Breast Cancer Res Treat (2013) 138:753–760 DOI 10.1007/s10549-013-2502-5
PRECLINICAL STUDY
Involvement of IGF-1R regulation by miR-515-5p modifies breast cancer risk among BRCA1 carriers Avital Gilam • Liat Edry • Efrat Mamluk-Morag • Dalia Bar-Ilan Camila Avivi • David Golan • Yael Laitman • Iris Barshack • Eitan Friedman • Noam Shomron
•
Received: 29 July 2012 / Accepted: 23 March 2013 / Published online: 3 April 2013 Ó Springer Science+Business Media New York 2013
Abstract Several lines of evidence indicate that sequence alterations within microRNA (miRNA)-binding sites can modify the binding to its target gene resulting in altered expression patterns. We hypothesized that a single nucleotide polymorphism (SNP) located in the miR-515-5p binding site of igf-1r gene may alter IGF-1R regulation, with consequent effects on breast cancer risk in BRCA1 mutation carriers. Computational prediction revealed that the rs28674628 SNP in the igf-1r 30 UTR is located within a predicted binding site for miR-515-5p. The effect of this SNP on breast cancer risk was evaluated by genotyping 115 Jewish Ashkenazi carriers of the 185delAG mutation in the BRCA1 gene using the Sequenom platform followed by Kaplan–Meier analysis. Additional data set of 378 Electronic supplementary material The online version of this article (doi:10.1007/s10549-013-2502-5) contains supplementary material, which is available to authorized users. A. Gilam L. Edry E. Mamluk-Morag D. Bar-Ilan I. Barshack E. Friedman (&) N. Shomron (&) Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel e-mail:
[email protected] N. Shomron e-mail:
[email protected] E. Mamluk-Morag Y. Laitman E. Friedman Susanne Levy Gertner Oncogenetics Unit, Institute of Human Genetics, The Chaim Sheba Medical Center, Tel-Hashomer, 52621 Ramat Gan, Israel D. Bar-Ilan C. Avivi I. Barshack Department of Pathology, The Chaim Sheba Medical Center, Tel-Hashomer, 52621 Ramat Gan, Israel D. Golan Department of Statistics and Operations Research, Tel Aviv University, 69978 Tel Aviv, Israel
Jewish BRCA1 carriers was analyzed to validate our results. MiRNA transfection, Western blot analysis, luciferase reporter assay, real time PCR, and immunohistochemistry were performed to assess direct regulation of igf-1r by miR515-5p. We show direct regulation of IGF-1R by miR-5155p. We identified that disrupting miR-515-5p and igf-1r 30 UTR binding by SNP may cause elevated IGF-1R protein levels. Interestingly, miR-515-5p is downregulated in tumor tissue compared to its non-neoplastic surrounding tissue while IGF-1R levels are elevated. This igf-1r SNP was found to be significantly associated with age at diagnosis of breast cancer in Jewish Ashkenazi BRCA1 mutation carriers. These findings support the hypothesis that a SNP located in igf-1r gene may alter miRNA regulation of IGF1R, with a putative effect on BRCA1 penetrance and breast cancer risk. Keywords Breast cancer microRNA miRNA Single nucleotide polymorphism IGF-1R BRCA1 penetrance Modifier factors Abbreviations IGF-1R Insulin-like growth factor-1 receptor miR miRNA-microRNA SNP Single nucleotide polymorphism UTR Untranslated region
Introduction Breast cancer is the most common cancer diagnosed among women in westernized societies and is the leading cause of feminine cancer deaths [1]. The BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) genes have important roles in the maintenance of genomic stability by facilitating repair
123
754
of DNA double strand breaks [2]. Germline mutations in these genes substantially increase risk for developing breast and ovarian cancers in female carriers, with an estimated X7- and X30-fold lifetime increased risk compared with the average risk population, respectively [3, 4]. Yet, penetrance is incomplete. Combined with the observed variability in age at diagnosis even among identical mutation carriers, it is likely that genetic, epigenetic, and environmental modifier factors affect mutant BRCA allele penetrance. While multiple factors have been evaluated as putative modifiers of BRCA1 and BRCA2 mutations, few emerged as ‘‘true modifiers’’ [5, 6]. We [7] and others [8–11] have shown that potential aberrant gene silencing by microRNAs (miRNAs) may play a role in modifying mutant BRCA allele penetrance or disease phenotype in sporadic and familial breast cancer cases. MiRNAs are small, naturally occurring, non-coding, single-stranded RNA molecules, about 22 nucleotides (nt) long that negatively regulate gene expression [12, 