Original Article FREM2 Is an Independent Predictor of Poor Survival in Clear Cell Renal Cell Carcinoma-Evidence from the Cancer Genome Atlas (TCGA)

Int J Clin Exp Med 2019;12(12):13741-13748 www.ijcem.com /ISSN:1940-5901/IJCEM0076963

Original Article FREM2 is an independent predictor of poor survival in clear cell renal cell carcinoma-evidence from the cancer genome atlas (TCGA)

Weiping Huang, Yongyong Lu, Xixi Huang, Feng Wang, Zhixian Yu

Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, Zhejiang Province, China Received February 26, 2018; Accepted October 7, 2018; Epub December 15, 2019; Published December 30, 2019

Abstract: Fraser syndrome protein 1 (FRAS1) and FRAS1 related extracellular matrix protein 1 and 2 (FREM1, FREM2) are a novel group of basement membrane proteins. The relationship between the three gene (FRAS1, FREM1, FREM2) and renal clear cell carcinoma is completely unclear. Thus, in this research, we used the mRNA sequencing data derived from TCGA kidney renal clear cell carcinoma cohort to assess the association of FRAS1, FREM1 and FREM2 with different clinical features. FRAS1, FREM1 and FREM2 mRNA levels were downregulated in KIRC (kidney renal clear cell carcinoma) tissues than normal tissues (FRAS1, P < 0.0001; FREM1, P < 0.0001, FREM2, P = 0.0001), respectively. FRAS1, FREM1 and FREM2 were significantly different in histologic grade, pathologic stage and pathologic T (all P < 0.001). Low FRAS1, FREM1 and FREM2 expression were correlated to worsen overall survival (all P < 0.01), and Low FREM1 and FREM2 expression had worse relapse-free survival (FREM1, P = 0.0113; FREM2, P = 0.0424). Multivariate Cox regression analysis revealed that FREM2 was an independent prognostic factor for overall survival. Taken together, FREM2 expression is an independent predictor of poor survival in renal clear cell carcinoma and is positively associated with advanced stage, high histologic grade.

Keywords: FREM2, FRAS1, renal cell carcinoma, KIRC, TCGA

Introduction sion of RCC [6]. With the further study of the molecular mechanism of RCC, finding a diag- Renal cell carcinoma (RCC), the third most nostic and prognostic molecular biomarker or malignant tumors of urinary system, account- an effective treatment will be a vital future ing for 63990 newly diagnosed cancer cases direction. and 14400 cancer deaths in the United States in 2017 [1, 2]. In China, an estimated 66800 Fraser syndrome protein 1 (FRAS1) and FRAS1 patients newly diagnosed with RCC and 23400 related extracellular matrix protein 1 and 2 patients died of RCC have occurred in 2015 [3]. (FREM1, FREM2) are a novel group of base- Clear cell RCC (ccRCC), the most common RCC ment membrane proteins [7]. Many research- histological subtype, consist of 70%-80% renal es have revealed that FRAS1/FREM proteins cancer [1, 4]. Surgery treatment is the first were mutated in patients with Fraser syndrome choice for patients with early stage RCC [4]. As [8, 9]. The deficiency of FRAS1/FREM proteins for patients with late stage RCC orrecurrent or could lead to several diseases, such as renal metastatic renal carcinoma, no effective treat- agenesis, congenital diaphragmatic hernia [10- ment was accessible, so the 3 and 5-year can- 12]. Furthermore, FRAS1 could mediates both cer-specific survival rates still remain poor [5]. the initiation of the mammalian kidney and the The occurrence and development of RCC is a integrity of renal glomeruli [7]. However, there multifactor, multistep and multistage complex are few studies about the functions of FRAS1/ biological process. Amounts of genes were FREM proteins in different cancers. So, the re- involved in the complex biological process, con- lationship between the three gene (FRAS1, tributing to tumorigenesis and tumor progres- FREM1, FREM2) and renal clear cell carcinoma FREM2 is an independent predictor of OS in ccRCC

