Kimihiro Shimizu

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.

The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein1

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of molecular weight 78 000 (gp78), mediating cell motility signaling in vitro and metastasis in vivo. Here, we cloned the full‐length cDNAs for both human and mouse AMFR genes. Both genes encode a protein of 643 amino acids containing a seven transmembrane domain, a RING‐H2 motif and a leucine zipper motif and showed a 94.7% amino acid sequence identity to each other. Analysis of the amino acid sequence of AMFR with protein databases revealed no significant homology with all known seven transmembrane proteins, but a significant structural similarity to a hypothetical protein of Caenorhabditis elegans, F26E4.11. Thus, AMFR is a highly conserved gene which encodes a novel type of seven transmembrane protein.

l-type amino acid transporter 1 and CD98 expression in primary and metastatic sites of human neoplasms.

The significance of l‐type amino acid transporter (LAT) 1 expression remains unclear in the metastatic process of human neoplasms, whereas experimental studies have demonstrated that LAT1 is associated with the metastatic process of cancer cells. We compared the immunohistochemical expression of LAT1 and CD98 between the primary site and a concordant pulmonary metastatic site in 93 cancer patients, all of whom had undergone thoracotomy. LAT1, CD98, Ki‐67 labeling index, vascular endothelial growth factor (VEGF), CD31, and CD34 were analyzed by immunohistochemical staining in the resected tumors of 93 cancer patients: 45 colon cancers; nine breast cancers; eight head and neck cancers; 11 genital cancers; 14 soft‐tissue sarcomas; and six other cancers. The expression of these markers was significantly higher in the metastatic sites than in the primary sites. In total, the positive rates of LAT1, CD98, Ki‐67, VEGF, CD31, and CD34 were 40, 24, 56, 41, 45, and 39%, respectively, in the primary sites and 65, 45, 84, 67, 73, and 61%, respectively, in the metastatic sites. LAT1 expression was closely correlated with CD98 expression, angiogenesis, and cell proliferation. The association between LAT1 and CD98 expression was strongest in the primary and metastatic sites. The present study suggests that overexpression of LAT1 and CD98 has an important role to play in the metastatic process of variable human neoplasms. Moreover, LAT1 expression was significantly correlated with cell proliferation and angiogenesis. (Cancer Sci 2008; 99: 2380–2386)

Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines

Two different proteins, p16(INK4a) and p14(ARF), encoded by the INK4a/ARF locus play important roles in the RB and p53 pathways, respectively. This study was performed to determine genetic and epigenetic alterations in the INK4a/ARF locus and their effects on the growth of osteosarcoma. Among six cell lines examined, both p16(INK4a) and p14(ARF) exons were homozygously deleted in two cell lines, MG63 and HOS, and both p16(INK4a) and p14(ARF) promoters were methylated in one cell line, U2OS. Wild-type mRNA and proteins for p16(INK4a) and p14(ARF) were expressed in three other cell lines, SaOS2, HuO9, and G292. Transfection studies were performed using two cell lines, U2OS and MG63. Both the RB and p53 genes were wild types in U2OS, whereas p53 but not RB was mutated in MG63. Both p16(INK4a) and p14(ARF) suppressed the growth of U2OS, whereas p16(INK4a) but not p14(ARF) suppressed the growth of MG63. p53 only did not suppress the growth of MG63 either; however, coexpression of p14(ARF) with p53 increased the fraction of the G0/G1 phase in MG63 cells. The data presented here demonstrate the importance of genetic and epigenetic alterations in the INK4a/ARF locus for the growth of osteosarcoma and thus will be useful to further understand the biologic behavior of osteosarcoma in association with the defects in the p53 and RB pathways.

Fluorine-18-α-Methyltyrosine Positron Emission Tomography for Diagnosis and Staging of Lung Cancer: A Clinicopathologic Study

Purpose:l-[3-18F]-α-Methyltyrosine ([18F]FMT) is an amino acid tracer for positron emission tomography (PET). We evaluated the diagnostic usefulness of [18F]FMT PET in non–small-cell lung cancer (NSCLC) patients. Tumor uptake of [18F]FMT was compared with that of 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]FDG) and correlated with L-type amino acid transporter 1 (LAT1) expression. Experimental Design: Fifty NSCLC patients were enrolled in this study, and a pair of PET study with [18F]FMT and [18F]FDG was done. LAT1 expression and Ki-67 labeling index of the resected tumors were analyzed by immunohistochemical staining. Results: For the primary tumor detection, [18F]FMT PET exhibited a sensitivity of 90% whereas the sensitivity for [18F]FDG PET was 94%. For lymph node staging, the sensitivity and specificity of [18F]FMT PET were 57.8% and 100%, and those of [18F]FDG PET were 65.7% and 91%, respectively. The expression of LAT1 in squamous cell carcinoma and large cell carcinoma was significantly higher than that in adenocarcinoma. [18F]FMT uptake was also higher in squamous cell carcinoma and large cell carcinoma than in adenocarcinoma. Uptake of [18F]FMT in the tumor is closely correlated with LAT1 expression (ρ = 0.890). Conclusion: [18F]FMT PET had no false-positives in the detection of primary tumor and lymph node metastasis and could improve the diagnostic performance in NSCLC. Uptake of [18F]FMT correlated with the expression of LAT1 that showed a significant association with cellular proliferation.

Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma

Epidermal growth factor receptor (EGFR) gene mutations have been reported to be clinically significant in non-small cell lung cancer (NSCLC). However, because most previous studies focused only on adenocarcinomas, EGFR mutations in other histotypes are poorly investigated. We evaluated the frequency of EGFR gene mutations in squamous cell carcinoma (SCC) and its clinicopathological features. In total, 89 frozen tumor specimens that had been first diagnosed as SCCs, were examined for EGFR mutations in exons 19 and 21 using direct sequencing, PNA-enriched sequencing and SmartAmp2. Additionally, pathological investigation, including immunostaining for p63 and TTF-1, alcian blue staining and EGFR mutation-specific immunohistochemistry in mutation-positive samples was also performed. The frequency of EGFR mutations was 5.6% (5/89); all mutations were deletions in EGFR exon 19. Immunohistological investigation of these samples revealed that two of five were positive for p63 and TTF-1 staining, and showed production of mucin, as evidenced by alcian blue staining. Consequently, three of the samples were considered to be true SCC at final pathological diagnosis, while the remaining two samples were revised to adenosquamous carcinoma and adenocarcinoma. The final frequency of the EGFR mutations in true SCC was 3.4% (3/87). In conclusion, EGFR mutations were found in a small, but significant, number of SCC tumor samples and thus EGFR mutational analysis was useful in the accurate diagnosis of SCC. Our data demonstrate that EGFR mutational analysis should be performed not only in adenocarcinoma, but also in SCC to allow accurate diagnosis and treatment.

Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer

Background:The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression.Methods:A total of 97 consecutive patients with surgically resected pathological stage I–IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53.Results:L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis.Conclusion:L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

Prognostic significance of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (CD98) expression in stage I pulmonary adenocarcinoma

BACKGROUND The purpose of this study was to evaluate the prognostic value of l-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (CD98) expression in patients with stage I pulmonary adenocarcinoma. METHODS A total of 139 consecutive patients with pathologic stage I pulmonary adenocarcinoma were retrospectively reviewed. Expression of LAT1, CD98, Ki-67 labeling index, vascular endothelial growth factor (VEGF) and microvessel density (MVD) was also evaluated immunohistochemically and correlated with the prognosis of patients after complete resection of the tumor. RESULTS LAT1 and CD98 expression were positive in 23% (32/139) and 22% (31/139), respectively (p=0.887). LAT1 with CD98 expression was recognized in 15% (21/139). LAT1 expression was significantly correlated with CD98, Ki-67 labeling index, VEGF, and MVD. The 5-year survival rates of LAT1-positive and -negative patients and CD98-positive and -negative patients, were 60.2 and 95.2% (p<0.0001) and 72.5 and 93.4% (p=0.0008), respectively. Univariate analysis disclosed that cellular proliferation and MVD in the tumor were significant prognostic factors. However, multivariate analysis confirmed that positive expression of LAT1 and CD98 was an independent factor for predicting a poor prognosis. CONCLUSION The overexpression of LAT1 and CD98 is a pathological factor to predict the prognosis in patients with resectable stage I pulmonary adenocarcinoma.

Clinicopathological features of lung adenocarcinoma with KRAS mutations.

KRAS and epidermal growth factor receptor (EGFR) mutations are thought to play an important role in the carcinogenesis of lung adenocarcinoma. However, clinicopathological findings of KRAS mutated adenocarcinoma cases have not yet been fully clarified. The authors analyzed the relationship between the KRAS mutation and corresponding clinicopathological findings, focusing on nonmucinous and mucinous bronchioloalveolar elements.

Oncogenic KRAS-induced interleukin-8 overexpression promotes cell growth and migration and contributes to aggressive phenotypes of non-small cell lung cancer.

The CXC chemokine interleukin‐8 (IL‐8) is an angiogenic growth factor that is overexpressed in various cancers, including non‐small cell lung cancer (NSCLC). Previously, IL‐8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS‐driven NSCLC. Using microarray analysis, we identified IL‐8 as the most downregulated gene by shRNA‐mediated KRAS knockdown in NCI‐H1792 NSCLC cells where IL‐8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL‐8, while IL‐8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL‐8 expression was downregulated by shRNA‐mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL‐8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL‐8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL‐8 expression, while the p38 inhibitor increased IL‐8 expression. Attenuation of IL‐8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL‐8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL‐8 expression in NSCLC; IL‐8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS‐mutated adenocarcinomas; and IL‐8 plays a role in cell growth and migration in oncogenic KRAS‐driven NSCLC.