Most malignant ovarian tumors in adult women are epithelial ovarian tumors. These neoplasms are classified into different morphological categories according to the appearance of epithelial cells in serous, mucinous, endometrial, clear cell carcinomas, mixed, squamous, transient, and non-differentiated tumors [1
]. Serous adenocarcinomas are the most common and make up 75% of all ovarian cancer. Each histological type of ovarian tumor and the degree of differentiation of the cells is associated with certain molecular-genetic changes.
The Hedgehog signaling pathway is a developmental pathway involved in formation of various tissues and organs, including the ovary. In mammals, canonical signal transduction is triggered by binding of the ligand Hedgehog (HH) to the transmembrane receptor Patched (PTCH). This leads to receptor internalization and exposure of the protein Smoothened (SMO) on the cell surface. Activation of SMO triggers the release of transcription factors GLI (GLI1-3) from Suppressor of Fused (SUFU) and the translocation of GLI to the nucleus. GLI, then, activates transcription of HH target genes which are involved in cell cycle regulation, proliferation, adhesion, epithelial-mesenchymal transition, self-renewal, and pathway autoregulation [2
]. Recently, survivin has also been identified as a novel HH target gene. Vlčková et al demonstrated that expression of survivin is directly regulated by GLI2, more specifically by the GLI2ΔN isoform [4
]. A study by Brun et al showed that survivin is overexpressed in HH-driven medulloblastoma. They suggest it may represent a novel therapeutic target for this disease [5
Survivin is the smallest member of the inhibitors of apoptosis (IAP) family of proteins in mammals [6
]. The gene for survivin, BIRC5
(baculoviral IAP repeat containing 5), is located on human chromosome 17q25 [7
]. The BIRC5
gene has four dominant exons (1, 2, 3, and 4) and two cryptic exons (2B and 3B). Alternative splicing of its pre-mRNA produces at least five different mRNAs encoding five different proteins with different intracellular distribution and anti-apoptotic properties: wild type survivin (S WT), survivin 2α (S 2α), survivin 2B (S 2B), survivin 3B (S 3B), and survivin-ΔEx3 (S ΔEx3) (Figure 1
S WT, discovered in 1997, contains a BIR domain necessary for its anti-apoptotic function [9
]. It is expressed during development but is not expressed in most differentiated adult tissues. The overexpression of survivin is common in almost all tumors and is indicative of decreased overall survival, increased rate of recurrence, and resistance to therapy [6
S 2α is the smallest isoform with a truncated BIR domain [10
]. The reports about its function in cancer is conflicting, while some studies report that it binds to and attenuates anti-apoptotic activity of WT survivin or correlates with expression in lower stages of the disease [8
], other studies report its association with resistance to treatment [11
S 2B is the longest survivin isoform, but the insertion of cryptic exon 2B interrupts the BIR domain [13
]. Its function remains unclear. While some studies report that it promotes cell death, or that its expression is inversely correlated with the tumor stage [8
], others report its expression being correlated with treatment resistant cancers [18
S 3B lacks the carboxyl-terminal coiled-coil domain of WT survivin [19
]. It is considered cytoprotective [20
] and its overexpression has been correlated with shorter overall survival and resistance to therapy [11
S Δex3 contains a bipartite nuclear localization signal (NLS) and localizes to nucleus in cancer cells [23
]. Its expression in tumors is associated with aggressive disease and unfavorable prognosis [24
Survivin exhibits cell-cycle-dependent expression that is mostly controlled at transcription level. Its accumulation during mitosis is also influenced by posttranslational modifications that affect its stability. When expressed during mitosis, it is located in various sections of the mitotic apparatus such as centrosomes, microtubules, and anaphase spindles, and remains of the mitotic apparatus [6
]. The homologous deletion of survivin results in early embryo death, which shows its crucial role in cellular development, differentiation, and homeostasis. It is selectively expressed in cancer cells, but not in healthy tissues. Excess accumulation of survivin is associated with the development of disease, disease recovery, and prognosis in various cancers, including bladder cancer, cervix, head and neck, prostate, skin, and ovarian [7
]. A global deregulation of the BIRC5
gene mediated by oncogenes (including STAT3, E2F or mutated RAS) or by the loss of tumor suppressors such as p53 or APC, accounts for the selective expression of survivin in cancer [26
]. Since survivin is expressed exclusively in cancer cells, it is an interesting target for targeted therapy and new methods for detection of survivin, as well as new inhibitors, are constantly being developed [18
polymorphisms have been studied and have been associated with susceptibility to lung [29
], gastric [30
], bladder [31
], oral [32
], and liver cancer [33
] as well as age of onset in ovarian cancer [7
] and survival in colorectal cancer [8
] and breast cancer [34
X-ray crystallography has shown that survivin is organized as a dimer [6
]. Since its isoforms exhibit various apoptosis-related properties, it is believed that the formation of survivin heterodimers with its isoforms may be important for regulating the function of survivin [8
]. Since survivin isoforms can affect the activity of wild type survivin, and the transcription of this gene is regulated by HH-GLI signaling, it is important to investigate which factors contribute to the expression of certain isoforms. In this paper our goal was to examine the role of individual GLI transcription factors in the transcriptional regulation of survivin isoforms.
