Abstract
Background/Aim: Since pathways involving LRRC17 are related to the survival of patients with various cancers, we analyzed LRRC17 as a prognostic gene in serous ovarian cancer. Materials and Methods: Data were collected from Gene Expression Omnibus (GSE9891, GSE13876, and GSE26712) and The Cancer Genome Atlas (TCGA). We performed survival analyses using C statistics, area under the curve, survival plot with optimal cutoff level, and cox proportional regression. Zebrafish embryos were used as an in vivo model. Results: The prognosis of patients with high LRRC17 expression was poorer than that of patients with low LRRC17 expression. Multivariate regression analysis showed that LRRC17 expression, age, and stage were independently related with survival. Knockdown of lrrc17 reduced survival rate and delayed development in zebrafish embryos. We also found that lrrc17 is important for cell viability by protecting from p53-dependent apoptosis. Conclusion: LRRC17 could be a prognostic gene in ovarian cancer as it regulates cancer cell viability through the p53 pathway.
Ovarian tumor is the most severe type of tumor among gynecologic cancers in the United States (1). Around 22,200 patients were diagnosed with ovarian cancer and about 14,000 women died due to ovarian cancer in 2018 in the Unites States (2). In 90% of ovarian cancer patients, the tumor has an epithelial origin, and is classified in 5 histological types, namely, serous, clear cell, endometrioid, mucinous, and undifferentiated. Serous ovarian carcinoma (SOC) constitutes about 70% of epithelial ovarian cancers (3). Since SOCs are usually diagnosed at stage III or IV (51% or 29%), the 5-year disease specific survival of SOC patients is 43%. However, 5-year disease specific survival of other histological types (endometrioid, mucinous, and clear cell carcinoma) of ovarian cancer are 82%, 71%, and 66%, respectively (2).
The 5-year survival of SOC patients varies significantly with the stage of cancer, ranging from 92% for early stage cancer to 27% for late stage cancer (4). Even though significant progresses have been made in both surgical and pharmaceutical treatment strategies, survival rates remain poor (5). One of the major factors associated with poor outcomes of ovarian cancer include the lack of clinically relevant and accurate prognostic biomarkers. Yet, cancer stage, histologic type, tumor grade, debulking status, and cancer antigen 125 (CA-125) constitute the current mainstay for predicting prognosis of SOC (6).
Patient characteristics in GSE9891, GSE13876, GSE26712, TCGA, GSE6008, and GSE27651 cohorts.
Leucine-rich-containing 17 (LRRC17), a secreted protein containing five Leucine-rich-containing domains, has been identified by cDNA subtraction experiments in the S-type neuroblastoma cell line (7). LRRC17 is an inhibitor of receptor activator of NF-ĸB ligand (RANKL)-induced osteoclastogenesis (8). Decreased LRRC17 has been shown to be a serum risk marker for osteoporotic fracture in the spines of postmenopausal women (9). Besides bone metabolism, Christopher et al. have reported that the expression of LRRC17 is particularly high in both oocyte and granulosa cells of follicles (10). Although LRRC17 is highly expressed in female tissues according to Human Protein Atlas, correlation between LRRC17 and ovarian cancer is still unveiled.
In this study, to develop and verify the LRRC17 gene as a potential prognostic and diagnostic factor for SOC, four large scale cohorts [GSE9891, GSE13876, GSE26712 from the Gene Expression Omnibus (GEO), and The Cancer Genome Atlas (TCGA)] were analyzed. In addition, we investigated the role of lrrc17 in zebrafish.
Materials and Methods
Patient data. Data from four cohorts were obtained from GEO (11) and TCGA (12) in March 2019 (13-18). Patients with insufficient clinical and/or genomic information were excluded as described before (19, 20). The characteristics of the patients in each cohort are presented in Table I.
Zebrafish maintenance and MO injection. Adult wild-type AB zebrafish were maintained in an automatic circulation system (Genomic-Design) at 28.5°C. All experiments with zebrafish were performed in accordance with the guidelines of Ulsan National Institute of Science and Technology (UNIST) Institutional Animal Care and Use Committee (IACUC) (IACUC approval number: UNISTIACUC-15-15) and ARRIVE/NC3R. Zebrafish embryos were maintained with E3 solution in incubators at 28°C. A translation-blocking morpholino targeting lrrc17 (Gene Tools, Philomath, OR, USA) was dissolved in DEPC-treated water at the stock concentration of 25 ng/nl. The sequence of lrrc17-MO is 5’-AGCCGCATGGCAAAAAGCAGCAGCT-3’. Thirty embryos were injected with 1.0, 2.5 and 5.0 ng of morpholino at the 1 or 2 cell stage of development for each experimental group. All experiments were performed in three biological replicates for validation. Survival rate of embryos was compared between groups by t-test. Microinjections were performed with a Femtojet 4i microinjector (Eppendorf, Hamburg, Germany).
