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Expression of Smad and its signalling cascade in osteosarcoma
Pathology (April 2010) 42(3), pp. 242–247
ANATOMICAL PATHOLOGY
Expression of Smad and its signalling cascade in osteosarcoma KYU YEOUN WON, YOUN WHA KIM
AND
YONG-KOO PARK
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 05/12/15 For personal use only.
Department of Pathology, College of Medicine, Kyung Hee University, Seoul, Korea
Summary Aims: Smad signalling is important for inducing the expression of cell cycle inhibitors such as p21/WAF1, thereby facilitating transforming growth factor (TGF)-b mediated inhibition of cell growth. The role and clinical implications of Smad signalling in osteosarcoma remain unclear. Methods: We assessed the immunohistochemical expression profiles of Smad2, P-Smad2, Smad4, and p21/WAF1 proteins in 34 cases of osteosarcoma. We also investigated whether the expression of these proteins was correlated with clinicopathological parameters. Results: The p21/WAF1 expression showed a significant relationship with tumour extension (p ¼ 0.018). Smad4 expression was significantly correlated with Smad2 (p ¼ 0.019) and P-Smad2 (p ¼ 0.006) expression in osteosarcoma. Kaplan-Meier analysis showed that tumour extension (p ¼ 0.0038), TNM stage (p ¼ 0.0096), and p21/WAF1 expression (p ¼ 0.0023) were significantly correlated with overall survival. Cox multivariate analysis showed that tumour extension (p ¼ 0.048) and p21/WAF1 expression (p ¼ 0.022) were independent poor prognostic factors. Conclusions: Smad signalling in osteosarcoma is partially operative. The high p21/WAF1 expression in osteosarcoma is significantly correlated with tumour extension. Our results suggest that overexpression of p21/WAF1 in osteosarcoma might be one of the poor prognostic factors. Key words: Osteosarcoma, Smad2, P-Smad2, Smad4, p21/WAF1. Received 21 July, revised 20 October and 19 November, accepted 23 November 2009
INTRODUCTION Osteosarcoma is the most common primary bone malignancy. Many genetic markers have proven to have prognostic value in osteosarcoma, and studies are underway to determine their potential applications as specific therapeutic targets. Smads act as well-conserved components in the transforming growth factor (TGF)-b family of signal transduction pathways. TGF-b receptor-phosphorylated Smads such as Smad2 oligomerise with the common mediator Smad4, and after nuclear import, they regulate gene expression positively or negatively by binding to DNA and by interacting with DNA sequence-specific transcription factors.1 As transcription factors, Smads are important for inducing the expression of cell cycle inhibitors such as p21/WAF1, thereby facilitating TGF-b mediated inhibition of cell growth.2 Smad signalling acts as a tumour suppressor by inhibiting cellular proliferation and inducing apoptosis.3
Loss of heterozygosity (LOH) at 18q, the locus of Smad2 and Smad4, is a common mechanism for inactivation of Smad4 in pancreatic tumours.4 Mutations in Smads have been associated with cancers, particularly those of the colon and gastrointestinal tract.5–8 Specifically, Smad4 was first cloned as a candidate tumour suppressor gene on chromosome 18q21.1, and was found to have been deleted in pancreatic carcinoma locus 4 (DPC4), while being lost or functionally inactivated in nearly 40% of pancreatic carcinomas.4 Loss of nuclear Smad4 expression in gastric cancer is correlated with poor survival.9 In colorectal and breast cancers, loss of Smad4 protein expression correlated with a negative outcome or the presence of lymph node metastasis.10,11 Furthermore, Smad4 is implicated in the regulation of p21/WAF1.1,12 However, the role and clinical implications of Smad signalling in osteosarcoma remain unclear. In the present study, we assessed the expression profile of Smad2, P-Smad2, Smad4, and p21/WAF1 proteins in osteosarcoma tissues. In addition, we investigated whether the expression of the four proteins was correlated with clinicopathological parameters including survival.
MATERIALS AND METHODS Patients and tissue specimens Between 1983 and 2005, we selected 34 osteosarcoma patient cases that had been treated by surgical resection at Kyung Hee University Hospital, Korea. The 34 formalin-fixed, paraffin-embedded samples were collected. For each case, paraffin blocks were retrieved and original haematoxylin and eosin (H&E) stained sections reviewed. The clinicopathological data of 34 patients is summarised in Table 1. Of the patients, 12 (35.3%) underwent neoadjuvant chemotherapy (methotrexate and leucovorin). Their treatment response was classified according to the Huvos grading system.13 In this system, four grades have been described: grade I, little or no evidence of necrosis; grade II, 50–89% necrosis; grade III, 90–99% necrosis; and grade IV, 100% necrosis. Based on the Huvos grade, seven (58.3%) of the 12 patients were classified as Huvos grades III and IV (good responder). Overall survival ranged from 1 month to 291 months. The records of seven patients were not obtained. Patients were classified as being alive (14 cases) or dead (13 cases) at the end of the study. This study was approved by the Institutional Review Board at Kyung Hee University Hospital.
Immunohistochemistry Immunohistochemistry was performed on 4 mm tissue sections using the Bond Polymer Intense Detection system (Vision BioSystems, Australia) according to the manufacturer’s instructions with minor modifications. In brief, 4 mm sections of formalin-fixed, paraffin-embedded tissues were deparaffinised by Bond Dewax Solution (Vision BioSystems), and an antigen retrieval procedure was carried out using Bond ER Solution (Vision BioSystems) for 30 min at 1008C. Endogenous peroxidases were
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2010 Royal College of Pathologists of Australasia DOI: 10.3109/00313021003631288
SMAD SIGNALLING IN OSTEOSARCOMA
Table 1
Clinicopathological data of 34 osteosarcoma patients
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Parameter Age (years) 27 527 Gender Male Female Tumour site Femur Tibia Humerus Pelvis Facial bone Skull Not identified Tumour size 8 cm 58 cm Histological grade High Low TNM stage I II III IV Tumour extension Present Absent Histological type Osteoblastic Chondroblastic Fibroblastic Periosteal Telangiectatic Neoadjuvant chemotherapy Yes No Huvos grade I–II III–IV
Patients, n (%)
12 (35.3) 22 (64.7) 19 (55.9) 15 (44.1) 14 4 3 6 5 1 1
(11.6) (11.6) (8.7) (17.4) (14.5) (2.9) (2.9)
16 (47.1) 18 (52.9) 22 (64.7) 12 (35.2) 19 7 1 7
(55.9) (20.6) (2.9) (20.6)
8 (23.5) 26 (76.5) 24 5 3 1 1
(70.5) (14.7) (8.8) (2.9) (2.9)
12 (35.3) 22 (64.7) 5 (41.7) 7 (58.3)
quenched by incubation with hydrogen peroxide for 5 min. Sections were incubated for 15 min at ambient temperature with primary polyclonal antibodies for Smad2 (mouse monoclonal, 1:200, clone no. YZ-13; Santa Cruz Biotechnology, USA), Smad4 (mouse monoclonal, 1:200, clone no. B8; Santa Cruz Biotechnology), P-Smad2 (rabbit monoclonal, 1:50, clone no. 138D4; Cell Signaling, USA), and p21/WAF1 (mouse monoclonal, 1:200, clone no. 4D10; Novacastra, UK) using the biotin-free polymeric horseradish peroxidase (HRP)-linker antibody conjugate system in a Bond-max automatic slide stainer (Vision BioSystems). Nuclei were counterstained with haematoxylin. Our positive controls for Smad2, Smad4, P-Smad2, and p21/WAF1 staining were squamous cell carcinoma from uterine cervix. Evaluation and scoring Immunohistochemical staining was evaluated according to intensity and proportion.14 Intensities were scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). Proportions were scored as 0 (510% of tumour cells), 1 (10% to 530% of tumour cells), 2 (30% to 560% of tumour cells), or 3 (60% of tumour cells). The intensity and proportion scores were added for a total score. Total scores were regarded as follows: 0–2 (negative), 3–4 (weak positive), and 5–6 (strong positive).
