Imaging, Diagnosis, Prognosis
Prognostic Value of CCN3 in Osteosarcoma Bernard Perbal,1,3 Monia Zuntini,1 Diana Zambelli,1 Massimo Serra,1 Marika Sciandra,1 Lara Cantiani,1 Enrico Lucarelli,2 Piero Picci,1 and Katia Scotlandi1
Abstract
Purpose: Osteosarcoma, the most common bone tumor, lacks prognostic markers that could distinguish patients before therapy and drive treatment choices.We assessed the prognostic value of CCN1, CCN2, and CCN3 genes, involved in fundamental biological processes. Experimental Design: Expression of CCN1, CCN2, and CCN3 was measured by quantitative PCR in 45 newly diagnosed osteosarcomas. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations with osteoblastic differentiation and/or drug response genes were assessed in tumor cells using Spearman correlation and Fisher’s exact tests. Results: CCN1 and CCN2 expression was associated with genes involved in commitment of mesenchymal stem cells toward osteoblasts and in early phases of osteoblastic differentiation (RUNX family genes; cadherin 4, 11, and 13; jun and fos; collagen I and SPARC). Although CCN3 is barely expressed in normal proliferating osteoblasts and mesenchymal stem cells, its expression was generally high in osteosarcoma and its level of expression did not correlate with any specific osteoblastic differentiation genes. High expression of CCN3 significantly correlated with worse prognosis in osteosarcoma. This may be only partly explained by the association with the expression of multidrug resistance ^ related protein 1and 4, two ATP-binding cassette transporters that also acted as predictors of worse outcome in our study. Conclusions: Our study showed temporal and coordinated expression of CCN1, CCN2, and CCN3 genes during osteoblastic differentiation and highlighted significant differences between human normal and osteosarcoma cell differentiation in vitro. CCN1 and CCN2 expression shows no prognostic relevance in osteosarcoma. In contrast, assessment for CCN3 expression levels at diagnosis may represent a useful molecular tool to early identification of patients with different prognosis.
Osteosarcomas constitute a heterogeneous group of malignant neoplasms, the great majority of which are represented by high-grade tumors with advanced phenotypes at the time of diagnosis. In spite of their relatively low incidence, 12 Site Femur Tibia Humerus Other Histologic subtype Osteoblastic Chondroblastic Fibroblastic Telangiectatic Not specified Surgery Resection Amputation Surgical margins Adequate Inadequate Histologic response Good (necrosis z90%) Poor (necrosis 75% of these neoplasms (28). By using quantitative PCR that allows relative quantification of osteocalcin, which is more precise and sensitive than immunohistochemistry, we reached the same conclusion. In our series of patients, 80% of osteosarcomas did not achieve terminal osteoblastic differentiation, as indicated by undetectable expression of osteocalcin. In vitro data support the view that osteoblastic differentiation is antagonistic to oncogenic processes and, accordingly, the level of differentiation has powerful prognostic relevance in osteosarcoma, with well-differentiated tumors being classified as low grade with better prognosis (28). In fact, the level of osteoblastic differentiation, usually evaluated by osteoid detection in H&E-stained slides, is one of the few biological prognostic markers that is widely recognized in osteosarcoma, the others being more related to treatment response (2 – 5, 29). In this context, the CCN genes are of particular interest because they encode proteins that have been implicated in the regulation of osteoblast and chondrocyte differentiation during intramembranous bone formation and endochondral ossification (13, 30). In addition, CCN genes play important roles in
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Imaging, Diagnosis, Prognosis
contrast, CCN3 expression levels may be of help in discriminating patients with different probability of relapse. Indeed, osteosarcoma patients with high expression of CCN3 have a worse outcome, an observation that agrees with the capacity of CCN3 to maintain an undifferentiated status in osteoblastic cells (35). The prognostic relevance of CCN3 applied to eventfree survival but not overall survival. We considered the possibility that the correlation with event-free survival might be due to an increased resistance to chemotherapeutic agents. Although no direct relationship could be drawn in this study between CCN3 expression and chemoresistance, we observed that high levels of CCN3 paralleled increased expression of MRP in osteosarcoma cells with significant correlations between CCN3 expression and members of the ABC family of membrane transporters. CCN3 high expression was significantly associated with MRP1 and MRP4, whose high expression was also a predictor of poor outcome. However, our data do not address whether there exists any functional relationship between CCN3 expression and levels of MRPs. Furthermore, inasmuch as ABC family of transporter expression correlated with CCN1 and CCN2, it cannot entirely account for the prognostic value of the CCN3 gene. Our data show that high levels of CCN3 expression at the time of diagnosis are associated with shorter times of event-free survival following treatment of osteosarcoma patients. This finding may seem paradoxical in light of the well-characterized antiproliferative activity of CCN3 because tumor expansion and metastasis are coupled to cellular proliferation. However, high CCN3 expression may reduce tumor cell proliferation sufficiently to provide partial protection against chemotherapeutic agents, which preferentially target the most rapidly proliferating cells. We speculate that the correlation between high CCN3 expression and lower survival may be explained by the ability of CCN3 to confer partial resistance to cytotoxic drug therapy, along with its capacity to antagonize osteoblast differentiation. Although the functional significance of elevated CCN3 expression in osteosarcoma remains to be determined, our data indicate that CCN3 levels can be a useful early indicator of outcome to conventional chemotherapeutic treatment. Our study shows that quantitative mRNA evaluation of the CCN1 and CCN2 as well as of osteoblastic differentiation markers with which they are significantly associated has no prognostic relevance in human osteosarcoma. In contrast, assessment of CCN3 expression levels in primary osteosarcomas at diagnosis may represent a useful molecular tool to early identify subgroups of patients with different prognosis.