Acridine orange (AO) stain using zebrafish embryos. Embryos were incubated with 50 μg/ml of Acridine orange solution (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 28°C in the dark. After staining, embryos were washed three times with E3 buffer. Then, zebrafish embryos were anesthetized using tricaine and observed using confocal microscope (LSM880, Carl Zeiss, Oberkochen, Germany).
Whole-mount in situ hybridization (WISH). For fixation, embryos were anesthetized with 76 mM of tricaine, and sacrificed with 0.76 M of tricaine. Then, embryos were fixed using 4% paraformaldehyde in PBS, and dehydrated with methanol at −20°C overnight. Samples were incubated with cold acetone at −20°C for permeabilization. Then, samples were hybridized with a digoxigenin (DIG)-labeled antisense RNA probe in hybridization buffer (50% formamide, SSC solution (0.75 M sodium chloride, 0.075 M trisodium citrate), 500 μg/ml Torula yeast tRNA, 50 μg/ml heparin, 0.1% Tween-20, and 9 mM citric acid (pH 6.5) for 3 days. The samples were washed with diluted SSC solution (30 mM sodium chloride, 3 mM trisodium citrate). Washed samples were blocked with normal goat serum and bovine serum albumin, and incubated with alkaline phosphate-conjugated DIG antibodies (1:5,000) (Roche, Mannheim, Germany) overnight at 4°C. Samples were incubated with the alkaline phosphatase reaction buffer [100 mM Tris (pH 9.5), 50 mM MgCl2, 100 mM NaCl, and 0.1% Tween-20] and the NBT/BCIP substrate (Promega, Madison, WI, USA) for visualization of the signal.
Statistical methods. We performed survival analyses to predict prognosis as described previously (21-23). To discriminate the risk of patients, we performed several survival analyses using Uno's C statistics in the time-dependent area under the curve (AUC), AUCs in the receiver operating characteristic (ROC) curve at 3 years, a survival plot with optimal cutoff, and cox proportional regression. The LRRC17 cutoff level in this study was the maximal Uno's C-index in each dataset. In survival analyses, we evaluated the prognostic significances of LRRC17 as a categorical value. We used Wilcoxon signed rank sum test to identify the expression differences of LRRC17 between healthy individuals and cancer patients. All statistical methods were performed using R software 3.5.1 (The R foundation for Statistical Computing, 2018).
Results
Patient information. We collected the information regarding the characteristics of all patients from four cohorts (GSE9891, n=242; GSE13876, n=157; GSE26712, n=185; TCGA, n=306) (Table I). Two cohorts had information regarding the patients' stage of SOC.
Evaluating the prognostic significance of LRRC17. To assess whether LRRC17 is a prognostic biomarker for SOC, we performed Kaplan–Meier analysis to estimate the relationship between the expression values of LRRC17 and patient survival. Optimal cutoff values of LRRC17 in GSE9891, GSE13876, GSE26712, and TCGA were 4.356, 548.058, 5.870, and 53.908, respectively. LRRC17 was notably related to prognosis of patients in all cohorts (Figure 1). The overall survival of SOC patients with high LRRC17 expression was lower than that of patients with low LRRC17 expression; TCGA (p=0.0022), GSE9891 (p=0.00088), GSE13876 (p=0.013), and GSE26712 (p<0.0001) (Figure 1A-D). To evaluate the prognostic significance of LRRC17 expression in SOC, we used Uno's C-index and the AUCs (Figure 1E-F). The C-indexes in the time-dependent curve were 0.593, 0.602, 0.619, and 0.578 for TCGA, GSE9891, GSE13876, and GSE26712, respectively (Figure 1E). In the 3-year ROC curves, the AUC values for TCGA, GSE9891, GSE13876, and GSE26712 were 0.643, 0.592, 0.629, and 0.546, respectively (Figure 1F).
To examine whether LRRC17 expression value is a self-sufficient prognostic factor for SOC, we used uni- and multi-variate COX proportional regressions. In univariate COX regression, LRRC17 expression (p=0.001), age (p=0.046) and stage (p=0.014) were related to overall survival in GSE9891, while LRRC17 expression was related to survival in GSE13876 (p=0.013) and GSE26712 (p≤0.001); LRRC17 expression (p=0.002) and age (p<0.001) were related to overall survival in TCGA (Table II). After adjustment for the significant parameter, multivariate regression analyses showed that LRRC17 expression (p<0.001), age (p=0.044), and stage (p=0.029) were independently related to overall survival in GSE9891; LRRC17 expression (p=0.011) in GSE13876; and LRRC17 expression (p=0.011) in TCGA (Table II).