Table 1. Characteristics of KIRC patients in and the data of clinicopathological characteris- TCGA database tics for patients with kidney cancer were do- Variables Case, n (%) wnloaded from UCSC Xena (http://xena.ucsc. edu/). Sample type Primary Tumor 533 (87.95%) FRAS1, FREM1 and FREM2 expression in Solid Tissue Normal 72 (11.88%) KIRC patients Additional-New Primary 1 (0.17%) Age at diagnosis Compared to normal controls, RSEM (RNA-Seq by Expectation-Maximization) expression val- ≤ 60 264 (49.53%) ues of FRAS1, FREM1 and FREM2 were used > 60 269 (50.47%) for statistical analysis. We further analyzed the Sex association of FRAS1, FREM1 and FREM2 with Female 188 (35.27%) different clinical features, including age at ini- Male 345 (64.73%) tial pathologic diagnosis, gender, size (longest Tumor size dimension), histologic grade, pathologic stage, < 2 cm 305 (57.22%) pathologic T, pathologic N, pathologic M. ≥ 2 cm 121 (22.7%) Prognosis analysis NA 107 (20.08%) Histologic grade Differences in overall survival (OS) and rela- G1 + G2 243 (45.59%) pse-free survival (RFS) between “high” and G3 + G4 282 (52.91%) “low” expression groups were compared using GX 5 (0.94%) Kaplan-Meier curves, with p-values calculated NA 3 (0.56%) via log-rank test. Univariate and multivariate Cox regression models were used to identify Pathologic stage the prognostic effects of clinical features and I + II 324 (60.79%) the three genes. III + IV 207 (38.84%) NA 2 (0.38%) Statistical analysis Pathologic T All data were analyzed by using SPSS 23.0 T1 + T2 342 (64.17%) (SPSS, Inc., Chicago, IL, USA). The Mann-Whit- T3 + T4 191 (35.83%) ney U test was applied to compare the expres- Pathologic N sion of the three genes in terms of differ- N0 240 (45.03%) ent clinicopathological characteristics includ- N1-2 16 (3%) ing age at initial pathologic diagnosis, gender, NX 277 (51.97%) size (longest dimension), histologic grade, Pathologic M pathologic stage, pathologic T, pathologic N, M0 422 (79.17%) pathologic M. RSEM (RNA-Seq by Expectation- M1 79 (14.82%) Maximization) expression values were used for MX 30 (5.63%) statistical analysis. Univariate logistic regres- NA 2 (0.38%) sion analysis of the three genes expression as a categorical dependent variable was used, with a median expression of the three genes, in is completely unclear. Thus, in this research, we association with clinicopathologic characteris- used the mRNA sequencing data derived from tics. Differences in overall survival between TCGA kidney renal clear cell carcinoma cohort “high” and “low” expression groups (defined by to assess the association of FRAS1, FREM1 median value of the three genes expression) and FREM2 with different clinical features. were compared using Kaplan-Meier curves, with p-values calculated via log-rank test. Uni- Materials and methods variate Cox regression analysis was used to Data of RNA-Seq expression and clinicopatho- estimate survival based on the three genes logical characteristics from TCGA database expression and clinicopathologic factors. Mul- tivariate Cox analysis was used to compare The mRNA sequencing data (combining level 3 the influence of the three genes expression data from Illumina GA and HiSeq platforms) on survival along with clinicopathologic charac-

13742 Int J Clin Exp Med 2019;12(12):13741-13748 FREM2 is an independent predictor of OS in ccRCC

Figure 1. FRAS1, FREM1 and FREM2 levels in KIRC and normal controls. A. FRAS1 (P < 0.0001); B. FREM1 (P < 0.0001); C. FREM2 (P < 0.0001).

