J Mol Hist (2013) 44:645–652 DOI 10.1007/s10735-013-9511-x

ORIGINAL PAPER

Loss of RUNX3 expression may contribute to poor prognosis in patients with chondrosarcoma Zhe Jin • Ya-Xin Han • Xiao-Rui Han

Received: 7 March 2013 / Accepted: 6 May 2013 / Published online: 12 May 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Chondrosarcoma is the second most common type of bone cancer. Loss of RUNX3 expression has been demonstrated in many other cancers. However, no studies have shown the relationship between RUNX3 expression and chondrosarcoma. In this study, we detected RUNX3 expression in the progression of chondrosarcoma. In patient samples, the levels of RUNX3 mRNA and protein were lower in cancer tissues than in normal tissues. Downregulation of RUNX3 mRNA in tumor tissues was associated with an increase in RUNX3 promoter methylation. Loss of RUNX3 expression was significantly associated with more aggressive chondrosarcoma types and decreased survival time of patients. To examine the effects of exogenous expression of RUNX3 in vitro, chondrosarcoma cells were transfected with the pcDNA3.1-RUNX3 expression vector. Relative to control cells, RUNX3-expressing cells exhibited lower proliferation and higher apoptosis rates as assessed by colony formation and Annexin V-FITC/PI double staining, respectively. Taken together, these results suggest that RUNX3 acts a tumor suppressor in chondrosarcoma and that RUNX3 promoter methylation may be the molecular mechanism for its decreased expression. Keywords Chondrosarcoma  RUNX3  Clinicopathological parameters  Methylation  Apoptosis

Z. Jin (&)  Y.-X. Han  X.-R. Han Department of Orthopaedics, First Affiliated Hospital of China Medical University, Nanjing North Street 155, Heping District, Shenyang 110001, China e-mail: [email protected]

Introduction Chondrosarcoma is a rare but deadly form of bone cancer and is the second most common type of bone cancer following osteosarcoma (Jemal et al. 2011). Chondrosarcomas of the bone are malignant cartilage-forming tumors that are highly resistant to conventional chemotherapy and radiotherapy, giving rise to poor patient outcomes (Johnson et al. 1986; York et al. 1999; Gelderblom et al. 2008; Bovee et al. 2010). Tumor resection is still the only effective treatment for these tumors (Rozeman et al. 2006). Therefore, there is an urgent need for new therapeutic options for those patients with chondrosarcoma. The Runt family of transcription factors consists of three members, RUNX1 (PEBP2aB/CBF2/AML1), RUNX2 (PEBP2aA/CBFA1/AML3), and RUNX3 (PEBP2aC/CBFA3/ AML2). All three RUNX factors play important roles in both normal developmental processes and carcinogenesis of bone (Lund and van Lohuizen 2002; Bai et al. 2013). RUNX3 was identified as a core-binding factor alpha subunit 3 by Levanon et al. (1994) and was first reported as a tumor suppressor gene for gastric cancer (Li et al. 2004a, b). RUNX3 hemizygous deletion and concomitant decrease in RUNX3 expression are also prevalent in hepatocellular carcinoma (Xiao and Liu 2004), pancreatic cancer (Li et al. 2004a, b), giant cell tumor of the bone (Han and Liang 2011), and breast cancer (Lau et al. 2006). Previous studies have demonstrated that restoration of RUNX3 induced cell cycle arrest and apoptosis (Han and Liang 2011; Chi et al. 2005). Aberrant methylation of CpG rich areas (or islands) in or near the promoter region has been associated with transcriptional inactivation of tumor suppressor genes in human cancers (Merlo et al. 1995; Zochbauer-Muller et al. 2001). A reduction in the expression of the RUNX3 gene caused by aberrant methylation has been frequently reported (Han and Liang 2011; Ku et al. 2004; Baylin et al. 2001).