13]. They exert their functional role by binding their ‘‘seed’’ region (nucleotides 2–8 of the miRNA) to a short conserved complementary sequence in 30 untranslated regions (UTRs) of downstream target mRNA [14], which follows by translation inhibition or mRNA degradation. With the potential to target hundreds of genes it is expected to be under tight and dynamic regulation, especially during developmental transitions or changes in cellular environment. Several studies indicate significant changes in miRNA profiles in different cancer types, compared to normal tissues [15–17]. Other studies have demonstrated a direct involvement of miRNAs in cancer processes or pathways [18, 19]. In addition, miRNA expression profiles enable successful classification of poorly differentiated tumors, whereas mRNA profiles were highly inaccurate in the same tumor types [20]. It has been shown that single nucleotide polymorphisms (SNPs) located within miRNA binding sites can modify (increase/decrease) miRNA binding to its target gene, thereby enabling the mRNA to escape (or gain) negative control, with resultant effect on expression levels and patterns [7, 19, 21, 22]. Several studies have identified SNPs that are located within predicted miRNA binding site in gene’s 30 UTRs [23]. Moreover, a few studies support a potential association between SNPs located in miRNA binding sites and human cancers [24, 25]. In this study, we observed that the rs28674628 SNP, located in the igf-1r 30 UTR, was associated with increased risk and earlier age at diagnosis of breast cancer in Jewish BRCA1 mutation carriers. Computational analysis revealed that the rs28674628 SNP is located within a predicted ‘‘seed’’ region of the binding site for miR-515-5p. IGF-1R (insulin-like growth factor-1 receptor) is a tyrosine kinase receptor that is activated by ligand (IGF-1)
123
Breast Cancer Res Treat (2013) 138:753–760
binding. The IGF-1R is highly expressed in most transformed cells, where it displays potent antiapoptotic, cellsurvival, and transforming activities [26]. In breast cancer, IGF, mediated by IGF-1R, promotes mitogenic, metastatic, and antiapoptotic phenotypes and it is intimately associated with the pathogenesis of this disease via its signaling transduction cascade proteins and its cross talk with the BRCA1 protein [27–30]. In addition, significant associations were found between genetic variants of IGF-1R and the risk of breast cancer diagnosis among BRCA1 carriers [31]. This study tested if and to what extent a genetic variability (in the form of single nucleotide polymorphisms— SNP) in a gene’s miRNA predicted binding sites might affect breast cancer risk in Jewish BRCA1 mutation carriers. In addition, it attempted to decipher the molecular mechanism underlying this effect.
Materials and methods Patients characteristics and SNP genotyping Overall, 115 Jewish Ashkenazi breast cancer patients, all carriers of the 185delAG BRCA1 mutation, were genotyped in this study. Median age at breast cancer diagnosis was 44.5 ± 12.2 years (mean ± SD) (range 19–81 years). The A/G polymorphism (rs28674628) in the 30 UTR of the igf-1r gene was determined by mass spectrometry (MALDI-TOF) using the MassARRAY by Sequenom Inc (La-Jolla, CA) as described previously [32]. SNP allele distribution was as follows: 108 were homozygotes for the common (A) allele and seven were heterozygotes (G/A). No homozygotes for the rare (G) allele were included in this data set. To verify our hypothesis, a larger additional data set was collected and analyzed. Here, 378 Jewish Ashkenazi breast cancer patients, carriers of the 185delAG BRCA1 mutation were genotyped. Median age at breast cancer diagnosis was 43.3 ± 9.5 years (mean ± SD) (range 25–82 years). SNP allele distribution was as follows: 362 were homozygotes for the common (A) allele and 16 were heterozygotes (G/A). No homozygotes for the rare (G) allele were included in this data set. The study was approved by the Institutional Review Board (IRB) of the Sheba Medical center and the high Ethics committee of the Ministry of Health in Israel, and each participant gave an informed consent approved by both committees. Cell cultures growth HeLa cells (cervical carcinoma), HEK-293T cells (transformed human embryonic kidney cells), and breast cancer cell lines (MCF7, T47D, MDA-231, Hs578, BT-549) were
Breast Cancer Res Treat (2013) 138:753–760
