Abstract
Objectives
To investigate insulin-like growth factor binding protein 7 (IGFBP7) mRNA levels in human glioma and normal brain tissue, and to determine their clinical significance.
Methods
In this retrospective study, IGFBP7 mRNA was quantified by real-time reverse transcription–polymerase chain reaction in brain tissue samples from patients with glioma and normal control subjects. Kaplan–Meier and Cox proportional hazards analyses were performed to determine any clinical and prognostic associations.
Results
IGFBP7 mRNA levels were significantly lower in glioma tissue (n = 120) than in normal brain tissue (n = 20). Low (i.e. below the median, 5.9) IGFBP7 mRNA levels were significantly associated with larger tumour size (≥5 cm, compared with <5 cm, diameter). Patients with high (above median) IGFBP7 had longer overall survival than those with low IGFBP7. Tumour grade and IGFBP7 mRNA level were independent predictors of overall survival.
Conclusions
IGFBP7 downregulation is associated with poor prognosis in glioma, and this molecule may represent both a prognostic marker and a potential therapeutic target.
Introduction
Gliomas are the most common primary tumours of the central nervous system. Around 70% of gliomas are malignant, and these have high recurrence and mortality rates. 1 Despite advances in diagnosis and therapeutic strategies, glioma remains one of the deadliest human cancers: the median survival time of patients with glioblastoma (the most aggressive form of glioma) is only 12 months. 1 Early diagnosis and improving survival in these patients remain challenges for neuro-oncologists. Prognostic factors for glioma patients include age, preoperative duration of symptoms, Karnofsky performance status (KPS) score, 2 histological grade, tumour necrosis, extent of surgical resection, use of postoperative radiation therapy and adjuvant chemotherapy.3,4 These clinical parameters do not completely account for the observed variations in survival rates, however.5–7 Biomarkers are therefore required in order to stratify patients into different risk categories and allow more specific treatment regimens to be developed and used.
Insulin and insulin-like growth factors (IGFs) are key regulators of energy metabolism and growth, with important roles in neoplasia. 8 The IGF axis comprises two growth factors (IGF-I and IGF-II), their receptors (IGF-IR and IGF-IIR) and a group of insulin-like growth factor-binding proteins (IGFBPs). 9 The 16 members of the IGFBP superfamily bind to and sequester IGFs, thereby regulating their bioavailability. 9 IGFBP7 differs from other members of the IGFBP family as it has 100-times lower affinity for IGF-I than other IGFBPs. 10 The functions of IGFBP7 include regulation of cell proliferation and differentiation, and induction of apoptosis and cellular senescence. 10 Downregulation of IGFBP7 is associated with poor postoperative survival in various tumour types, including colorectal, 11 breast 12 and liver 13 cancers, suggesting that IGFBP7 acts as a tumour suppressor with an important role in the development and progression of malignancies. To our knowledge, there are no data available on the association between IGFBP7 expression and prognosis in human glioma.
The current study retrospectively examined IGFBP7 mRNA levels in glioma and normal brain tissue via real-time reverse transcription–polymerase chain reaction (RT–PCR). In addition, the relationship between IGFBP7 expression and prognosis in patients with glioma was assessed.
Patients and methods
Study population
This retrospective study recruited patients undergoing surgical resection of glioma at the Department of Neurosurgery, Xinxiang Central Hospital, Xinxiang, China, between January 2002 and December 2012. Inclusion criteria were: (i) primary diagnosis of glioma with no history of other tumours; (ii) complete clinical data available including: age, sex, clinical manifestations, mean tumour diameter (MTD, defined as the geometric mean of the three diameters on magnetic resonance scan), extent of resection, tumour stage and adjuvant therapy; (iii) tissue sample of sufficient quality for experimental use. Normal control brain tissue samples were obtained from trauma patients undergoing partial resection of normal brain as decompression treatment for severe head injury; these patients were treated at the Department of Neurosurgery, Xinxiang Central Hospital, between January 2011 and November 2011.
Tumours were classified by two independent pathologists, according to World Health Organization (WHO) criteria for classification of central nervous system tumours. 14 A 5-year follow-up was performed and overall survival was calculated from date of initial surgery to death. Patients who died as a result of accidents or conditions unrelated to glioma were censored.
The study protocol and acquisition of tissue specimens were approved by the Medical Ethics Committee of Xinxiang Central Hospital, Xinxiang, China. All experimental procedures were performed in accordance with the Declaration of Helsinki. All study participants or their legal surrogates provided written informed consent to surgical procedures and the use of resected tissue specimens.
