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Expression of Pirh2, a Newly Identified Ubiquitin Protein Ligase, in Lung Cancer

Wenrui Duan, Li Gao, Lawrence J. Druhan, Wei-Guo Zhu, Carl Morrison, Gregory A. Otterson, Miguel A. Villalona-Calero

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)


    ABSTRACT
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Maintenance of p53 function is important for normal cell growth and development, and loss of p53 function contributes directly to malignant tumor development. The recently discovered Pirh2 protein is an ubiquitin–protein ligase that negatively regulates p53 through activity by targeting it for degradation. To determine how Pirh2 may mediate lung tumorigenesis, we evaluated Pirh2 expression in human and mouse lung tumor samples and paired normal lung tissues using immunoblot analysis and immunohistochemistry. Pirh2 protein expression was higher in 27 (84%) of 32 human lung neoplasms than in matched normal lung tissue and in 14 of 15 mouse lung tumors evaluated. In addition, p53 protein was more ubiquitinated in the mouse lung tumors than in normal tissue, and overall p53 expression was lower than that in normal tissue. These results are consistent with the hypothesis that increased Pirh2 expression affects lung tumorigenesis by reducing p53 activity. To our knowledge, this is the first description of altered Pirh2 expression in human and mouse tumors.


The tumor suppressor protein p53 is a key regulator of cell cycle control, apoptosis, and genomic stability in response to various cellular stresses (16), and mutation of the p53 gene is the most frequently reported genetic defect in human cancers (78). Tight regulation of p53 function is critical for normal cell growth and development. Genomic stress (such as exposure to ionizing radiation or DNA-damaging agents) leads to posttranslational modifications of the p53 protein that enhance its transcriptional activity and extend its half-life (5,6,9). In contrast, in unstressed cells, interactions between p53 and its negative regulators, such as MDM2 and JNK (1015), dramatically reduce p53 protein levels and decrease its half-life. MDM2 was first discovered as an amplified oncogene in sarcomas (16) and was subsequently found to encode a ubiquitin ligase that promotes the rapid degradation of p53 (12,13,17,18). A recently identified ubiquitin–protein ligase, Pirh2, has also been observed to promote p53 degradation (19). The Pirh2 gene, which is also regulated by p53, encodes a RING-H2 domain–containing protein with intrinsic ubiquitin–protein ligase activity (19). Pirh2 interacts physically with p53 and promotes ubiquitination of p53 independently of MDM2 (19).

To investigate whether Pirh2 protein expression is altered in human lung cancers, we analyzed 32 human non–small-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 non–small-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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Table 1. Pirh2 expression in human non–small-cell lung cancer

 


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Fig. 1. Pirh2 and MDM2 expression in human and mouse lung tumors and matched normal lung tissues. A) Immunoblot analysis of Pirh2 expression in human lung tumors and matched lung tissues. One hundred micrograms of lung tumor (LT) or lung tissue (L) lysate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred onto nitro cellulose membranes. Membranes were probed with anti–Pirh2 antibody (clone BL588). {beta}-Actin was used as a control for equal loading. Representative data for two subjects of the 32 analyzed are shown. B) SPC-p53(273H) transgenic mice were treated with ionizing radiation (5 Gy/mouse); lung tumors and lung tissues from 15 mice were harvested at 24 hours postirradiation. Two hundred fifty micrograms of mouse lung tumor lysate or lung tissue lysate for each sample was separated and electrophoretically transferred for immunoblot analysis with the BL588 antibody. CE) Immunohistochemical analysis of Pirh2 protein with the BL588 antibody on formalin-fixed, paraffin-embedded, large-cell carcinoma (C), squamous-cell carcinoma (D), and normal lung tissue (E). Brown staining indicates the presence of the Pirh2 protein. Twenty human non–small-cell lung cancers and matched normal lung tissues were analyzed. FG) Real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis of Pirh2 (F) and MDM2 (G) mRNA expression in lung adenocarcinomas and matched normal lung samples from irradiated SPC-p53(273H) mice. Ribosomal RNA was used as an internal loading control. The mean value of Pirh2 or MDM2 RNA from normal lung tissues was used to normalize (value set to 1) the mean values of Pirh2 or MDM2 RNA from tumor samples. Panels F and G show the mean and upper 95% confidence interval (CI) obtained after normalization of 12 amplifications from four lung tumors (LT) and four matched lung tissues (L) (Pirh2 of LT = 11.3, 95% confidence interval [CI] = 11.2 to 11.4; Pirh2 of L = 1.0, 95% CI = 0.97 to 1.03; * = P<0.001, two-sided t test; MDM2 of LT = 1.2, 95% CI = 1.19 to 1.21; MDM2 of L = 1.0, 95% CI = 0.99 to 1.01; P = 0.6, two-sided t test). Primers used for the real-time RT–PCR analysis were designed using mouse mRNA sequences (GenBank Accession numbers: NM_026557 for mPirh2, NM_010786 for MDM2). Primers Pirh2F102 (5'-GCTGCGAGCACTATGACAGA) and mPirh2R195 (5'-TGATCTTCATTGGTATCGTGACA) were used to amplify a 94-bp fragment from the mouse Pirh2 RNA. Primers MDM2F1220 (5'-CTTCGTGAGAACTGGCTTCC) and MDM2R1346 (5'-CTGTCAGCTTTTTGCCATCA) were used to amplify a 127-bp fragment from the mouse MDM2 RNA. pcDNA3–mPirh2 vectors (provided by Dr. Samuel Benchimol, University of Toronto, Toronto, Ontario, Canada) were used as a positive control. Real time RT–PCR amplification was conducted in a 25-µL reaction using the QuantiTect Sybr Green RT–PCR kit (Qiagen, Valencia, CA) according to the protocol supplied by the manufacturer. Reactions were carried out in 96-well plates using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA).