grown in DMEM medium supplemented with 10 % FBS and antibiotic (Streptomycin 100 mg/ml, Penicillin 100 mg/ml). HeLa cells and HEK-293T cells were purchased from ATCC. All other cell lines were received from Prof. Ilan Tsarfaty (Tel-Aviv University). Cloning and site-directed mutagenesis Fragments of IGF-1R 30 UTR spanning the miR-515-5p binding site were cloned into the XhoI–NotI restriction site downstream to the Renilla luciferase Reporter of the psiCHECK-2 plasmid (Promega, USA) that contain a firefly luciferase reporter (used as control) under a different promoter. For this purpose, the 30 UTR fragment was PCR amplified using Phusion High-Fidelity DNA Polymerase (Finnzymes) from genomic DNA of patients that carried the desired SNP genotypes, and XhoI–NotI restriction sites were added. Three nucleotides in the seed of miR-515-5p were mutated using the QuickChange site-directed mutagenesis procedure (Stratagene). The vectors were then sequenced to verify the correct SNP genotype (see Fig. 2). Transfection Cells were plated in 12-well plates at a concentration of 0.5 9 106 cells/well. Transfection was done using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Three plasmids were used in the transfections: psiCHECK-2 relevant clone, miR-Vec-515-5p—a miR-Vec retroviral vector that contains the genomic region of the pre-miRNA under a strong CMV promoter (Dr Reuven Agami, The Netherlands Cancer Institute), and pEGFP. Forty-eight hours after transfection RNA extraction or luciferase reporter assay was performed. Luciferase reporter assay HEK293 cells were seeded in 12-well plates in DMEM supplemented with 10 % FBS and 1 % Pen/Strep. Cells were transfected with 1 lg DNA, 10 ng psiCHECK-2 containing the desired IGF-1R 30 UTR with or without sitedirected mutations, 15 ng pEGFP vector, and 795 ng miRVec containing the desired pre-miR-515-5p or an empty miR-vec vector. Forty-eight hours after transfection, the transfection efficiency was measured by green fluorescent protein (GFP) fluorescence, indicating a transfection efficiency of [50 % repeatedly, and Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay kit (Promega, USA). The Veritas Luminometer (Promega) was used to read the intensities. The Renilla luciferase results were normalized to the values of the firefly luciferase.
755
RNA extraction Total RNA was isolated from cell ines using Trizol (Invitrogen). In addition, RNA was extracted from paraformaldehyde-fixed paraffin-embedded (FFPE) tissues using the RecoverAll Total Nucleic Acid Isolation Kit according to manufacturer’s protocol (Applied Biosystems). qRT-PCR Reverse transcription reaction for mRNA was done using random-primer and SuperScript III reverse transcriptase (Invitrogen). Reverse transcription reaction for specific mature miRNA was done using TaqMan miRNA Assays according to manufacturer’s protocol (Applied Biosystems). Single miRNAs/mRNAs expression was tested similarly using TaqMan Universal PCR Master Mix (No AmpErase UNG; Applied Biosystems) or SYBR green PCR master mix (Applied Biosystems), respectively. The PCR amplification and reading was done using the StepOne or ABI Prism 7900HT Sequence Detection Systems under the following thermal cycler conditions: 2 min at 50 °C, 10 min at 95 °C, and 40 amplification cycles (30 s at 95 °C and 1 min at 60 °C). Specific primers for mRNA expression detection were ordered from Sigma: IGF-1R Forward primer—AAAAACCTTCGCCTCATCC; IGF-1R reverse primer—TGGTTGTCGAGGACGTAGAA. Expression values were calculated based on the comparative threshold cycle (Ct) method. MiRNA levels were normalized to U6 and mRNA expression levels were normalized to GAPDH. Western blot analysis HeLa cells were homogenized with lysis buffer containing 50 mM Tris HCl (pH = 7.6), 20 mMMgCl2, 150 mM NaCl, 0.5 % NP40, 5 units/mL Aprotinin, 5 lg/mL Leupeptin, 5 lg/mL Pepstatin, 1 mM Benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 200 lM sodium orthovanadate. Debris was removed by centrifugation, and protein levels in the lysates were determined with the aid of the Bio-Rad protein assay (Bio-Rad Laboratories). Lysates were resolved by SDS—polyacrylamide gel electrophoresis (PAGE) through 7.5 % gels and electrophoretically transferred to nitrocellulose membrane. Membranes were blocked for 2 h in TBST buffer (0.02 M Tris HCl, pH 7.5, 0.15 M NaCl, and 0.05 % Tween 20) containing 5 % milk, blotted with 1 lg/mL primary antibodies (IGF-1R, ACTIN) for 18 h, followed by 0.5 lg/mL secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected with the enhanced chemiluminescence reagent (Amersham Corp).