RNA extraction and RT–PCR
Tissue was frozen immediately after resection and stored in liquid nitrogen. Total RNA was extracted using TRIzol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, and 2-µl aliquots were reverse transcribed with M-MLV Reverse Transcriptase (Promega, Madison, WI, USA), also according to the manufacturer’s instructions. IGFBP7 and glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA were amplified by quantitative real-time PCR using the following primers: IGFBP7, forward 5′-TGCGGCTGCTGCCCTATGTG-3′ and reverse 5′-CTGCTGCCCCGGCTTTACCC-3′; GAPDH, forward 5′-CTCCTCCTGTTCGACAGTCAGC-3′ and reverse 5′-CCCAATACGACCAAATCCGTT-3′. The PCR reaction mix comprised 0.5 μl cDNA, 7.5 μl 2 × SYBR® Green master mix (Invitrogen) and 200 nM primer, in a total volume of 15 µl. Amplification was performed using an ABI 7900HT real-time PCR system (Life Technologies, Carlsbad, CA, USA). The cycling conditions were initial denaturation at 95℃ for 10 min followed by 45 cycles of 95℃ for 30 s and 60℃ for 1 min. The resolution curve was measured at 95℃ for 15 s, 60℃ for 15 s and 95℃ for 15 s. The threshold cycle (CT) of each sample was determined, and the relative expression of IGFBP7 mRNA was normalized to the GAPDH value using the 2−ΔΔ
C
Statistical analyses
Data were presented as median ± SD or n (%), unless otherwise indicated. Differences in mRNA levels between cancerous and normal brain tissue were evaluated with Mann–Whitney U-test; χ2-test was used to analyse the relationships between IGFBP7 mRNA level and clinicopathological parameters. Survival was calculated by the Kaplan–Meier method and compared using a log-rank test. Univariate and multivariate Cox proportional hazards regression models were used to calculate the hazard ratio (HR) and 95% confidence intervals (CI), for the effects of clinicopathological parameters and IGFBP7 mRNA level on survival. P-values < 0.05 were considered statistically significant. All statistical tests were two-sided. Statistical analyses were performed using SPSS® version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows®.
Results
Demographic and clinicopathological characteristics of patients with glioma, and observed associations between high or low insulin-like growth factor binding protein 7 (IGFBP7) mRNA levels in tumour tissues.
Data presented as n (%).
KPS, Karnofsky performance status; IICP, increased intracranial pressure; WHO, World Health Organization; NS, not statistically significant (P ≥ 0.05; χ2-test).
Low <median value; high ≥median value. Median level in patients was 5.9.
Median patient age, 43 years.
Median ± SD levels of IGFBP7 mRNA were significantly lower in glioma tissue than in normal brain tissue (5.9 ± 4.5 versus 35.2 ± 14.3; P < 0.001). Low IGFBP7 mRNA levels were significantly associated with larger mean tumour size (≥5 cm MTD; P = 0.032. Table 1). There were no significant associations between IGFBP7 mRNA level and any other clinicopathological characteristic, including age, sex, KPS, increased intracranial pressure (IICP) or WHO grade.
Figure 1 shows the results of Kaplan–Meier survival analysis of patients with glioma, stratified according to the median IGFBP7 mRNA level. Patients with low IGFBP7 mRNA levels had significantly shorter overall survival duration than those with high IGFBP7 mRNA levels (P = 0.025).
Kaplan–Meier survival analysis of patients with glioma, stratified according to insulin-like growth factor binding protein 7 (IGFBP7) mRNA levels. Patients with low (<median [i.e., 5.9]) IGFBP7 levels (n = 60) had significantly shorter overall survival duration than those with high (≥median) levels (n = 60).
Univariate and multivariate Cox regression analyses of factors affecting overall survival in patients with glioma (n = 120).
KPS, Karnofsky performance status; IICP, Increased intracranial pressure; WHO, World Health Organization; IGFBP7, insulin-like growth factor binding protein 7.
Discussion
Although IGFBP7 has been found to have tumour suppressor functions in a number of malignancies, little is known about its role in glioma. Since the prognosis of patients with glioma is extremely poor, there is an urgent need for specific and effective prognostic markers. These would allow the development of individualized treatment plans targeting specific pathophysiological and molecular tumour properties. The current study investigated IGFBP7 mRNA levels in human glioma and normal brain tissue samples, and found that IGFBP7 mRNA levels were lower in glioma than in normal tissue. In addition, low IGFBP7 mRNA levels (i.e. below the median of 5.9) were significantly associated with larger tumour size and reduced postoperative survival time. To the authors’ knowledge, this is the first study to examine the expression and clinical significance of IGFBP7 in glioma.
