The association of inhibitor of Growth 4 with its prognostic value in osteogenic sarcoma

Abstract

Background: Osteosarcoma is one of the first two cause of cancer-related death in children and young adolescents. Inhibitor of Growth 4 (ING4) is a member of the ING tumor suppressor family and play an important role in many cellular processes. The purpose of this study was to explore the correlation between ING4 expression and the prognosis of osteosarcoma patients. Methods: ING4 mRNA and protein expressions in osteosarcoma and normal tissues were detected by quantitative real-time transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) method, respectively. Chi-square test was adopted to estimated the relationship of ING4 expression and clinical parameters of osteosarcoma patients. Besides, the overall survival of osteosarcoma patients was evaluated by Kaplan-Meier method. The potential of ING4 as a prognostic marker gene was addressed by Cox regression analysis. Results: Down-regulated expression of ING4 mRNA and protein were observed in osteosarcoma tissues. ING4 expression was significantly associated with metastasis (P = 0.030) and recurrence (P = 0.008), but not other clinical features (P > 0.05). Cox regression analysis indicated that ING4 can be used as an independent prognotic biomarker for osteosarcoma, in univariate and multivariate analysis (P = 0.004, HR = 3.945, 95 % CI = 1.565-9.940; P = 0.001, HR = 4.213, 95 % CI = 1.747-10.161). Conclusion: Taken together, ING4 was down-regulated in osteosarcoma tissues. ING4 can act as an independent prognostic factor for osteosarcoma.

Introduction

Osteosarcoma, the most common primary bone malignancy [1-4], is one of the first two cause of cancer-related death in children and young adolescents [5]. Osteosarcoma is a complex disease. The development of osteosarcoma is a multistep and multifactorial process, implicating many factors [6-8]. Since it was discovered, the therapy method of osteosarcoma was continuously explored. However, the survival rate of the osteosarcoma patients is quite poor. Distant metastases to the lung and the local recurrence are the most common phenomenon after the surgery for osteosarcoma. In order to improve the prognosis of osteosarcoma, many treatment methods were carried out. Recently, molecular target therapy for tumors has been employed in the clinical treatment [9], which will promote and broaden the underlying application prospects of tumor target therapy in future.

Inhibitor of Growth 4 (ING4) is a member of ING family, which comprises 5 members, including ING1, ING2, ING3, ING4 and ING5. ING4 encode by ING4 gene, it is localized at chromosome 12p12-13 region [10] and encodes a 249-amino acid protein containing a highly conserved C-terminal plant homeodomain finger motif (PHD) and 2 nuclear localization signals [11]. It is reported that ING4 play an important role in many cancer-related cellular processes including cell proliferation, apoptosis, migration, angiogenesis, contact inhibition, DNA damage response, and hypoxia [12-19]. Numerous studies have revealed the suppressive role of ING4 in various cancers, including breast cancer, gastric carcinoma, colon cancer, non-small cell lung cancer, ovarian carcinoma, head and neck squamous cell carcinoma, melanoma, hepatocellular carcinoma [20-27]. Although it has been demonstrated that ING4 gene significantly inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cell [28], there were few studies on the correlation between ING4 gene and prognosis of osteosarcoma.

In the present study, we aimed to explore the potential role of ING4 gene in the prognosis of osteosarcoma patients.

Materials and methods

Patients and tissue samples

89 osteosarcoma tissues and adjoining normal tissue samples were obtained from patients who were diagnosed as osteosarcoma in The First Affiliated Hospital of Chongqing Medical University from 2005 to 2009. All patients had not received chemotherapy or radiotherapy prior surgery. A 5-years’ follow-up was conducted and the information was updated via a telephone or questionnaire. The death of the participants was ascertained by a report from the family and verified by the review of public records. The study was approved by the ethics committee of The First Affiliated Hospital of Chongqing Medical University. Written informed consent was obtained from all participants. The samples were quickly frozen in liquid nitrogen and stored at -80°C until use.

RNA preparation, and quantitative real-time transcriptase polymerase chain reaction (qRT-PCR)

Total RNAs were extracted from tumor tissue and paired normal tissue samples using Trizol (Takara, Dalian, China). First-strand cDNA synthesis was performed using the Superscript III kit (Life Technologies, USA). qRT-PCR was performed using a One Step SYBR^® PrimeScript^® RT-PCR Kit (Takara) following the manufacturer’s instruction. Human β-actin was amplified as internal PCR control. Sequences of the primers were as follows: ING4, 5’-TCG TGC TCG TTC CAA AGG-3’ and 5’-GGC AAT AGG TGG GTT CGT T-3’ [29]; human β-actin forward 5’-TGA CGT GGA CAT CCG CAA AG-3’ and reverse 5’-CTG GAA GGT GGA CAG CGA GG-3’ [30]. The real-time PCR was performed with an Applied Biosystems 7900 (Applied Biosystems, USA). The expression level of ING4 was evaluated by comparative cycle threshold (CT) method.