123
756
IGF-1R immunostaining Formalin-fixed tissues from patients were dehydrated, embedded in paraffin, and sectioned at 4 lm. The slides were warmed up to 60 °C for 1 h, dewaxed in xylene, and rehydrated. Antigen retrieval was performed using a pressure cooker (Milestone, Microwave Laboratory Systems) at 120 °C for 5 min in 0.1 M citrate buffer pH 6. After cooling for 10 min, the slides were rinsed in TBS buffer and an endogenous peroxidase block was performed for 10 min in 3 % H2O2/MeOH. After rinses in TBS, sections were blocked with 10 % goat serum for 30 min at room temperature, and incubated overnight at 4 °C with the primary antibody to IGF-1R (Abcam, ab54274, 1:750). Detection was performed with the Histostain SP Broad Spectrum kit (Invitrogen). Briefly, sections were incubated for 30 min at room temperature with a biotinylated second antibody. After TBS rinses, sections were submitted to additional 30 min room temperature incubation with HRP-streptavidin. The antibody binding was visualized with the substratechromogen AEC. Sections were counterstained with hematoxylin and cover-slipped with an aqueous mounting fluid (glycergel). The stained sections were reviewed with a light microscope and were analyzed by a pathologist. Statistical analysis Data are presented as mean ± SEM. Unless otherwise noted, Student’s t test was used, with p \ 0.05 considered significant. The Kaplan–Meier analysis demonstrates the risk of BRCA1 mutation carrier to develop breast cancer at a certain age. The difference of Kaplan–Meier curves was calculated using Log-rank test and gave a significant p value as mentioned. Calculations were done using SPSS software version 15 and R version 2.15.2 [33].
Breast Cancer Res Treat (2013) 138:753–760
the Wilms’ tumor protein-1 (WT1) [29]. Several evidences suggest igf-1r as a downstream target for BRCA1 action. First, BRCA1 induces a large reduction in endogenous IGF-1R levels [35]. Second, a significant elevation in IGF1R levels was found in tumors from BRCA1 mutation carriers compared with non-carriers [35]. Third, Wild type BRCA1 was shown to repress the activity of co-transfected igf-1r promoter in a number of cell lines [36]. Furthermore, loss-of-function mutation of BRCA1 and/or p53 in familial and/or sporadic breast cancer may result in aberrant regulation of igf-1r gene expression [29]. Analysis of the igf-1r-SNP effect on age at breast cancer diagnosis (Fig. 1) showed that the rare G containing allele (in a heterozygous form) was significantly associated with an earlier age at diagnosis (p value 0.00165): All BRCA1 carriers who also carried the G allele of the rs28674628 SNP were diagnosed with breast cancer by age 45 years, whereas almost 50 % of the wild type (A) allele homozygotes were cancer free at that age. To test reproducibility of our results, we analyzed an additional larger data set, which comprise 378 Jewish Ashkenazi breast cancer patients, all carriers of the 185delAG BRCA1 mutation. We have determined the patient’s genotype of the A/G SNP (rs28674628) in the 30 UTR of the igf-1r gene and analyzed the age at breast cancer diagnosis. This data set included two groups: homozygotes for the common (A) allele (362 patients) and heterozygotes (G/A, 16 patients). No homozygotes for the rare (G) allele were included in this data set. This new data set analysis supports
Results and discussion This study aimed to test if and to what extent, SNPs within miRNA binding sites might affect breast cancer risk in BRCA1 mutation carriers. To do so, we determined the genotype of the A/G SNP (rs28674628) in the 30 UTR of the igf-1r gene for 115 Jewish Ashkenazi breast cancer patients, all carriers of the 185delAG BRCA1 mutation. This mutation is the most frequent mutation of BRCA1 alleles predisposing to hereditary breast cancer among the Ashkenazim [34]. IGF-1R was a prime candidate for this analysis due to its well-established role in breast cancer pathways and its reported association with BRCA1. The igf-1r gene is negatively regulated by BRCA1 during transcription, as well as by other tumor suppressor genes, including p53 and
123
Fig. 1 igf-1r-SNP effect on age at breast cancer diagnosis. Kaplan– Meier analysis of breast cancer appearance (age of diagnosis), among Jewish women who carry the 185delAG BRCA1 mutation, with the genotype of rs28674628 SNP. Common homozygote (AA) Black; Heterozygote (GA) Red. p = 0.00165
Breast Cancer Res Treat (2013) 138:753–760
