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 1 and 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, <10% of all tumors, these sarcomas represent a considerable burden because they are one of the most frequent pediatric bone tumors and remain prominent among both teenagers and young adults.

Although new therapies that combine high-dose chemotherapy with local control of the tumor have led to a significant benefit in terms of patient survival (1, 2), there is a need for molecular prognostic markers that would permit, at the time of diagnosis, identification of poorly responsive tumors that may benefit from modified treatment regimen.

To date, aside from metastasis at presentation, the other most accepted prognostic factors are based on the response to treatment (percentage of necrosis or P-glycoprotein expression). However, P-glycoprotein overexpression still suffers from an undisputed recognition as a prognostic factor (24) and of lack of suitable drugs that are available for clinical practice, impairing its action. Histologic response to chemotherapy (i.e., the percentage of necrosis following adjuvant chemotherapy) is the most dependable and reproducible prognostic indicator of the probability of event-free survival (1, 2). This indicator can only be assessed after therapy has already been given, whereas optimal prognostic markers should distinguish patients before therapy. Especially for patients who display poor histologic response to chemotherapy, it is crucial to determine the biological factors that are responsible for the lack of response at the time of diagnosis to identify potential target candidates to customize therapy. Indeed, there is no widely accepted treatment for patients with tumors unresponsive to standard therapy. Therefore, identification of crucial molecular prognostic markers has a double value in osteosarcoma patients and will lead to a better understanding of the pathways that need to be targeted for future drug development.

Impairment of osteoblastogenesis is a constant biological feature of osteosarcoma and seems to severely influence its clinical outcome. In addition, recent publications have highlighted the importance of the tumor-microenvironment interactions in osteosarcoma and a specific expression signature involving genes integral to bone remodeling was reported to clearly distinguish between chemoresistance and chemoresponsive osteosarcoma patients (5). In this study, we assessed the prognostic value of CCN1, CCN2, and CCN3 genes, essential matricellular signaling modulators that play crucial roles in bone formation.

The CCN family of proteins (6) contains six members that possess specific, nonredundant functions (79) in spite of their high degree of structural identity (see ref. 10 for a detailed review). Insights into specific physiologic roles of CCN1 and CCN2 come from mutant mice lacking one of the two genes (11), which exhibited impaired chondrocyte proliferation and differentiation and reduced endochondral ossification (12). CCN proteins are expressed in chondrocytes and osteoblasts and are induced during fracture repair, further indicating that CCNs are key participants in connective tissue regeneration. In cellular models, CCN1 and CCN2 were shown to promote proliferation of fibroblasts, chondroblasts, osteoblasts, and vascular endothelial cells. However, the same proteins also promoted differentiation of these cells (see ref. 13 for a detailed review). These apparently opposite effects reflect the ability of CCN proteins to act as biological modulators. Consistent with complex role/s of adaptor molecules, CCN proteins have also been reported to be related either positively or negatively to development of cancer. Because CCN proteins are involved in the regulation of crucial distinct biological processes, such as cellular proliferation, differentiation, adhesion, migration, chemotaxis, and angiogenesis (7), their dysregulation likely contributes to tumorigenesis and cancer progression. Indeed, alterations in CCN1, CCN2, and CCN3 were associated with tumor development and progression in several tumors, including glioma, lung cancer, renal cell carcinoma and prostate carcinoma, endometrial carcinoma, uterine leiomioma, and breast carcinoma (7, 8, 1422).

In sarcomas, dual roles of CCN proteins have been described. Altered expression of CCN2 and CCN3 was associated to tumor development and progression in chondrosarcoma (17), whereas CCN3 was found highly expressed in rhabdomyosarcoma and associated with myoblastic differentiation. In contrast, most of the Ewing's sarcoma samples were negative for CCN3 and expression of CCN3 in these tumors was associated with higher risk of developing metastasis (23). With respect to osteosarcoma, little is known apart from a previous study showing variable expression of CCN3 in 60% of cell lines (23).