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In this study, we first examined RUNX3 expression in resected specimens from patients with chondrosarcoma. Second, we evaluated the association of RUNX3 expression with clinicopathological information from patients. Finally, we analyzed the anti-tumor activity of RUNX3 expression in a chondrosarcoma cell line, SW1353. Collectively, we provide evidence that RUNX3 has a tumor suppressor function in chondrosarcoma tissues and cells.

Materials and methods Chondrosarcoma patient specimens Tissue specimens were obtained from 63 patients with no chemotherapy or radiotherapy prior to resection at the Department of Orthopaedics, First Affiliated Hospital of China Medical University between September 2001 and September 2011. All patients approved the use of their tumor tissues for clinical research and the China Medical University Ethical Committee approved the research protocols.

Cell lines and cell culture The human chondrosarcoma cell line (SW1353) was obtained from ATCC (Rockville, MD, USA). The cells were maintained at 37 °C in a 5 % CO2 incubator in DMEM/a-MEM medium (Hyclone, Logan, UT, USA) containing 10 % heat-inactivated FBS and 1 % penicillin– streptomycin.

Quantitative real-time PCR Total tissue RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Quantitative real-time PCR analysis was performed on the ABI prism 7,500 sequence detection system (Applied Biosystems, Foster, CA, USA) using the SYBR Green PCR Master mixture (Takara, Dalian, China) and the following specific primers: RUNX3 sense, 50 -GAGTTTCACCCTGACCAT CACTGTG-30 , antisense, 50 -GCCCATCACTGGTCTT GAAGGTTGT-30 ; GAPDH sense, 50 -GAAGGTGAAGGT CGGAGT-30 , antisense, 50 -CATGGGTGGAATCATATT GGAA-30 . The PCR conditions were as follows: one cycle at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Relative quantitation was calculated by the DD Ct method. Each reaction was repeated independently in triplicate.

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Methylation-specific PCR (MSP) Genomic DNA was extracted from chondrosarcoma specimens and cell lines using a TissueGen DNA Kit (CWbiotech, Beijing, China). Genomic DNA (2 lg) was denatured with 0.2 M NaOH followed by the addition of 10 mM hydroquinone (Sigma-Aldrich, Carlsbad, CA, USA) and 3 M sodium-bisulfite (Sigma). The solution was incubated at 55 °C for 16 h. DNA samples were then purified using a WizardDNA purification resin (Promega Biotech, Beijing, China). In this procedure, unmethylated (but not methylated) cytosines are converted to uracil, which are then converted to thymidine during subsequent PCR reactions resulting in sequence differences between methylated and unmethylated DNA. The modified DNA was used as a template both for MSP and unmethylated-specific PCR (USP). The primer sequences for the methylated RUNX3 gene were 50 -TTACGAGGGGCGGTCGTACGCGGG-30 (sense) and 50 -AAAA CGACCGACGCGAACGCCTCC-30 (antisense) and for the unmethylated allele were 50 -TTATGAGGGGTGGTTGTATGTGGG-30 (sense) and 50 -AAAACAACCAACACAAACACCTCC-30 (antisense). The PCR products were separated in a 2 % agarose gel with ethidium bromide and visualized under UV illumination. To examine the effect of demethylation, cells were incubated with medium containing 10 lM 5-aza-20 -deoxycytidine (5-aza-dC, Sigma) for 36 h. Then DNA was isolated and MSP carried out as described above. Transfection and creation of stable cell lines The expression vector, pcDNA3.1-RUNX3, was constructed in our previous study (Han and Liang 2011). SW1353 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Overexpression of exogenous RUNX3 was achieved by transfecting with the pcDNA3.1-RUNX3 expression vector. G418-resistant clones were selected using G418 (450 ng/ll). The stably-expressing RUNX3 positive cell lines were named T1, T2, T3, T4 and T5. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) Total cellular RNA was isolated using an RNeasy Mini Kit (Biomed, Beijing, China). First strand cDNA was reverse transcribed with 1 lg of total RNA, using TaKaRa Reverse Transcription Kit (Takara) and oligo (dT) primers (Takara). Primers specific for RUNX3 and GAPDH were used as described in the previous section. PCR amplification of cDNA was performed in 20 ll mixtures. Amplified products were separated by electrophoresis in 2 % agarose gel

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with ethidium bromide and visualized under UV illumination.

