Characterization of WWOX tumor suppressor gene in osteosarcoma

Jilong Yang, M.D., Ph.D.

Attending Doctor

Department of Bone and Soft Tissue Tumor

Tianjin Cancer Hospital and Institute, Tianjin, China, 300060

Wei Zhang, PhD

Professor

Department of Pathology, Unit 85

Director, Cancer Genomics Core Lab

M. D. Anderson Cancer Center

1515 Holcombe Blvd. Houston, Texas 77030

[Editor's Note: This article gives an overview of a research study that was announced June 15, 2008. Click here to read the formal announcement.]

Introduction

Osteosarcoma is the most common primary malignancy of bone in children and adolescents. It is an aggressive neoplasm composed of spindle cells producing osteoid, and can occur in any bone, but most frequently occurs in the long bones of the arms and legs near the growth plates, such as the thigh bone above the knee, the shin bone below the knee, and the upper arm bone. Currently, the standard therapy for osteosarcoma patients includes surgery with pre- and post-surgery chemotherapy consisting of a combination of adriamycin, cisplatin, ifosfamide, and methotrexate. The combination of these drugs and surgery has significantly improved the 5-year disease-free survival rate up to 60-70% during the last 30 years. However, the total survival and disease-free survival is still very low.

Background

The causes of osteosarcoma are not known. Aqeilan et al demonstrated that the loss of both alleles of WWOX gene results in osteosarcomas in some early postnatal mice, while loss of one allele significantly increases the incidence of spontaneous and chemically induced tumors. Histological examination of the knockout mice showed focal lesions along the bone diaphysis with increased numbers of osteoblast and enlarged cells in cartilage matrix with multiple nucleoli, suggesting proliferation of the progenitors arising from the periosteum. These tumors developed in young postnatal mice in the absence of any carcinogenic treatment, suggesting a role of WWOX as a tumor suppressor in the bone. Although WWOX gene alteration has been implicated in chromosomal alterations in many types of human malignancies including breast, lung, prostate, esophageal, pancreatic, and stomach carcinoma, there is no report on the role of the WWOX gene in osteosarcoma.

Figure 1: Loss of both alleles of WWOX gene results in osteosarcoma and the death of the mice, while loss of one allele significantly increases the incidence of spontaneous cancers such as lung cancer.

The WWOX gene encodes a WW domain-containing oxidoreductase. The gene is located at chromosome 16q23.1-16q23.2, a region that spans the second most common human fragile site, FRA16D. The WWOX protein contains two N- terminal WW domains and a central short chain oxidoreductase-like domain. The two N-terminal WW domains are important for protein–protein interactions that form protein complexes involved in a variety of cellular processes such as transcriptional regulation and protein stability. Analysis of several WWOX interacting candidates revealed that WWOX, via its first WW domain, associates with the proline-rich motif PPxY found in a number of transcription factors including p73, AP2gamma, and Jun. WWOX associates with its targets in the cytoplasm and prevents their translocation into the nucleus, thus regulating their transcriptional activity. The recent observations indicate that loss of WWOX expression in human tumors may contribute to their pathogenesis. Bednarek et al. showed that ectopic WWOX expression strongly inhibits anchorage independent growth of breast cancer cell lines in soft agar. WWOX overexpression induces a dramatic inhibition of tumorigenicity of MDA-MB-435 breast cancer cells in mice. Subsequently, Fabbri et al. utilized a high-titer adenoviral vector that encodes WWOX (Ad-WWOX) to restore WWOX expression in lung cancer cell lines that lack expression of endogenous WWOX. Using this technique, overexpression of WWOX inhibited cell growth in three different WWOX-negative human lung cancer cell lines, but not in a WWOX-positive lung cell line. Similar experiments in pancreatic, prostate and breast cancer cell lines supported these results. WWOX overexpression in these studies resulted in caspase-mediated apoptosis. Thus, multiple lines of evidence, both in cell culture and in athymic mice, suggested that WWOX functions as a tumor suppressor. Aqeilan et al recently generated mice carrying a targeted deletion of the WWOX gene. Using conventional gene targeting methods, a mouse lacking exons 2, 3, and 4 of the mouse WWOX gene was generated. Analysis of the WWOX mutant mice demonstrated that WWOX functions as a tumor suppressor. Four of 13 juvenile WWOX_/_ knockout mice developed osteosarcoma. These tumors developed in young postnatal mice in the absence of any carcinogenic treatment, suggesting a role of WWOX as a tumor suppressor in the bone. Altogether these data indicate that WWOX is a bona fide tumor suppressor and that inactivation of one WWOX allele is sufficient for tumorigenesis. 

Figure 2: The function of the WWOX: WWOX gene is known to regulate transcription as a protein partner of transcription factors such as c-Jun, TNF, p53, p73, AP-2gamma, and E2F-1. WWOX gene can be induced by carcinogens and regulate apoptosis.

Purpose of the specific investigation

In our study, we propose to test the hypothesis that the WWOX gene is an osteosarcoma tumor suppressor gene that is inactivated in human osteosarcoma. We will perform the following experiments in this study. 1. To examine homozygous deletion, loss of heterogeneity (LOH), methylation of WWOX promoter, and mutation of WWOX in osteosarcoma cell lines and patient tumor tissues. 2. To investigate the role of WWOX in cell apoptosis and proliferation by correlating WWOX gene deletion, mutation, and expression status with expression for P73, BAX, Bcl-2, Bcl-xL, casepase-3, casepase-9, Ki-67 and PCNA. We will use standard molecular and pathological methods including PCR analysis, sequencing, Immunoblotting and immunohistochemistry, methylation-specific PCR (MSP), DHPLC and flow cytometric analysis. The tissue bank of our hospital has accumulated 35 frozen osteosarcoma tissues and there are about 150 paraffin-embedded archival blocks available for this investigation. Our preliminary investigations in human osteosarcoma tissues detected WWOX gene deletion and promoter methylation. These studies support our hypothesis. We anticipate that this study will improve our understanding of the molecular mechanisms of osteosarcoma development by determination whether the WWOX gene is key tumor suppressor gene and a new target for gene therapy development for osteosarcoma.

Research and experimental design

We will test our hypothesis through the following research steps:

1.  To determine the status of homozygous deletions, loss of heterozygosity, and mutation of WWOX gene in bone and osteosarcoma cell lines, S180S37, U-2OS, SW 1353, and MG-63.

2.  To determine the methylation status of WWOX promoter region in bone and osteosarcoma cell lines.

3.  To determine gene deletion and mutation of WWOX gene in osteosarcoma tissues.

4.  To determine expression levels of WWOX protein and related proteins such as P73, BAX, Bcl-2, Bcl-xL, casepase-3, casepase-9, Ki-67 and PCNA on a tissue microarray. We will correlate the WWOX status with clinical information of the patients as well as the apoptosis and proliferation markers.

The potential significance of this investigation

Osteosarcoma is among the most common non-hematologic primary malignant tumors of adults and children. Although an increased number of molecular studies have been initiated in recent years, no clear results or new therapeutic treatments have been obtained. This first study of the biological effects of WWOX expression in osteosarcoma cells will determine the role of the reduced WWOX signal in osteosarcoma pathobiology and whether the WWOX gene is an important tumor suppressor gene in osteosarcoma. Our proposed study will explore whether drugs capable of reactivating the epigenetically silenced WWOX gene could be effective in treatment of osteosarcoma. Restoration of WWOX protein expression may lead to induction of apoptosis and suppression of tumorigenicity. The delineation of this signal pathway will not only help to clarify the role of a significant signal mediator in osteosarcoma but also may lead to identification of therapeutic targets for osteosarcoma.

References

1.   Aqeilan RI, Trapasso F, Hussain S, et al. 2007. Targeted deletion of Wwox reveals a tumor suppressor function. Proc Natl Acad Sci USA,104:3949–3954.

2.   Bednarek AK, Laflin KJ, Daniel RL, Liao Q, Hawkins KA, Aldaz CM. (2000). WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3-24.1, a region frequently affected in breast cancer. Cancer Res. 60, 2140-2145.

3.   Fabbri M, Iliopoulos D, Trapasso F, et al. 2005.WWOXgene restoration prevents lung cancer growth in vitro and in vivo. Proc Natl Acad Sci USA, 102:15611–15616.

V5N3 ESUN Copyright © 2008 Liddy Shriver Sarcoma Initiative.