Deletion of the WWOX Gene and Frequent Loss of its |
An ESUN Article
Introduction
The WWOX gene has been proposed as a tumor suppressor gene, and loss of its expression has been found in many cancers. Loss of the WWOX expression has been associated with more aggressive phenotypes and poorer prognosis in breast cancer, non-small cell lung cancer, bladder cancer, gastric cancer, and sporadic meningioma (Płuciennik E, et al., 2006; Donati V, et al., 2007; Ramos D, et al., 2008; Aqeilan RI, et al., 2004; Aarhus M, et al., 2008; Lewandowska U, et al., 2009; Gourley C, et al., 2009). Loss of WWOX expression has also been found in pancreatobiliary cancers, but there is no correlation with prognosis, suggesting that loss of WWOX expression may be an early event in these cancers (Bloomston et al., 2009). Restoration of WWOX expression in breast and lung cancers cells that lack endogenous WWOX expression resulted in significant caspase-mediated apoptosis, growth inhibition, and blocked tumor development in athymic nude mice (Bednarek AK, et al., 2001; Fabbri M, et al., 2005). In ovarian cancer WWOX expression abolishes the tumorigenicity in vivo and decreases attachment to fibronectin via integrin alpha3 (Gourley C, et al., 2009). The WWOX protein has been shown to interact with c-Jun, p53, p73, AP-2, and E2F-1 and may play a central role in tumor suppression through transcriptional regulation of apoptosis pathway (Yang J, et al., 2008).
Supporting evidence for the WWOX gene as a tumor suppressor gene was obtained in a genetically engineered mouse model where targeted deletion of the WWOX gene resulted in development of osteosarcomas in juvenile WWOX _/_ mice and lung papillary carcinomas in adult WWOX _/_ mice (Aqeilan RI, et al., 2007). This finding also implicated the WWOX gene in the development of osteosarcoma, but there is no information regarding the status of the WWOX gene in human osteosarcoma.
Osteosarcoma is the most common primary non-haemopoietic malignant bone tumor with complex karyotypes and a highly unstable genome exhibiting both numerical- and structural-chromosomal instability. In this study, which was supported by the Liddy Shriver Sarcoma Initiative, we investigated the role of the WWOX gene in the pathogenesis of osteosarcoma by examining the gene copy number status of the WWOX gene and WWOX protein expression in primary osteosarcoma tissues. We collected fresh osteosarcoma tissues, isolated genomic DNA from these tissues, and performed whole-genome array Comparative Genomic Hybridization (aCGH) to evaluate the copy number changes of the WWOX gene. We also collected FFPE tissues with clinical information to detect the expression of the WWOX protein by immunohistochemistry. The results from this study are in press in an upcoming issue of the journal Cancer Letters (Yang et al, In press).
Results
To gain insight into the global genetic alterations and specifically the gene copy number status of the WWOX gene in osteosarcoma, we carried out a high-density genome-wide aCGH profiling with genomic DNA isolated from 10 fresh osteosarcoma tissues from 9 patients. The aCGH results were analyzed for recurrent alterations.
When all ten samples were analyzed together for recurrent gene copy number alterations, we observed several major regions of deletions and amplifications (Fig.1A). These regions included 1,162 genes with high level amplifications and 146 genes with deletion (FDR <10%). To determine whether the recurrent gene copy alterations we observed are representative, we downloaded an independent aCGH data (GSE9654) (Fig 1B) from a cohort of ten osteosarcoma patients (Squire AJ, et al., 2003). Strikingly, the overall recurrent gene copy alteration patterns of these two independent populations in two different countries (China and Canada) were very similar, suggesting distinct genetic alterations underlying the pathogenesis of osteosarcoma.