In this study, we analyzed the temporal expression of CCN1, CCN2, and CCN3 during terminal osteoblastic differentiation that was induced in human mesenchymal stem cells and in osteoblast-like cell lines and therefore providing for the first time insights into the expression of these genes during the process of membranous ossification. In addition, we explored the relationship/s between the expression of the three CCN and osteoblastic differentiation genes as well as clinical outcome in a series of osteosarcoma patients.

Cell lines. A panel of 13 human osteosarcoma cell lines was analyzed. The osteosarcoma cell lines Saos-2, U-2 OS, and MG63 were obtained from the American Type Culture Collection. All of the other cell lines (SARG, IOR/MOS, IOR/OS7, IOR/OS9, IOR/OS10, IOR/OS14, IOR/OS15, IOR/OS17, IOR/OS18, and IOR/OS20) were established at the Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli (Bologna, Italy) and previously characterized (24). Drug-resistant cell lines were obtained as previously described (25). Human osteoblasts were obtained at the Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment, University of Turin, Candiolo (Turin, Italy) and previously characterized (26). All cell lines were routinely cultured in Iscove's modified Dulbecco's medium supplemented with 20 units/mL penicillin, 100 μg/mL streptomycin (Sigma), and 10% heat-inactivated fetal bovine serum (Biowhittaker Europe) and maintained at 37°C in a humidified 5% CO2 atmosphere. Human primary bone marrow–derived mesenchymal stem cells were obtained after informed consent from bone marrow aspirates (iliac crest) of two patients undergoing hip replacement surgery. Nucleated cells were plated in α-MEM modification (Li StarFish) containing 20% fetal bovine serum (Cambrex Bioscience), 100 units/mL penicillin (Life Technologies), 100 mg/mL streptomycin (Life Technologies), and 2 mmol/L glutamax (Life Technologies). Confluent cells were harvested by trypsin/EDTA and seeded at 1:3 density. Cells were phenotyped as bone marrow–derived mesenchymal stem cells by flow cytometry (CD45 and CD34 negative and CD166, CD105, CD44, and CD90 positive) and induced to differentiate as specified in osteoblastic differentiation section.

Clinical series. The study included 45 newly diagnosed osteosarcoma patients who were referred to the Rizzoli Institute and entered the neoadjuvant chemotherapy protocols that are based on the administration of doxorubicin, high-dose methotrexate, cisplatin, and ifosfamide (1). All patients underwent surgery and surgical margins were classified according to the Enneking score system (27). Based on the evaluation of tumor necrosis after preoperative chemotherapy, a good histologic response was considered when the extent of tumor necrosis was ≥90%. Postoperative chemotherapy used the same drugs of the preoperative phase, with the addition of ifosfamide for patients with a poor histologic response. After surgery, specimens were evaluated to define the surgical margins. Radical or wide surgical margins were considered as adequate, whereas marginal or intralesional margins were classified as inadequate. Patients were followed and clinical data were updated for at least 5 years. Adverse events were defined as recurrence of the tumor at any site or death. Event-free survival was calculated from the date of initial diagnosis. Clinicopathologic features of the present series of osteosarcoma patients are shown in Supplementary Table S1.

RNA extraction and cDNA synthesis. H&E staining method was used to select representative osteosarcoma specimens. Total RNA from frozen osteosarcoma clinical samples was extracted using the Trizol extraction kit (Invitrogen Ltd.) and the quality of the RNA samples was determined by electrophoresis through 1% agarose gel. Total RNA (500 ng) for each sample was reverse transcribed to cDNA in a 50 μL reaction mixture using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

Taqman low-density arrays and real-time reverse transcription-PCR analysis. For clinical samples, Taqman low-density arrays were used. Predesigned Taqman probe and primer sets for target genes were chosen from an online catalogue (Applied Biosystems). Array format was customized online with three replicates per target gene. Expression levels of target genes were normalized to that of GAPDH and/or 18S genes. Samples were analyzed using the 7900HT system with a Taqman LDA Upgrade (Applied Biosystems) according to the manufacturer's instructions. The quantitative detection of CCN genes in osteosarcoma cell lines was done using specific Taqman gene expression assay from Applied Biosystems. For the relative quantification of genes specific for osteoblastic differentiation, specific SYBR Green primers were designed by using Primer Express software (Applied Biosystems). All PCRs were done by using ABI PRISM 7900 Sequence Detection System (Applied Biosystems) as recommended by the supplier. Gene expression values were calculated based on the ΔΔCt method, where a pooled cDNA from three human osteoblast primary cultures or from four normal muscle tissues was used as calibrator.