our conclusion drawn from the first set (a one-sided hypothesis gave a significant p value of 0.04 using the logrank test) that the rare G containing allele is significantly associated with an earlier age at diagnosis of breast cancer in BRCA1 mutation carriers (see Supplementary Fig. 1). Based on computational prediction, by searching for conserved bindings sites complementary to the miRNA ‘‘seed’’ region, we determined that miR-515-5p recognizes the sequence contained within the rs28674628 SNP in the igf-1r gene (Fig. 2). We investigated the experimental interaction between igf-1r 30 UTR and miR-515-5p. IGF-1R expression levels were significantly downregulated (29 %) when miR-5155p was constitutive expressed in HeLa cell line (Fig. 3a). Moreover, we used luciferase reporter assay to confirm miRNA linking to its mRNA binding site. For this assay, the 30 UTR of interest is cloned downstream to a reporter gene, and then this vector is co-transfected with the miRNA. If the miRNA binds its target site, it negatively regulates the reporter gene expression and a decreased glowing is detected. Here, we used three UTR constructs (Fig. 2)—one having the common (A) allele of the igf-1r gene, one having the rare (G) allele, and one knockdown control (ACT). Three nucleotides were substituted in the knockdown control, to ensure prevention of miR-515-5p binding to its target site. This assay confirmed a direct functional regulation of IGF-1R by miR-515-5p but only when the common allele (A) is present (Fig. 3b).
757
To validate the specificity of IGF-1R regulation by miR515-5p, we tested this regulation using two additional constructs for other miRNAs, miR-210 and miR-222, which lack any predicted binding site on the igf-1r 30 UTR (Targetscan). As expected, this direct regulation on igf-1r expression was demonstrated only when using miR-515-5p construct, but not while using control miRNA constructs (see Supplementary Fig. 2). These results demonstrate that IGF-1R expression is directly regulated by miR-515-5p, and that disrupting miR515-5p-igf-1r 30 UTR binding by A to G nucleotides substitution allows avoidance of this regulation. We note that both rs28674628 SNP alleles are prevalent in the population with Minor Allele Frequency (MAF) of 0.015 (according to dbSNP). To evaluate to what extent the identified interaction has any clinical relevance, we determined igf-1r mRNA and miR-515-5p levels by RT-PCR (Fig. 4) in different human breast cancer cell lines. We found an inverse expression between igf-1r mRNA and miR-515-5p expression levels. In other words, in cell lines which showed high IGF-1R expression, the miR-515-5p expression was low (for example, cell lines T47D and MCF7), whereas cell lines which exhibited low IGF-1R expression also demonstrated high miR-515-5p expression (specifically Hs578 cell line). We should note here that the IGF-1R SNP genotype of all human breast cancer cell lines that we used in this experiments was tested and found to be homozygote for the common (A) allele. Therefore, it seems that differences in
Fig. 2 MiR-515-5p and igf-1r sequences alignment. The rs28674628 SNP position is marked in yellow. MiR-515-5p ‘‘seed’’ region is in bold. Three igf-1r forms that were used for the reporter assay are presented: the common allele (A), the rare allele (G), and knockdown control (ACT)
Fig. 3 IGF-1R direct regulation by miR-515-5p. a Western blot analysis of IGF-1R and actin in HeLa cells constitutively expressing miR-515-5p. Bands quantification was done using ImageJ software and the relative expression of IGF-1R is indicated below in percent. b Assessment of luciferase reporter activity in HEK-293T cells cotransfected with miR-515-5p in combination with either the
Ranilla/firefly luciferase psiCHECK2 constructs under regulation of igf-1r 30 UTR: the common allele (A), the rare allele (G), and knockdown control (ACT). The data presented is the relative expression level of firefly luciferase expression standardized to Renilla luciferase (n C 3, *p \ 0.05). Construct’s sequences are shown in Fig. 2
123
758
Breast Cancer Res Treat (2013) 138:753–760
Fig. 4 Inverse expression of igf-1r mRNA and miR-515-5p in breast cancer cell lines. RNA was extracted from T47D, MCF7, BT-549, MDA-231, and Hs578 cell lines, and the fold change in expression levels of miRNA-515-5p and igf-1r mRNA were determined by
quantitative RT-PCR analysis (n = 3). IGF-1R expression is presented relative to the expression in MCF7 cell line, and miR-515-5p expression is presented relative to the expression in MDA-231 cell line
