Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas

Tarek G Gharib,Sharon L.R Kardia,Dafydd G Thomas,Rork Kuick,David G Beer,Mark B Orringer,Samir M Hanash,Guoan Chen,Jeremy M.G Taylor,Mark D Iannettoni,Kerby A Shedden,Thomas J Giordano,Chiang-Ching Huang,Hong Wang,David E Misek

doi:10.1074/mcp.m200055-mcp200

Tarek G Gharib, Sharon L.R Kardia + Show 13 more

Open Access

https://doi.org/10.1074/mcp.m200055-mcp200

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Abstract

We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays. Three phosphorylated forms and one unphosphorylated form of the Op18 protein were identified and found to be overexpressed in lung adenocarcinomas as compared with normal lung. The percentage of phosphorylated to total Op18 protein isoforms increased from 3.2% in normal lung to 7.9% in lung tumors. Both the phosphorylated and unphosphorylated Op18 proteins were significantly increased in poorly differentiated tumors as compared with moderately or well differentiated lung adenocarcinomas (p<0.03), suggesting that up-regulated expression of Op18 reflects a poor differentiation status and higher cell proliferation rates. This was further verified in A549 and SKLU1 lung adenocarcinoma cell lines by examining Op18 levels and phosphorylation status following treatment that altered either cell proliferation or differentiation. The increased expression of Op18 protein was significantly correlated with its mRNA level indicating that increased transcription likely underlies elevated expression of Op18. The overexpression of Op18 proteins in poorly differentiated lung adenocarcinomas and the elevated expression of the phosphorylated forms of Op18 may offer a new target for drug- or gene-directed therapy and may have potential utility as a tumor marker.

Highlights

We examined the expression of oncoprotein 18 (Op18) in 93 lung adenocarcinomas and 10 uninvolved lung samples using quantitative two-dimensional PAGE analysis with confirmation by mass spectrometry and two-dimensional Western blot analysis. mRNA expression was examined using oligonucleotide microarrays, and the cellular localization of the Op18 protein was examined using immunohistochemical analysis of tissue microarrays
Spots corresponding to Op18, Op18a, and Op18b were identified as different isoforms of oncoprotein 18 by mass spectrometry (Fig. 2) and 2D Western analysis [19, 30]
Op18 has been previously identified as the unphosphorylated form, and Op18a and Op18b have been identified as representing phosphorylated forms of the Op18 protein (12, 26 – 29). 2D Western blot analysis identified an additional isoform, Op18c, of similar molecular weight but more acidic and likely to be a triphosphorylated form in the lung tumors [19, 30]

Summary

The abbreviations used are

Op18, oncoprotein 18; 2D, twotems under different names including stathmin [1, 2], metablastin [3], phosphoprotein 19 [4], and LAP18 [5]. The unphosphorylated form of Op18, which predominates in the interphase portion of the cell cycle, promotes the depolymerization of microtubules by increasing the catastrophe frequency at pH 7.5 or by sequestering tubulin at pH 6.8 [8]. The incidence of lung adenocarcinoma is increasing among both men and women in the United States [11]; the expression status of Op18 protein and its specific isoforms or its mRNA have not been examined. Dimensional; MS/MS, tandem mass spectrometry; IL-6, interleukin 6; IFN-␣, interferon ␣; DX, dexamethasone; PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION