Katsumi Koshikawa

A Novel Target Gene, SKP2, within the 5p13 Amplicon That Is Frequently Detected in Small Cell Lung Cancers

We investigated DNA copy-number aberrations in 22 cell lines derived from small cell lung cancers (SCLCs) using comparative genomic hybridization. A minimal common region at 5p13, within the 5p11-p13 amplicon that was most frequently involved, harbored the CDH6, PC4, and SKP2 genes. These three genes showed amplification and consequent overexpression in the SCLC cell lines. SKP2 positively regulates progression of cell cycle by targeting several regulators, such as the cell-cycle inhibitor p27(KIP1), for ubiquitin-mediated degradation. SKP2 was amplified in 7 (44%) of 16 primary SCLC tumors, and consequently overexpressed in 10 (83%) of the 12 of those tumors we examined. Expression levels of SKP2 protein were cell cycle-dependent in SCLC cells as well as in normal cells, and were correlated with the DNA copy-number of the gene. There was an inverse correlation between the expression of SKP2 and p27(KIP1) proteins. Down-regulation of SKP2 using an anti-sense oligonucleotide remarkably suppressed the growth of SCLC cells. Our results indicate that SKP2 is likely to be a target of the 5p13 amplification and to play an important role in the growth of SCLC cells.

Gene expression-based, individualized outcome prediction for surgically treated lung cancer patients

Individualized outcome prediction classifiers were successfully constructed through expression profiling of a total of 8644 genes in 50 non-small-cell lung cancer (NSCLC) cases, which had been consecutively operated on within a defined short period of time and followed up for more than 5 years. The resultant classifier of NSCLCs yielded 82% accuracy for forecasting survival or death 5 years after surgery of a given patient. In addition, since two major histologic classes may differ in terms of outcome-related expression signatures, histologic-type-specific outcome classifiers were also constructed. The resultant highly predictive classifiers, designed specifically for nonsquamous cell carcinomas, showed a prediction accuracy of more than 90% independent of disease stage. In addition to the presence of heterogeneities in adenocarcinomas, our unsupervised hierarchical clustering analysis revealed for the first time the existence of clinicopathologically relevant subclasses of squamous cell carcinomas with marked differences in their invasive growth and prognosis. This finding clearly suggests that NSCLCs comprise distinct subclasses with considerable heterogeneities even within one histologic type. Overall, these findings should advance not only our understanding of the biology of lung cancer but also our ability to individualize postoperative therapies based on the predicted outcome.

Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma.

Background/aims: Alteration in transforming growth factor‐β signaling pathway is one of the main causes of hepatocellular carcinoma (HCC). The human runt‐related transcription factor 3 gene (RUNX3) is an important component of this pathway. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including HCC. Therefore, we examined genetic and epigenetic alterations of RUNX3 in human HCC.

Expression of CD109 in human cancer

It was recently reported that the human CD109 gene encodes a glycosyl-phosphatidylinositol-anchored glycoprotein that is a member of the α2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. In this study, we found that the expression of mouse CD109 gene was upregulated in NIH3T3 cells expressing RET tyrosine kinase with a multiple endocrine neoplasia 2B mutation. Northern blot analysis showed a high level of expression of the CD109 gene only in the testis in normal human and mouse tissues. In addition, its expression was high in some human tumor cell lines, which included squamous cell carcinoma and glioblastoma cell lines, whereas it was undetectable in neuroblastoma and small-cell lung carcinoma cell lines. When CD109 expression was examined in 33 cases of human lung cell carcinomas by quantitative RT–PCR, a significant high expression of CD109 was detected in about half of squamous cell carcinomas examined, but not in adenocarcinoma, large-cell carcinoma and small-cell carcinoma. Similarly, upregulation of CD109 was observed in nine out of 17 esophageal squamous cell carcinomas. Thus, these results suggested that CD109 might be a useful molecular target for the development of new therapeutics for malignant tumors, such as squamous cell carcinoma.

Multi-faceted analyses of a highly metastatic human lung cancer cell line NCI-H460-LNM35 suggest mimicry of inflammatory cells in metastasis.

This study established and characterized low-metastatic revertant and parental clones of a highly metastatic human lung cancer cell line, NCI-H460-LNM35 (hereafter referred to as LNM35). Expression-profiling analysis revealed that up-regulation of various proinflammatory cytokines and angiogenic chemotactic chemokines was present in LNM35. Further, while COX-2 itself is known to be inducible in inflammation, COX-2 expression levels correlated well with the capabilities of these clones for not only in vitro motility and invasion but also in vivo metastasis, and COX-2 inhibitors were shown for the first time to reduce lung cancer metastasis in vivo. These findings suggest that lung cancer cells may mimic inflammatory cells in the process of metastasis.

Frequent promoter methylation and gene silencing of CDH13 in pancreatic cancer

It has recently been reported that CDH13 expression is silenced by aberrant methylation of the promoter region in several cancers. We examined the methylation status of the CDH13 gene in pancreatic cancer using methylation‐specific PCR (MSP), and detected aberrant methylation of CDH13 in all 6 pancreatic cancer cell lines examined. To confirm the status of the CDH13 gene in relation to the methylation pattern, we next examined CDH13 expression in these cell lines using reverse transcription (RT)‐PCR. As expected, no CDH13 expression was detected in any of the 6 pancreatic cancer cell lines. Moreover, 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) treatment of CDH13‐methylated cell lines led to restoration of CDH13 expression. Among primary pancreatic cancers, 19 of 33 (58%) cases exhibited CDH13 methylation, while no cases exhibited it in corresponding normal pancreatic tissues. CDH13 methylation was detected even in relatively early pancreatic cancers, such as stage II cancers and cancers less than 2 cm in diameter. Our results suggest that the aberrant methylation of CDH13 occurs frequently in pancreatic cancer, even at a relatively early stage.

