DNA AND CELL BIOLOGY Volume 33, Number 11, 2014 ª Mary Ann Liebert, Inc. Pp. 1–5 DOI: 10.1089/dna.2014.2527

ORIGINAL RESEARCH ARTICLE

Transforming Growth Factor-Beta Polymorphisms and Serum Level in the Development of Osteosarcoma Shaogang Xu,1,* Shufeng Yang,2,* Guojing Sun,3 Wanxin Huang,1 and Yelong Zhang1

The transforming growth factor-beta (TGF-b) signaling pathway plays critical roles in the development of various diseases. The current study investigated whether TGF-b was involved in the pathogenesis of osteosarcoma from the genetic polymorphism perspective and serum level perspective. We first examined two TGFb1 polymorphisms, rs1800469C/T and rs1800470T/C, in 202 osteosarcoma patients and 216 healthy controls. Data revealed that the prevalence of rs1800470TT genotype and T allele was significantly elevated in patients than in controls (odds ratio [OR] = 2.28, 95% confidence interval [CI]: 1.30–3.98, p < 0.001, and OR = 1.49, 95% CI: 1.14–1.96, p < 0.001). Function analyses showed that healthy controls carrying rs1800470TT genotype had a significantly higher serum level of TGF-b than those carrying the rs1800470CC genotype (191.1 – 15.7 pg/mL vs. 129.4 – 10.9 pg/mL, p = 0.003). We then compared the serum level of TGF-b between osteosarcoma patients and healthy controls. Results demonstrated a significantly increased serum level of TGF-b in patients than in controls. Further analyses identified that patients with metastasis had augmented levels of serum TGF-b than those without metastasis. These data indicate that TGF-b may be closely involved in the pathogenesis of osteosarcoma.

stasis in an autocrine manner (Izumchenko et al., 2014). Conversely, TGF-b has also emerged as a key player in the maintenance of self-renewal and stemness, ensuring that embryonic and somatic stem cells retain their undifferentiated state (Alcaraz et al., 2014; Fouad et al., 2014). Studies have shown that TGF-b may play critical roles in bone tissues. In general, TGF-b is growth stimulatory for mesenchymal cells such as osteoblasts (Tu et al., 2014a). Bone cells produce large amounts of TGF-b, and the bone matrix is the major reservoir for TGF-b storage in the body (Schedlich et al., 2013). Osteosarcomas arise from cells that are predominantly osteoblastic in origin, and several lines of evidence suggest a role for TGF-b in the malignant progression of osteosarcomas (Lv et al., 2014). TGF-b signals through transmembrane serine/threonine kinase receptors comprising the TGF-b type II receptor (TbRII) and TGF-b type I receptors (TbRIs) (Chen et al., 2013). Activated TbRI phosphorylates the receptor-activated Smads, which include Smad2 and Smad3, on serine residues in the carboxy-terminal SSXS motif (Tu et al., 2014a). Single-nucleotide polymorphisms (SNPs) can affect the development of different diseases (Bai et al., 2013; Cha et al., 2014). However, no literature reported TGF-b polymorphism with susceptibility to osteosarcoma. In addition, correlations between the serum level of TGF-b and the progression of the cancer remain elusive. In the current study, we investigated

Introduction

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steosarcoma is the most common pediatric bone malignancy in many countries such as China and the United States (Botter et al., 2014). Fifty years ago, surgery was the only available treatment and survival was abysmal at less than 20% (Nugent, 2014). Treatment of patients with osteosarcoma was transformed in the 1980s and 1990s with advances in chemotherapy and orthopedic surgical techniques, leading to long-term survival rates that now approach 70% (Luetke et al., 2014). Unfortunately, further increase in survival has not occurred in the last decade despite multiple attempts at intensification of chemotherapy and trials utilizing new agents (Luetke et al., 2014). Therefore, it is necessary to understand the natural history and biology of osteosarcoma to improve our therapeutic approaches (Liu et al., 2012). Transforming growth factor-beta (TGF-b) is one of the most abundant cytokines in the tumor microenvironment (Tu et al., 2014b). Three alternative TGF-b isoforms exist in mammals: TGF-b1, TGF-b2, and TGF-b3. TGF-b1 has become the focal point for many mechanistic studies in vitro and in vivo because it is more abundant than TGF-b2 or TGF-b3 (Matsuda et al., 2014). The precise effect of TGF-b on tumors is not well understood; it can act as both a tumor suppressor and a tumor promoter ( Jiang et al., 2014). Recent studies have shown that TGF-b promotes tumor progression and meta1