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analysis was performed using CellQuest software (BD Biosciences, Heidelberg, Germany).

Western blot analysis Measurement of caspase-3 and -9 activities Chondrosarcoma specimens and cell lines were lysed in 20 mM Tris–HCl buffer (pH 7.4) containing 150 mM NaCl, 2 mM EDTA, 1 % Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 1 mM Na2MoO4, 10 lM aprotinin and 10 lM leupeptin. The cell lysate was centrifuged at 15,0009g for 5 min, and the supernatant was removed. Equal amounts of cell lysate protein extracts (60 lg) were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose (NC) membrane. The membranes were incubated with anti-RUNX3 (Santa Cruz Biotechnology, sc-23576, Santa Cruz, CA, USA) and anti-b-actin antibodies (Santa Cruz, sc-47778) for 24 h at 4 °C and then incubated with peroxidase-conjugated secondary antibodies for 2 h at room temperature. Chemiluminescent signals were detected using an ECL Kit (Takara).

Caspase activities were measured by colorimetric assay according to the manufacturer’s instructions. After harvesting, cells were washed in ice-cold PBS and lysed; proteins were extracted and stored at -80 °C until use. Cell lysates (20 ll) were added to a buffer containing a p-nitroaniline (pNA)-conjugated substrate (80 ll) for caspase-3 (Ac-DEVD-pNA; KeyGEN, KGA203), or -9 (LEHD-pNA; KeyGEN, KGA402). Incubation was performed at 37 °C with shaking (500 rpm for 1 min) and then at room temperature for 2 h. The released pNA in each well was measured using a plate-reading luminometer (Thermo Scientific, Beijing, China). Data were collected from three independent experiments.

Statistical analysis Immunofluorescence Cells were washed with PBS, fixed in 4 % paraformaldehyde, permeabilized in 1 % Triton X-100 for 15 min at room temperature and blocked with 5 % bovine serum albumin in PBS containing 0.5 % Triton X-100 for 1 h. RUNX3 and His-tag were detected using anti-RUNX3 (Santa Cruz, sc-23576) or anti-His (Santa Cruz, sc-803) antibodies for 1 h at room temperature. Cells were washed with PBS and incubated with the appropriate fluorophoreconjugated secondary antibody, either Alexa FluorÒ 488 Donkey Anti-Goat IgG (H ? L) or Alexa FluorÒ 594 Donkey Anti-Rabbit IgG (H ? L), for 1 h at room temperature, washed with PBS, and mounted using Prolong Anti-fade (Sigma).

All values are presented as the mean ± SEM. The student’s paired t test was used to identify statistical significances. Kaplan–Meier survival plots were generated and comparisons were made with log-rank statistics. The Cox’s proportional hazard model was used to identify significant factors correlated with prognosis in a multivariate analysis. P values less than 0.05 were considered significant. Statistical analyses were performed using SPSS Software for Windows (version 16.0; SPSS, Inc., Chicago, IL, USA).