Immunohistochemistry (IHC) analysis

IHC method was utilized to detect the ING4 protein expression in osteosarcoma samples. Immunohistochemistry procedure was performed as described previously [27]. Tumor and normal tissues were cut into 4 μm sections after formalin-fixed, paraffin-embedded blocks. Then, tissue sections were dewaxed at 55°C for 30 min and washed three times with xylene and rehydrated in graded alcohol, followed by two washes in distilled water. Antigen retrieval was performed by heating the samples at 95°C for 30 min in citrate buffer (10 mM, pH = 6). Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide in methanol for 30 min. Blocking with universal blocking serum for 30 min was used to reduce background non-specific staining. Sections were incubated overnight at 4°C with a goat anti-human polyclonal antibody against ING4 (Abcam; 1:200 dilution). Second antibody (rabbit anti-goat antibody; MaiXin Bio) was applied for 45 min at 37°C, and then incubated with DAB (Golden Bridge Int.) to visualize ING4 expression. Formalin-fixed paraffin-embedded human brain tissues, which express ING4 in glial cells, were used as internal positive controls. Negative staining controls were obtained by omitting the primary antibody.

Intensity of cytoplasmic staining was scored as 1 to 4, by comparison to the positive internal controls. Percentage of ING4-positive stained cells was: 1 (0-25%), 2 (26-50%), 3 (51-75%) and 4 (76-100%). This scoring system has been previously validated [31,32]. Diffuse, moderate to strong cytoplasmic staining characterized ING4-positive cells (scores 3 and 4). ING4-negative cells were devoid of any cytoplasmic staining or contained faint, equivocal staining (scores 1 and 2).

Statistical analysis

All statistical analysis were carried out using the software of SPSS version 18.0 for Windows (SPSS Inc, IL, CA, USA). Differential expression of ING4 between osteosarcoma tissues and paired normal tissues was evaluated by paired sample t test. Chi-square test was used to analyze the relationship between ING4 protein expression and the clinicopathological characteristics. Kaplan-Meier analysis was used to estimated the overall survival rate of the osteosarcoma patients with different ING4 expression, differences were calculated using the log rank test. Hazard ratios (HRs) with 95% confidence intervals (95% CI) for the time-to-event endpoint were estimated using the multivariate Cox regression analysis in a forward stepwise method to evaluate the effect of multiple independent prognostic factors on survival outcome. Differences were considered statistically significant when P value < 0.05.

Results

ING4 expression is decreased in osteosarcona

QRT-PCR was used for evaluating ING4 mRNA level in 89 paired tumor and normal tissues. The relative expression of ING4 mRNA was 0.653 ± 0.029 in osteosarcoma tissues and 0.731 ± 0.019 in the paired normal tissues. The difference was significant (Figure 1, P = 0.022). IHC assay was used for evaluating ING4 protein expression, 52.81 % osteosarcoma samples were ING4-positive (47 of 89), that was significantly less than normal samples (67.42%, 60 of 89, P = 0.047, Table 1).

Figure 1.

The relative ING4 mRNA expression level in osteosarcoma and normal tissue.

Table 1.

Expressions of ING4 protein in osteosarcoma and normal tissues

Tissues	Number	ING4 protein expression		X2	P value
		Positive	Negative
Normal	89	60 (67.42%)	29 (32.58%)	/	/
OS	89	47 (52.81%)	42 (47.19%)	3.960	0.047

Correlation between ING4 expression and clinicopathologic features of osteosarcoma

According to the results of IHC, the osteosarcoma cells were divided into two groups: ING4-positive group and ING4-negative group. The IHC data from 89 osteosarcoma specimens were analyzed for the correlation of ING4 levels with clinicopathologic features. The results were summarized in Table 2. Most of the non-metastasis osteosarcoma tissues performed as ING4-positive tissues (62.75%), meanwhile only 39.47% metastasis osteosarcoma tissues were performed as ING4-positive (P = 0.030). About 38.64% local recurrence osteosarcoma tissues were ING4-positive tissues, 66.67% non-recurrence osteosarcoma tissues were performed as ING4-positive tissues (P = 0.008). No significant association was found between ING4 protein expression levels and the following clinical features in the osteosarcoma tissues, such as age, gender, WHO grade and tumor site (all, P > 0.05).

Table 2.