LRRC17 expression as a marker for advancement of cancer stage. To determine whether changes in the expression indicate advancement of cancer stage, we compared the expression of LRRC17 between early stages (I & II) and advanced stages (III & IV) of cancer from TCGA and GSE9891. In both cohorts, the expression of LRRC17 was higher at early stages compared to that of advanced stages (Figure 2).
LRRC17 is evolutionally conserved in vertebrates. Although LRRC17 is known to be a negative regulator of the RANKL pathway, this cannot explain the correlation between LRRC17 expression and prognosis of SOC patients. To investigate the novel function of LRRC17, we used zebrafish embryos as an in vivo model. We compared the amino acid sequence of human LRRC17 and zebrafish paralog, lrrc17. Using Pubmed protein blast, we confirmed that 53% of amino acids residues were exactly the same, and 15% of the other amino acid residues had similar structure. This indicates that LRRC17 is evolutionally conserved in vertebrates and may have other functions in addition to bone differentiation.
Zebrafish paralog, lrrc17 promotes cell viability by protecting from p53-dependent apoptosis. Since lrrc17 has only one exon, we used translation blocking morpholino instead of splicing-blocking morpholino. To optimize the dosage of morpholino, we injected 1, 2.5 and 5 ng of translation blocking morpholino to 30 zebrafish embryos in biological triplicates. As the concentration of morpholino increased, the development of embryos delayed (Figure 3A). Then, the survival rate was compared between uninjected embryos and morpholino-injected embryos by t-test. The survival rate of embryos was reduced following injection with 5 ng morpholino compared to uninjected embryos (Figure 3B). These results suggest that lrrc17 is linked to viability in addition to bone differentiation.
While LRRC17 is known to be a negative regulator of the RANKL pathway (8), another LRR family protein, LRRN1, is known as a negative regulator of apoptosis through the regulation of Fas/FasL (24). Therefore, we examined the hypothesis that, similar to LRRN1, lrrc17 could be related to apoptosis, by using AO staining of lrrc17-morphants. Injection with 2.5 ng and 5 ng of morpholino delayed the development of embryos (Figure 3A). These concentrations were considered high and that they may have off-target effects. To avoid off-target effects of morpholino, we used 1 ng of morpholino for AO staining.
Compared to uninjected control embryos, lrrc17-morphants show a high number of AO-positive cells in embryos (Figure 4A). Since p53 is the most common and important key molecule in apoptosis (25), expression of p53 was examined using WISH in lrrc17-morphants. As the signal of AO was increased in lrrc17-morphants, the signal of p53 was also increased (Figure 4B). These results suggest that lrrc17 supports cell viability by protecting from p53-dependent apoptosis.
Survival analysis of patients with SOC. (A-D) Kaplan–Meier survival analysis of patients with SOC with respect to LRRC17 gene expression. Survival analysis was performed in patients with SOC from GSE9891 (A), GSE13876 (B), GSE26712 (C), and TCGA (D). The p-value was calculated using log-rank test and is provided at the bottom left corner of each dataset. (E-F) Time-dependent area under the curve (AUC) analysis and receiver operating characteristic (ROC) curves at 5 years with respect to LRRC17 gene expression. Time-dependent AUC (E) and ROC curve at 3 years (F) in the GSE9891, GSE13876, GSE26712, and TCGA. C-index values and AUC values at 3 years are provided at the bottom right corner of each cohort.
Univariate and multivariate analysis of overall survival in each cohort.
Discussion
In this study, by analyzing large-scale cohorts (GSE9891, GSE13876, GSE26712, and TCGA), we found that LRRC17 is a potent prognostic factor in SOC as much as cancer stage in the FIGO system. In addition to bone differentiation, we found that lrrc17 plays an important role in cell viability by protecting from p53-dependent apoptosis in zebrafish embryos.
The cancer antigen 125 (CA 125) was used to screen and monitor ovarian cancer as a serum marker. Since the efficacy of CA 125 as a biomarker is low, major efforts have been made to enhance the performance of CA 125 by combining it with ultrasonography in several studies (26-29). However, the US Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial has not shown mortality benefit (27). In the United Kingdom Collaborative Trial of Ovarian Cancer Screening, multimodal screening resulted in significantly increased detection of early stage invasive epithelial ovarian/tubal/peritoneal cancers compared to the control arm (29). They also showed a mortality benefit; however, further follow-up is needed to assess whether screening results in a definitive mortality reduction. Human epididymis protein 4 (HE4) has been investigated as a biomarker for ovarian cancer. HE4 has higher expression in the patients with ovarian cancer compared to healthy persons (30). Furthermore, several studies have examined whether the combination of HE4 with CA 125 is a biological marker (31, 32). However, several factors influence serum HE4 concentration, such as age, smoking, kidney function, and non-gynecologic malignancies (33).