Osteoblastic differentiation in human mesenchymal stem cells and osteosarcoma cell lines. Cells were seeded in 10% fetal bovine serum-Iscove's modified Dulbecco's medium at a density of 3.5 × 105 per plate in 60-mm plates. After 4 days, cells were incubated in differentiating medium (Iscove's modified Dulbecco's medium containing 2% fetal bovine serum supplemented with 5 mmol/L β-glycerophosphate and 50 μg/mL ascorbic acid) and maintained in differentiative conditions for up to 18 days. Medium was renewed every 4 days. Cultures were harvested at various time points to collect RNAs. Duplicate plates were stained with 2% Alizarin Red S to visualize bone mineralization. Bromodeoxyuridine incorporation (10 μmol/L, 1 h, 37°C) was used to highlight cell proliferation.

Statistics. Two-tailed Fisher's exact test was used to evaluate the statistical association between two variables. Kaplan-Meier and log-rank methods were used, respectively, to draw and evaluate the significance of survival curves. Correlations were established according to Spearman test.

CCN1, CCN2, and CCN3 expression during osteoblast differentiation. Osteosarcomas are thought to originate from mesenchymal cells having osteoblastic features. However, the great majority of osteosarcomas are poorly differentiated and loss of differentiation has prognostic significance, with well-differentiated tumors being classified as low grade. To evaluate temporal expression of CCN1, CCN2, and CCN3 genes in a context that can be considered to represent normal physiologic conditions, we analyzed CCN1, CCN2, and CCN3 expression profile in human mesenchymal stem cells that were induced to differentiate into osteocytes (Fig. 1A). Terminal differentiation was confirmed by the expression of specific markers and Alizarin Red S staining (Fig. 1A). We observed a decrease in CCN1 and CCN2 expression during the mesenchymal stem cell commitment toward osteoblastic lineage and terminal differentiation in osteocytes, whereas expression of CCN3 remained undetectable in mesenchymal stem cell during differentiation. Analysis of CCN1, CCN2, and CCN3 expression during induction of differentiation was also done in Saos-2 and OS7 osteoblastic-like cells (Fig. 1B and C). In both cell lines, the expression of CCN1 and CCN2 decreased at the beginning of differentiation, whereas CCN3 increased remarkably and progressively during the osteoblastic differentiation, indicating that the expression of the three genes is differentially, although coordinately, regulated during the progression toward postmitotic mature osteoblasts or osteocytes. These data suggest that, in a normal context, the antiproliferative activity of CCN3 is not necessary for osteoblastic differentiation to proceed, whereas in the pathologic context of osteosarcoma cells, where regulatory pathways for cell proliferation are disrupted, CCN3 might be required to induce the growth arrest that occurs during terminal osteoblastic differentiation.

Fig. 1.

Expression of CCN genes during osteoblastic differentiation. A, human mesenchymal stem cells were cultured in osteogenic differentiation medium and terminal differentiation was monitored by temporal modulation in expression of two markers of osteoblastic differentiation and the appearance of mineralized bone nodules. Relative mRNA expression of CCN1, CCN2, CCN3, collagen I, and osteocalcin was normalized to a calibrator (undifferentiated cells at time 0, mRNA fold change = 1). Collagen I and osteocalcin were selected as examples of early and late, respectively, specific markers of osteoblastic differentiation. Bottom, Alizarin Red S staining visualized the formation of mineralized bone matrix during osteoblastic differentiation. B, osteoblastic differentiation of osteosarcoma Saos-2 and OS7 cells cultured in osteogenic differentiation medium. Terminal differentiation is indicated by markers of osteoblastic differentiation and by mineralized bone nodules. Bromodeoxyuridine incorporation (top) was used to highlight cell proliferation. Anti-bromodeoxyuridine staining showed how, starting from 14 d of differentiation, the cells appeared in a stationary state. Bottom, Alizarin Red S staining visualized the formation of mineralized bone matrix. C, temporal expression of CCN1, CCN2, and CCN3 mRNA along the terminal osteoblastic differentiation in osteosarcoma cells.

