A novel target gene, SKP2, within the 5p13 amplicon that is frequently detected in small cell lung cancers

Abstract

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.

Figure 1.

Summary of genetic imbalances detected by CGH in 22 SCLC cell lines. The 22 autosomes and X chromosome are represented by ideograms showing G-banding patterns. As judged by the computerized green-to-red profiles, vertical lines on the left (red) of each chromosome ideogram show losses of genomic material in cell lines, whereas those on the right (green) correspond to copy-number gains. HLGs are represented as yellow rectangles. The number at the top of each vertical line corresponds to the cell line in which each change was recorded (see Table 1 ▶ ).

Figure 2.

Map of the amplicon at 5p13 and representative images of two-color FISH in SCLC cell lines. A: Represented to the right of the chromosome 5 ideogram are STS markers selected according to the GeneMap 1999 database (http://www.ncbi.nlm.nih.gov/genemap99/), the positions of eight genes (CDH12, CDH10, CDH6, PC4, SKP2, RAD1, ZNF131, and GHR), and the 10 BACs used as probes for FISH experiments. The RP11-prefix is omitted here for all BACs. The graph at far right represents the copy number per cell and the extent of HSR determined by FISH on metaphase chromosomes from two SCLC cell lines, ACC-LC-5 (open circles) and ACC-LC-172 (filled circles). B–E: Two-color FISH using BAC RP11–36A10 (green) and RP11–309K8 (red) on metaphase chromosomes from cell lines SBC-5 (B), ACC-LC-173 (C), Lu-134A (D), and ACC-LC-172 (E). BAC RP11–36A10 generated strong signals as a HSR on a marker chromosome in ACC-LC-172 (E); BAC RP11–309K8 generated three signals in the same cell line. In other lines both RP11–36A10 and RP11–309K8 generated two signals in SBC-5 (B), three in ACC-LC-173 (C), and eight in Lu-134A (D).

Figure 3.

Amplification and overexpression of three genes (SKP2, PC4, and CDH6) lying within the 5p13 amplicon in SCLC cell lines and primary tumors. Asterisks indicate cell lines and primary tumors bearing amplification or overexpression of these genes. A: Representative Southern blots containing 5 μg of genomic DNA derived from cell lines and normal peripheral lymphocytes. B: Northern-blot analyses of SKP2 and PC4 using 10 μg of total RNA from the same SCLC cell lines; GAPDH served as a quantity-control probe. C: Semiquantitative RT-PCR analyses of CDH6 in the same cell lines, using GAPDH as control. D: Representative Southern blots analysis of SKP2 containing 5 μg of genomic DNA derived from 10 primary SCLC tumors (T) and nontumorous counterparts (N) of three of them. E: Northern-blot analysis of SKP2 using 10 μg of total RNA from each of the same 10 primary SCLC tumors and three nontumorous counterparts. Two rRNA species served as controls.

Figure 4.

Expression of SKP2 protein during cell-cycle progression. A: Flow-cytometric analysis. ACC-LC-172 cells were synchronized to the M phase with nocodazole as described in Materials and Methods, and harvested at the indicated times after release from the nocodazole block. The x axis shows DNA content and the y axis shows the number of cells. B: Protein extracts of ACC-LC-172 cells analyzed by Western blotting with antibodies to SKP2, p27, and cyclin E during the M phase (0 hours) and the S phase (24 hours). C: Immunoblots of protein extracts from M phase showing expression of SKP2 and p27 in four SCLC cell lines that had shown different SKP2 DNA copy-numbers in FISH. Experiments using the SKP2-specific BAC 36A10 (Figure 2) ▶ had shown the gene amplification as HSR in ACC-LC-172, three-copy signals in ACC-LC-173 and ACC-LC-80, and two-copy signals in SBC-5. The results of FISH analyses also agreed with those of Southern blotting (Figure 3A) ▶ .

Figure 5.

Growth inhibition by the anti-sense oligonucleotide targeting SKP2 mRNA. ACC-LC-172 cells, exhibiting amplification and overexpression of SKP2, were treated with an anti-sense oligonucleotide targeting SKP2 (AS) or a scramble oligonucleotide (SC) by using Oligofectamine. A: Levels of SKP2 determined by Western blotting. Cells were treated with 200 nmol/L of oligonucleotides (anti-sense or scrambled) or Oligofectamine alone, and then harvested at 48 hours after transfection. Untreated cells were maintained under identical experimental conditions. Six μg of whole cell lysate were separated by polyacrylamide gel electrophoresis and analyzed by immunoblotting. B and C: The effects of anti-sense oligonucleotide on the viability of ACC-LC-172 cells. Cells were treated with the indicated concentrations of oligonucleotides (anti-sense or scrambled) and cell viability was determined by MTT assay at 4 days after transfection (B). Cells were treated with 200 nmol/L of oligonucleotides (anti-sense or scrambled) and cell viability was determined at the indicated times after transfection (C). The percentage was calculated against the absorbance of the control cells treated with Oligofectamine alone. The values were shown on the plot represent the means ± SD of four independent experiments.