Sana Yokoi

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.

Correlation between interleukin 6 production and tumor proliferation in non-small cell lung cancer

Interleukin 6 (IL-6) facilitates the differentiation of B cells to immunoglobulin-secreting cells and is reported to be a proliferative factor in some tumors. In this study, we examined IL-6 production in non-small cell lung carcinoma (NSCLC) and the proliferation of tumor cells following IL-6 treatment in vitro and in vivo. We analyzed the expression of IL-6 mRNA and protein in a series of 15 human lung cancer cell lines (four adenocarcinomas, five squamous cell carcinomas, two large cell carcinomas, and four small cell carcinomas) by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). We established an IL-6-producing cell line (ABC-1#IL-6) by transfecting a human IL-6 cDNA into a human non-IL-6-producing NSCLC cell line (ABC-1). These two cell lines were used to determine tumor cell proliferation both in vivo and in vitro in order to clarify the effect of IL-6 on tumor growth and metastasis. Athymic nude mice, SCID mice, and BALB/c mice were subcutaneously inoculated with these two cell lines, and body weight, tumor growth, and tumor doubling time were measured. The presence of IL-6 and tumor-infiltrating lymphocytes (TILs) within tumor tissues was examined by immunohistochemical staining. Results: Eight of 15 (53%) lung cancer cell lines expressed both IL-6 mRNA and protein. Tumor lesions of both cell lines developed in nude and SCID mice, although no such lesions of either cell lines developed in BALB/c mice. The tumor doubling time in nude and SCID mice was 2.97±1.22xa0days and 2.45±1.32xa0days, respectively, in mice inoculated with the cell line ABC-1#IL-6. These doubling times were statistically significantly shorter than those evident in mice inoculated with the control original ABC-1 cell line (nude, p=0.0337; SCID, p=0.0119; unpaired t-test). The rates of cell proliferation in vitro of the ABC-1#IL-6 and original ABC-1 cells lines were comparable (p=0.1441, unpaired t-test). Immunohistochemical staining revealed strong IL-6 expression in tumors derived from the IL-6-producing cell line but not in tumors derived from the original ABC-1 cell line (both in nude and SCID mice). Conclusion: 53% of lung cancer cell lines produce IL-6 mRNA and protein. Although IL-6 itself does not influence tumor cell proliferation in vitro, an association between IL-6 expression and tumor proliferation was found in vivo in nude and SCID mice. An anti-IL-6 reagent could provide a novel therapeutic strategy in patients with IL-6-producing lung tumors.

Overexpression of Collagen XVIII Is Associated with Poor Outcome and Elevated Levels of Circulating Serum Endostatin in Non–Small Cell Lung Cancer

Purpose: The aim of this study was to determine whether collagen XVIII expression is correlated with circulating serum endostatin and whether this has any prognostic value in patients with non–small cell lung cancer (NSCLC). Experimental Design: Serum endostatin levels were measured quantitatively by a competitive enzyme immunoassay, and collagen XVIII expression in tumor tissue was investigated with an immunohistochemical method in a series of 94 patients who underwent surgery for NSCLC. Results: Sixty cases (63.8%) had positive immunohistochemical staining with anticollagen XVIII polyclonal antibodies, including strongly positive staining in 11 (11.7%) cases. The mean (± SD) serum endostatin level was 41.6 ± 34.4 ng/ml in the patient group and 16.3 ± 10.3 ng/ml in the control group (P < 0.0001). The 11 cases who were strongly collagen XVIII-positive had significantly higher serum endostatin levels than the cases who were negative or weakly positive (P = 0.0297). The 5-year survival rates of negative, weakly positive, and strongly positive patients were 77.8%, 56.9%, and 43.8%, respectively. The cases with strongly positive collagen XVIII expression had a significantly poorer outcome than cases with negative expression (P = 0.0027). A multivariate analysis with Cox proportional hazards model for disease-specific survival revealed that expression of collagen XVIII (strongly positive versus negative; weakly positive versus negative), tumor classification, and regional lymph node classification were independent prognostic factors. Conclusions: Our results suggest that expression of collagen XVIII in tumor tissue is strongly associated with a poorer outcome in NSCLC and correlates with elevated levels of circulating serum endostatin.

Down-regulation of SKP2 induces apoptosis in lung-cancer cells.

S‐Phase kinase associated protein 2 (SKP2), an F‐box protein constituting the substrate‐recognition subunit of the SCFSKP2 ubiquitin ligase complex, targets cell‐cycle regulators, such as the cyclin‐de‐pendent kinase inhibitor p27KIP1, for ubiquitin‐mediated degradation. Our earlier studies indicated frequent amplification and over‐expression of the SKP2 gene in primary small‐cell lung cancers (SCLCs) and cell lines derived from this type of tumor, and showed that down‐regulation of SKP2 expression by means of an antisense oligonucleotide inhibited the growth of SCLC cells in culture (Yokoi et al. Am J Pathol, 161, 207‐216, 2002). The anti‐sense effect was confirmed in two cell lines of non‐small cell lung cancer (NSCLC) that also exhibited over‐expression of the gene. In the work reported here, we examined the mechanism(s) responsible for antisense‐mediated growth inhibition of SCLC‐ and NSCLC‐derived cultures. SKP2‐antisense treatment not only suppressed DNA synthesis, as determined by [3H]thymidine incorporation, but also induced spontaneous apoptosis characterized by an increase in the sub‐G1 population, fragmentation of nuclei, and activation of caspase‐3. Our results suggest that since down‐regulation of SKP2 appears to induce apoptosis in lung‐cancer cells directly, targeting this molecule could represent a promising new therapeutic approach for this type of cancer, and possibly other tumors that over‐express SKP2. (Cancer Sci 2003; 94: 344–349)

A case of multiple synchronous lung adenocarcinomas with differing EGFR mutations

We report a case of four synchronous lung cancers, each with a different epidermal growth factor receptor (EGFR) mutation in the tyrosine kinase domain. Bilateral double lung cancer was diagnosed clinically, and a left upper lobectomy and partial left S6 resection were performed. Right S1 segmental resection was performed later. Pathological examination revealed three lung adenocarcinomas in the left lung (S1xa0+xa02, S3, and S6) and one in the right. Three different mutations of EGFR were identified with the polymerase chain reaction and direct sequencing of DNA from the three left lung tumors. The EGFR mutation on the right also differed from that of the left lung tumors.