Statistical analysis The SPSS statistical package (SPSS, USA) was used for statistical analysis. The Pearson’s chi-square test was used to evaluate the association between Smad2, Smad4, P-Smad2, and p21/WAF1 expression and various clinicopathological parameters. The Kaplan-Meier test was used to
243
determine the probability of survival, and the data were analysed by the log-rank test. A p value of 50.05 was considered significant. To examine whether the expression of these four proteins were correlated with various clinicopathological parameters, cases were divided into two groups as follows: low expression (negative or weak positive staining) or high expression (strong positive staining). Multivariate analysis was performed using the Cox regression model to study different variables and overall survival.
RESULTS In osteosarcoma tissue, immunohistochemical localisation of Smad2, P-Smad2, and p21/WAF1 was evaluated by nuclear staining. Smad4 expression was localised in both the nucleus and cytoplasm. Smad2 expression in osteosarcoma was high in 12 cases (35.3%) (Fig. 1A) and low in 22 cases (64.7%). Smad4 expression in osteosarcoma was high in 16 cases (47.1%) (Fig. 1B) and low in 18 cases (52.9%). P-Smad2 expression in osteosarcoma was high in 27 cases (79.4%) (Fig. 1C) and low in seven cases (20.6%). p21/ WAF1 expression in osteosarcoma was high in six cases (17.6%) (Fig. 1D) and low in 28 cases (82.4%). Various morphological characteristics of our osteosarcoma cases were shown in H&E staining (Fig. 2), including lace-like or trabecular eosinophilic osteoid produced by surrounding pleomorphic bizarre cells (Fig. 2A) and plasmacytoid tumour cells with blue-grey myxoid cartilaginous appearing matrix (Fig. 2B). Tumour cells within lacuna-like chondrocyte showed marked anaplasia (Fig. 2C). Osteoclast-like multinucleated giant cells were mixed with anaplastic tumour cells (Fig. 2D). Correlating the expression of Smad2, Smad4, P-Smad2, and p21/WAF1 with clinicopathological parameters, the p21/WAF1 expression showed a significant relationship with tumour extension (p ¼ 0.018) and Smad4 expression with histological type (p ¼ 0.043) (Table 2). Smad4 expression was significantly correlated with Smad2 expression (p ¼ 0.019) and P-Smad2 expression (p ¼ 0.006) in osteosarcoma (Table 3). p21/WAF1 expression was not correlated with Smad2 (p ¼ 0.351), P-Smad2 (p ¼ 0.640), or Smad4 (p ¼ 0.611) expression in osteosarcoma. KaplanMeier analysis showed that tumour extension (p ¼ 0.0038), TNM stage (p ¼ 0.0096) and p21/WAF1 expression (p ¼ 0.0023) were significantly correlated with overall survival (Table 4, Fig. 3A–C). Cox multivariate analysis showed that p21/WAF1 expression (p ¼ 0.022) and tumour extension (p ¼ 0.048) were independent poor prognostic factors (Table 5).
DISCUSSION The Smad signalling pathway controls growth, apoptosis, and differentiation of diverse cell types and plays important roles during developmental patterning, morphogenesis, and disease pathogenesis.15 Smad signalling can function both as a tumour suppressor and a tumour promoter in a wide array of cancers. Mutations in Smads have been associated with cancers, particularly those of the colon and gastrointestinal tract.6,7 However, the role of Smad signalling and p21/WAF1 in osteosarcoma remains unclear. In the present study, we found that Smad signalling (Smad2, Smad4, P-Smad2, p21/WAF1) was partially operative in osteosarcoma. Positive correlations between Smad2 and Smad4, and between P-Smad2 and Smad4,
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WON et al.
Pathology (2010), 42(3), April
Fig. 1 (A) Strong positive nuclear staining of Smad2 in osteosarcoma. (B) Strong positive nuclear and cytoplasmic staining of Smad4 in osteosarcoma. (C) Strong positive nuclear staining of P-Smad2 in osteosarcoma. (D) Strong positive nuclear staining of p21/WAF1 in osteosarcoma.
Fig. 2 (A–D) Various morphological characteristics are shown in H&E staining. (A) Lace-like or trabecular eosinophilic osteoid produced by surrounding pleomorphic bizarre cells. (B) Plasmacytoid tumour cells with blue-grey myxoid cartilaginous appearing matrix. (C) Tumour cells within lacuna-like chondrocyte show marked anaplasia. (D) Osteoclast-like multinucleated giant cells are mixed with anaplastic tumour cells.