genes were differentially expressed, CCN1 and CCN2 expression being strongly repressed (33). Importantly, in contrast to CCN1, we were unable to detect CCN3 expression in human mesenchymal stem cells during any phase of their differentiation toward terminal osteoblasts. Mouse osteoblasts derived from calvaria showed only a slight increase in CCN3 expression during their progression to postmitotic osteocytes (33). Therefore, down-regulation of CCN1 and CCN2 seems to be a key element in controlling the balance between proliferation and differentiation in osteoblasts. In contrast, CCN3 upregulation seemed to be specific for osteosarcoma cells (>20fold increase at mRNA level). These observations in cultured cells correlate with our findings in osteosarcoma samples. Despite variable levels of expression for CCN1, CCN2, and CCN3 in osteosarcoma cell lines and tissue samples, the relative expression of CCN1 and CCN2 in osteosarcoma is similar to that in normal reference cells and tissues (normal osteoblasts and muscle tissues), whereas expression of CCN3 is much higher, indicating aberrant expression of CCN3 in this tumor. In addition, whereas CCN1 and CCN2 seem to be highly correlated in each sample (correlation coefficient >0.85 in clinical samples), CCN3 expression is less correlated with CCN1 or CCN2 (correlation coefficient f0.50), raising the possibility that regulation and function of CCN3 are independent of CCN1 and CCN2. In line with the characterized antiproliferative actions of CCN3 (30 – 33), we speculate that CCN3 overexpression in osteosarcoma may reflect the compensatory activation of an antiproliferative pathway, which attempts to allow terminal osteoblastic differentiation and the requirement of loss of proliferation during differentiation (34). In contrast, up-regulation of CCN3 may not be essential in normal osteoblasts, whose regulatory pathways are not altered by oncogenic transformation. Consistent with our observations of undetectable expression of CCN3 during mesenchymal stem cell differentiation to osteocytes is the recent report that CCN3 acts as a negative regulator of osteoblastic differentiation (35). Forced expression of CCN3 in normal osteoblasts resulted in Notch pathway-driven inhibition of RUNX2, with subsequent alteration of osteoblast differentiation (35). High expression of CCN3 in osteosarcoma might therefore activate Notch signaling pathway, thereby contributing to maintaining cells in an early stage of osteoblastic differentiation. Osteosarcoma cells in fact show proved impairment in osteoblastic differentiation (36) and are generally unable to express markers of terminal osteoblastic differentiation. We confirmed these findings in clinical samples, showing that the great majority of osteosarcomas express only markers of early osteoblastic differentiation. Therefore, differentiation markers studied here had no obvious significant correlation with better prognosis. Accordingly, CCN1 and CCN2, whose expression is associated with expression of early differentiation genes, show no statistically significant prognostic effect. In
Acknowledgments We thank Gary Fisher (Department of Dermatology, University of Michigan, Ann Arbor, MI) for dedicating us his time to critically revise the manuscript.
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Prognostic Value of CCN3 in Osteosarcoma Bernard Perbal, Monia Zuntini, Diana Zambelli, et al. Clin Cancer Res 2008;14:701-709.
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