Table 2. Association between FRAS1 expression and logical and the three gene expression clinical characteristics in KIRC patients data were analyzed in January 2018. FRAS1 The median age at diagnosis was 61 Variables Numbers U value P value expression years (range, 26-90 years). All of the Age at diagnosis patients were evaluated according to the system for TNM described in the ≤ 60 264 32342 9.49 ± 1.55 0.75 AJCC cancer staging manual. The medi- > 60 269 9.27 ± 1.67 an of overall survival (OS) was 39.23 Sex months (range, 0.06-151.23 months) Female 188 31091.5 9.47 ± 1.54 0.431 and the median of relapse-free survival Male 345 9.33 ± 1.65 (RFS) was 40.27 months (range, 0.1- Tumor size 151.23 months). The clinicopathologi- < 2 cm 305 17499 9.45 ± 1.62 0.405 cal characteristics of the TCGA data- ≥ 2 cm 121 9.34 ± 1.58 base are summarized in Table 1. Histologic grade FRAS1, FREM1 and FREM2 expression G1 + G2 243 29124.5 9.62 ± 1.46 0.003 and association with clinicopathologi- G3 + G4 282 9.22 ± 1.67 cal characteristics Pathologic stage I + II 324 25507.5 9.59 ± 1.57 < 0.001 A total of 533 KIRC samples with the III + IV 207 9.04 ± 1.62 three genes expression and clinicopathological data were analyzed from Pathologic T TCGA. Solid normal tissue samples T1 + T2 342 25595.5 9.56 ± 1.57 < 0.001 were excluded. As shown in Figure 1, T3 + T4 191 9.06 ± 1.64 FRAS1, FREM1 and FREM2 mRNA lev- Pathologic N els were downregulated in KIRC tis- N0 240 1068 9.34 ± 1.56 0.003 sues than normal tissues (FRAS1, P < N1-2 16 8.25 ± 1.90 0.0001; FREM1, P < 0.0001, FREM2, P Pathologic M = 0.0001), respectively. Further, the M0 422 14379 9.42 ± 1.57 0.052 associations between FRAS1, FREM1 M1 79 9.03 ± 1.78 and FREM2 expression and clinicopathological characteristics in KIRC patients were explored. FRAS1 was signifi- teristics. A P-value of less than 0.05 was con- cantly different in histologic grade (P = 0.003), sidered statistically significant. pathologic stage (P < 0.001), pathologic T (P < 0.001), pathologic N (P = 0.003) (Table 2). Results FREM1 expression was found to be significantly Patient characteristics from TCGA database different in sex (P < 0.001), histologic grade (P < 0.001), pathologic stage (P < 0.001), patho- From the TCGA KIRC data, 533 primary tumors logic T (P < 0.001), pathologic M (P = 0.001) and 72 normal controls with both clinicopatho- (Table 3). FREM2 expression was also signifi-