 
We also analyzed Pirh2 expression in a mouse lung tumor model that recapitulates the molecular events occurring in human lung cancer (20). This transgenic mouse model, SPC-p53(273H), expresses a mutant human p53 gene encoding the amino acid histidine at position 273 (273H) under the transcriptional control of the human surfactant protein C (SP-C) promoter (20,21). SPC-p53(273H) mice develop malignant lung tumors, which have been histologically characterized as adenocarcinomas, earlier and at a higher rate than their nontransgenic littermates (20,21). All animal protocols were approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee. Similar to the results seen in human lung cancer, the Pirh2 protein was elevated in 14 of 15 mouse lung tumors compared with matched normal lung tissue (Fig. 1, B).

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, C–E).

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 {gamma}-irradiation by exposing them to a 137Cs {gamma}-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 transcription–polymerase chain reaction (real-time RT–PCR) 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. Anti–mouse p53 antibody (Pab246; BD PharMingen, San Diego, CA) was used to detect the mouse wild-type p53, and anti–human 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 17–29 [Fig. 2, A]) and DO-1 (which recognizes amino acids 11–25 [data not shown]), suggesting that it is from the N-terminal portion of the mutant p53.



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Fig. 2. p53 status in lung tumors and matched normal lung tissues. SPC-p53(273H) transgenic mice were treated with ionizing radiation (5 Gy/mouse); lung tumors (LT) and normal lung tissues (L) were harvested from 10 mice at 24 hours postirradiation. A) Immunoblot analysis was used to detect the expression of the wild-type (WT) p53 and mutant p53 (273H) proteins in mouse samples with the Pab246 (BD PharMingen, San Diego, CA) and DO-7 (BD PharMingen) antibodies, respectively. The antibody DO-7 also binds to a 45-kd p53(273H) fragment, p53(273H)-D. Immunoblots from three tumor-bearing mice and one tumor-free mouse of the 10 mouse lung tumors and matched normal tissues analyzed are shown. B) Reverse transcription–polymerase chain reaction (RT–PCR) analysis of the p53 mRNA expression in the mouse lung tumors and lung tissues used in the previous experiment. Representative RT–PCR amplifications are shown. CD) Analysis of ubiquitination of p53 protein in lung tumors and lung tissues; mouse lung tumor or matched normal lung tissue total protein (1000 µg) was immunoprecipitated with Pab 246 (C) or with DO-7 (D). Protein G agarose beads were used to collect the protein–antibody complexes, which were washed five times with washing buffer (250 mM NaCl, 1% igepal, 50 mM Tris-HCl) and were used for immunoblot analysis. A rabbit polyclonal anti–p53 antibody (FL-393) was used to detect p53 and ubiquitinated p53 protein. The long exposure shows ubiquitinated p53 (upper bracketed portion), and the short exposure shows nonubiquitinated p53 protein expression. Immunoblots from two of the 10 mouse tumors and matched normal lung tissues analyzed are shown.

 
To determine if the marked reduction of wild-type p53 protein was due to transcriptional or posttranscriptional events, we performed RT–PCR to analyze the expression of mouse p53 mRNA. The RT–PCR results showed that mouse p53 mRNA was expressed in all lung tumors (Fig. 2, B).

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 p53–273H (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 5–9 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 ubiquitin–protein 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 MDM2–p53 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 Pirh2–p53 interaction may be of value in the treatment of lung cancer.


    NOTES
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The mouse Pirh2 vector was kindly provided by Dr. Samuel Benchimol (University of Toronto). We thank Erinn Hade for the assistance in statistics. We thank the Transgenic Core Facility, Histology Core Facility, Real-Time PCR Shared Resource, The Midwestern Division of the Cooperative Human Tissue Network, The Ohio State University, Columbus, Ohio, and the National Cancer Institute (NCI), Bethesda, MD, for their assistance.

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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