Results Expression levels of RUNX3 protein and mRNA in chondrosarcoma specimens

Colony formation assay Cells were seeded at 200 cells per well in six-well tissue culture plates. Plates were incubated for 3 weeks in a humidified incubator at 37 °C. 3 weeks after seeding, colonies were stained with 0.05 % crystal violet containing 50 % methanol. The colonies were counted in four to five random fields for each of the duplicate samples using a microscope at 1009 magnification. Measurement of apoptotic cell death Cells were harvested and immunostained with Annexin-V FITC and PI according to the manufacturer’s instructions (Apoptosis Detection Kit; KeyGEN, Nanjing, China). Data

In order to determine the protein expression level of RUNX3 in chondrosarcoma specimens, western blot analysis was performed. RUNX3 protein expression in cancer tissue was significantly lower than in normal tissue (P \ 0.05, Fig. 1a). To determine whether RUNX3 transcript was also reduced, real-time PCR analysis for RUNX3 expression was performed. Results showed that the level of RUNX3 mRNA expression was also decreased in tumor tissues relative to normal tissues (P \ 0.05, Fig. 1b) and corresponded with the observed levels of protein expression. Additionally, immunostaining of tissue sections demonstrated a loss of RUNX3 expression in tumor tissue compared to normal tissue and RUNX3 localization in the cytoplasm (Fig. 1c).

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Fig. 1 a Representative RUNX3 protein expression in paired cancer and corresponding normal tissues by Western blot analysis. b-Actin was used as an internal control. b RUNX3 mRNA expression in specimens using quantitative real-time PCR. GADPH was used as an

internal control. c Representative immunohistochemical staining for RUNX3 protein in tissue specimens. RUNX3 was stained brown with granules localized to the cytoplasm. The nuclei were counterstained with hematoxylin. N normal, C cancer, Neg negative control

RUNX3 promoter methylation analysis

potent demethylating agent, we found that the RUNX3 mRNA expression was restored and methylation disappeared (Fig. 2b).

To investigate the mechanism of RUNX3 down-regulation in chondrosarcoma, the methylation status of RUNX3 was measured by methylation-specific PCR (MSP) in chondrosarcoma samples and cell lines. All patients with downregulated RUNX3 expression had substantial methylation of the RUNX3 promoter region. Representative examples are illustrated in Fig. 2a. A correlation was also observed between RUNX3 promoter methylation and decreased RUNX3 mRNA levels in chondrosarcoma cell lines (Fig. 2b). After the cells were treated with 5-aza-dC, a

Fig. 2 a Methylation-specific PCR (MSP) analysis of CpG island methylation within the RUNX3 promoter region in specimens. PCR products specific for unmethylated (U) and methylated (M) CpG sites were analyzed using electrophoretic separation in a 2 % agarose gel.

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RUNX3 expression and clinicopathological parameters in patients RUNX3 protein was weakly expressed in chondrosarcoma specimens, but highly expressed in normal tissue areas. Based on that observation, we analyzed the potential relationship between the expression of RUNX3 and the

b MSP was used to analyze the CpG island methylation status in chondrosarcoma cells transfected with pcDNA3.1-RUNX3, clones T1–T5. RUNX3 mRNA expression was restored in chondrosarcoma cells after treatment with 5-aza-dC, a demethylating agent

J Mol Hist (2013) 44:645–652 Table 1 Relationship between RUNX3 expression and clinicopathological parameters of patients with chondrosarcoma

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Clinicopathological features

n

RUNX3 expression -

?

??

???

PR (%)

v2

Sex Female

21

13

3

2

3

38.1

Male

42

27

6

5

4

35.7

0.37

0.426

2.76

0.324

5.68

0.257

3.60

0.241

3.52

0.413

11.71

0.047

Age (years) \50

18

10

4

1

3

44.4

C50

45

30

5

6

4

33.3

-

22

15

5

0

2

31.8

?

41

25

4

7

5

39.0

Recurrence

Metastatic ?