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| The x-axis denotes chromosome numbers. The y-axis denotes logRatio for every aCGH probe (scatter plot) and circular binary segmentation (blue line plot). (A and B) Recurrent gene copy alteration patterns in our data and Squire’s data (Squire JA, et al, 2003). The recurrence of CNAs is shown in our data in (A) and in Squire’s data in (B). The y-axis shows recurrence of gains (positive axis) and losses (negative axis) for each measured sequence aligned evenly in chromosomal order on the x-axis. The dashed line indicates the threshold for a significant number of aberrations. Recurrence rates that exceed this threshold are color-coded to emphasize the locations of significantly recurrent aberrations. Red color denotes significantly recurrent amplification and green denotes significantly recurrent deletion. Gray color represents nonsignificant recurrence of aberrations. The overall recurrent gene copy alteration patterns of these two datasets were very similar, suggesting distinct genetic alterations underlying the pathogenesis of osteosarcoma. [This figure appears in an article by Yang et al. (in press) and is used with permission of Cancer Letters.] |
We next zeroed in on the WWOX gene, which is located on chromosome 16q23.3-q24.1. The WWOX gene was not on the recurrent copy number alteration list from our analysis. We then examined the WWOX gene locus on aCGH data. Deletion was only observed in 3 of 10 samples (Fig.2A). Our analysis of the Squire et al dataset (Squire AJ, et al., 2003) showed that the WWOX gene was deleted in 3 of ten samples and amplified in 1 sample (Fig. 2B). The data suggested that deletion of the WWOX gene occurs in 30% of human osteosarcomas.
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| Scatters denote the copy number change of 14 WWOX probes. The line-plots denote the segmented value of WWOX copy number change. The black lines denote significant amplifications or deletions; grey lines denote nonsignificant amplification or deletion. (A) 3 samples deletions (S6276, S6277, and S6283) of WWOX gene in our data; (B) 3 samples (OS9, OS13, and OS15) deletions and 1 sample amplification (OS11) of WWOX gene in data from Squire (Squire JA, et al., 2003). [This figure appears in an article by Yang et al. (in press) and is used with permission of Cancer Letters.] |
We evaluated whether the expression of WWOX protein is altered in osteosarcoma by immunohistochemistry. For this purpose, we acquired 55 cases of FFPE tissues from patients whom we also obtained clinical information. The normal cutaneous, muscular, and skeletal tissues exhibited strong positive staining in the cytoplasm (Fig. 3A, 3B). The WWOX protein was negative in 61.8% (34/55) cases (Fig. 3I) and positive in 38.2% (21/55) (Fig. 3C-3H). The counts of weak positive (Fig. 3G, 3H) and moderate positive (Fig. 3E, 3F) were 10 (18.2%, 10/55) and 6 (10.9%, 6/55), respectively. Consistently with what had been observed, for the positive cases, the WWOX protein was located predominantly in the cytoplasm. The staining was very strong in 5 cases and in these case both cytoplasm and nucleus were positive (Fig. 3C, 3D). In contrast to what was reported in some cancer types such as breast cancer, the WWOX expression in osteosarcoma had no correlation with the prognosis and other clinical information including age, tumor site, pathological type, PTNM staging, tumor recurrence, metastasis, disease free survival, or the overall survival. This pattern is similar to results in pancreatobiliary cancers where the loss or reduced WWOX expression did not predict tumor progression or patient survival (Bloomston M, et al., 2009).
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| The WWOX protein expression in normal and osteosarcoma tissues. (A and B): The positive expression in cutaneous and muscular tissues. (C and D): Strong expression in 5 cases. Original magnification for (C) is 20× and for (D) is 40×. (E and F): Moderate expression in osteosarcoma tissues. Original magnification for (E) is 20× and for (F) is 40×. (G and H): Weak expression in osteosarcoma tissues. Original magnification for (G) is 20× and for (H) is 40×. (I): Negative for WWOX expression in 61.8% osteosarcoma. [This figure appears in an article by Yang et al. (in press) and is used with permission of Cancer Letters.] |
Our results showed that the WWOX protein is lost in more than half of the osteosarcoma tissues and in accordance with the gene deletion. Thus, the WWOX gene deletion and loss of expression appear to be the mechanism for loss of function of this tumor suppressor gene. All these results suggest that loss of WWOX through deletion and loss of expression is likely involved in the early stage of pathogenesis of human osteosarcoma.
Conclusion and Future Directions
As far as we can tell, this is the first investigation regarding the role of the WWOX gene in the pathogenesis of human osteosarcoma. We are reporting this new finding in an upcoming issue of Cancer Letters (Yang et al., In press). Because the loss of WWOX expression is common but the gene deletion is not so frequent, epigenetic changes of the WWOX gene such as promoter methylation might also be involved in the pathogenesis of osteosarcoma. Future investigation into the epigenetic regulation of the WWOX gene will shed more light into the early event leading to the loss of the WWOX tumor suppressor gene and provide new therapeutic opportunities for osteosarcomas with the emerging drugs that reverse the cancer associated epigenetic alteration.
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