were found in osteosarcoma. However, Smad2, Smad4, and P-Smad2 expressions were not significantly correlated with any clinicopathological parameters. Thus, we still do not know whether Smad signalling influences the progression of
osteosarcoma including the degree of malignancy, potency of soft tissue extension and metastasis. In addition, our novel finding is that high p21/WAF1 expression in osteosarcoma is significantly related with
SMAD SIGNALLING IN OSTEOSARCOMA
Table 2
Correlation of Smad2, P-Smad2, Smad4, and p21/WAF1 expression with clinicopathological variables in osteosarcoma Smad2, n (%)
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Tumour extension Present Absent Tumour size 8 cm 58 cm Age (years) 27 527 Gender Male Female Tumour site Long bone Flat bone Histological type Osteoblastic Non-osteoblastic Histological grade High Low TNM stage I–III IV Huvos grade I–II III–IV
High
Low
4 (11.8) 8 (23.5)
4 (11.8) 18 (52.9)
5 (14.7) 7 (20.6)
11 (32.4) 11 (32.4)
9 (26.5) 9 (26.5)
3 (8.8) 13 (38.2)
7 (20.6) 5 (14.7)
12 (35.3) 10 (29.4)
8 (24.2) 4 (12.1)
13 (39.4) 8 (24.2)
6 (17.6) 6 (17.6)
17 (50.0) 5 (14.7)
6 (17.6) 6 (17.6)
16 (47.1) 6 (17.6)
9 (26.5) 3 (8.8) 3 (25.0) 2 (16.7)
P-Smad2, n (%) p value
High
Low
7 (20.6) 20 (58.8)
1 (2.9) 6 (17.6)
14 (41.2) 13 (38.2)
2 (5.9) 5 (14.7)
10 (29.4) 17 (50.0)
2 (5.9) 5 (14.7)
13 (38.2) 14 (41.2)
6 (17.6) 1 (2.9)
18 (54.5) 9 (27.3)
3 (9.1) 3 (9.1)
18 (52.9) 9 (26.5)
5 (14.7) 2 (5.9)
0.171
19 (55.9) 8 (23.5)
3 (8.8) 4 (11.8)
18 (52.9) 4 (11.8)
0.479
21 (61.8) 6 (17.6)
2 (16.7) 5 (41.7)
0.311
3 (25.0) 6 (50.0)
Smad4, n (%) High
Low
3 (8.8) 11 (32.4)
5 (14.7) 15 (44.1)
9 (26.5) 7 (20.6)
7 (20.6) 11 (32.4)
5 (14.7) 11 (32.4)
7 (20.6) 11 (32.4)
9 (26.5) 7 (20.6)
10 (29.4) 8 (23.5)
11 (33.3) 5 (15.2)
10 (30.3) 7 (21.2)
8 (23.5) 8 (23.5)
15 (44.1) 3 (8.8)
0.180
10 (29.4) 6 (17.6)
12 (35.3) 6 (17.6)
6 (17.6) 1 (2.9)
0.550
11 (32.4) 5 (14.7)
2 (16.7) 1 (8.3)
0.364
3 (25.0) 2 (16.7)
0.279
p value
High
Low
4 (11.8) 2 (5.9)
4 (11.8) 24 (70.6)
2 (5.9) 4 (11.8)
14 (41.2) 14 (41.2)
3 (8.8) 3 (8.8)
9 (26.5) 19 (55.9)
5 (14.7) 1 (2.9)
14 (41.2) 14 (41.2)
4 (12.1) 1 (3.0)
17 (51.5) 11 (33.3)
3 (8.8) 3 (8.8)
20 (58.8) 8 (23.5)
0.541
4 (11.8) 2 (5.9)
18 (52.9) 10 (29.4)
0.649
16 (47.1) 2 (5.9)
0.153
3 (8.8) 3 (8.8)
24 (70.6) 4 (11.8)
0.086
2 (16.7) 5 (41.7)
0.311
1 (8.3) 1 (8.3)
4 (33.3) 6 (50.0)
0.682
0.465
0.459
0.389
0.351
0.620
0.374
0.108
0.018*
0.459
0.085
0.544
p value
0.252
0.521
0.561
p value 0.276
0.252
0.294
p21/WAF1, n (%)
0.150
0.410
0.596
0.388
0.288
0.043*
Statistical analysis: chi-square test. *Significant difference (%).
Table 3 Positive correlation of Smad4 with Smad2 and P-Smad2 expression in osteosarcoma
Table 4 Analysis of clinicopathological variables for overall survival rate in osteosarcoma
Smad4, n (%) Variables
Smad2 High Low P-Smad2 High Low
High
Low
p value
9 (26.5) 7 (20.6)
3 (8.8) 15 (44.1)
0.019*
16 (47.1) 0 (0)
11 (32.4) 7 (20.6)
0.006*
Statistical analysis: chi-square test. *Significant difference (%).
poor prognosis. Increased expression of p21/WAF1 is significantly correlated with more frequent tumour extension including soft tissue extension and distant metastasis (p ¼ 0.018). High TNM stage is correlated with high p21/ WAF1 expression (p ¼ 0.086). Although it is not of significant value, the result supports a relationship between high p21/WAF1 expression and poor prognosis. The role of p21/WAF1 in osteosarcoma has not been thoroughly understood. In contrast to our results, Kao et al. reported that overexpression of p21/WAF1 served as an independent and good survival factor in hepatocellular carcinoma.16 Chen et al. reported that the induction of p21/ WAF1 by RNAs had anti-tumour activity in vitro in bladder cancer cells by promoting cell cycle arrest and enhancing apoptotic cell death.17 Zhang et al. also reported
Age Tumour site Tumour size (8 cm vs 58 cm) Histological type Histological grade Tumour extension TNM stage (I–III vs IV) Neoadjuvant chemotherapy Chemotherapy response (Huvos grade) Smad2 expression Smad4 expression P-Smad2 expression p21/WAF1 expression
Overall survival rate (p value) 0.2501 0.2554 0.1882 0.5543 0.9875 0.0038* 0.0096* 0.6357 0.1485 0.1107 0.1404 0.9394 0.0023*
*Significantly different by Kaplan-Meier test.
that with the increase in degree of malignancy, the expression of p21/WAF1 mRNA and p21/WAF1 protein in osteosarcoma tended to decrease, and the expression of p21/WAF1 mRNA had definite value in judging prognosis in osteosarcoma.18 However, our present study of the p21/ WAF1 expression in osteosarcoma showed a discrepancy with these previous studies. The p21/WAF1 is a wellknown cyclin-dependent kinase (CDK) inhibitor that suppresses cell proliferation by interfering with cyclin/ CDK2 activity and blocks DNA replication by binding to proliferating cell nuclear antigen (PCNA).19 The p21/ WAF1 protein induces G1 arrest of the cell cycle, in which
246
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Fig. 3
WON et al.
Pathology (2010), 42(3), April
Tumour extension, TNM stage, and high p21/WAF1 expression are significantly correlated with poor overall survival in osteosarcoma.