Fig. 5 IGF-1R is highly expressed and miR-515-5p levels are low in cancerous tissues. a Cytoplasmic positive immunoexpression of IGF1R was found in the carcinoma cells, while not in the non-neoplastic surrounding tissue from BRCA1 mutation carrier. b Quantitative RTPCR analysis of miR-515-5p expression level. RNA was extracted from paraformaldehyde-fixed paraffin-embedded (FFPE) non-
neoplastic tissue (Average, Av) and cancerous tissues from four BRCA1 mutation carriers (1–4). The data were normalized to U6 snRNA and analyzed with SDS software (ABI). The comparative threshold cycle (Ct) method was used for quantization. P value, 1–3 asterisks, refer to \0.05, \0.01, and \0.005, respectively
IGF-1R expression levels were due to its regulation by miR-515-5p, and not due to the presence of the SNP which may influence this regulation. Subsequently, immunohistochemistry was used to assess protein expression of IGF-1R in tumor samples from BRCA1 carriers. Immunostaining for IGF-1R was found to be positive in tumor cells in contrast to negative nonneoplastic surrounding tissue (Fig. 5a, also see Supplementary Fig. 3). We also monitored the levels of miR-5155p expression levels in the same tissue samples by RT-PCR and observed that miR-515-5p is downregulated in breast cancer tissue relative to non-neoplastic surrounding tissue (Fig. 5b). This indicates that IGF-1R increase can take place either by mutating the miRNA binding site or by
reducing the regulating miRNA levels. Further experiments would be required to determine whether these mechanisms are related. This study is consistent with a modifier effect of the rs28674628 SNP on age at diagnosis of breast cancer in Jewish Ashkenazi BRCA1 mutation carriers. Naturally, the results should be viewed with caution given the genetic homogeneity of the analyzed individuals. Validation in a larger group of BRCA1 carriers of different ethnicities with a range of BRCA1 mutations is required prior to any concrete conclusions in terms of clinical utility of this marker. MiRNAs are known to be highly regulated in cancer cells by a variety of mechanisms, including epigenetic alterations [37]. One such mechanism is miRNA silencing
123
Breast Cancer Res Treat (2013) 138:753–760
by CpG island hypermethylation in the promoter region of the miRNA gene [38, 39]. In our study, it is not probable that miR-515-5p expression is controlled by hypermethylation as there are no CpG islands upstream of the gene (data now shown). Further studies should be carried out to closely monitor the downregulation of this miRNA by other mechanisms such as transcription factors or histone modifications including acetylation, phosphorylation, ubiquitylation, or sumoylation. The miRNA tested here, miR-515-5p, was shown to be highly effective in regulating IGF-1R. Interestingly, we have reached this conclusion by first looking into patient data and then testing this effect in cell lines. Personalized medicine attempts to treat patients based on their genomic make up. It is tempting to speculate that 1-day miR-515-5p and its analogs could be used to potentially regulate IGF1R levels on a personal genomic level, namely for those patients with matching polymorphisms.
Conclusions In this study, we examined the direct regulatory role of miR-515-5p on IGF-1R mRNA and protein expression. We conclude that a substantial proportion of IGF-1R expression could be relieved of this regulation via a miRNA polymorphic site in its 30 UTR. In addition, the low level of miR515-5p in tumor versus normal tissue indicates that lowering miR-515-5p levels might allow increase of IGF-1R expression. Thus, our study further supports the role of miRNAs during the cancer transformation process, suggests a possible interplay between miR-515-5p and IGF-1R, proposes miRNAs as modifiers on BRCA1 mutant allele penetrance, and strengthens miRNAs’ putative role in modulating molecular pathways in familial breast cancer. Acknowledgments The study was in part funded by grants from the Israel Cancer association and Rosetta Genomics to EF. The Shomron laboratory is supported by the Chief Scientist Office, Ministry of Health, Israel; Israel Cancer Association; Wolfson Family Charitable Fund; I-CORE Program of the Planning and Budgeting Committee, The Israel Science Foundation (Grant Number 41/11). DG is supported in part by a fellowship from the Edmond J Safra Center for Bioinformatics at Tel Aviv University. Conflict of interest interests.
All authors declare they have no competing
References 1. Althuis MD, Doizer JM, Anderson WF, Devesa SS, Brinton LA (2005) Global trends in breast cancer incidence and mortality 1973–1997. Int J Epidemiol 34:405–412 2. Turnbull C, Rahman N (2008) Genetic predisposition to breast cancer: past, present, and future. Annu Rev Genomics Hum Genet 9:321–345
759 3. Wacholder S, Struewing JP, Hartge P, Greene MH, Tucker MA (2004) Breast cancer risks for BRCA1/2 carriers. Science 306: 2187–2191 4. Robles-Diaz L, Goldfrank JD, Kauff ND, Robson M, Offit K (2004) Hereditary ovarian cancer in Ashkenazi Jews. Fam Cancer 3:259–264 5. Narod SA (2006) Modifiers of risk of hereditary breast cancer. Oncogene 25:5832–5836 6. Levy-Lahad E, Friedman E (2007) Cancer risks among BRCA1 and BRCA2 mutation carriers. Br J Cancer 96:11–15 7. Kontorovich T, Levy A, Korostishevsky M, Nir U, Friedman E (2010) SNPs in miRNA binding sites and miRNA genes as breast/ovarian cancer risk modifiers in Jewish high risk women. Int J Cancer 127(3):589–597 8. Shen J, Ambrosone CB, Zaho H (2008) Novel genetic variants in microRNA genes and familial breast cancer. Int J Cancer 124: 1178–1182 9. Tchatchou S, Jung A, Hemminki K, Sutter C, Wappenschmidt B, Bugert P, Weber BH, Niederacher D, Arnold N, Varon-Mateeva R, Ditsch N, Meindl A, Schmutzler RK, Bartram CR, Burwinkel B (2009) A variant affecting a putative miRNA target site in estrogen receptor one (ERS1) is associated with breast cancer risk in premenopausal women. Carcinogenesis 30:59–64 10. Hu Z, Liang J, Wang Z, Tian T, Zhou X, Chen J, Miao R, Wang Y, Wang X, Shen H (2009) Common genetic variants in premicroRNAs were associated with increased risk of breast cancer in Chinese women. Hum Mutat 30:79–84 11. Brendle A, Lei H, Brandt A, Johansson R, Enquist K, Henriksson R, Hemminki K, Lenner P, Fo¨rsti A (2008) Polymorphisms in predicted microRNA-binding sites in integrin genes and breast cancer: ITGB4 as prognostic marker. Carcinogenesis 29:1394–1399 12. Bushati N, Cohen SM (2007) microRNA functions. Annu Rev Cell Dev Biol 23:175–205 13. Shomron N, Golan D, Hornstein E (2009) An evolutionary perspective of animal microRNAs and their targets. J Biomed Biotechnol 2009:594738 14. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233 15. Calin GA, Croce CM (2006) MicroRNA signatures in human cancers. Nat Rev Cancer 6:857–866 16. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Me´nard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65(16):7065–7070 17. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 103(7):2257–2261 18. Esquela-Kerscher A, Slack FJ (2006) Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6(4):259–269 19. Kwak PB, Iwasaki S, Tomari Y (2010) The microRNA pathway and cancer. Cancer Sci 101(11):2309–2315 20. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature 435:834–838 21. Nicoloso MS, Sun H, Spizzo R, Kim H, Wickramasinghe P, Shimizu M, Wojcik SE, Ferdin J, Kunej T, Xiao L, Manoukian S, Secreto G, Ravagnani F, Wang X, Radice P, Croce CM, Davuluri RV, Calin GA (2010) Single-nucleotide polymorphisms inside microRNA target sites influence tumor susceptibility. Cancer Res 70(7):2789–2798
123