13743 Int J Clin Exp Med 2019;12(12):13741-13748 FREM2 is an independent predictor of OS in ccRCC

Table 3. Association between FREM1 expression and clini- [95% CI: 1.312-2.376], P = 0.0002, cal characteristics in KIRC patients Figure 2B). Besides, low FREM2 ex- FREM1 pression was also identified to be Variables Numbers U value P value expression correlated with worse OS in KIRC Age at diagnosis patients (HR = 2.187 [95% CI: 1.625- Figure 2C ≤ 60 264 34147 4.58 ± 2.47 0.444 2.942], P < 0.001, ). Next, RFS in low FREM1 expression group > 60 269 4.41 ± 2.34 was significantly lower than the high Sex FREM1 expression group (HR = 2.954 Female 188 26571 4.96 ± 2.22 < 0.001 [95% CI: 1.296-6.736], P = 0.0113, Male 345 4.25 ± 2.47 Figure 2E). RFS in low FREM2 expres- Tumor size sion group was also poorer than the < 2 cm 305 17679 4.55 ± 2.49 0.5 high FREM2 expression group (HR = ≥ 2 cm 121 4.36 ± 2.20 2.432 [95% CI: 1.07-5.489], P = Histologic grade < 0.001 0.0243, Figure 2F). However, low FR- G1 + G2 243 25380 5.01 ± 2.34 AS1 expression was found no associa- G3 + G4 282 3.97 ± 2.29 tion with RFS in KIRC patients (HR = Pathologic stage < 0.001 1.289 [95% CI: 0.5693-2.92], P = 0.544, Figure 2D). I + II 324 24653.5 4.93 ± 2.53 III + IV 207 3.83 ± 2.02 To evaluate whether FRAS1, FREM1 Pathologic T < 0.001 and FREM2 could be the independ- T1 + T2 342 25139.5 4.83 ± 2.56 ent prognostic factors for KIRC pati- T3 + T4 191 3.88 ± 1.96 ents, the univariate and multivariate Pathologic N 0.117 Cox regression analysis was conduct- N0 240 1470 4.42 ± 2.52 ed, including age at initial patholo- N1-2 16 3.39 ± 1.93 gic diagnosis, gender, size (longest Pathologic M dimension), histologic grade, patho- M0 422 12909.5 4.66 ± 2.43 0.001 logic stage, pathologic T, pathologic N, pathologic M, FRAS1, FREM1 and M1 79 3.74 ± 2.18 FREM2 expression levels (Table 5). In the univariate Cox regression analy- cantly different in sex (P < 0.001), histologic sis, higher FRAS1 (HR = 0.571 [95% CI: 0.42- grade (P < 0.001), pathologic stage (P < 0.001), 0.775], P < 0.001), FREM1 (HR = 0.566 [95% pathologic T (P < 0.001), pathologic M (P < CI: 0.417-0.766], P < 0.001), and FREM2 (HR = 0.001) (Table 4). However, no significant differ- 0.453 [95% CI: 0.331-0.62], P < 0.001) expres- ence was observed in age at diagnosis, tumor sion was correlated with longer OS in KIRC size, and pathologic N for FREM1 and FREM2, patients. Furthermore, age (HR = 1.807 [95% and no significant difference in age, gender, CI: 1.323-2.467], P < 0.001), tumor size (HR = tumor size and pathologic M for FRAS1 expres- 1.499 [95% CI: 1.076-2.088], P = 0.0168), his- sion (all P > 0.05). tologic grade (HR = 2.623 [95% CI: 1.869- 3.68], P < 0.001), pathologic stage (HR = Prognostic performances of FRAS1, FREM1 3.909 [95% CI: 2.854-5.355], P < 0.001), pa- and FREM2 in KIRC patients thologic T (HR = 3.216 [95% CI: 2.38-4.346], P < 0.001), pathologic N (HR = 3.532 [95% CI: According to the median of FRAS1, FREM1 and 1.919-6.503], P < 0.001), pathologic M (HR = FREM2, the patient’s OS and RFS were ana- 4.302 [95% CI: 3.158-5.862], P < 0.001) were lyzed via Kaplan-Meier analysis by using Log- all related with OS in KIRC patients. Multivari- rank (Mantel-Cox) test. As presented in Figure ate COX regression analysis indicated that 2, Kaplan-Meier survival curve revealed that FREM2 (HR = 0.402 [95% CI: 0.246-0.656], patients with low FRAS1 expression had poor- P < 0.001), age (HR = 1.737 [95% CI: 1.099- er OS in KIRC patients (hazard ratio [HR] = 2.745], P = 0.018), pathologic T (HR = 1.897 1.752 [95% CI: 1.303-2.357], P = 0.0003, Fig- [95% CI: 1.187-3.033], P = 0.007) and patho- ure 2A). Low FREM1 expression was associat- logic M (HR = 2.63 [95% CI: 1.604-4.315], P < ed with worse OS in KIRC patients (HR = 1.766 0.001) were independent prognostic factors

