Skp2 Gene Copy Number Aberrations Are Common in Non-Small Cell Lung Carcinoma, and Its Overexpression in Tumors with ras Mutation Is a Poor Prognostic Marker

Abstract

Purpose: Skp2 plays a critical role in cell cycle progression, especially at the G 1 -S transition, putatively through its control of several cell cycle regulator proteins. The Skp2 gene is located on a region of chromosome 5p that is commonly overrepresented in lung cancer. The present study aimed to evaluate Skp2 abnormalities and their prognostic value in non-small cell lung cancer (NSCLC).Experimental Design: In total 16 NSCLC cell lines and 163 primary tumors were included in studies to measure Skp2 relative gene copy number, mRNA abundance, and protein level. The tumors were also evaluated for p27 protein expression level and ras mutation. These values were correlated with the clinical and pathological features of the patients.Results: Skp2 relative gene copy number aberrations were found in 88 and 65% of NSCLC cell lines and primary tumors, respectively. Overrepresentation was especially common among squamous cell carcinoma (74%). Both gene copy overrepresentation (13%) and loss (35%) were found in adenocarcinoma. Skp2 relative gene copy number was significantly correlated with mRNA and protein levels, but none of these were correlated with p27 protein levels. Neither high Skp2 protein expression nor ras mutation was prognostically significant. In NSCLCs with ras mutation, however, high Skp2 protein expression was a significant independent poor prognostic marker.Conclusion: There appears to be a synergistic interaction between high Skp2 protein expression and ras mutation with negative impact on the survival of NSCLC patients.

Amplification of telomerase (hTERT) gene is a poor prognostic marker in non-small-cell lung cancer

“…In addition, DNA was also extracted from 19 non-neoplastic lung samples. The tumour cells micro-dissected using LCM system were incubated in DNA extraction buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.1 mg ml À1 gelatin, 0.45% Nonidet P-40, 0.45% Tween 20 and 0.4 mg ml À1 proteinase K. DNA was subsequently extracted by the phenol -chloroform method (Zhu et al, 2004).…”

“…The primer sequences were: hTERT sense 5 0 -taa aat tat cca cat ggc tca cgt-3 0 , antisense 5 0 -ctt ggg aac cag gac aaa gg-3 0 ; PIK3R1 sense 5 0 -atc tgc cac tgg ctt ccc tt-3 0 , antisense 5 0 -cag tct ttc cct gat cat tga acc-3 0 . The PCR conditions were optimized as reported (Zhu et al, 2004). The PIK3R1 (5q13.1) gene is used as the reference nonamplified gene in NSCLC (Massion et al, 2002), and the hTERT gene copy number was estimated using comparative CT method.…”

“…The mRNA expression was assayed using reverse transcription (RT) -qPCR on total RNA of 130 primary NSCLC and 18 corresponding non-neoplastic lung tissues using the ABI PRISM 7700 Sequence Detection System (Zhu et al, 2004). Total cellular RNA was isolated from the frozen tissues, as previously described (Tsao et al, 1998) and purified by the RNeasy Mini kit (Qiagen Inc., Mississauga, ON, Canada).…”

“…The primers for hTERT were: sense 5 0 -cgtcgagctgctcaggtctt-3 0 , antisense 5 0 -agt gctgtctgattccaatgctt-3 0 . The DCT method was used to normalize the sample-to-sample variation in RNA/cDNA quantity using the 18s ribosomal RNA as the housekeeping gene (Zhu et al, 2004).…”

Ras oncogene mutations and survival in patients with lung cancer

“…Patients with K-ras mutant NSCLC showed poorer clinical outcomes when treated with erlotinib and chemotherapy (Eberhard et al, 2005). Furthermore, in NSCLCs with ras mutation, the overexpression of Skp2, a protein that plays a critical role in cell cycle progression, was a significant independent poor prognostic marker of survival (Zhu et al, 2004).…”