17 46

14 26

1 8

1 6

1 6

17.6 43.5

\5 (cm)

24

18

2

1

3

25.0

C5 (cm)

39

22

7

6

4

43.6

P value

Tumor size

Typing

PR positive rate; v2 value, Chi-square distribution

Conventional type

11

6

1

1

3

45.5

I

13

7

3

2

1

46.1

II

15

7

4

2

2

53.3

III

12

10

0

1

1

16.7

Other type

12

10

1

1

0

16.7

Table 2 Multivariate analysis of clinical variables for chondrosarcoma Clinicopathological parameters

Relative risk (95 % CI)

P value

Sex (male)

1.38 (0.92–2.09)

0.402

Age ([50 years)

1.35 (0.90–2.05)

0.428

Differentiation

1.22 (0.81–1.85)

0.291

Metastatic

1.32 (0.88–2.01)

0.278

Typing

0.45 (0.30–0.68)

0.456

Tumor size (C5 cm)

0.34 (0.23–0.52)

0.347

RUNX3 expression (? to ???)

6.23 (4.17–9.47)

0.032

CI confidence interval Fig. 3 Kaplan–Meier curves of the cumulative survival rate of patients with chondrosarcoma based on their RUNX3 expression

clinicopathological characteristics of the sampled patients. The results are summarized in Table 1. No correlation was found with sex, age, recurrence, metastasis and tumor size (P [ 0.05). However, RUNX3 expression was significantly associated with pathological typing of chondrosarcoma (P \ 0.05). Follow-up information was available for 63 patients for periods ranging from 1 month to 5 years (median = 32 months). A Kaplan–Meier analysis showed that RUNX3 expression was closely correlated with the favorable prognosis of patients with chondrosarcoma, whereas negative RUNX3 expression was correlated with a poor prognosis (P \ 0.01, Fig. 3). Cox’s proportional

hazard analysis indicated that RUNX3 is an independent prognostic factor for chondrosarcoma (P \ 0.05, Table 2). RUNX3 expression in SW1353 chondrosarcoma cells We next investigated the consequence effect of exogenous RUNX3 expression in SW1353 cells. SW1353 cells were transfected with the pcDNA3.1-RUNX3 expression vector, and the level of RUNX3 protein and mRNA was measured by Western blot, immunofluorescence, and RT-PCR. As shown in Fig. 4a, b, the results of RT-PCR and Western blot analysis confirmed exogenous expression of RUNX3 in SW1353 cells after transfection. Immunofluorescence

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Fig. 4 Confirmation of exogenous RUNX3 expression in SW1353 cells transfected with pcDNA3.1-RUNX3. T1–T5 each represents a separate clone. a, b RT-PCR and Western blot analysis of RUNX3 mRNA and RUNX3 protein levels in SW1353 cells transfected with pcDNA3.1RUNX3. c Immunofluorescence demonstrating localization of RUNX3 and His-tag in RUNX3-expressing SW1353 cells

analysis showed the localization of RUNX3 and His-tag in RUNX3-expressing SW1353 cells (Fig. 4c). Effect of RUNX3 expression on proliferation and apoptosis in vitro Colony formation assays were performed to detect the antitumor activities of RUNX3 in SW1353 cells. The growth curves obtained demonstrate that RUNX3 inhibits the growth of SW1353 cells (Fig. 5a). Next, the ratio of apoptotic cells present was determined using flow cytometry. As shown in Fig. 5b, the percentage of apoptotic SW1353 cells in the control, untransfected group was 0.34 ± 0.07 %, whereas the percentage of apoptotic SW1353 cells in the pcDNA3.1-RUNX3-transfected group (T1 clone) was 2.31 ± 0.12 % (P \ 0.05). Results using the T2, T3, and T4 clones were comparable to the results using the T1 clone (Fig. 5b). Furthermore, the activities of caspase-3 and -9 were significantly increased in cells transfected with pcDNA3.1-RUNX3 compared to untreated cells (P \ 0.05, Fig. 5c), indicating a high rate of apoptosis with RUNX3 expression.