Department of Orthopedics, Zhengzhou Orthopedics Hospital, Zhengzhou, Henan, China. Department of Orthopedics, The 81st Hospital of PLA, Nanjing, Jiangsu, China. Department of Orthopedics, Jinling Hospital, Nanjing, Jiangsu, China. *These authors contributed equally to the work.

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two TGF-b1 polymorphisms, rs1800469C/T and rs1800470T/ C, with the risk of osteosarcoma and assessed the effects of these SNPs on gene expression. We further examined the serum level of TGF-b in the development of osteosarcoma. Materials and Methods Study population

The study population consisted of 202 osteosarcoma patients (10–67 years of age) and 216 healthy controls (12–70 years of age) recruited from Zhengzhou Orthopedics Hospital. The diagnosis of osteosarcoma was established by histological examination in all cases. This study included 137 high-grade, 60 low-grade, and 5 intermediate-grade osteosarcomas. These intermediate-grade tumors are treated as if they are low-grade osteosarcomas. Therefore, we put them under the low-grade category. In the same period, subjects who underwent regular physical examinations at the same hospital were recruited as controls. Relatives of study participants were excluded from this study. Those with a history of familial cancer syndromes were also excluded from this study. All the control subjects were matched with the patient population in terms of age, sex, and residence area (urban or rural). All subjects were of Han Chinese ethnicity and were unrelated to each other. Each study participant provided a peripheral blood sample. The study was approved by the Review Board of Zhengzhou Orthopedics Hospital. Written informed consent was obtained from each participant or guardians on behalf of the children participants involved in the study. SNP genotyping

Genomic DNA was extracted from a 5-mL peripheral blood sample, which was obtained from patients before they were given chemotherapy or radiotherapy, to avoid any influence of these treatments on the outcomes. Genotypes were analyzed using PCR-based methods, as described below (Hu et al., 2012). The genotypes of rs1800469C/T and rs1800470T/C were analyzed by the PCR-restriction frag-

Table 1. General Characteristics of the Subjects Characteristics Age £ 20 > 20 Gender Male Female Tumor location Long tubular bones Axial skeleton Metastasis With Without Grade Low High Pathological fracture Present Absent

Osteosarcoma Control (n = 202) (%) (n = 216) (%) p-Value > 0.05 137 (67.8) 65 (32.2)

150 (69.4) 66 (30.6)

125 (61.9) 77 (38.1)

127 (58.8) 89 (41.2)

142 (70.3) 60 (29.7)

— —

53 (26.2) 149 (73.8)

— —

65 (32.2) 137 (67.8)

— —

34 (16.8) 168 (83.2)

— —

> 0.05

ment length polymorphism method on the basis of that reported previously. The primers used for rs1800469C/T were F, 5¢-CAG ACT CTA GAG ACT GTC AG-3¢ and R, 5¢-GTC ACC AGA GA A AGA GGA C-3¢. The primers used for rs1800470T/C were F, 5¢-CCT CCC CAC CAC ACC AG-3¢ and R, 5¢-CGG CAC CTC CCC CTG GCT CG-3¢. Amplification was accomplished with a 25-mL reaction mixture containing 100 ng template DNA, 0.5 mM each primer, 0.2 mM each dNTP, 1.5 mM MgCl2, and 1.2 U of Taq DNA polymerase with a 1· reaction buffer (Promega). The PCR profile consisted of an initial melting step of 2 min at 94C, followed by 35 cycles of 30 s at 94C, 30 s at 56C, 30 s at 72C, and a final elongation step of 7 min at 72C. The PCR products were then subject to the digestion with Bsu36I (rs1800469C/T) and