Fig. 1.

Expression of CCN genes during osteoblastic differentiation. A, human mesenchymal stem cells were cultured in osteogenic differentiation medium and terminal differentiation was monitored by temporal modulation in expression of two markers of osteoblastic differentiation and the appearance of mineralized bone nodules. Relative mRNA expression of CCN1, CCN2, CCN3, collagen I, and osteocalcin was normalized to a calibrator (undifferentiated cells at time 0, mRNA fold change = 1). Collagen I and osteocalcin were selected as examples of early and late, respectively, specific markers of osteoblastic differentiation. Bottom, Alizarin Red S staining visualized the formation of mineralized bone matrix during osteoblastic differentiation. B, osteoblastic differentiation of osteosarcoma Saos-2 and OS7 cells cultured in osteogenic differentiation medium. Terminal differentiation is indicated by markers of osteoblastic differentiation and by mineralized bone nodules. Bromodeoxyuridine incorporation (top) was used to highlight cell proliferation. Anti-bromodeoxyuridine staining showed how, starting from 14 d of differentiation, the cells appeared in a stationary state. Bottom, Alizarin Red S staining visualized the formation of mineralized bone matrix. C, temporal expression of CCN1, CCN2, and CCN3 mRNA along the terminal osteoblastic differentiation in osteosarcoma cells.

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Differential expression of CCN1, CCN2, and CCN3 genes in osteosarcoma. Coordinated and regulated expression of the three CCN genes was also observed in osteosarcoma. Quantitative PCR done on a panel of 13 osteosarcoma cell lines and 45 clinical samples indicated variable levels of expression for all the three genes (Fig. 2A and B). Their expression was correlated both in osteosarcoma cell lines and clinical samples, although the correlation among CCN1 and CCN2 was stronger than the correlation between CCN3 and CCN1 or CCN2 (CCN1 versus CCN2, Spearman test: r = 0.88, P < 0.001; CCN3 versus CCN1 or CCN2, Spearman test: r = 0.53, r = 0.59, P < 0.001, respectively).

Fig. 2.

Expression of CCN1, CCN2, and CCN3 in a panel of 13 osteosarcoma cell lines and 45 osteosarcoma biopsies. To compare gene levels in normal and pathologic conditions, a pooled cDNA derived from three human osteoblast primary cultures was used as calibrator (mRNA fold change = 1). Although variable levels of expression are observed for each gene, CCN3 expression seems generally high in the majority of osteosarcomas, both in cell lines (A) and clinical samples (B), in contrast with what was observed during osteoblast differentiation.

Fig. 2.

Expression of CCN1, CCN2, and CCN3 in a panel of 13 osteosarcoma cell lines and 45 osteosarcoma biopsies. To compare gene levels in normal and pathologic conditions, a pooled cDNA derived from three human osteoblast primary cultures was used as calibrator (mRNA fold change = 1). Although variable levels of expression are observed for each gene, CCN3 expression seems generally high in the majority of osteosarcomas, both in cell lines (A) and clinical samples (B), in contrast with what was observed during osteoblast differentiation.