13744 Int J Clin Exp Med 2019;12(12):13741-13748 FREM2 is an independent predictor of OS in ccRCC

Table 4. Association between FREM2 expression and Discussion clinical characteristics in KIRC patients FREM2, located on chromosome 3q- FREM2 Variables Numbers U value P value 13.3, encodes FRAS1 related extracel- expression lular matrix protein 2 [13]. Lots of extra- Age at diagnosis cellular matrix (ECM)-related molecules ≤ 60 264 34057 8.34 ± 2.08 0.414 could be associated with the ability of > 60 269 8.07 ± 2.32 cancer cells migration and invasion. Sex Zhan et al [14] found that FRAS1 kno- Female 188 24566.5 8.70 ± 2.05 < 0.001 ckdown reduced A549 cells migration Male 345 7.93 ± 2.24 and invasion through downregulation of Tumor size FAK signaling. Butz et al [15] identified < 2 cm 305 16466 8.3 ± 2.26 0.83 that the expression of FRAS1 could be ≥ 2 cm 121 8.03 ± 2.26 regulated by miRNA-1. MiR-1 could act Histologic grade as a tumor suppressor to regulate sev- G1 + G2 243 25771 8.79 ± 1.69 < 0.001 eral genes, including MET, Slug, CCND2, G3 + G4 282 7.77 ± 2.41 CXCR4, in order to inhibit cancer cell cycle progression and metastasis [16]. Pathologic stage Besides, amplification of the FREM2 I + II 324 23250.5 8.63 ± 1.97 < 0.001 gene is correlated with mesenchymal III + IV 207 7.55 ± 2.39 differentiation in gliosarcoma [17], sug- Pathologic T gesting FREM2 was associated with T1 + T2 342 23242.5 8.56 ± 2.01 < 0.001 the ability of cancer cells migration and T3 + T4 191 7.56 ± 2.38 invasion. However, there are few studies Pathologic N about the functions of FRAS1/Frem pro- N0 240 1224 8.19 ± 2.31 0.15 teins in cancers. So, the relationship N1-2 16 6.80 ± 2.70 between the three gene (FRAS1, FREM1, Pathologic M FREM2) and renal clear cell carcinoma M0 422 11625 8.37 ± 2.13 < 0.001 is completely unclear. Thus, in this re- M1 79 7.27 ± 2.53 search, we used the mRNA sequencing data derived from TCGA kidney renal clear cell carcinoma cohort to assess for OS. Subsequent univariate and multivariate the association of FRAS1, FREM1 and FREM2 Cox regression models were implemented to with different clinical features. determine the independence of the prognostic value of the three genes in KIRC patients’ From the TCGA KIRC data, 533 primary tumors RFS. In the univariate Cox regression analysis, and 72 normal controls with both clinicopatho- higher FREM1 (HR = 0.409 [95% CI: 0.176- logical and the three gene expression data 0.948], P = 0.037), and FREM2 (HR = 0.418 were analyzed in January 2018. The median [95% CI: 0.18-0.968], P = 0.042) expression age at diagnosis was 61 years (range, 26-90 was correlated with longer OS in KIRC pati- years). All of the patients were evaluated ac- ents. Moreover, gender (HR = 4.241 [95% CI: cording to the system for TNM described in the 1.259-14.289], P = 0.02), histologic grade (HR AJCC cancer staging manual. The median of = 3.97 [95% CI: 1.471-10.716], P = 0.006), overall survival (OS) was 39.23 months (range, pathologic stage (HR = 6.728 [95% CI: 2.65- 0.06-151.23 months) and the median of dis- 17.078], P < 0.001), pathologic T (HR = 4.813 ease free survival (DFS) was 40.27 months [95% CI: 2.038-11.363], P < 0.001), pathologic (range, 0.1-151.23 months). The clinicopatho- M (HR = 5.252 [95% CI: 2.012-13.709], P < logical characteristics of the TCGA database 0.001) were correlated with RFS in KIRC pa- are summarized in Table 1. FRAS1, FREM1 and tients. However, multivariate Cox regression FREM2 mRNA levels were downregulated in suggested that only pathologic stage (HR = KIRC tissues than normal tissues (FRAS1, P < 6.531 [95% CI: 2.391-17.839], P < 0.001) could 0.0001; FREM1, P < 0.0001, FREM2, P = be an independent prognostic factor for DFS 0.0001), respectively. FRAS1, FREM1 and FR- for KIRC patients. EM2 were significantly different in histologic

13745 Int J Clin Exp Med 2019;12(12):13741-13748 FREM2 is an independent predictor of OS in ccRCC

Figure 2. Kaplan-Meier survival curves for OS and RFS in KIRC patients stratified by median of FRAS1, FREM1 and FREM2. A. OS-FRAS1; B. OS-FREM1; C. OS-FREM2; D. RFS-FRAS1; E. RFS-FREM1; F. RFS-FREM2.

