Affiliations of authors: Comprehensive Cancer Center and Department of Internal Medicine (WD, LG, LJD, W-GZ, GAO, MAV-C) and Department of Pathology (CM), The Ohio State University College of Medicine and Public Health, Columbus
Correspondence to: Miguel A. Villalona-Calero, MD, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, B406 Starling-Loving Hall, 320 W. 10th Ave., Columbus, OH 43210-1240 (e-mail: villalona-1{at}medctr.osu.edu)
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ABSTRACT |
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To investigate whether Pirh2 protein expression is altered in human lung cancers, we analyzed 32 human nonsmall-cell lung cancers and 32 matched normal lung tissues from the same patients by immunoblot with the Pirh2 antibody (clone BL588; Bethyl, Montgomery, TX). Human lung tumors and matched normal lung tissue samples were obtained from The Cooperative Human Tissue Network, Midwestern Division, at Ohio State University, after Ohio State University Institutional Review Board approval. Written informed consent was obtained from each human subject. Appropriate pathologic tissue evaluation was performed for each sample. Table 1 describes the histologic characteristics of the lung tumors; there were eight adenocarcinomas, 13 squamous-cell carcinomas, six large-cell carcinomas, and five poorly differentiated nonsmall-cell carcinomas. Semiquantitative analysis of the Pirh2 immunoblot signal by densitometry showed that Pirh2 protein expression was elevated (by at least twofold) in 27 (84%) of the 32 human lung tumors when compared with matched uninvolved lung tissues (Fig. 1, A and supplementary figure online at http://jncicancerspectrum.oupjournals.org/jnci/content/vol96/issue22).
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The localization of Pirh2 protein in human lung tumors was evaluated immunohistochemically, using a rabbit polyclonal Pirh2 antibody (clone BL588; Bethyl). Pirh2 protein was found primarily in the cytoplasm and plasma membrane of malignant cells (Fig. 1, CE).
The level of MDM2 protein is very low in unstressed human and animal tissues, including the lung tumors in SPC-p53(273H) mice (data not shown). To investigate if genotoxic stress would lead to increased MDM2 protein expression in lung tumors, we treated 10 tumor-bearing SPC-p53(273H) mice with -irradiation by exposing them to a 137Cs
-source at a dose of 5 Gy/mouse. At 24 hours after irradiation, tumor and normal lung tissues were harvested for analysis of MDM2 expression. Immunoblot analysis showed that MDM2 protein was expressed at a similar level in lung tumors and in normal lung tissue (data not shown). To compare Pirh2 and MDM2 mRNA expression, we performed real-time reverse transcriptionpolymerase chain reaction (real-time RTPCR) analysis on irradiated mouse lung tumor and tissue samples. Pirh2 mRNA expression was higher in lung tumors (11.3, 95% confidence interval [CI] = 11.2 to 11.4) than in matched normal lung tissues (1.0, 95% CI = 0.97 to 1.03; P<.001 [Fig. 1, F]). However, MDM2 mRNA expression in lung tumor and normal tissue was not statistically significantly different (Fig. 1, G).
To determine if p53 protein expression is decreased in mouse lung adenocarcinomas compared with normal tissue, we performed immunoblot analysis to examine the level of mouse wild-type p53 protein and the level of the human mutant p53(273H) protein. Because p53 protein is not detectable without being induced by genotoxic stress (data not shown), we used the same irradiated mice as were used to evaluate MDM2 mRNA expression to evaluate p53 expression after genotoxic stress. Antimouse p53 antibody (Pab246; BD PharMingen, San Diego, CA) was used to detect the mouse wild-type p53, and antihuman p53 antibody (DO-7; BD PharMingen) was used to detect the mutant p53(273H). Levels of the mouse wild-type p53 protein was reduced in 10 of 10 tumor samples compared with matched normal lung tissue. In addition, the level of mutant p53(273H) protein was markedly lower in tumor tissue than in matched normal lung tissue (Fig. 2, A). We also detected a short (45 kd) form of p53 with two different antibodies, DO-7 (which recognizes amino acids 1729 [Fig. 2, A]) and DO-1 (which recognizes amino acids 1125 [data not shown]), suggesting that it is from the N-terminal portion of the mutant p53.