2. Materials and Methods
2.1. Generation of Knockout Cell Lines
SKOV-3 cell line [3
] was maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin. The CRISPR/Cas9 system was used to generate the knock-out cell lines. sgRNA molecules targeting the area surrounding the STG site of GLI1, GLI2, and GLI3 genes were generated using the online tool at crispr.mit.edu web site. Five top ranking sgRNA molecules were selected, and the one closest to the ATG site of each gene was selected. The sgRNA oligos were annealed, phosphorylated and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 (plasmid #42230, Addgene, Watertown, MA, USA) [35
] into the BbsI restriction site, and the resulting vectors were sequenced on the ABI Prism 310 sequencing machine. The oligo sequences are listed in Supplementary Table S1
. The SKOV-3 cell line was transfected with the generated vectors using the Xfect transfection reagent (Clontech, Mountain View, CA, USA). At 24 h post-transfection, the cells were trypsinized and plated into 96-well plates at the density of 1 cell/well. Several cell lines were propagated from single cells, and protein expression for each of the three GLI proteins was examined by Western blot to select the lines with the best knock-out for each protein.
2.2. Cell Culture Experiments
Cells were plated in 6-well plates, treated with two concentrations of GANT-61 (10 and 20 µM). The cells were collected 24 h after treatment. For the transfection of GLI1 (pcDNA4NLSMTGLI1, a kind gift from Prof. F. Aberger, Austria), GLI2 (p4TO6MTGLI2, a kind gift from Prof. M. Stevanovic, Serbia), GLI3 (pcDNA4/TO/GLI3richtig, a kind gift from Prof. M. Stevanovic, Serbia), and GLI3R (EGLI3-PHS, a kind gift from Prof. R. Toftgard, Sweden) the cells were seeded in 6-well plates and transfected the next day with 5 µg of plasmid DNA using the X-fect reagent (Clontech) following the manufacturer’s instructions.
2.3. Clinical Samples
Forty ovarian carcinoma (OC) samples and nine healthy fallopian tubes (FT) tissue samples (excised for reasons other than malignant transformations) were collected at the Department of Obstetrics and Gynaecology, University Hospital Centre Zagreb, School of Medicine, University of Zagreb. Patient blood samples were also collected. Blood samples collected from 74 healthy elderly women with no personal history of ovarian cancer were used as control for comparing polymorphism frequencies.
All patients gave their informed consent before the samples were taken, and samples were collected with the approval of the hospital’s Ethical Committee (number of approval 02/21 AG, issued by the University Hospital Centre Zagreb on March 7th, 2017). The study was carried out following the rules of the Declaration of Helsinki Principles. All tissue samples taken during surgery were immediately placed in a vial containing 1 mL RNALater solution (Invitrogen, Carlsbad, CA, USA), kept at 4 °C overnight, and DNA and RNA were extracted the following day. DNA from tissue samples was extracted by the standard phenol-chloroform method and from blood by the salting out method.
2.4. Western Blot
Proteins were extracted by sonication in RIPA buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Western blot was performed as previously described [36
] using the following primary antibodies: GLI1 (V812, Cell Signaling Technology, 1:1000, Danvers, MA, USA), GLI2 (ARP31885_T100, Aviva Systems Biology, 1:1000, San Diego, CA, USA), GLI3 (19949-1-AP, ProteinTech, 1:600, Rosemont, IL, USA), GFP for detection of GFP tagged GLI3R (Abcam ab6556, 1:1000, Cambridge, UK), and β-actin (60008-1-Ig, ProteinTech, 1:4000) and γ-tubulin (Santa Cruz Biotechnology, sc-7396, 1:500, Dallas, TX, USA) as loading controls.