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Quantitative PCR analysis was used to draw relationships between CCN genes and a set of representative genes involved in osteoblastic differentiation, whose expression levels are shown in Supplementary Fig. S1 as referred to normal osteoblasts. Notably, similar results were obtained when a different calibrator (normal muscle) was used (data not shown). Expression of both CCN1 and CCN2 was found to be associated with the expression of 10 of 15 genes considered (Table 1). These correlations are drawn on a case by case basis. Of note, most of the significant correlations for both CCN1 and CCN2 were found with genes involved in the commitment of mesenchymal stem cells toward osteoblasts and in the early phases of osteoblastic differentiation (RUNX family genes; cadherin 4, 11, and 13; jun and fos; collagen I and SPARC) when differentiating osteoblasts are still characterized by intense proliferation. In contrast, CCN3 expression was not significantly correlated with these genes, apart from RUNX1 and cadherin 13, which are not specifically involved in osteoblast differentiation (Supplementary Fig. S2). Therefore, whereas a relationship can be drawn between CCN1 and CCN2 expression and osteoblast differentiation in osteosarcoma, CCN3 expression seems to be substantially high, with great variations among cases or cell lines and without any relationships with osteoblastic differentiation genes.

Table 1.

Statistical correlations between CCN genes and genes of osteoblastic differentiation in 45 osteosarcoma clinical samples (Spearman test; significant correlations were highlighted and showed a correlation rate ≥0.5 and P < 0.05)

 
 

Relationship between CCN1, CCN2, and CCN3 genes and osteosarcoma clinical features and outcome. Based on CCN1, CCN2, and CCN3 expression levels measured by quantitative PCR (Fig. 2B), patients were stratified according to median values into CCN high-expressing or CCN low-expressing groups. High expression of CCN genes at diagnosis was detected in 23 of 45 cases for CCN1 (51%), 24 of 45 cases for CCN2 (53%), and 23 of 45 cases for CCN3 (51%). Two-tailed Fisher's exact test further confirmed the correlation of CCN1 and CCN2, but not of CCN3, with genes involved in the early steps of osteogenic differentiation (data not shown).

About relationships with clinicopathologic variables, expression of CCN1 and CCN2 was significantly associated with age, osteosarcomas from adult patients being a group in which levels of CCN1 and CCN2 were significantly higher (Table 2). However, because CCN1 and CCN2 are both associated with skeletal growth (13), such difference may be observed even in normal calcifying tissues and may not have pathobiological significance. A significantly higher incidence of CCN1 high-expressing cases was also found in male patients, whereas CCN3 did not correlate with any clinicopathologic features. To assess the correlations with clinical outcome, both event-free survival and overall survival were analyzed. The majority of adverse events (21 of 24) occurred within the first 3 years of follow-up and were related to development of lung metastases (21 patients) or local recurrences (3 patients). Neither clinicopathologic features or differentiation genes (data not shown) nor CCN1 and CCN2 expression (Fig. 3A) showed statistically significant association versus survival. On the contrary, high expression of CCN3 significantly correlated with worse prognosis (P < 0.05; Fig. 3A).

Table 2.

Correlation between CCN protein expression level and clinicopathologic features in the 45 high-grade osteosarcoma patients included in this study

