% of primary melanomas (PMs), but to the authors' knowledge no tissue studies to date have assessed GPC3 expression in MM. In this study, GPC3 protein expression was investigated in FNAs from MM, and in corresponding histologic sections from the primary tumors.

METHODS:

Sixty archival, direct FNA smears or CytoLyt‐fixed samples from 50 patients with MM were retrieved together with formalin‐fixed, paraffin‐embedded specimens available from 17 corresponding PMs. All cases were stained with anti‐GPC3 antibody. FNA and core biopsy specimens from HCCs and benign liver were used as positive and negative controls. GPC3 expression was divided into 2 categories: negative (negative or weak cytoplasmic staining) and positive (moderate or strong cytoplasmic with membranous accentuation).

RESULTS:

All FNAs from MM cases were negative (0 of 60) for GPC3. The exact 95% Clopper‐Pearson confidence interval was 0.0% to 5.96%. Only 1 case of PM (1 of 17; 5.9%) demonstrated weak focal cytoplasmic staining (regarded as negative).

CONCLUSIONS:

In the current study, all MM and PM cases in archival FNAs and tissue sections were found to be negative for GPC3. These data suggest that GPC3 is not expressed in melanoma using the 1G12 clone. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

Thymidylate synthase gene expression in primary colorectal cancer and metastatic sites

No Abstract     

L-type amino acid transporter 1 as a potential molecular target in human astrocytic tumors

L-type amino acid transporter 1 (LAT1) is a Na+-independent neutral amino acid transport agency and essential for the transport of large neutral amino acids. LAT1 has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells. We have investigated for the first time, the expression of the transporter in the human primary astrocytic tumor tissue from 60 patients. LAT1 is unique because it requires an additional single membrane spanning protein, the heavy chain of 4F2 cell surface antigen (4F2hc), for its functional expression. 4F2hc expression was also determined by immunohistochemistry. Kaplan-Meier analyses demonstrated that high LAT1 expression correlated with poor survival for the study group as a whole (p<0.0001) and for those with glioblastoma multiforme in particular (p=0.0001). Cox regression analyses demonstrated that LAT1 expression was one of significant predictors of outcome, independent of all other variables. On the basis of these findings, we also investigated the effect of the specific inhibitor to LAT1, 2-aminobicyclo-2 (2,2,1)-heptane-2-carboxylic acid (BCH), on the survival of C6 glioma cells in vitro and in vivo using a rat C6 glioma model. BCH inhibited the growth of C6 glioma cells in vitro and in vivo in a dose-dependent manner. Kaplan-Meier survival data of rats treated with BCH were significant. These findings suggest that LAT1 could be one of the molecular targets in glioma therapy.     

Adenoviral modulation of the tumor-associated system L amino acid transporter, LAT1, alters amino acid transport, cell growth and 4F2/CD98 expressionwith cell-type specific effects in cultured hepatic cells

Altered expression of metabolite transporters is observed frequently in tumor cell lines and primary neoplasms. The extent to which these may to contribute to the growth autonomy associated with cancer is not clear. LAT1 is a major L-type amino acid transporter over-expressed in a variety of cancer types and a light chain component of the CD98 heterodimer. We utilized an adenoviral expression system to modulate the level of LAT1 in a hepatic in vitro model to examine phenotypic changes associated with short-term exogenous and blocked expression. LAT1 levels were increased three fold and resulted in increased L-type amino acid transport as a result of adenoviral expression in murine hepatocytes. The protein was expressed on the cell surface and complexed with the CD98 heavy chain known as 4F2. Surprisingly, levels of the total CD98 protein complex were increased 2.4-fold as a result of adenoviral expression of light chain only, suggesting coordinate regulation. Exogenous overexpression was less effective in normal rat liver cells relative to mouse. LAT1 antisense expression in hepatic tumor cells resulted in a modest though statistically significant decrease in cell number, viability and S-phase cells over a 5-day period relative to controls despite the absence of a significant decrease in L-type transport over this period. These studies are preparatory to in vivo efforts focusing on LAT1/CD98 as a potential therapeutic target.     

Expression of neutral amino acid transporter ASCT2 in human prostate

The neutral amino acid transporter ASCT2 has been associated with increased metabolism in malignant tumors. Its biological significance in tumor proliferation, progression, and its impact on cancer patients' survival remains largely unknown. Tissue microarray (TMA) technology was used to build arrays from 640 cases of radical prostatectomies (triplicate normal prostate, benign prostatic hyperplasia (BPH), and prostate adenocarcinoma (PCa)). Slides were immunostained with an antibody to ASCT2 and scored using a 0-3+ semi-quantitation scoring system for both intensity and percentage. Correlation of ASCT2 expression with patients' clinical and pathological variables was analyzed by the Spearman correlation test. Kaplan-Meier analysis and log rank test were used to determine the probability of disease recurrence. Cox regression model was also used for multivariate analysis. 497 PCa had accessible data. ASCT2 was localized in the cytoplasm of epithelial cells of normal prostate, BPH and PCa. High-level expression of ASCT2 was significantly higher in normal tissues (49%) as compared to BPH (25.8%) or cancer (25.3%) (p < 0.001). ASCT2 expression was weakly but significantly correlated with preoperative PSA (Pre-PSA), Gleason score (GS), lymph node status (LN) (p = 0.019). ASCT2 expression was significantly associated with shorter time to biochemical recurrence only on univariate analysis (p = 0.046). ASCT2 appears to be required for the glutamine metabolism in both nonmalignant and malignant prostate. ASCT2-positive PCa seems to be related to a more aggressive biological behavior. ASCT 2 seems to be involved in tumor progression.     