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Discussion In our previous study, we confirmed that the levels of RUNX3 protein and mRNA were lower in cancer tissues than in normal tissues of giant cell tumor of the bone (GCTB) specimens (Han and Liang 2011). Other reports have indicated that RUNX3 expression is decreased by about 45–60 % in human gastric cancer (Li et al. 2004a, b). Consistent with previous studies, we report the down-regulation of RUNX3 expression in chondrosarcoma tissues. The methylation status of the RUNX3 promoter appears to play a major role in the degree to which it is expressed. Little or no expression of RUNX3 due to CpG island hypermethylation has been observed in GCTB, gastric cancer, lung cancer, breast cancer, and prostate cancer (Li et al. 2004a, b; Han and Liang 2011; Yanagawa et al. 2007; Kim et al. 2004; Sato et al. 2006). We confirmed an association between decreased RUNX3 expression and methylation of the RUNX3 promoter region in both chondrosarcoma primary tissues and in an established cell line. RUNX3 methylation status has also been found to be an

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Fig. 5 Analysis of proliferation and apoptosis in RUNX3-overexpressing cells. a The proliferation ratio was determined by colony formation assay. RUNX3 expression showed an inhibitory effect on cell growth (P \ 0.05). b The apoptosis ratio was analyzed by double-

staining with Annexin-V/PI. c The activity of caspase-3 and -9 was detected in each group (P \ 0.05). Control: untransfected SW1353 cells, T1–5: SW1353 cells transfected with pcDNA3.1-RUNX3

independent prognostic factor for lung cancer (Yanagawa et al. 2007). To our knowledge, little evidence exists showing the relationship between RUNX3 expression and clinicopathological characteristics of chondrosarcoma patients. In this study, we found an inverse relationship between RUNX3 expression and the aggressiveness of the chondrosarcoma tumor. In addition, we also found that the survival time of patients with higher levels of RUNX3 expression was significantly longer than patients who lacked RUNX3 expression. These results are consistent with previous studies of RUNX3 expression and survival in other tumor types. In a previous report, reduced expression of RUNX3 was associated with significantly decreased survival in patients with gastric cancer (Wei et al. 2005). Araki et al. (2005) reported that decreased expression of RUNX3 was an indicator of poor survival in pulmonary alveolar carcinoma. We also confirmed RUNX3 was an independent prognostic factor for chondrosarcoma. Our results are consistent with other studies and suggest that RUNX3 is functioning as a tumor suppressor. To confirm the anti-tumor activities of RUNX3 in chondrosarcoma cells in vitro, we overexpressed RUNX3 in the chondrosarcoma cell line, SW1353, by transfecting with pcDNA3.1-RUNX3. Our results indicate that expression of RUNX3 inhibits proliferation and enhances apoptosis in chondrosarcoma cells. Similarly, Chi et al. (2005) has

shown that RUNX3 overexpression inhibited proliferation and induced cell cycle arrest in gastric cancer cell lines. Lee et al. (2011) and Yamamura et al. (2006) also confirmed a tumor suppressive role of RUNX3 in colorectal cancer cell lines and neuroblastoma cell lines, respectively. In our previous study, we found that RUNX3 inhibited cell growth and induced apoptosis in GCTB cell lines (Han and Liang 2011). Taken together, these results indicate that loss of RUNX3 expression in chondrosarcoma may contribute to tumorigenesis given its anti-proliferative properties. In conclusion, this study provides evidence that RUNX3 acts as a suppressor gene in chondrosarcomas. Loss of RUNX3 expression was detected in both chondrosarcoma tissues and in an established cell line. Furthermore, the RUNX3 promoter was heavily methylated in cancer tissues but not in normal tissues, suggesting that hypermethylation is the prominent silencing mechanism. Most importantly, down-regulation of RUNX3 was associated with a significantly decreased survival rate in patients with chondrosarcoma. These results indicate that inactivation of the RUNX3 gene plays an important role in the pathogenesis of chondrosarcoma. Acknowledgments assistance. Conflict of interest interest to declare.

We thank Dr. Miao Yu for her technical

The authors have no financial conflicts of

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