760 22. Bao BY, Pao JB, Huang CN, Pu YS, Chang TY, Lan YH, Lu TL, Lee HZ, Juang SH, Chen LM, Hsieh CJ, Huang SP (2011) Polymorphisms inside microRNAs and microRNA target sites predict clinical outcomes in prostate cancer patients receiving androgen-deprivation therapy. Clin Cancer Res 17(4):928–936 23. Chen K, Song F, Calin GA, Wei Q, Hao X, Zhang W (2008) Polymorphisms in microRNA targets: a gold mine for molecular epidemiology. Carcinogenesis 29(7):1306–1311 24. Yu Z, Li Z, Jolicoeur N, Zhang L, Fortin Y, Wang E, Wu M, Shen SH (2007) Aberrant allele frequencies of the SNPs located in microRNA target sites are potentially associated with human cancers. Nucleic Acids Res 35(13):4535–4541 25. Landi D, Gemignani F, Barale R, Landi S (2008) Catalog of polymorphisms falling in microRNA-binding regions of cancer genes. DNA Cell Biol 27(1):35–43 26. Werner H, Bruchim I (2009) The insulin-like growth factor-I receptor as an oncogene. Arch Physiol Biochem 115(2):58–71 27. Helle SI (2004) The insulin-like growth factor system in advanced breast cancer. Best Pract Res Clin Endocrinol Metab 18(1):67–79 28. Pollak MN (1998) Endocrine effects of IGF-I on normal and transformed breast epithelial cells: potential relevance to strategies for breast cancer treatment and prevention. Breast Cancer Res Treat 47(3):209–217 29. Sarfstein R, Maor S, Reizner N, Abramovitch S, Werner H (2006) Transcriptional regulation of the insulin-like growth factor-I receptor gene in breast cancer. Mol Cell Endocrinol 252(1–2):241–246 30. Maor S, Papa MZ, Yarden RI, Friedman E, Lerenthal Y, Lee SW, Mayer D, Werner H (2007) Insulin-like growth factor-I controls BRCA1 gene expression through activation of transcription factor Sp1. Horm Metab Res 39(3):179–185 31. Neuhausen SL, Brummel S, Ding YC, Singer CF, Pfeiler G, Lynch HT, Nathanson KL, Rebbeck TR, Garber JE, Couch F, Weitzel J, Narod SA, Ganz PA, Daly MB, Godwin AK, Isaacs C,
123
Breast Cancer Res Treat (2013) 138:753–760
32.
33.
34.
35.
36.
37.
38.
39.
Olopade OI, Tomlinson G, Rubinstein WS, Tung N, Blum JL, Gillen DL (2009) Genetic variation in insulin-like growth factor signaling genes and breast cancer risk among BRCA1 and BRCA2 carriers. Breast Cancer Res 11(5):R76 Ragoussis J, Elvidge GP, Kaur K, Colella S (2006) Matrixassisted laser desorption/ionisation, time-of-flight mass spectrometry in genomics research. PLoS Genet 2(7):e100 R Development Core Team (2008) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. http://www.R-project.org. ISBN 3-90005107-0 Roa BB, Boyd AA, Volcik K, Richards CS (1996) Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2. Nat Genet 14(2):185–187 Maor S, Yosepovich A, Papa MZ, Yarden RI, Mayer D, Friedman E, Werner H (2007) Elevated insulin-like growth factor-I receptor (IGF-IR) levels in primary breast tumors associated with BRCA1 mutations. Cancer Lett 257(2):236–243 Maor SB, Abramovitch S, Erdos MR, Brody LC, Werner H (2000) BRCA1 suppresses insulin-like growth factor-I receptor promoter activity: potential interaction between BRCA1 and Sp1. Mol Genet Metab 69(2):130–136 Lujambio A, Esteller M (2009) How epigenetics can explain human metastasis: a new role for microRNAs. Cell Cycle 8(3):377–382 Lujambio A, Esteller M (2007) CpG island hypermethylation of tumor suppressor microRNAs in human cancer. Cell Cycle 6(12):1455–1459 Formosa A, Lena AM, Markert EK, Cortelli S, Miano R, Mauriello A, Croce N, Vandesompele J, Mestdagh P, Finazzi-Agro` E, Levine AJ, Melino G, Bernardini S, Candi E (2013) DNA methylation silences miR-132 in prostate cancer. Oncogene 32(1):127–134