Decreased IGFBP7 expression occurs in a number of malignancies including melanoma, 10 colorectal cancer (CRC), 11 breast cancer, 12 hepatocellular carcinoma (HCC), 13 nonsmall cell lung cancer (NSCLC), 16 pancreatic ductal adenocarcinoma 17 and acute leukaemia. 18 In accordance with these findings, levels of IGFBP7 mRNA were significantly lower in patients with glioma than in normal controls, in the present study. The relatively small number of control subjects, however, limited the value of these data; further studies with larger control groups are required in order to confirm the findings.
The molecular mechanisms of IGFBP7 downregulation in cancer remain unclear, but may include promoter methylation. The IGFBP7 promoter has been found to be hypermethylated in NSCLC, 19 CRC 20 and leukaemia, 18 resulting in reduced IGFBP7 expression in these malignancies. In addition, 5-aza-2′-deoxycytidine treatment (a demethylating agent), may restore IGFBP7 expression in a gastric cancer cell line. 21 These data collectively suggest the important role of promoter methylation in the regulation of IGFBP7 expression. The IGFBP7 promoter methylation status in glioma warrants further investigation.
Low IGFBP7 mRNA levels were associated with larger tumour size in the current study, suggesting that downregulation may facilitate the growth and invasion of glioma cells. In accordance with these findings, IGFBP7 downregulation has been positively correlated with the progression of HCC, 13 NSCLC 16 or CRC. 22 IGFBP7 is involved in various cellular processes through its regulatory role in IGF-I signalling. 23 Ectopic IGFBP7 expression in cancer cells induces cell senescence and apoptosis by inhibition of AKT1/SMARCB1/SNF5 and BRAF/MPA2K1/MPARK1 signalling pathways.24,25 In addition, IGFBP7 has been found to inhibit angiogenesis, by suppressing vascular endothelial growth factor and inducing apoptosis, in human umbilical vein endothelial cells 26 and a mouse melanoma model. 27 Exogenous IGFBP7 repressed glioma cell proliferation via cellular senescence and vessel stabilization in the glioblastoma cell lines U87MG and T98G. 28 Taken together with the findings of the current study, these data suggest that IGFBP7 may exert its tumour suppressing effects both directly by the inhibition of cell proliferation, and indirectly by the inhibition of angiogenesis.
Downregulation of IGFBP7 expression has been associated with poor prognosis in NSCLC, 16 breast cancer, 12 HCC, 13 leukaemia 18 and pancreatic ductal adenocarcinoma. 17 In accordance with these findings, the present study found that low IGFBP7 mRNA levels were associated with shorter survival duration in patients with glioma. In addition, low IGFBP7 mRNA was an independent risk factor for glioma survival in the current study, suggesting that IGFBP7 may serve as a prognostic biomarker for glioma patients after surgical resection. The impact of IGFBP7 expression on cancer survival may stem from its direct tumour-suppressive effects, as well as indirect effects via inhibition of angiogenesis. In mouse models, tumour cells with higher IGFBP7 expression exhibited slower growth and lower vascular density than those with lower IGFBP7 levels.27,29 In addition, IGFBP7 expression may be involved in the sensitivity of cancer cells to anticancer therapeutics. It has been reported that IGFBP7 transcription was decreased in cisplatin-resistant human NSCLC cancer cell lines and xenografts, and small interfering RNA targeting IGFBP7 increased cellular resistance to cisplatin. 30 Others have shown that short hairpin RNA directed against IGFBP7 increased resistance to interferon-α/5-fluorouracil treatment, and IGFBP7 transfection restored treatment sensitivity in HCC. 31 Intravenous recombinant IGFBP7 treatment has shown good tumour-suppressive effects in mouse models of several cancers, including glioblastoma.28,32 The potential anticancer effects of IGFBP7 in glioma therefore warrant further investigation.
In conclusion, the present study reports that decreased levels of IGFBP7 mRNA are found in human glioma compared with normal brain. Our findings reveal a correlation between reduced IGFBP7 levels and less-favourable clinical outcome; IGFBP7 may represent both a prognostic marker and a potential therapeutic target in glioma. These findings contribute to the current understanding of the relationship between IGFBP7 expression and outcome of glioma. To our knowledge, this is the first report to correlate IGFBP7 expression with clinicopathological characteristics in patients with glioma.
Footnotes
Declaration of conflicting interest
The authors declare that there are no conflicts of interest.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Acknowledgements
We would like to thank Dr Shaogui Wan, Department of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA, for his kind help in the language revision of our work.