Introduction
Ovarian cancer is one of the most common and lethal gynecologic cancer in women worldwide, and the incidence of this cancer has been increasing in many countries (1–3). More than 200,000 new cases of ovarian cancer were diagnosed in 2011 worldwide (4). At present, most ovarian cancer patients are diagnosed at an advanced stage due to the lack of effective detection at early stages (5). Although advances in surgery and chemotherapy have been developed, only ~30% of patients achieve a 5-year survival after diagnosis (6). To date, surgery is still of importance for improving the effect of chemotherapy and the survival rate. Chemotherapy is an important strategy and the combination of platinum and taxane has been used as the reference standard for the chemotherapy of ovarian cancer (7). This standard combination shows effectiveness with a response rate of ~80% in advanced ovarian cancer patients, while unfortunately most of these patients relapse owing to drug resistance (8,9). Therefore, identification of molecular biomarkers with clinicopathologic and prognostic significance is critically important for improving therapeutic methods and prolonging the survival of ovarian cancer patients.
Insulin-like growth factor 2 (IGF2) is a mitogenic peptide hormone, which is expressed in most tissues (10). Serum IGF2 is usually low in newborns, increasing in childhood and remaining at a similar concentration in adults, although it may decrease slightly in healthy elders (11,12). Many studies have shown that IGF2 regulates cell growth, differentiation and metabolism (13). Particularly, it is highly expressed during embryogenesis, and is important in promoting fetal growth (14). Growing evidence has shown that IGF2 can promote cancer development and progression (15). In hepatocellular carcinoma, the expression of IGF2 is usually elevated in patients (16). In esophageal cancer, high IGF2 expression is associated with reduced disease-free survival (17). The differential expression of IGF2 between African-American and Caucasian patients is believed to contribute to breast cancer survival disparities (18). However, the clinical significance of IGF2 expression remains unclear in ovarian cancer.
In the present study, we also identified IGF2 as a differentially expressed gene between tumor and non-tumor ovarian tissues (19), and we confirmed its expression in normal, corresponding non-cancerous and cancerous ovarian tissues by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RT-qPCR), western blotting and IHC. Furthermore, the prognostic significance of IGF2 expression was analyzed in human ovarian cancers. Our study showed that IGF2 expression is frequently higher in ovarian cancer tissues, and the expression pattern may be an important prognostic factor for ovarian cancer patients.
Materials and methods
Patient samples
A total of 72 tumor, 15 para-carcinoma and 10 normal ovarian tissues were obtained from the Southwest Hospital in Chongqing, China. The present study was approved by the ethics Committee of the Southwest Hospital in Chongqing, China. Informed consent was signed by all of the recruited patients.
Isolation of total RNA
Total RNAs were extracted from frozen tissues. Approximately 2.0 µg of total RNAs was treated with DNase I to eliminate the genomic DNA contamination, and then were reverse-transcribed to generate cDNAs.
Analysis of IGF2 expression by RT-PCR and RT-qPCR
IGF2 expression was determined by RT-PCR. A series of PCRs with different cycles was performed. Based on the pilot experiments, the appropriate cycles were chosen. Human β-actin was amplified as an endogenous control. The primers for IGF2 and β-actin were (5′-3′): IGF2-F, TAC TTC AGC AGG CCC GCA AG and IGF2-R, GGT GAC GTT TGG CCT CCC TG; β-actin-F, TTC TAC AAT GAG CTG CGT GTG and β-actin-R, GGG GTG TTG AAG GTC TCA AA. RT-qPCR was performed using an iq5 Real-Time Detection system (Bio-Rad Laboratories, Hercules, CA, USA) and GoTaq® qPCR Master Mix (Promega). The relative gene expression was calculated by the equation 2−ΔΔCT. The sequences of the primers were (5′-3′): IGF2-F1, GAT GCT GGT GCT TCT CAC CT and IGF2-R1, CAG ACG AAC TGG AGG GTG TC; β-actin-F2, TGA CGT GGA CAT CCG CAA AG and β-actin-R2, CTG GAA GGT GGA CAG CGA GG. All RT-qPCRs were performed in triplicate.
Tissue microarray generation
To construct the tissue microarray (TMA) slides, two cores were obtained from each representative tumor and adjacent non-cancerous tissue (within a distance of 20 mm). The non-cancerous adjacent tissues were stained with hematoxylin and eosin, and then reviewed histologically by two pathologists, and compared with normal tissue. Duplicate cylinders from intratumoral and peritumoral areas were obtained, and the TMA containing 72 tumor and 15 peritumoral ovarian tissues was constructed (20).