Table 2. The Distribution of Transforming Growth Factor Beta-1 Single-Nucleotide Polymorphisms Between Patients and Controls Polymorphisms rs1800469C/T Genotype CC CT TT Allele C T rs1800470T/C Genotype TT TC CC Allele T C

Patients (n = 202) (%)

Controls (n = 216) (%)

OR (95% CI)

p-Value

60 (29.7) 104 (51.5) 38 (18.8)

69 (31.9) 109 (50.5) 38 (17.6)

1.00 1.10 (0.71–1.71) 1.15 (0.65–2.03)

0.678 0.629

224 (55.4) 180 (44.6)

247 (57.2) 185 (42.8)

1.00 1.07 (0.82–1.41)

0.614

42 (20.8) 102 (50.5) 58 (28.7)

66 (30.6) 110 (50.9) 40 (18.5)

1.00 1.46 (0.91–2.34) 2.28 (1.30–3.98)

0.117 0.05) between patients and healthy controls. Genotype and allele frequencies of the TGF-b1 rs1800469C/ T and rs1800470T/C polymorphisms are summarized in Table 2. The genotype distributions of these two polymorphisms among the controls were in agreement with the Hardy– Weinberg equilibrium ( p > 0.05). As for the rs1800469C/T SNP, frequencies of C allele versus T allele were 57.2% versus 42.8% in controls and 55.4% versus 44.6% in patients ( p = 0.614). In addition, percentages of the polymorphic CT genotype and TT genotype did not observe any significant changes in patients compared with controls (Table 2). As for the rs1800470T/C polymorphism, frequencies of T allele versus C allele were 56.0% versus 44.0% in controls and 46.0% versus 54.0% in patients (OR = 1.49, 95% CI: 1.14–1.96, p < 0.001). Prevalence of the rs1800470CC genotype was significantly higher in patients than in controls (OR = 2.28, 95% CI: 1.30–3.89, p < 0.001). In addition, we analyzed the linkage disequilibrium of the two SNPs, and data showed no linkage between these two polymorphisms (D¢ < 0.1). These results suggested that TGF-b1 rs1800470T/C polymorphism was associated with an increased susceptibility to osteosarcoma in the Chinese population. Comparison of TGF-b polymorphisms on protein expression

To learn the mechanism of the association between TGF-b polymorphism and the increased risk of osteosarcoma, we analyzed the effect of rs1800469C/T and rs1800470T/C polymorphisms on the serum level of TGF-b in healthy controls. As for the rs1800469C/T SNP, 20 healthy subjects car-

FIG. 1. The serum level of transforming growth factorbeta (TGF-b) in healthy subjects carrying different genotypes of (A) rs1800469C/T polymorphism or (B) rs1800470T/C polymorphism.

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FIG. 2. (A) The serum level of TGF-b between osteosarcoma patients and controls. (B) The serum level of TGF-b in osteosarcoma patients with different tumor locations. (C) The serum level of TGF-b in osteosarcoma patients with different metastatic status. (D) The serum level of TGF-b in osteosarcoma patients with different tumor grades.

Fig. 1B). These results indicated that the rs1800470T/C polymorphism could increase the TGF-b serum level in healthy subjects. Serum level of TGF-b in patients and controls

To understand the effect of TGF-b on the development of osteosarcoma, we compared the serum level of TGF-b in patients and controls (Fig. 2A). A total of 60 patients and 60 controls were included. Since rs1800470T/C SNP may affect the TGF-b expression, subjects carrying this polymorphism were excluded. As shown in Figure 2A, an increased TGF-b expression was observed in osteosarcoma patients (151.3 – 11.4 pg/ mL) than in healthy controls (124.5 – 4.4 pg/mL) ( p = 0.031). To learn whether the TGF-b level is correlated with the progression of the disease, we analyzed serum TGF-b in patients with different locations (Fig. 2B), metastatic status (Fig. 2C), and grade status (Fig. 2D). Data showed that the serum TGF-b level was not greatly altered between patients with different tumor locations ( p = 0.918). However, patients with metastatic tumors presented a significantly higher TGF-b level than those without metastasis (216.5 – 24.2 pg/mL vs. 129.6 – 11.3 pg/mL, p < 0.001, Fig. 2C). The serum level of TGF-b did not reveal a significant difference between patients with high-grade and lowgrade osteosarcomas (136.6 – 15.3 pg/mL vs. 159.8 – 15.7 pg/ mL, p = 0.333, Fig. 2D). These results indicated that serum TGFb may play roles in the progression of osteosarcoma. Discussion