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To investigate if the decreased mouse p53 expression is associated with ubiquitin-dependent degradation in mouse lung tumors, we immunoprecipitated mouse wild-type p53 and mutant p53(273H) protein and analyzed ubiquitinated forms by immunoblot analysis with a rabbit polyclonal antibody against p53 (22,23). Levels of both ubiquitinated mouse wild-type p53 (Fig. 2, C) and ubiquitinated mutant p53273H (Fig. 2, D) were increased in lung tumors.
To determine whether the overexpression of Pirh2 in lung tumors is related to p53 mutational status, we immunoprecipitated mutant and wild-type p53 with the DO-7 antibody and detected p53 by immunoblot analysis with the FL-393 antibody (Santa Cruz, Santa Cruz, CA). Twelve human lung tumor samples, seven with Pirh2 overexpression and five without Pirh2 overexpression, were compared with their matched normal lung tissue. P53 protein was detected in four of the 12 lung tumors, and was undetectable in all 12 uninvolved lung tissues. Because exons 59 contain the majority of the known p53 mutations, we sequenced these exons of the p53 gene on DNA from all 12 tumors. Five of the 12 lung tumors contained mutant p53, including the four tumors with detectable p53 protein. Among the seven tumors with Pirh2 overexpression, four tumors contained a p53 gene mutation, whereas among the five tumors without Pirh2 expression, one contained a p53 gene mutation.
In summary, we have shown for the first time, to our knowledge, that Pirh2, a ubiquitinprotein ligase that promotes p53 protein degradation, is overexpressed in lung tumors in both a mouse model and in human samples compared with matched normal lung tissues. The overexpression of Pirh2 in mouse tumor samples was accompanied by low p53 protein expression, which was due to a posttranscriptional mechanism. In addition, p53 ubiquitination was increased in the tumor tissue compared with normal lung tissues, indicating that ubiquitin-dependent p53 degradation may be increased in lung tumors.
The data in our study, in conjunction with the model of Pirh2 acting as a ubiquitin ligase, suggest the possibility that inhibition of Pirh2 activity to increase wild-type p53 function and activity may represent an attractive strategy for cancer therapy. The potential use of proteasome inhibitors as therapeutic agents is currently being investigated (24,25). Specifically, PS-341 (bortezomib) has recently been approved for the treatment of multiple myeloma and is under intensive investigation as the first of its class as a novel cancer therapeutic (24,25). Testing the ability of bortezomib and other proteasome inhibitors in this system will be of great interest. Furthermore, small-molecule compounds that stabilize the active conformation of the p53 DNA binding domain (e.g., CP-31398) have been reported to block p53 ubiquitination and degradation and to restore wild-type p53 function in p53 mutant cells (22,26). In addition, chalcone derivatives (compounds derived from 1,3-diphenyl-2-propen-1-one) have been reported to act as MDM2 inhibitors by disrupting the MDM2p53 protein complex (27). Together, this evidence supports the potential role of Pirh2 as a therapeutic target.
Our observation that p53 mutational status and Pirh2 overexpression were not linked points out an important distinction between MDM2 and Pirh2 in that MDM2 overexpression and amplification is seen exclusively in tumors with wild-type p53. Possible explanations for this observation are that p53 is not the sole target of Pirh2 ubiquitin ligase activity and that mutant as well as wild-type p53 is a target of this activity. These hypotheses need further experimental investigation.
We hypothesize that the functional interaction between p53 and Pirh2 has a critical role in lung tumor progression. Agents that stabilize wild-type p53 by altering ubiquitination or by interfering with the Pirh2p53 interaction may be of value in the treatment of lung cancer.
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NOTES |
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The work was supported by NCI grant CA76970 to Miguel A. Villalona-Calero and NCI grant CA16058, to the Ohio State University Comprehensive Cancer Center.
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Manuscript received January 29, 2004; revised August 20, 2004; accepted August 26, 2004.
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