Table 5
Cox multivariate analysis in osteosarcoma
Variables Age Tumour site Tumour size (8 cm vs 58 cm) Histological type Tumour extension Histological grade TNM stage Neoadjuvant chemotherapy Smad2 expression Smad4 expression P-Smad2 expression p21/WAF1 expression
Relative risk ratio
95% confidence limits
P value
0.763 2.746 2.575 0.551 9.410 0.274 0.156 0.413 1.691 2.781 0.303 16.086
0.076–7.689 0.227–33247 0.146–45.288 0.049–6.226 1.018–86.945 0.021–3.521 0.011–2.162 0.038–4.487 0.404–7.088 0.287–26–945 0.015–6.228 1.480–174.783
0.818 0.427 0.518 0.630 0.048* 0.320 0.166 0.467 0.472 0.377 0.439 0.022*
*Significant difference.
action is essential for differentiation and apoptosis.20 Therefore, the cells that have lost the activity to express p21/WAF1 cannot regulate those cell cycles to attain G1 arrest for apoptosis and this may lead to unregulated cell proliferation as a step toward carcinogenesis.21 Some authors reported that p21/WAF1 expression might represent a form of CDK inhibitor dysfunction involved in tumorigenicity.21,22 From these previous opposite suggestions, some possibilities may be raised. One possible explanation of our results is that overexpression of p21/ WAF1 in osteosarcoma might represent CDK inhibitor dysfunction involved in tumorigenesis. This dysfunction of p21/WAF1 protein may lead to unregulated cell proliferation such as tumour extension. The other explanation is that the overexpression of p21/WAF1 controls the abnormal cell-cycle progression and suppresses replication of tumour cells. Under high proliferating tumour states, increased p21/WAF1 is just a reactive event to inhibit cell growth.16 Based on this hypothesis, the overexpression of p21/WAF1 in osteosarcoma might be a reactive response of highly proliferative tumour cells that easily invade to other tissue. Additionally, as chemotherapy impact may influence patient survival, we excluded the 12 cases that underwent neoadjuvant chemotherapy, and performed statistical analysis. In these results, p21/WAF1 expression was also
significantly correlated with tumour extension (p ¼ 0.024, not shown in table). However, p21/WAF1 expression was not significantly correlated with overall survival in the Kaplan-Meier test (p ¼ 0.1232, not shown in table). These findings suggest that p21/WAF1 expression is significantly correlated with tumour extension, regardless of chemotherapy. Although we investigated the relationship between chemotherapy response and four Smad signalling cascades, no significant findings were observed (Table 2). None of the Smad signalling downstream protein components was significantly related with any clinicopathological parameters; however, our results suggest that Smad signalling might play a partial role in osteosarcoma tumorigenesis due to the fact that a positive correlation between Smad2, PSmad2, and Smad4 in osteosarcoma was found. Our study has limitations due to the small number of cases and further studies of Smad signalling in osteosarcoma are needed. These findings will assist in the development of a novel therapeutic modality. In conclusion, Smad signalling in osteosarcoma is partially operative. High p21/WAF1 expression in osteosarcoma is significantly correlated with tumour extension. Our results suggest that overexpression of p21/WAF1 in osteosarcoma might be one of the poor prognostic factors. Acknowledgements: This work was supported by a grant from the Kyung Hee University in 2009 (KHU20090568). Address for correspondence: Dr Y-K. Park, Department of Pathology, Kyung Hee Medical Center, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, 130-702 Seoul, Korea. E-mail: [email protected]
References 1. Miyazawa K, Shinozaki M, Hara T, et al. Two major smad pathways in tgf-beta superfamily signalling. Genes Cells 2002; 7: 1191–204. 2. Ten Dijke P, Goumans MJ, Itoh F, et al. Regulation of cell proliferation by smad proteins. J Cell Physiol 2002; 191: 1–16. 3. Bierie B, Moses HL Tumour microenvironment: TGFbeta: The molecular jekyll and hyde of cancer. Nat Rev Cancer 2006; 6: 506–20. 4. Hahn SA, Schutte M, Hoque AT, et al. Dpc4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996; 271: 350–3. 5. Fukushima T, Mashiko M, Takita K, et al. Mutational analysis of TGF-beta type II receptor, smad2, smad3, smad4, smad6 and
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6. 7.
8.
9.
10.
11.
12.
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 05/12/15 For personal use only.
13.
smad7 genes in colorectal cancer. J Exp Clin Cancer Res 2003; 22: 315– 20. Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 2000; 103: 295–309. de Caestecker MP, Piek E, Roberts AB. Role of transforming growth factor-beta signaling in cancer. J Natl Cancer Inst 2000; 92: 1388–402. Eppert K, Scherer SW, Ozcelik H, et al. Madr2 maps to 18q21 and encodes a TGFbeta-regulated mad-related protein that is functionally mutated in colorectal carcinoma. Cell 1996; 86: 543–52. Wang LH, Kim SH, Lee JH, et al. Inactivation of smad4 tumor suppressor gene during gastric carcinoma progression. Clin Cancer Res 2007; 13: 102–10. Xie W, Mertens JC, Reiss DJ, et al. Alterations of smad signaling in human breast carcinoma are associated with poor outcome: A tissue microarray study. Cancer Res 2002; 62: 497–505. Xie W, Rimm DL, Lin Y, et al. Loss of smad signaling in human colorectal cancer is associated with advanced disease and poor prognosis. Cancer J 2003; 9: 302–12. Shi Y, Massague J Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003; 113: 685–700. Rosen G, Caparros B, Huvos AG, et al. Preoperative chemotherapy for osteogenic sarcoma: Selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 1982; 49: 1221–30.
247
14. Kloth JN, Kenter GG, Spijker HS, et al. Expression of smad2 and smad4 in cervical cancer: Absent nuclear smad4 expression correlates with poor survival. Mod Pathol 2008; 21: 866–75. 15. Piek E, Heldin CH, Ten Dijke P Specificity, diversity, and regulation in TGF-beta superfamily signaling. Faseb J 1999; 13: 2105–24. 16. Kao JT, Chuah SK, Huang CC, et al. P21/waf1 is an independent survival prognostic factor for patients with hepatocellular carcinoma after resection. Liver Int 2007; 27: 772–81. 17. Chen Z, Place RF, Jia ZJ, et al. Antitumor effect of dsrna-induced p21(waf1/cip1) gene activation in human bladder cancer cells. Mol Cancer Ther 2008; 7: 698–703. 18. Zhang CL, Liao WM, Li FB, et al. Prognostic significance of p21(waf1) expression in osteosarcoma. (Chinese.) Zhonghua Bing Li Xue Za Zhi 2005; 34: 524–7. 19. Waga S, Hannon GJ, Beach D, et al. The p21 inhibitor of cyclindependent kinases controls DNA replication by interaction with PCNA. Nature 1994; 369: 574–8. 20. el-Deiry WS, Harper JW, O’Connor PM, et al. Waf1/cip1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994; 54: 1169–74. 21. Kobayashi S, Matsushita K, Saigo K, et al. P21waf1/cip1 messenger RNA expression in hepatitis B, C virus-infected human hepatocellular carcinoma tissues. Cancer 2001; 91: 2096–103. 22. Qin LF, Ng IO. Expression of p27(kip1) and p21(waf1/cip1) in primary hepatocellular carcinoma: Clinicopathologic correlation and survival analysis. Hum Pathol 2001; 32: 778–84.