Western blot analysis
Western blotting (WB) was performed using the IGF2 antibody (Santa Cruz Biotechnology) as previously described (21). Protein (100 µg) was run on 8% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked and incubated with the primary antibody (IGF2, 1:1,000; Santa Cruz Biotechnology). Then, the membranes were washed, incubated with the secondary antibody (1:3,000; Jackson ImmunoResearch Laboratories), and developed with SuperSignal West Pico chemiluminescent substrate (Pierce). The same membrane was stripped and incubated with β-actin (Sigma) serving as an internal control.
Immunohistochemical analysis
Immunohistochemistry was performed using the IGF2 antibody (1:50; Santa Cruz Biotechnology) as previously described (22). Tumor cell staining was evaluated and considered positive when immunoreactivity was ≥10%. Positive staining was quantified and classified into 5 categories: <10% positive cells as 0; 10 to 25% as 1; 26 to 50% as 2; 51 to 75% as 3 and ≥76% as 4. Staining intensity was graded as negative (0), weak (1), moderate (2) or strong (3). All core biopsies were independently reviewed by two pathologists, and the expression level was defined by the sum of positive staining and intensity.
Statistical analysis
Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Results are expressed as the mean ± standard deviation (SD). Measurement data were analyzed by a Student’s t-test, and the Chi-square test was used to analyze the differences between categorical variables. A Kaplan-Meier survival database that contained the survival information of ovarian cancer patients and gene expression data obtained by using affymetrix microarrays was used (23). The probe set was 202409_at (there are 3 IGF2 probe sets: 210881_s_at, 202410_x_at and 202409_at). Although the probe sets are different, the result was similar. The patients were grouped according to the median or auto selection of the best cut-off value. P<0.05 was considered to be statistically significant.
Results
IGF2 is frequently increased in human ovarian cancer tissues
To investigate the level of IGF2 expression in human ovarian cancers, RT-PCR and RT-qPCR assays were performed in 10 normal ovarian (10 different normal ovaries) and 35 ovarian cancer tissues (35 different ovarian cancers). The expression of IGF2 in most cases (30/35) of tumor tissues was upregulated compared to the mean expression of the 10 normal ovarian tissues, and the mean expression of IGF2 in cancer tissues (2.86±0.72) was markedly higher than that in the normal ovarian tissues (1.00±0.18) (P=0.000, Fig. 1A and B). The data showed that IGF2 was frequently increased in the tumor tissues compared to the frequency in the normal ones, which was further confirmed by WB at the protein level (Fig. 1C).
IGF2 expression is correlated with histological grade of ovarian cancer patients
To confirm whether the IGF2 protein level is altered in cancer, we also conducted IHC for IGF2 on a TMA containing 72 cancer and 15 para-carcinoma ovarian tissues. The IGF2 protein level was increased in most (61/72) cancer tissues compared to the level in the para-carcinoma ovarian tissues, and the mean expression of IGF2 in cancer tissues (2.25±0.37) was evidently higher than that in the para-carcinoma ovarian tissues (0.90±0.12) (P=0.000, Fig. 2A–C). After investigating the associations between IGF2 expression and clinicopathologic features of the ovarian cancer patients, IGF2 expression was found to be significantly correlated with histological grade (P=0.047). However, IGF2 expression was not correlated with age, histological type, tumor size or location (Table I).
High expression of IGF2 is significantly associated with poor overall survival (OS) of ovarian cancer patients
To investigate the correlation between IGF2 expression and survival of the tumor patients, we examined the contribution of IGF2 expression to the OS of the ovarian cancer patients in a clinical microarray database (23). This database collected gene expression data obtained by using affymetrix microarrays and the OS information of 1,648 ovarian cancer patients. The OS analysis revealed that high expression of IGF2 predicts poorer survival of ovarian cancer patients (HR=1.44, P=0.000, Fig. 3A). In addition, the patients with high IGF2 expression also had a poorer progression-free survival (PFS) compared with the low expression group (HR=1.35, P=0.000, Fig. 3B).
High expression of IGF2 predicts poorer survival of the patients at clinical stage I+II and III
To determine the association between IGF2 expression and the OS of ovarian patients with different stages, we analyzed the survival data of patients with different stages stratifying the patients based on IGF2 expression. IGF2 expression was not associated with OS time of ovarian patients at stage IV (P=0.301). However, there was a statistically significant effect of high IGF2 expression on the poorer OS of the ovarian cancer patients at stage I+II (HR=2.60, P=0.014) and III (HR=1.30, P=0.003, Fig. 3C and D). These findings suggest that high expression of IGF2 is an unfavorable factor for the prognosis of ovarian cancers at stage I+II and III.