VariableCCN1 high expression
CCN2 high expression
CCN3 high expression
Cases/total (%)P*Cases/total (%)P*Cases/total (%)P
Gender  0.02     
    Male 18/27 (67%)  17/27 (63%)  16/27 (59%)  
    Female 5/18 (28%)  7/18 (39%)  7/18 (39%)  
Age (y)  0.02  0.01   
    ≤12 2/11 (18%)  2/11 (18%)  5/11 (45%)  
    >12 21/34 (62%)  22/34 (64%)  18/34 (53%)  
Site       
    Femur 13/25 (52%)  12/25 (48%)  16/25 (64%)  
    Tibia 2/6 (33%)  2/6 (33%)  1/6 (17%)  
    Humerus 5/8 (63%)  6/8 (75%)  4/8 (50%)  
    Other 3/6 (50%)  4/6 (67%)  2/6 (33%)  
Histologic subtype       
    Osteoblastic 8/19 (42%)  8/19 (42%)  8/19 (42%)  
    Chondroblastic 3/6 (50%)  3/6 (50%)  4/6 (67%)  
    Fibroblastic 8/11 (73%)  8/11 (73%)  8/11 (73%)  
    Telangiectatic 1/2 (50%)  1/2 (50%)  1/2 (50%)  
    Not specified 3/7 (43%)  4/7 (57%)  2/7 (29%)  
Surgery       
    Resection 14/32 (44%)  15/32 (47%)  17/32 (53%)  
    Amputation 9/13 (69%)  9/13 (69%)  6/13 (46%)  
Surgical margins       
    Adequate 23/43 (53%)  23/43 (53%)  23/43 (53%)  
    Inadequate 0/2  1/2 (50%)  0/2  
Histologic response       
    Good (necrosis ≥90%) 13/24 (54%)  13/24 (54%)  12/24 (50%)  
    Poor (necrosis <90%) 10/21 (48%)  11/21 (52%)  11/21 (52%)  
VariableCCN1 high expression
CCN2 high expression
CCN3 high expression
Cases/total (%)P*Cases/total (%)P*Cases/total (%)P
Gender  0.02     
    Male 18/27 (67%)  17/27 (63%)  16/27 (59%)  
    Female 5/18 (28%)  7/18 (39%)  7/18 (39%)  
Age (y)  0.02  0.01   
    ≤12 2/11 (18%)  2/11 (18%)  5/11 (45%)  
    >12 21/34 (62%)  22/34 (64%)  18/34 (53%)  
Site       
    Femur 13/25 (52%)  12/25 (48%)  16/25 (64%)  
    Tibia 2/6 (33%)  2/6 (33%)  1/6 (17%)  
    Humerus 5/8 (63%)  6/8 (75%)  4/8 (50%)  
    Other 3/6 (50%)  4/6 (67%)  2/6 (33%)  
Histologic subtype       
    Osteoblastic 8/19 (42%)  8/19 (42%)  8/19 (42%)  
    Chondroblastic 3/6 (50%)  3/6 (50%)  4/6 (67%)  
    Fibroblastic 8/11 (73%)  8/11 (73%)  8/11 (73%)  
    Telangiectatic 1/2 (50%)  1/2 (50%)  1/2 (50%)  
    Not specified 3/7 (43%)  4/7 (57%)  2/7 (29%)  
Surgery       
    Resection 14/32 (44%)  15/32 (47%)  17/32 (53%)  
    Amputation 9/13 (69%)  9/13 (69%)  6/13 (46%)  
Surgical margins       
    Adequate 23/43 (53%)  23/43 (53%)  23/43 (53%)  
    Inadequate 0/2  1/2 (50%)  0/2  
Histologic response       
    Good (necrosis ≥90%) 13/24 (54%)  13/24 (54%)  12/24 (50%)  
    Poor (necrosis <90%) 10/21 (48%)  11/21 (52%)  11/21 (52%)  
*

Only significant P values by two-tailed Fisher's exact test have been reported.

Fig. 3.

Event-free and overall survival curves in 45 patients with high-grade osteosarcoma according to expression level of CCN1, CCN2, or CCN3 genes (A) or to the ABC family members MDR1, MRP1, and MRP4 (B). Comparison of survival curves was done by the log-rank test. Time scale refers to months from diagnosis. Significant P values are in bold. C, quantitative evaluation of CCN1, CCN2, and CCN3 expression by real-time PCR in U-2 OS variants resistant to methotrexate (MTX), doxorubicin (DX), and cisplatin (CDDP).

Fig. 3.

Event-free and overall survival curves in 45 patients with high-grade osteosarcoma according to expression level of CCN1, CCN2, or CCN3 genes (A) or to the ABC family members MDR1, MRP1, and MRP4 (B). Comparison of survival curves was done by the log-rank test. Time scale refers to months from diagnosis. Significant P values are in bold. C, quantitative evaluation of CCN1, CCN2, and CCN3 expression by real-time PCR in U-2 OS variants resistant to methotrexate (MTX), doxorubicin (DX), and cisplatin (CDDP).

