Amplification and overexpression of SKP2 are associated with metastasis of non-small-cell lung cancers to lymph nodes - PubMed (original) (raw)

Amplification and overexpression of SKP2 are associated with metastasis of non-small-cell lung cancers to lymph nodes

Sana Yokoi et al. Am J Pathol. 2004 Jul.

Abstract

SKP2, an F-box protein constituting the substrate recognition subunit of the SCF(SKP2) ubiquitin ligase complex, is implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27(KIP1). Our earlier studies revealed SKP2 as a target gene within the 5p13 amplicon that is often seen in small-cell lung cancers. In the present study we examined amplification status and expression levels of SKP2 in non-small-cell lung cancer (NSCLC) and investigated its clinicopathological significance in this type of tumor because amplification of DNA at 5p13 is observed frequently in NSCLCs as well as in small-cell lung cancers. SKP2 exhibited amplification in 5 (20%) of 25 cell lines derived from NSCLC, and the transcript was overexpressed in 11 (44%) of the 25 lines. Moreover, expression of SKP2 was up-regulated significantly in 60 primary NSCLC tumors as compared to nontumorous lung tissues (P < 0.0001). Elevated expression of SKP2 correlated significantly with positive lymph node metastasis (P = 0.007), with stage II or higher of the international TNM classification (P = 0.014), with poor or moderate differentiation (P < 0.001), and with the presence of squamous cell carcinoma (P = 0.037). Reduction of SKP2 expression by transfection of an anti-sense oligonucleotide inhibited invasion and migration of NSCLC cells in culture. Our results suggest that SKP2 may be involved in progression of NSCLC, and that targeting this molecule could represent a promising therapeutic option.

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Figures

Figure 1

Figure 1

Representative images of FISH for SKP2 on metaphase chromosomes from two NSCLC cell lines, PC-10 (A) and HS-24 (B). Twin-spot FISH signals on sister chromatids numbered eight in PC-10 and five in HS-24.

Figure 2

Figure 2

Amplification and overexpression of SKP2 in NSCLC cell lines, indicated by asterisks. A: Southern blots containing genomic DNA derived from 18 of the 25 cell lines examined and normal peripheral lymphocyte (N1). GAPDH was used as a control probe to eliminate loading differences on the blots. B: Relative copy number of SKP2 determined by real-time PCR in the same cell lines shown in A and normal peripheral lymphocytes (N1 to N5). Results are presented as the ratio between SKP2 and a reference gene (RNase P), and normalized in such a manner that the average ratio in five normal DNAs (N1 to N5) equals a value of 2. Two copies are indicated by a dotted line. More than four copies are defined as amplification. C: Northern blot analyses using total RNA from the same NSCLC cell lines; GAPDH again served as a quantity control probe. D: Relative expression levels of SKP2 mRNA determined by real-time RT-PCR in the same cell lines and normal lung tissues (NL1, NL2). Results are presented as the ratio between SKP2 and a reference gene (GAPDH), and normalized in such a manner that the average ratio in two normal lung tissues (NL1, NL2) equals a value of 1. A value of 4 was used to determine the cutoff for overexpression, shown as a dotted line. E: Significant correlation (r = 0.54, P = 0.0078) between copy numbers and relative expression levels of SKP2 was observed in 25 NSCLC cell lines.

Figure 3

Figure 3

Overexpression of SKP2 in primary NSCLC tumors. Levels of SKP2 mRNA were evaluated by real-time quantitative RT-PCR and normalized according to levels of GAPDH. A: Relative expression of SKP2 in paired tumor and nontumor tissues from 20 patients with NSCLC. B: Expression of SKP2 in 60 primary NSCLC tumors relative to expression in 20 nontumorous lung tissues. Solid horizontal lines indicate the means of expression levels. The mean + 2 × SD of nontumorous tissues was used to determine the cutoff value for overexpression, shown as a dotted line.

Figure 4

Figure 4

Inhibition of invasion and migration of PC-10 cells by anti-sense oligonucleotide mediates reduction of SKP2 expression. A: Western blot analysis of SKP2 and β-actin as an internal control. PC-10 cells were treated with an anti-sense oligonucleotide (AS) targeting SKP2, a control oligonucleotide with scrambled sequence (SC), or oligofectamine alone (oligofectamine). Cells were harvested 48 hours, 60 hours, and 72 hours after transfection. Ten-μg aliquots of each whole cell lysate were separated by 12.5% dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting. B: Invasive and migratory ability of PC-10 cells treated with AS, SC, or oligofectamine alone (control). The cells were placed in Matrigel-coated (for invasion assay; top) or uncoated (for migration assay; bottom) chambers containing 8-μm pores. Invaded or migrated cells were stained with Giemsa and counted. C, D, E: Effect of AS or SC on the invasion (C), migration (D), and viability (E) of PC-10 cells. Results are expressed as percentages of oligofectamine alone (control), and values are represented as the mean ± SD of at least three independent experiments.

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