Overexpression of pituitary tumor transforming gene 1 in HCC is associated with angiogenesis and poor prognosis

The pituitary tumor transforming gene 1 (PTTG1) protein is cell‐cycle regulated and is identified as a human securin that inhibits sister chromatid separation and is involved in transformation and tumorigenesis. PTTG1 has very low or undetectable expression in most normal human tissues, but it is abundantly expressed in malignant cell lines and pituitary tumors. In this study, we investigated human PTTG1 expression in 62 hepatocellular carcinoma (HCC) specimens using quantitative real‐time reverse transcription polymerase chain reaction analysis. We found that, compared with corresponding noncancerous liver tissues, PTTG1 was remarkably overexpressed in HCCs (PTTG1/β‐actin; 0.443 ± 0.073 vs. 0.068 ± 0.007; P < .0001). Furthermore, we found a significant correlation between PTTG1 expression and serum alpha‐fetoprotein level (P < .001). Univariate and multivariate analyses revealed that the PTTG1 messenger RNA (mRNA) expression was an independent prognostic factor for disease‐free (odds ratio 2.70; P = .037) and overall (odds ratio 5.35; P = .007) survival. Moreover, we discovered a significant relationship between PTTG1 expression and intratumoral microvessel density. Our data supported an important role for PTTG1‐mediated upregulation of fibroblast growth factor (FGF)–2, one of angiogenesis and modulation of tumor progression, in hepatocarcinogenesis. In conclusion, PTTG1 might be critically involved in the development of HCCs through the promotion of angiogenesis. PTTG1 is overexpressed in HCC and our results suggest that PTTG1 mRNA expression has prognostic significance for the survival of postoperative patients with HCC. (HEPATOLOGY 2006.)

Significant up-regulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivo

Most lung cancer patients are unfortunately uncurable and die because of widespread metastases, thus indicating the importance of identification of molecules with a crucial role in this process. Our previous expression profiling analysis of a highly metastatic lung cancer cell line, NCI-H460-LNM35, and its parental low metastatic line, NCI-H460-N15, revealed significant up-regulation of both known and unknown genes in LNM35. In this study, we describe the isolation and detailed characterizations of a novel gene, named CLCP1, which corresponds to one of such expression sequence tags with up-regulated expression in LNM35. The CLCP1 gene was found to encode a protein with 775 amino acids with structural similarities to, but distinct from neuropilins, cell surface receptors for VEGF165 and semaphorins. Notably, CLCP1 was shown to be up-regulated not only in LNM35 in association with its acquisition of metastatic phenotype during in vivo selection, but also in a significant fraction of lung cancers in vivo with high frequency in metastatic lesions, warranting future studies for a better understanding of the molecular mechanisms of lung cancer metastasis.

A phase II study of radical surgery followed by postoperative chemotherapy with S-1 for gastric carcinoma with free cancer cells in the peritoneal cavity (CCOG0301 study)

BACKGROUND Patients with gastric cancer who have positive cytologic results for cancer cells in peritoneal washings (CY1) have poor outcomes, even in the absence of other distant metastases. A standard treatment for such patients remains to be established. METHODS We conducted a phase II trial with the 2-year survival rate as the primary endpoint. Patients who had gastric cancer with CY1 status but no other residual disease received postoperative chemotherapy with S-1 (1M tegafur-0.4M gimestat-1M otastat potassium) at a daily dose of 80mg/m(2) for 4 weeks, followed by 2 weeks of rest. This cycle was continued until disease progression or intolerable adverse events. D2 dissection was the recommended surgical procedure; splenectomy could be omitted at the discretion of the surgeon. Accrual of 50 patients was planned, and a 2-year survival rate of more than 36% was needed to exceed the historical control. RESULTS Forty-eight patients were enrolled, among whom 47 were assessable for survival and 46 for adverse reactions. Median overall survival was 705 days, and progression-free survival was 376 days. The 2-year survival rate was 47%. Median time to treatment failure was 288 days. Neutropenia was the commonest > or = grade 3 toxicity (6 patients), and anorexia was the most frequent > or = grade 2 non-hematologic toxicity (10 patients). CONCLUSIONS Gastrectomy followed by S-1 monotherapy resulted in survival that surpassed historical data and can serve as an active control treatment for future trials in patients who have gastric cancer with CY1 status in the Far East.

Identification of a metastasis signature and the DLX4 homeobox protein as a regulator of metastasis by combined transcriptome approach

Although widespread metastasis is the major cause of human lung cancer-related deaths, its underlying mechanism remains largely unclear. Our genome-wide comparison of the expression profiles of a highly metastatic lung cancer cell line, NCI-H460-LNM35 (LNM35), and its parental clone, NCI-H460-N15 (N15), resulted in the identification of a cancer metastasis signature composed of 45 genes. Through gene ontology analysis, our study also provided insights into how this 45-gene metastasis signature may contribute to the acquisition of metastatic potential. By applying the signature to datasets of human cancer cases, we could demonstrate significant associations with a subset of cases with poor prognosis not only for the two datasets of cancers of the lung but also for cancers of the breast. Furthermore, we were able to show that enforced expression of the DLX4 homeobox gene, which was identified as a gene with significant downregulation in LNM35 as well as with significant association with favorable prognosis for lung cancer patients, markedly inhibited in vitro motility and invasion as well as in vivo metastasis via both hematogenous and lymphogenous routes. Taken together, these findings indicate that our combined transcriptome analysis is an efficient approach in the search for genes possessing both clinical usefulness in terms of prognostic prediction in human cancer cases and clear functional relevance for studying cancer biology in relation to metastasis.