Correlation between ING4 expression and clinicopathologic features of osteosarcoma

Characteristics	Numbe of case	ING4 expression				P value
		Positive (n = 47)	%	Negative (n = 42)	%
Age						0.708
< 20	49	25	51.02	24	48.98
≥ 20	40	22	55.00	18	45.00
Gender						0.822
Male	54	28	51.85	26	48.15
Female	35	19	54.29	16	45.71
Tumor site						0.986
Femur	27	15	55.56	12	44.44
Tibia	23	12	52.17	11	47.83
Humerus	21	11	52.38	10	47.62
Other	18	9	50.00	9	50.00
WHO grade						0.074
Low (I, II )	45	28	62.22	17	37.78
High (III, IV)	44	19	43.18	25	56.82
Metastasis						0.030
Absent	51	32	62.75	19	37.25
Present	38	15	39.47	23	60.23
Recurrence						0.008
Absent	45	30	66.67	15	33.33
Present	44	17	38.64	27	61.36

Association of ING4 expression with prognosis of osteosarcoma patients

Correlation between ING4 protein expression and survival time of the patients with osteosarcoma was evaluated by Kaplan-Meier survival analysis. Mean follow-up period of all patients with osteosarcoma in this study was 38.18 months (range from 3 to 60 months). During the follow-up period, 20 of 42 patients with ING4 negative expression (47.62%) had died, whereas 10 of 47 ING4 positive expression patients (21.28%) had died. The result of Kaplan-Meier survival analysis indicated that patients with ING4-negative expression had worse overall survival than those with ING4-postive expression, log-rank test suggested that the difference was significant between ING4-positive and ING4-negative group (P < 0.001) (Figure 2). Multivariate Cox analysis indicated that ING4 was an independent prognostic factor (Table 3, P = 0.001, HR = 4.213, 95% CI = 1.747-10.161).

Figure 2.

Kaplan-Meier analysis for patients with osteosarcoma based on ING4 expression.

Table 3.

Univariate and multivariate analysis of prognostic factors in osteosarcoma

Characteristics	Univariate analysis			Multivariate analysis
	HR	95% CI	P	HR	95% CI	P
ING4 negative expression	3.945	1.565-9.940	0.004	4.213	1.747-10.161	0.001
Age	1.137	0.506-2.554	0.756	-	-	0.408
Gender	1.268	0.594-2.709	0.539	-	-	0.488
Tumor site	1.432	0.596-3.441	0.422	-	-	0.604
WHO grade	1.251	0.555-2.821	0.589	-	-	0.431
Metastasis	2.283	0.999-5.216	0.050	-	-	0.088
Recurrence	1.998	0.875-4.559	0.100	-	-	0.252

Discussion

Osteosarcoma is a most common malignancy in children and young adolescents. This tumor has a poor prognosis, such as the rapid growth, lower cure rate and easy relapse. Recent years, many investigators devote themselves to looking for an effective therapy method for osteosarcoma, so as to enhance the survival rate of patients. A novel prognosis factor will contribute to solving this issue. ING4 as a novel member of ING family has potential tumor-suppressive effects. ING4 is frequently mutated and its expression is decreased in various types of human cancers [33]. In previous study, the role of overexpression of ING4 in osteosarcoma cell and ING4 expression level in osteosarcoma tissues were studied by Li M et al. [28] However, its prognostic value in osteosarmonain patients was still unclear.

We carried out this study to investigate the correlation between ING4 protein expression level and prognosis of osteosarcoma. QRT-PCR and IHC analysis results indicated that ING4 expression was down-regulated in osteosarmona tissue than that in normal tissue. This findings are consistent with these earlier publications [28]. So ING4 is an tumor suppressor gene. Then, we thought that ING4 might has a potential role in the prognosis of osteosarcoma, and carried out a further study to solve this issue.

In the following work, we explored the correlation of the ING4 protein expression and clinicopathologic characteristics of osteosarcoma patients. The results demonstrated that ING4 protein expression level and distant metastasis of the tumor had an inverse correlation. Meanwhile, ING4 protein expression were significantly lower in osteosarcoma patients with recurrence compared to patients without recurrence. This result was consistent with the previous study in in head and neck squamous cell carcinomas, brain tumour, ovarian carcinoma and lung cancer [10,15,24,29]. The results provided a further evidence to testify that ING4 can be used as a marker to determine the progression of osteosarcoma.

In the present study, correlation between ING4 expression and overall survival of the patients with osteosarcoma was evaluated by Kaplan-Meier survival analysis. Patients with ING4 positive expression had longer survival time and higher survival rate than ING4 negative expression patients. The results showed that ING4 could be a prognostic factor, so we did further Cox regression analysis. The result indicated that ING4 was an independent prognostic factors.

In conclusion, the ING4 can be an independent prognostic marker to predict the unfavorable prognosis of osteosarcoma patients. Although we get a specific result, but our study sample size is too small, so the obtained results may be not accurate. Therefore, further studies with a large sample size are required.

Disclosure of conflict of interest

None.