ANATOMICAL PATHOLOGY
Expression of Smad and its signalling cascade in osteosarcoma KYU YEOUN WON, YOUN WHA KIM
AND
YONG-KOO PARK
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 05/12/15 For personal use only.
Department of Pathology, College of Medicine, Kyung Hee University, Seoul, Korea
Summary Aims: Smad signalling is important for inducing the expression of cell cycle inhibitors such as p21/WAF1, thereby facilitating transforming growth factor (TGF)-b mediated inhibition of cell growth. The role and clinical implications of Smad signalling in osteosarcoma remain unclear. Methods: We assessed the immunohistochemical expression profiles of Smad2, P-Smad2, Smad4, and p21/WAF1 proteins in 34 cases of osteosarcoma. We also investigated whether the expression of these proteins was correlated with clinicopathological parameters. Results: The p21/WAF1 expression showed a significant relationship with tumour extension (p ¼ 0.018). Smad4 expression was significantly correlated with Smad2 (p ¼ 0.019) and P-Smad2 (p ¼ 0.006) expression in osteosarcoma. Kaplan-Meier analysis showed that tumour extension (p ¼ 0.0038), TNM stage (p ¼ 0.0096), and p21/WAF1 expression (p ¼ 0.0023) were significantly correlated with overall survival. Cox multivariate analysis showed that tumour extension (p ¼ 0.048) and p21/WAF1 expression (p ¼ 0.022) were independent poor prognostic factors. Conclusions: Smad signalling in osteosarcoma is partially operative. The high p21/WAF1 expression in osteosarcoma is significantly correlated with tumour extension. Our results suggest that overexpression of p21/WAF1 in osteosarcoma might be one of the poor prognostic factors. Key words: Osteosarcoma, Smad2, P-Smad2, Smad4, p21/WAF1. Received 21 July, revised 20 October and 19 November, accepted 23 November 2009
INTRODUCTION Osteosarcoma is the most common primary bone malignancy. Many genetic markers have proven to have prognostic value in osteosarcoma, and studies are underway to determine their potential applications as specific therapeutic targets. Smads act as well-conserved components in the transforming growth factor (TGF)-b family of signal transduction pathways. TGF-b receptor-phosphorylated Smads such as Smad2 oligomerise with the common mediator Smad4, and after nuclear import, they regulate gene expression positively or negatively by binding to DNA and by interacting with DNA sequence-specific transcription factors.1 As transcription factors, Smads are important for inducing the expression of cell cycle inhibitors such as p21/WAF1, thereby facilitating TGF-b mediated inhibition of cell growth.2 Smad signalling acts as a tumour suppressor by inhibiting cellular proliferation and inducing apoptosis.3
Loss of heterozygosity (LOH) at 18q, the locus of Smad2 and Smad4, is a common mechanism for inactivation of Smad4 in pancreatic tumours.4 Mutations in Smads have been associated with cancers, particularly those of the colon and gastrointestinal tract.5–8 Specifically, Smad4 was first cloned as a candidate tumour suppressor gene on chromosome 18q21.1, and was found to have been deleted in pancreatic carcinoma locus 4 (DPC4), while being lost or functionally inactivated in nearly 40% of pancreatic carcinomas.4 Loss of nuclear Smad4 expression in gastric cancer is correlated with poor survival.9 In colorectal and breast cancers, loss of Smad4 protein expression correlated with a negative outcome or the presence of lymph node metastasis.10,11 Furthermore, Smad4 is implicated in the regulation of p21/WAF1.1,12 However, the role and clinical implications of Smad signalling in osteosarcoma remain unclear. In the present study, we assessed the expression profile of Smad2, P-Smad2, Smad4, and p21/WAF1 proteins in osteosarcoma tissues. In addition, we investigated whether the expression of the four proteins was correlated with clinicopathological parameters including survival.
MATERIALS AND METHODS Patients and tissue specimens Between 1983 and 2005, we selected 34 osteosarcoma patient cases that had been treated by surgical resection at Kyung Hee University Hospital, Korea. The 34 formalin-fixed, paraffin-embedded samples were collected. For each case, paraffin blocks were retrieved and original haematoxylin and eosin (H&E) stained sections reviewed. The clinicopathological data of 34 patients is summarised in Table 1. Of the patients, 12 (35.3%) underwent neoadjuvant chemotherapy (methotrexate and leucovorin). Their treatment response was classified according to the Huvos grading system.13 In this system, four grades have been described: grade I, little or no evidence of necrosis; grade II, 50–89% necrosis; grade III, 90–99% necrosis; and grade IV, 100% necrosis. Based on the Huvos grade, seven (58.3%) of the 12 patients were classified as Huvos grades III and IV (good responder). Overall survival ranged from 1 month to 291 months. The records of seven patients were not obtained. Patients were classified as being alive (14 cases) or dead (13 cases) at the end of the study. This study was approved by the Institutional Review Board at Kyung Hee University Hospital.
Immunohistochemistry Immunohistochemistry was performed on 4 mm tissue sections using the Bond Polymer Intense Detection system (Vision BioSystems, Australia) according to the manufacturer’s instructions with minor modifications. In brief, 4 mm sections of formalin-fixed, paraffin-embedded tissues were deparaffinised by Bond Dewax Solution (Vision BioSystems), and an antigen retrieval procedure was carried out using Bond ER Solution (Vision BioSystems) for 30 min at 1008C. Endogenous peroxidases were
Print ISSN 0031-3025/Online ISSN 1465-3931 # 2010 Royal College of Pathologists of Australasia DOI: 10.3109/00313021003631288
SMAD SIGNALLING IN OSTEOSARCOMA
Table 1
Clinicopathological data of 34 osteosarcoma patients
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Parameter Age (years) 27 527 Gender Male Female Tumour site Femur Tibia Humerus Pelvis Facial bone Skull Not identified Tumour size 8 cm 58 cm Histological grade High Low TNM stage I II III IV Tumour extension Present Absent Histological type Osteoblastic Chondroblastic Fibroblastic Periosteal Telangiectatic Neoadjuvant chemotherapy Yes No Huvos grade I–II III–IV
Patients, n (%)
12 (35.3) 22 (64.7) 19 (55.9) 15 (44.1) 14 4 3 6 5 1 1
(11.6) (11.6) (8.7) (17.4) (14.5) (2.9) (2.9)
16 (47.1) 18 (52.9) 22 (64.7) 12 (35.2) 19 7 1 7
(55.9) (20.6) (2.9) (20.6)
8 (23.5) 26 (76.5) 24 5 3 1 1
(70.5) (14.7) (8.8) (2.9) (2.9)
12 (35.3) 22 (64.7) 5 (41.7) 7 (58.3)
quenched by incubation with hydrogen peroxide for 5 min. Sections were incubated for 15 min at ambient temperature with primary polyclonal antibodies for Smad2 (mouse monoclonal, 1:200, clone no. YZ-13; Santa Cruz Biotechnology, USA), Smad4 (mouse monoclonal, 1:200, clone no. B8; Santa Cruz Biotechnology), P-Smad2 (rabbit monoclonal, 1:50, clone no. 138D4; Cell Signaling, USA), and p21/WAF1 (mouse monoclonal, 1:200, clone no. 4D10; Novacastra, UK) using the biotin-free polymeric horseradish peroxidase (HRP)-linker antibody conjugate system in a Bond-max automatic slide stainer (Vision BioSystems). Nuclei were counterstained with haematoxylin. Our positive controls for Smad2, Smad4, P-Smad2, and p21/WAF1 staining were squamous cell carcinoma from uterine cervix. Evaluation and scoring Immunohistochemical staining was evaluated according to intensity and proportion.14 Intensities were scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). Proportions were scored as 0 (510% of tumour cells), 1 (10% to 530% of tumour cells), 2 (30% to 560% of tumour cells), or 3 (60% of tumour cells). The intensity and proportion scores were added for a total score. Total scores were regarded as follows: 0–2 (negative), 3–4 (weak positive), and 5–6 (strong positive).