2.5. Expression Analysis
RNA was extracted using TRIZol reagent (Thermo Fisher) and reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). qRT-PCR was performed on a CFX96 machine (Bio-Rad Laboratories, Hercules, CA, USA) using Sso Fast EVAGreen Supermix (Bio-Rad).
The PCR conditions were as follows: initial denaturation at 95 °C for 3 min, 40 cycles of 95 °C for 15 sec, 61 °C for 1 min, and finally melting curve from 70 °C to 95 °C. All experiments were performed at least in duplicate. Primer sequences are listed in Supplementary Table S2
. Expression levels were calculated using the 2−ΔΔCt
formula, relative to the housekeeping gene TBP
]. For samples with no expression of a specific target after 40 cycles of qRT-PCR, the Ct was set to 40 to enable the statistical analysis of data [38
Whole coding region (including alternate exons S 2α, S 2B, and S 3B) was genotyped, including the six SNPs in the BIRC5
promoter and four SNPs in the 3′UTR region selected from the National Center for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/snp
). Primers were designed using the Primer3 online tool (http://bioinfo.ut.ee/primer3/) [39
]. Thirteen PCR fragments were analyzed using high resolution melting analysis on High-Resolution Melter (HR-1, Idaho Technology, Salt Lake City, UT, USA) as described in Cvok et al, 2008 [40
] followed by Sanger sequencing (ABI PRISM 310 Genetic Analyzer, Applied Biosystems). Due to presence of four different polymorphisms in the PCR product of exon 4 and the beginning of 3′UTR, it was directly sequenced. PCR primer sequences and cycling conditions are listed in Supplementary Table S3
. Due to high GC content in the DNA sequence of promoter region, in all PCR fragments located in the promoter CG RICH buffer (Roche) was added.
2.7. Statistical Analysis
D'Agostino–Pearson test was used for testing distribution normality of the expression data. For variables which showed normal distribution after logarithmic transformation, independent sample t-test and one-way analysis of variance were used to test the differences in isoforms’ expressions. Otherwise Mann–Whitney test and Kruskal–Wallis test were used. Nonparametric Spearman rank correlation coefficient (ρ) was used to assess the correlation of expression between various isoforms. For comparing polymorphism frequencies Fisher’s exact test (2 × 2) and χ2 test (3 × 2) were used. Two-tailed p values less than 0.05 were considered statistically significant. Statistical analysis was performed using MedCalc, version 18.2.1 (MedCalc Software bvba, Ostend, Belgium).
We have detected survivin isoform expression (S WT, S 2α, S 2B, S 3B, and S Δex3) in the ovarian cancer cell line SKOV-3, that follows the trend of isoform expression levels in its GLI KO variants and in ovarian cancer samples (Figure 5
). The BIRC5
gene has been recognized as one of the transcriptional targets of the HH-GLI signaling pathway [4
]. The GLI2ΔN isoform has been indicated as the main transcription factor that regulates BIRC5
gene expression. We have previously detected activity of the HH-GLI pathway in ovarian cancer [36
] and therefore we wanted to examine the role of the GLI transcription factors not only in the regulation of survivin wild type expression, but also its isoforms. Because of the different roles of certain isoforms and their ability to regulate survivin function after heterodimer formation it is important to investigate how certain isoforms are regulated. Interestingly, our results confirm that survivin is regulated by the HH-GLI signaling pathway, but not all isoforms are regulated in the same manner. More specifically, all isoforms are regulated by GLI1, but isoforms S WT, S 2α, and S 2B can additionally be inhibited with the GLI1/2 inhibitor GANT-61, suggesting that these isoforms can also be regulated through other GANT-61 targets, most likely, GLI2.
Seven BIRC5 polymorphisms are also associated with the expression of survivin isoforms. Therefore, there is an intricate regulation of expression between genetic influence and transcription factors such as GLI proteins to determine the final expression of survivin isoform expression and their anti- and pro-apoptotic role that could determine the fate of the cells.