Factors responsible for the pathogenesis of osteosarcoma remain unclear. The current study investigated the effect of TGF-b on osteosarcoma from various angles. We first identified that TGF-b rs1800470T/C polymorphism could affect the susceptibility to osteosarcoma by increasing protein expression. Furthermore, we found that the serum TGF-b level was elevated in osteosarcoma patients and associated with metastasis. This study demonstrated that TGF-b may play critical roles in the development and progression of osteosarcoma.

Several SNPs have been identified in the TGF-b1 gene, in which rs1800469C/T and rs1800470T/C have been widely researched. The rs1800469C/T polymorphism, generally known as -509C/T, is located in the promoter region of TGF-b. Cruz et al. (2013) reported that the TGF-b1 rs1800469 SNP was significantly associated with increased risk of silent myocardial ischemia as compared to controls under dominant and additive models. Yuan et al. (2013) presented that rs1800469 could be useful for predicting distant metastasis-free survival among patients with nonsmall cell lung cancer treated with definitive radiation therapy. Hu et al. suggested that rs1800469T allele carriers were associated with a significantly reduced risk of nasopharyngeal carcinoma as compared with the noncarriers. However, some other studies reported no associations between this polymorphism and atrial fibrillation (Zheng et al., 2013) or late radiotherapy toxicity (Zhu et al., 2013). Our data did not show a significant association between different genotypes of rs1800469 and susceptibility to osteosarcoma in the Chinese population (Table 2). The function of the rs1800469 polymorphism remains elusive. The SNP was generally not thought to be part of a known consensus sequence for a promoter regulatory element and therefore may not affect TGF-b expression. However, the study showed that rs1800469CC genotype carriers had a higher TGF-b1 mRNA level than the TT genotype carriers did (Zheng et al., 2013). We found that serum levels of TGF-b were not significantly different among healthy subjects carrying TT, TC, or CC genotypes (Fig. 1A). Further research on the function of this polymorphism is required. The rs1800470T/C, generally known as + 869T/C, is a nonsynonymous mutation, which changes Leu to Pro. Frydecka et al. (2013) reported that the polymorphism could affect susceptibility to schizophrenia. Almeida et al. (2013) presented that the C allele and CC genotype of the rs1800470 SNP were both significantly associated with Crohn’s disease. We identified a strong association between this SNP and osteosarcoma (Table 2). The rs1800470 SNP is located in the signal peptide and may alter the production of TGF-b. Our

TGF-b AND OSTEOSARCOMA

data also showed that subjects with rs1800470CC genotype had elevated serum levels of TGF-b than those with the TT genotype, confirming the function of this polymorphism on gene expression from another angle. In summary, our study identified that the TGF-b polymorphism could affect the susceptibility to osteosarcoma by increasing TGF-b expression and found that the serum TGFb level was elevated in osteosarcoma patients, especially in those with metastasis. This research sheds light on understanding the pathogenesis of osteosarcoma and provides therapeutic strategies in bone cancers. Acknowledgment

This work was supported by the Nanjing Science and Technology Development Program (No. 201303039). Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Yelong Zhang, MD Department of Orthopedics Zhengzhou Orthopedics Hospital 58 Longhai Middle Road Zhengzhou 450052 Henan China E-mail: [email protected] Received for publication May 19, 2014; received in revised form June 20, 2014; accepted June 24, 2014.

Transforming growth factor-beta polymorphisms and serum level in the development of osteosarcoma.

The transforming growth factor-beta (TGF-β) signaling pathway plays critical roles in the development of various diseases. The current study investiga...
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