Breast cancer 1 (BRCA1) and insulin-like growth factor 1 receptor (IGF1R) are critical in ovarian cancer progression. However, the crosstalk between the BRCA1 and IGF1R signaling pathways in ovarian cancer remains largely unknown. The effects of BRCA1 on IGF1R were assessed in 121 serous ovarian cancer patients (BRCA1 mutation, n=30; non-BRCA1 mutation, n=32; hypermethylated BRCA1 promoter, n=28; and non-methylation, n=31). BRCA1 promoter methylation was analyzed via bisulfite sequencing using primers focused on the core promoter region. The expression levels of BRCA1 and IGF1R were assessed by immunohistochemistry and real-time polymerase chain reaction. Knockdown and overexpression of BRCA1 were achieved using a lentiviral vector in 293T and SKOV3 ovarian cancer cells, and primary non-mutated and BRCA1-mutated ovarian cancer cells. The present study demonstrated that IGF1R expression is increased in non-BRCA1-mutated ovarian cancer when compared with adjacent normal tissue. Furthermore, IGF1R levels are additionally significantly elevated in BRCA1 inactivation ovarian cancer (BRCA1 mutation or hypermethylated BRCA1 promoter). In addition, BRCA1 knockdown was found to be an effective method of activating IGF1R expression in non-BRCA1-mutated ovarian cancer cells. The observations of the current study indicate that BRCA1 may be a potential trigger that is involved in the transcriptional regulation of IGF1R in the development of ovarian cancer.
Ovarian cancer is characterized by a high mortality rate among the gynecological malignancies worldwide (
The present study was approved by the Institutional Review Board at the China Medical University (Shenyang, China). In total, 121 serous ovarian cancer patients (BRCA1 mutation, n=30; non-BRCA1 mutation, n=32; hypermethylated BRCA1 promoter, n=28; and non-methylation, n=31) were enrolled between 2010 and 2012, and all patients provided written informed consent. Fresh tumor samples, adjacent normal ovarian tissue, ascites and blood samples were obtained at the time of primary surgery prior to any chemotherapy or radiotherapy. Hematoxylin and eosin staining of the samples for histopathological diagnosis and grading were determined by three pathologists using the World Health Organization criteria (
Primary ovarian cancer cells were obtained from ascites of patients undergoing surgery for ovarian cancer and were cultured in RPMI-1640 with 10% fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, CA, USA), as described previously (
qPCR and immunohistochemistry were performed as previously described (
Genomic DNA extracted from ovarian cancer and normal ovarian tissue using a TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) was subjected to bisulfite conversion using the EZ DNA Methylation-Direct kit (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer’s instructions; the conversion efficiency was estimated to be ≥99.6%. The DNA was subsequently amplified by nested PCR and following gel purification, cloning and transformation into JM109 competent
Data are presented as means ± standard deviation. Statistical differences in the data were evaluated by Student’s t-test or one-way analysis of variance as appropriate. P<0.05 was considered to indicate a statistically significant difference.
qPCR and immunohistochemical analysis showed that the levels of IGF1R mRNA and protein were increased in non-mutated and BRCA1-mutated ovarian cancer tissue compared with their adjacent normal tissue. However, BRCA1-mutated ovarian cancer markedly increased the expression of IGF1R compared with the remaining three groups (
In mammals, promoter methylation is an epigenetic modification involved in regulating gene expression (
The present study reports the following associations between BRCA1 and IGF1R status in ovarian cancer cells: i) IGF1R expression is increased in non-BRCA1-mutated ovarian cancer; ii) BRCA1 inactivation (BRCA1 mutation and promoter hypermethylation) markedly increases the expression of IGF1R; and iii) BRCA1 knockdown is an effective method to activate the IGF1R gene. These results indicated that BRCA1 may be a potential regulator of IGF1R in ovarian cancer, although, a comparable phenomenon has been observed in breast cancer (
In conclusion, the present study emphasizes the convergence of the IGF1R-mediated cell proliferation pathway and a BRCA1-mediated antitumor mechanism. However, further clarification of the complex interactions between the BRCA1 and IGF1R signaling pathways, at the transcriptional, post-transcriptional and epigenetic levels, may improve the current understanding of the basic molecular mechanism of ovarian cancer.
IGF1R expression patterns in non-mutated and BRCA1-mutated ovarian cancer. (A) Relative IGF1R mRNA levels were measured in non-mutated and BRCA1-mutated ovarian cancer, and their adjacent normal tissue. (B) IGF1R protein levels were assessed by immunohistochemistry in non-mutated and BRCA1-mutated ovarian cancer, and their adjacent normal tissue. Bar graphs are presented as the mean ± standard deviation. The intensity of the staining was divided into 10 units. IGF1R, insulin-like growth factor 1 receptor; BRCA1, breast cancer 1.
IGF1R expression patterns in ovarian cancer with hypermethylated promoter-mediated BRCA1 inactivation. (A) Location of CpG sites in the core promoter region of IGF1R. Genomic coordinates are shown, along with the primer amplified fragments, GC percentage, location of individual CpG dinucleotides (dashes) and IGF1R RefSeq gene (exon 1 shown as a black box and intron shown as an arrowed line). The arrow indicates the direction of transcription. (B and C) Comparative analysis of methylation patterns in the core promoter region of BRCA1 and their adjacent normal tissue. The circles correspond to the CpG sites denoted by the black dashes in Fig. 2A. Closed circles indicate methylated BRCA1 and open circles indicate unmethylated BRCA1. In total, 10 individual clones were sequenced for each sample. (Da and Ea) Summary of the methylation levels of BRCA1 core promoter from the measurements shown in B and C. (Db and Eb) Relative BRCA1 mRNA levels were measured in ovarian cancer with the identified hypermethylated or unmethylated BRCA1 promoter and compared with their adjacent normal tissue. (Dc and Ec) Relative IGF1R mRNA levels were measured in ovarian cancer with or without identified BRCA1 inactivation. Bar graphs are presented as the mean ± standard deviation. *P<0.05 vs. normal. IGF1R, insulin-like growth factor 1 receptor; GC, guanine-cytosine; BRCA1, breast cancer 1.
Effects of BRCA1 on IGF1R expression. (A–D) Relative IGF1R mRNA levels following overexpression or knockdown of BRCA1 in 293T, human SKOV3 ovarian cancer, and primary non-mutated and BRCA1-mutated ovarian cancer cells. Bar graphs are presented as the mean ± standard deviation. Sh, short hairpin RNAs; Op, overexpression; IGF1R, insulin-like growth factor 1 receptor; BRCA1, breast cancer 1.