Statistical analysis The SPSS statistical package (SPSS, USA) was used for statistical analysis. The Pearson’s chi-square test was used to evaluate the association between Smad2, Smad4, P-Smad2, and p21/WAF1 expression and various clinicopathological parameters. The Kaplan-Meier test was used to
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determine the probability of survival, and the data were analysed by the log-rank test. A p value of 50.05 was considered significant. To examine whether the expression of these four proteins were correlated with various clinicopathological parameters, cases were divided into two groups as follows: low expression (negative or weak positive staining) or high expression (strong positive staining). Multivariate analysis was performed using the Cox regression model to study different variables and overall survival.
RESULTS In osteosarcoma tissue, immunohistochemical localisation of Smad2, P-Smad2, and p21/WAF1 was evaluated by nuclear staining. Smad4 expression was localised in both the nucleus and cytoplasm. Smad2 expression in osteosarcoma was high in 12 cases (35.3%) (Fig. 1A) and low in 22 cases (64.7%). Smad4 expression in osteosarcoma was high in 16 cases (47.1%) (Fig. 1B) and low in 18 cases (52.9%). P-Smad2 expression in osteosarcoma was high in 27 cases (79.4%) (Fig. 1C) and low in seven cases (20.6%). p21/ WAF1 expression in osteosarcoma was high in six cases (17.6%) (Fig. 1D) and low in 28 cases (82.4%). Various morphological characteristics of our osteosarcoma cases were shown in H&E staining (Fig. 2), including lace-like or trabecular eosinophilic osteoid produced by surrounding pleomorphic bizarre cells (Fig. 2A) and plasmacytoid tumour cells with blue-grey myxoid cartilaginous appearing matrix (Fig. 2B). Tumour cells within lacuna-like chondrocyte showed marked anaplasia (Fig. 2C). Osteoclast-like multinucleated giant cells were mixed with anaplastic tumour cells (Fig. 2D). Correlating the expression of Smad2, Smad4, P-Smad2, and p21/WAF1 with clinicopathological parameters, the p21/WAF1 expression showed a significant relationship with tumour extension (p ¼ 0.018) and Smad4 expression with histological type (p ¼ 0.043) (Table 2). Smad4 expression was significantly correlated with Smad2 expression (p ¼ 0.019) and P-Smad2 expression (p ¼ 0.006) in osteosarcoma (Table 3). p21/WAF1 expression was not correlated with Smad2 (p ¼ 0.351), P-Smad2 (p ¼ 0.640), or Smad4 (p ¼ 0.611) expression in osteosarcoma. KaplanMeier analysis showed that tumour extension (p ¼ 0.0038), TNM stage (p ¼ 0.0096) and p21/WAF1 expression (p ¼ 0.0023) were significantly correlated with overall survival (Table 4, Fig. 3A–C). Cox multivariate analysis showed that p21/WAF1 expression (p ¼ 0.022) and tumour extension (p ¼ 0.048) were independent poor prognostic factors (Table 5).
DISCUSSION The Smad signalling pathway controls growth, apoptosis, and differentiation of diverse cell types and plays important roles during developmental patterning, morphogenesis, and disease pathogenesis.15 Smad signalling can function both as a tumour suppressor and a tumour promoter in a wide array of cancers. Mutations in Smads have been associated with cancers, particularly those of the colon and gastrointestinal tract.6,7 However, the role of Smad signalling and p21/WAF1 in osteosarcoma remains unclear. In the present study, we found that Smad signalling (Smad2, Smad4, P-Smad2, p21/WAF1) was partially operative in osteosarcoma. Positive correlations between Smad2 and Smad4, and between P-Smad2 and Smad4,
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Fig. 1 (A) Strong positive nuclear staining of Smad2 in osteosarcoma. (B) Strong positive nuclear and cytoplasmic staining of Smad4 in osteosarcoma. (C) Strong positive nuclear staining of P-Smad2 in osteosarcoma. (D) Strong positive nuclear staining of p21/WAF1 in osteosarcoma.
Fig. 2 (A–D) Various morphological characteristics are shown in H&E staining. (A) Lace-like or trabecular eosinophilic osteoid produced by surrounding pleomorphic bizarre cells. (B) Plasmacytoid tumour cells with blue-grey myxoid cartilaginous appearing matrix. (C) Tumour cells within lacuna-like chondrocyte show marked anaplasia. (D) Osteoclast-like multinucleated giant cells are mixed with anaplastic tumour cells.