Effect of miR‐122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis

Control of liver metastasis is an important issue in the treatment of colorectal cancer (CRC). MicroRNAs have been shown to be involved in the development of many cancers, but little is known about their role in the process of colorectal liver metastasis. We compared miRNA expression between primary colorectal tumors and liver metastasis to identify those involved in the process of metastasis. Cancer cells were isolated from formalin‐fixed paraffin‐embedded primary CRC samples and their corresponding metastatic liver tumors in six patients using laser capture microdissection, and miRNA expression was analyzed using TaqMan miRNA arrays. The most abundant miRNA in liver metastasis compared with primary tumors was miR‐122. Immunohistochemical analysis revealed that the expression levels of cationic amino acid transporter 1 (CAT1), a negative target gene of miR‐122, were lower in liver metastases than primary tumors (P < 0.001). Expression levels of CAT1 in 132 primary tumors were negatively correlated with the existence of synchronous liver metastasis (P = 0.0333) and tumor stage (P < 0.0001). In an analysis of 121 colon cancer patients without synchronous liver metastasis, patients with CAT1‐low colon cancer had significantly shorter liver metastasis‐free survival (P = 0.0258) but not overall survival or disease‐free survival. Overexpression of miR‐122 and concomitant suppression of CAT1 in the primary tumor appears to play important roles in the development of colorectal liver metastasis. Expression of CAT1 in the primary CRC has the potential to be a novel biomarker to predict the risk of postoperative liver metastasis of CRC patients.     

Prognostic significance of L-type amino acid transporter 1 expression in resectable stage I-III nonsmall cell lung cancer

The clinical significance of L-type amino acid transporter 1 (LAT1) expression remains unclear, whereas many experimental studies have demonstrated that LAT1 is associated with the proliferation of cancer cells. The purpose of this study was to evaluate the prognostic value of LAT1 in patients with nonsmall cell lung cancer (NSCLC). A total of 321 consecutive patients with completely resected pathologic stage I-III NSCLC were retrospectively reviewed. Expression of LAT1 and proliferative activity, as determined by the Ki-67 labelling index, was also evaluated immunohistochemically and correlated with the prognosis of patients who underwent complete resection of the tumour. Expression of LAT1 was positive in 163 patients (51%) (29% of adenocaricnoma (58 of 200 patients), 91% of squamous cell carcinoma (91 of 100 patients), and 67% of large cell carcinoma (14 of 21 patients)). The 5-year survival rate of LAT1-positive patients (51.8%) was significantly worse than that of LAT1-negative patients (87.8%; P<0.001). L-type amino acid transporter 1 expression was significantly associated with lymph node metastasis and disease stage. Multivariate analysis confirmed that positive expression of LAT1 was an independent factor for predicting a poor prognosis. There was a significant correlation between LAT1 expression and Ki-67 labelling index. LAT1 expression is a promising pathological factor to predict the prognosis in patients with resectable stage I-III NSCLC.     

Antitumor activity of an anti‐CD98 antibody

CD98 is expressed on several tissue types and specifically upregulated on fast‐cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98‐specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell‐line derived xenograft models and was as efficacious as standard of care carboplatin in patient‐derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase‐3 and ‐7‐mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.     

αv‐Integrin isoform expression in primary human tumors and brain metastases

To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvβ3, αvβ5, αvβ6 and αvβ8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvβ3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non‐small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p < 0.01). Similarly, integrin αvβ8 expression was increased in brain metastases compared to primary tumors of breast cancer (p < 0.0001), lung cancer (p < 0.01) and renal cancer (p < 0.0001), with a similar trend in metastatic melanoma. Integrin αvβ5 was expressed in most primary tumors (98% breast cancer; 67% lung cancer; 90% renal cancer; 89% melanoma) and showed a stronger expression in brain metastases compared to primary tumors from lung cancer and melanoma (p < 0.05). Also integrin αvβ6 expression was increased in brain metastases compared to primary breast cancer (p < 0.001). Conclusions: The stronger αv‐integrin expression in brain metastases, especially of αvβ3 and αvβ8 integrins, suggests that certain αv integrin are involved in the process of brain metastasis. αv Integrins may be therapeutic targets for patients with metastatic cancer in brain.