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Association of CCN3 expression and outcome was observed when event-free survival, but not overall survival, was taken into account. Considering that the final patient outcome depends on both tumor intrinsic malignancy and response to treatments, which very likely determine overall and event-free survival, respectively, we studied the possible association between CCN3 expression and response to chemotherapy. In particular, because one of the major superfamilies of membrane transporter proteins that influence pharmacokinetics of drugs is the ATP-binding cassette (ABC) transporters, we analyzed expression of ABCB1 [multidrug resistance 1 (MDR1) or P-glycoprotein], ABCC-1 [MDR-related protein 1 (MRP1)], and ABCC-4 [MDR-related protein 4 (MRP4)] in the same series of patients by means of quantitative PCR analysis. We chose these transporters because they are involved in regulation of the bioavailability and therapeutic efficacy of the most effective drugs used in the treatment of osteosarcoma (doxorubicin, methotrexate, and cisplatin). A positive correlation was observed between high levels of CCN3 and an increased expression of MRP1 and MRP4 but not with MDR1 (Supplementary Table S2). Accordingly, whereas MDR1 mRNA expression did not show statistical associations with outcome, a worse prognosis was found to be associated with high expression of MRP1 and MRP4 (Fig. 3B). However, similar associations were also found between CCN1 or CCN2 and MRP1 or MRP4 expression (Supplementary Table S2).

To better characterize the potential relationship that might exist between CCN genes and chemoresistance-associated genes, we analyzed their expression in osteosarcoma cells (U-2 OS variants) resistant to methotrexate, doxorubicin, or cisplatin (25). A general increase in CCN3 expression was observed in these cell lines. CCN1 and CCN2 expression seemed to be associated with resistance to methotrexate but not to doxorubicin or cisplatin (Fig. 3C). Although these results suggest that expression of CCN genes in osteosarcoma may indeed be associated with drug resistance and/or increased metabolism, they provide only a partial support to the prognostic value of CCN3.

The data reported in this article establish that CCN1, CCN2, and CCN3 proteins are differentially expressed in osteosarcoma and that there is a statistically significant association of high levels of CCN3 with worse prognosis in primary osteosarcoma.

Although the precise origin of osteosarcoma is unknown, it is thought that osteosarcoma arises from primitive mesenchymal bone-forming cells and results from alterations in their differentiation program. The large majority of conventional, high-grade osteosarcomas are either poorly differentiated or undifferentiated and the late marker of osteogenic differentiation osteocalcin is undetectable by immunohistochemistry in >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 (25, 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 cellular processes, such as proliferation, adhesion, migration, and survival (7).

A differential temporal expression pattern was observed for CCN1, CCN2, and CCN3 on induction of osteoblastic differentiation in two osteosarcoma cell lines, which are able to undergo terminal osteocyte differentiation when exposed to a cocktail of differentiating agents. Progressive repression of CCN1 and CCN2 and remarkable increase of CCN3 expression are in line with previous data that showed opposite and complementary functions of CCN proteins in regulation of proliferation (7, 31). CCN1 and CCN2 proteins promote proliferation of various cell types, whereas CCN3 exhibits antiproliferative activity. In mesenchymal stem cells, CCN1 and CCN2 expression was reported to decrease during differentiation into osteoblasts, adipocytes, and chondrocytes (32). We observed reduced expression of CCN1, but not CCN2, during osteoblast differentiation, suggesting a specific role of CCN1 in maintenance of the mesenchymal stem cell phenotype. Similarly, gene expression patterns during osteoblast differentiation in cultures from calvaria have shown that the three CCN 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 up-regulation seemed to be specific for osteosarcoma cells (>20-fold 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 ∼0.50), raising the possibility that regulation and function of CCN3 are independent of CCN1 and CCN2. In line with the characterized antiproliferative actions of CCN3 (3033), 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 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 event-free 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.

Grant support: European Commission grant PROTHETS LSHC-CT-2004-50303306 and Italian Association for Cancer Research (K. Scotlandi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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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Supplementary data