were found in osteosarcoma. However, Smad2, Smad4, and P-Smad2 expressions were not significantly correlated with any clinicopathological parameters. Thus, we still do not know whether Smad signalling influences the progression of
osteosarcoma including the degree of malignancy, potency of soft tissue extension and metastasis. In addition, our novel finding is that high p21/WAF1 expression in osteosarcoma is significantly related with
SMAD SIGNALLING IN OSTEOSARCOMA
Table 2
Correlation of Smad2, P-Smad2, Smad4, and p21/WAF1 expression with clinicopathological variables in osteosarcoma Smad2, n (%)
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Tumour extension Present Absent Tumour size 8 cm 58 cm Age (years) 27 527 Gender Male Female Tumour site Long bone Flat bone Histological type Osteoblastic Non-osteoblastic Histological grade High Low TNM stage I–III IV Huvos grade I–II III–IV
High
Low
4 (11.8) 8 (23.5)
4 (11.8) 18 (52.9)
5 (14.7) 7 (20.6)
11 (32.4) 11 (32.4)
9 (26.5) 9 (26.5)
3 (8.8) 13 (38.2)
7 (20.6) 5 (14.7)
12 (35.3) 10 (29.4)
8 (24.2) 4 (12.1)
13 (39.4) 8 (24.2)
6 (17.6) 6 (17.6)
17 (50.0) 5 (14.7)
6 (17.6) 6 (17.6)
16 (47.1) 6 (17.6)
9 (26.5) 3 (8.8) 3 (25.0) 2 (16.7)
P-Smad2, n (%) p value
High
Low
7 (20.6) 20 (58.8)
1 (2.9) 6 (17.6)
14 (41.2) 13 (38.2)
2 (5.9) 5 (14.7)
10 (29.4) 17 (50.0)
2 (5.9) 5 (14.7)
13 (38.2) 14 (41.2)
6 (17.6) 1 (2.9)
18 (54.5) 9 (27.3)
3 (9.1) 3 (9.1)
18 (52.9) 9 (26.5)
5 (14.7) 2 (5.9)
0.171
19 (55.9) 8 (23.5)
3 (8.8) 4 (11.8)
18 (52.9) 4 (11.8)
0.479
21 (61.8) 6 (17.6)
2 (16.7) 5 (41.7)
0.311
3 (25.0) 6 (50.0)
Smad4, n (%) High
Low
3 (8.8) 11 (32.4)
5 (14.7) 15 (44.1)
9 (26.5) 7 (20.6)
7 (20.6) 11 (32.4)
5 (14.7) 11 (32.4)
7 (20.6) 11 (32.4)
9 (26.5) 7 (20.6)
10 (29.4) 8 (23.5)
11 (33.3) 5 (15.2)
10 (30.3) 7 (21.2)
8 (23.5) 8 (23.5)
15 (44.1) 3 (8.8)
0.180
10 (29.4) 6 (17.6)
12 (35.3) 6 (17.6)
6 (17.6) 1 (2.9)
0.550
11 (32.4) 5 (14.7)
2 (16.7) 1 (8.3)
0.364
3 (25.0) 2 (16.7)
0.279
p value
High
Low
4 (11.8) 2 (5.9)
4 (11.8) 24 (70.6)
2 (5.9) 4 (11.8)
14 (41.2) 14 (41.2)
3 (8.8) 3 (8.8)
9 (26.5) 19 (55.9)
5 (14.7) 1 (2.9)
14 (41.2) 14 (41.2)
4 (12.1) 1 (3.0)
17 (51.5) 11 (33.3)
3 (8.8) 3 (8.8)
20 (58.8) 8 (23.5)
0.541
4 (11.8) 2 (5.9)
18 (52.9) 10 (29.4)
0.649
16 (47.1) 2 (5.9)
0.153
3 (8.8) 3 (8.8)
24 (70.6) 4 (11.8)
0.086
2 (16.7) 5 (41.7)
0.311
1 (8.3) 1 (8.3)
4 (33.3) 6 (50.0)
0.682
0.465
0.459
0.389
0.351
0.620
0.374
0.108
0.018*
0.459
0.085
0.544
p value
0.252
0.521
0.561
p value 0.276
0.252
0.294
p21/WAF1, n (%)
0.150
0.410
0.596
0.388
0.288
0.043*
Statistical analysis: chi-square test. *Significant difference (%).
Table 3 Positive correlation of Smad4 with Smad2 and P-Smad2 expression in osteosarcoma
Table 4 Analysis of clinicopathological variables for overall survival rate in osteosarcoma
Smad4, n (%) Variables
Smad2 High Low P-Smad2 High Low
High
Low
p value
9 (26.5) 7 (20.6)
3 (8.8) 15 (44.1)
0.019*
16 (47.1) 0 (0)
11 (32.4) 7 (20.6)
0.006*
Statistical analysis: chi-square test. *Significant difference (%).
poor prognosis. Increased expression of p21/WAF1 is significantly correlated with more frequent tumour extension including soft tissue extension and distant metastasis (p ¼ 0.018). High TNM stage is correlated with high p21/ WAF1 expression (p ¼ 0.086). Although it is not of significant value, the result supports a relationship between high p21/WAF1 expression and poor prognosis. The role of p21/WAF1 in osteosarcoma has not been thoroughly understood. In contrast to our results, Kao et al. reported that overexpression of p21/WAF1 served as an independent and good survival factor in hepatocellular carcinoma.16 Chen et al. reported that the induction of p21/ WAF1 by RNAs had anti-tumour activity in vitro in bladder cancer cells by promoting cell cycle arrest and enhancing apoptotic cell death.17 Zhang et al. also reported
Age Tumour site Tumour size (8 cm vs 58 cm) Histological type Histological grade Tumour extension TNM stage (I–III vs IV) Neoadjuvant chemotherapy Chemotherapy response (Huvos grade) Smad2 expression Smad4 expression P-Smad2 expression p21/WAF1 expression
Overall survival rate (p value) 0.2501 0.2554 0.1882 0.5543 0.9875 0.0038* 0.0096* 0.6357 0.1485 0.1107 0.1404 0.9394 0.0023*
*Significantly different by Kaplan-Meier test.
that with the increase in degree of malignancy, the expression of p21/WAF1 mRNA and p21/WAF1 protein in osteosarcoma tended to decrease, and the expression of p21/WAF1 mRNA had definite value in judging prognosis in osteosarcoma.18 However, our present study of the p21/ WAF1 expression in osteosarcoma showed a discrepancy with these previous studies. The p21/WAF1 is a wellknown cyclin-dependent kinase (CDK) inhibitor that suppresses cell proliferation by interfering with cyclin/ CDK2 activity and blocks DNA replication by binding to proliferating cell nuclear antigen (PCNA).19 The p21/ WAF1 protein induces G1 arrest of the cell cycle, in which
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Fig. 3
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Tumour extension, TNM stage, and high p21/WAF1 expression are significantly correlated with poor overall survival in osteosarcoma.