With the compelling need to identify new biomarkers for personalized and predictive medicine, several molecular profiling studies have recently been performed. In the CLOVAR study, using the TCGA dataset, four distinct prognostically relevant subtypes were revealed (34). Due to advances in bioinformatics, through analyzing multiple expression profiles from GEO, Esra et al. have developed a co-regulated gene module in ovarian cancer consisting of 84 prognostic genes (35) and Chuan-Di et al. screened out 11 genes associated with prognosis of ovarian cancer (36). These transcriptomic studies provide insights for research towards the identification of biomarkers, yet the molecular pathways in modules consisting of gene clusters need to be validated experimentally. In addition to transcriptomic biomarkers, miRNAs have been widely studied. The role of miR-200 family as a prognostic biomarker in ovarian cancer has been reported by multiple studies (37, 38). Because of inconsistencies in miRNA-based biomarkers, they have not yet been brought to clinical practice (39).
In our study, high expression of LRRC17 in all patients with SOC from all cohorts was correlated with poor overall survival. The p value regarding the expression of LRRC17 in multivariate analysis in the GSE9891 cohort was lower than that in univariate analysis (from p=0.001 to p<0.001), while the p-value regarding stage was higher than that in univariate analysis (from p=0.014 to p=0.029). Moreover, in the TCGA dataset, cancer stage was not a risk factor for overall survival in univariate analysis. This implies that LRRC17 is a more potent prognostic factor than stage regarding the overall survival of patients with SOC. These results suggest that LRRC17 could be applicable as a diagnostic and prognostic biomarker for SOC.
Comparison of LRRC17 gene expression between stage I, II, III and IV from TCGA (A) and GSE9891 (B).
Zebrafish paralog, lrrc17 is important for the development and survival of embryos. (A) Representative images of uninjected embryos and lrrc17-morpholino injected embryos (1.0 ng, 2.5 ng, and 5.0 ng) at 24 h post fertilisation (hpf). Black arrows indicate delayed development of zebrafish embryos. (B) Survival rate of uninjected zebrafish embryos and lrrc17-morpholino injected embryos (1.0 ng, 2.5 ng, and 5.0 ng) at 24 hpf. Survival rate is measured using 30 embryos with three biological replicates.
lrrc17 is important for cell viability by protecting from p53-dependent apoptosis. (A) Lateral view of confocal imaging after AO staining using uninjected embryos and 1 ng of lrrc17-morpholino injected embryos at 26 24 h post fertilisation (hpf). Representative images selected from 30 uninjected embryos and lrrc17-morpholino injected embryos. (B) Lateral view of whole-mount in situ hybridization imaging using a probe for p53. Thirty embryos were fixed 26 hpf. All results are representative images from three biological replicates.
This is the first study to evaluate LRRC17 as a prognostic marker for SOC. By analyzing large-scale publicly accessible four cohorts, we gained high statistical power. Additionally, we found that lrrc17 in zebrafish embryos is important for cell viability by protecting from apoptosis. As LRRN1 is linked to negative regulation of apoptosis through Fas/FasL (24), lrrc17 was also found to be a negative regulator of p53-dependent apoptosis. In a previous study, we have shown that the basal levels of p53 are important for drug resistance in colon cancer (40). This means that high levels of LRRC17 can inhibit chemotherapy-induced apoptosis in SOC. However, for clinical application, identification of a more specific mechanism of LRRC17 function is necessary. In conclusion, LRRC17 could be applicable as a prognostic gene in addition to cancer stage.
Acknowledgements
This study was supported by Gachon University Gil Medical Center (FRD2019-09) and grants from the Medical Research Center (MRC) Program through the National Research Foundation of Korea (NRF) funded by the Korea government (NRF-2018R1A5A2023879). This work was supported by the Institute for Basic Science (IBS-R022-D1).
Footnotes
↵* These Authors contributed equally to this work.
Authors' Contributions
DSK and YHK made substantial contributions to the conception and design. HJH, JK, and EJK collected data and JWK, YK (Yoongjoo Kim), and YK (Yeji Ko) performed statistical analysis. JJP and MH analyzed and interpreted the data. CO produced data using zebrafish. DSK and CO wrote the manuscript. JMK, KH, and SZY made critical revision.
Conflicts of Interest
All Authors have declared that they have no conflicts of interest to disclose in relation to this study.
- Received August 7, 2020.
- Revision received August 24, 2020.
- Accepted August 25, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