High expression of IGF2 predicts poorer outcomes of the patients at grade 2 and 3
We then analyzed the survival data of patients at different grades by stratifying the patients based on IGF2 expression. We observed a statistically significant effect of high IGF2 expression on the poorer OS of the ovarian cancer patients at grade 2 (HR=1.59, P=0.004) and 3 (HR=1.31, P=0.002, Fig. 4A and B), but no effect on that of the patients at grade 1 (P=0.289). The results suggest that high expression of IGF2 is an unfavorable factor for the prognosis of ovarian cancers at grade 2 and 3.
High expression of IGF2 implies poorer outcomes of the patients treated with chemotherapy containing platin and taxol
We next aimed to ascertain whether high expression of IGF2 also predicts poorer survival of the ovarian cancer patients treated with different chemotherapeutic agents. We found a statistically significant effect of high IGF2 expression on the poorer OS of the ovarian cancer patients treated with chemotherapy containing platin (HR=1.48, P=0.000) and chemotherapy containing taxol (HR=1.46, P=0.00024, Fig. 4C and D). To further validate the results, we analyzed the association between IGF2 expression and OS times of the ovarian patients treated with chemotherapy containing platin and taxol. There was also a statistically significant effect of high IGF2 expression on the poorer OS of the patients treated with chemotherapy containing platin and taxol (HR=1.46, P=0.000, Fig. 4e). However, no effect of high IGF2 expression was observed on the OS of the patients treated with chemotherapy containing avastin, docetaxel, gemcitabine, paclitaxel or topotecan.
Discussion
Accumulating data reveal the activation of IGF2 in subsets of embryonic tumors, such as hepatoblastoma, neuroblastoma, Wilms tumors and rhabdomyosarcoma (24,25). Dysregulation of IGF 2 was also found in subsets of adult cancers, and overexpression of IGF2 was detected in many tumors, for example, in ~40% of colon carcinoma, 20% of hepatocarcinoma, 90% of liposarcoma and adrenocortical carcinoma (10,26–28). In the present study, we found that IGF2 expression was increased in ~85.7% (30/35) of the ovarian cancer cases at the mRNA level and 84.7% (61/72) at the protein level, and the mean expression of IGF2 in the tumor tissues was obviously higher compared to that in the non-tumor ovarian tissues (P=0.000).
Previous studies have shown that IGF2 mutations are associated with risk for oral, colon and hepatocellular carcinoma (29–31). Extensive evidence indicates that increased IGF2 expression in tumors is associated with poorer prognosis, for instance more rapid disease progression in chronic myeloid leukemia, shorter time to disease recurrence in esophageal cancer and higher mortality in breast cancer (17,18,32). Moreover, transgenic mice overexpressing IGF2 have a high risk of developing mammary gland adenocarcinoma and lung cancer, while the animals with low IGF2 expression usually live longer, and have a lower incidence of tumors (33–37). In the present study, we found that the ovarian cancer patients with high IGF2 expression had poorer OS and PFS compared with the low expression group, suggesting that the expression of IGF2 could act as a potential biomarker for prognostic evaluation of ovarian cancers.
To further determine the association between IGF2 expression and OS of ovarian cancer patients with different clinical stages, histological grades and chemotherapeutic treatments, survival data were analyzed by stratifying the patients based on the IGF2 levels. The results showed that high expression of IGF2 may be an unfavorable factor for the prognosis of ovarian cancer patients who were at clinical stage I+II and III, histological grade 2 and 3 or treated with chemotherapy containing platin and Taxol.
At present, the standard treatment for ovarian cancer is surgery and systemic chemotherapy, usually with the combination of taxol and platinum (6,7). Unfortunately, the majority of these patients still succumb to recurrent, progressive disease due to resistance to chemotherapy. A recent study has shown that the silencing of IGF 2 can restore taxol sensitivity in drugresistant ovarian cancer (38,39). This study is consistent with our result that high expression of IGF2 may be an unfavorable factor for the prognosis of ovarian cancer patients.
Taken together, the present study revealed that IGF2 is upregulated in ovarian cancer tissues, and its expression may be a potential marker for prognostic evaluation of ovarian cancers. However, further investigation of the potential of IGF2 as a therapeutic target is clearly warranted in ovarian cancer.