Table 5
Cox multivariate analysis in osteosarcoma
Variables Age Tumour site Tumour size (8 cm vs 58 cm) Histological type Tumour extension Histological grade TNM stage Neoadjuvant chemotherapy Smad2 expression Smad4 expression P-Smad2 expression p21/WAF1 expression
Relative risk ratio
95% confidence limits
P value
0.763 2.746 2.575 0.551 9.410 0.274 0.156 0.413 1.691 2.781 0.303 16.086
0.076–7.689 0.227–33247 0.146–45.288 0.049–6.226 1.018–86.945 0.021–3.521 0.011–2.162 0.038–4.487 0.404–7.088 0.287–26–945 0.015–6.228 1.480–174.783
0.818 0.427 0.518 0.630 0.048* 0.320 0.166 0.467 0.472 0.377 0.439 0.022*
*Significant difference.
action is essential for differentiation and apoptosis.20 Therefore, the cells that have lost the activity to express p21/WAF1 cannot regulate those cell cycles to attain G1 arrest for apoptosis and this may lead to unregulated cell proliferation as a step toward carcinogenesis.21 Some authors reported that p21/WAF1 expression might represent a form of CDK inhibitor dysfunction involved in tumorigenicity.21,22 From these previous opposite suggestions, some possibilities may be raised. One possible explanation of our results is that overexpression of p21/ WAF1 in osteosarcoma might represent CDK inhibitor dysfunction involved in tumorigenesis. This dysfunction of p21/WAF1 protein may lead to unregulated cell proliferation such as tumour extension. The other explanation is that the overexpression of p21/WAF1 controls the abnormal cell-cycle progression and suppresses replication of tumour cells. Under high proliferating tumour states, increased p21/WAF1 is just a reactive event to inhibit cell growth.16 Based on this hypothesis, the overexpression of p21/WAF1 in osteosarcoma might be a reactive response of highly proliferative tumour cells that easily invade to other tissue. Additionally, as chemotherapy impact may influence patient survival, we excluded the 12 cases that underwent neoadjuvant chemotherapy, and performed statistical analysis. In these results, p21/WAF1 expression was also
significantly correlated with tumour extension (p ¼ 0.024, not shown in table). However, p21/WAF1 expression was not significantly correlated with overall survival in the Kaplan-Meier test (p ¼ 0.1232, not shown in table). These findings suggest that p21/WAF1 expression is significantly correlated with tumour extension, regardless of chemotherapy. Although we investigated the relationship between chemotherapy response and four Smad signalling cascades, no significant findings were observed (Table 2). None of the Smad signalling downstream protein components was significantly related with any clinicopathological parameters; however, our results suggest that Smad signalling might play a partial role in osteosarcoma tumorigenesis due to the fact that a positive correlation between Smad2, PSmad2, and Smad4 in osteosarcoma was found. Our study has limitations due to the small number of cases and further studies of Smad signalling in osteosarcoma are needed. These findings will assist in the development of a novel therapeutic modality. In conclusion, Smad signalling in osteosarcoma is partially operative. High p21/WAF1 expression in osteosarcoma is significantly correlated with tumour extension. Our results suggest that overexpression of p21/WAF1 in osteosarcoma might be one of the poor prognostic factors. Acknowledgements: This work was supported by a grant from the Kyung Hee University in 2009 (KHU20090568). Address for correspondence: Dr Y-K. Park, Department of Pathology, Kyung Hee Medical Center, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, 130-702 Seoul, Korea. E-mail: [email protected]
References 1. Miyazawa K, Shinozaki M, Hara T, et al. Two major smad pathways in tgf-beta superfamily signalling. Genes Cells 2002; 7: 1191–204. 2. Ten Dijke P, Goumans MJ, Itoh F, et al. Regulation of cell proliferation by smad proteins. J Cell Physiol 2002; 191: 1–16. 3. Bierie B, Moses HL Tumour microenvironment: TGFbeta: The molecular jekyll and hyde of cancer. Nat Rev Cancer 2006; 6: 506–20. 4. Hahn SA, Schutte M, Hoque AT, et al. Dpc4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996; 271: 350–3. 5. Fukushima T, Mashiko M, Takita K, et al. Mutational analysis of TGF-beta type II receptor, smad2, smad3, smad4, smad6 and
SMAD SIGNALLING IN OSTEOSARCOMA
6. 7.
8.
9.
10.
11.
12.
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 05/12/15 For personal use only.
13.
smad7 genes in colorectal cancer. J Exp Clin Cancer Res 2003; 22: 315– 20. Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 2000; 103: 295–309. de Caestecker MP, Piek E, Roberts AB. Role of transforming growth factor-beta signaling in cancer. J Natl Cancer Inst 2000; 92: 1388–402. Eppert K, Scherer SW, Ozcelik H, et al. Madr2 maps to 18q21 and encodes a TGFbeta-regulated mad-related protein that is functionally mutated in colorectal carcinoma. Cell 1996; 86: 543–52. Wang LH, Kim SH, Lee JH, et al. Inactivation of smad4 tumor suppressor gene during gastric carcinoma progression. Clin Cancer Res 2007; 13: 102–10. Xie W, Mertens JC, Reiss DJ, et al. Alterations of smad signaling in human breast carcinoma are associated with poor outcome: A tissue microarray study. Cancer Res 2002; 62: 497–505. Xie W, Rimm DL, Lin Y, et al. Loss of smad signaling in human colorectal cancer is associated with advanced disease and poor prognosis. Cancer J 2003; 9: 302–12. Shi Y, Massague J Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003; 113: 685–700. Rosen G, Caparros B, Huvos AG, et al. Preoperative chemotherapy for osteogenic sarcoma: Selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 1982; 49: 1221–30.
247
14. Kloth JN, Kenter GG, Spijker HS, et al. Expression of smad2 and smad4 in cervical cancer: Absent nuclear smad4 expression correlates with poor survival. Mod Pathol 2008; 21: 866–75. 15. Piek E, Heldin CH, Ten Dijke P Specificity, diversity, and regulation in TGF-beta superfamily signaling. Faseb J 1999; 13: 2105–24. 16. Kao JT, Chuah SK, Huang CC, et al. P21/waf1 is an independent survival prognostic factor for patients with hepatocellular carcinoma after resection. Liver Int 2007; 27: 772–81. 17. Chen Z, Place RF, Jia ZJ, et al. Antitumor effect of dsrna-induced p21(waf1/cip1) gene activation in human bladder cancer cells. Mol Cancer Ther 2008; 7: 698–703. 18. Zhang CL, Liao WM, Li FB, et al. Prognostic significance of p21(waf1) expression in osteosarcoma. (Chinese.) Zhonghua Bing Li Xue Za Zhi 2005; 34: 524–7. 19. Waga S, Hannon GJ, Beach D, et al. The p21 inhibitor of cyclindependent kinases controls DNA replication by interaction with PCNA. Nature 1994; 369: 574–8. 20. el-Deiry WS, Harper JW, O’Connor PM, et al. Waf1/cip1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994; 54: 1169–74. 21. Kobayashi S, Matsushita K, Saigo K, et al. P21waf1/cip1 messenger RNA expression in hepatitis B, C virus-infected human hepatocellular carcinoma tissues. Cancer 2001; 91: 2096–103. 22. Qin LF, Ng IO. Expression of p27(kip1) and p21(waf1/cip1) in primary hepatocellular carcinoma: Clinicopathologic correlation and survival analysis. Hum Pathol 2001; 32: 778–84.