Table 5. Univariate and multivariate Cox regression analysis in KIRC patients Univariate analysis Multivariate analysis HR (95% CI) P value HR (95% CI) P value Overall survival (OS) FRAS1 level (high vs. low) 0.571 (0.42, 0.775) < 0.001 FREM1 level (high vs. low) 0.566 (0.417, 0.766) < 0.001 FREM2 level (high vs. low) 0.453 (0.331, 0.62) < 0.001 0.402 (0.246, 0.656) < 0.001 Age at initial pathologic diagnosis (> 60 vs. ≤ 60) 1.807 (1.323, 2.467) < 0.001 1.737 (1.099, 2.745) 0.0180 Gender (male vs. female) 0.955 (0.703, 1.296) 0.7662 Tumor size (longest dimension) (≥ 2 cm vs. < 2 cm) 1.499 (1.076, 2.088) 0.0168 Histologic grade (G3 + G4 vs. G1 + G2) 2.623 (1.869, 3.68) < 0.001 Pathologic stage (III + IV vs. I + II) 3.909 (2.854, 5.355) < 0.001 Pathologic T (T3 + T4 vs. T1 + T2) 3.216 (2.38, 4.346) < 0.001 1.897 (1.187, 3.033) 0.0070 Pathologic N (N1 vs. N0) 3.532 (1.919, 6.503) < 0.001 Pathologic M (M1 vs. M0) 4.302 (3.158, 5.862) < 0.001 2.63 (1.604, 4.315) < 0.001 Relapse-free survival (RFS) FRAS1 level (high vs. low) 0.918 (0.402, 2.096) 0.8390 FREM1 level (high vs. low) 0.409 (0.176, 0.948) 0.037 FREM2 level (high vs. low) 0.418 (0.18, 0.968) 0.042 Age at initial pathologic diagnosis (> 60 vs. ≤ 60) 0.933 (0.409, 2.129) 0.8690 Gender (male vs. female) 4.241 (1.259, 14.289) 0.02 Size (longest dimension) (≥ 2 cm vs. < 2 cm) 1.481 (0.582, 3.767) 0.41 Histologic grade (G3 + G4 vs. G1 + G2) 3.97 (1.471, 10.716) 0.006 Pathologic stage (III + IV vs. I + II) 6.728 (2.65, 17.078) < 0.001 6.531 (2.391, 17.839) < 0.001 Pathologic T (T3 + T4 vs. T1 + T2) 4.813 (2.038, 11.363) < 0.001 Pathologic N (N1 vs. N0) 0.048 (0, 1097630543) 0.803 Pathologic M (M1 vs. M0) 5.252 (2.012, 13.709) 0.001 grade, pathologic stage and pathologic T (all was observed in age at diagnosis, tumor size, P < 0.001). However, no significant difference and pathologic N for FREM1 and FREM2, and

13746 Int J Clin Exp Med 2019;12(12):13741-13748 FREM2 is an independent predictor of OS in ccRCC no significant difference in age, gender, tumor of Wenzhou Medical University, Shangcai Village, size and pathologic M for FRAS1 expression South White Elephant Street, Ouhai District, Wen- (all P > 0.05). Low FRAS1, FREM1 and FREM2 zhou 325035, Zhejiang Province, China. Tel: +86- expression were correlated to worsen overall 451 84881200; Fax: +86-451 84881200; E-mail: survival (all P < 0.01), and Low FREM1 and [email protected] FREM2 expression had worse disease free survival (FREM1, P = 0.0113; FREM2, P = 0.0424). References To evaluate whether FRAS1, FREM1 and FR- [1] Linehan WM and Rathmell WK. Kidney cancer. EM2 could be the independent prognostic fac- Urol Oncol 2012; 30: 948-951. tors for KIRC patients, the univariate and multi- [2] Siegel RL, Miller KD and Jemal A. Cancer sta- variate Cox regression analysis was conducted. tistics, 2017. CA Cancer J Clin 2017; 67: 7-30. In the univariate Cox regression analysis, high- [3] Chen W, Zheng R, Baade PD, Zhang S, Zeng H, er FRAS1 (HR = 0.571 [95% CI: 0.42-0.775], P < Bray F, Jemal A, Yu XQ and He J. Cancer statis- 0.001), FREM1 (HR = 0.566 [95% CI: 0.417- tics in China, 2015. CA Cancer J Clin 2016; 66: 0.766], P < 0.001), and FREM2 (HR = 0.453 115-132. [95% CI: 0.331-0.62], P < 0.001) expression [4] Jonasch E, Gao J and Rathmell WK. Renal cell was correlated with longer OS in KIRC pati- carcinoma. BMJ 2014; 349: g4797. ents. Multivariate COX regression analysis in- [5] Abe H and Kamai T. Recent advances in the treatment of metastatic renal cell carcinoma. dicated that FREM2 (HR = 0.402 [95% CI: Int J Urol 2013; 20: 944-955. 0.246-0.656], P < 0.001) was an independent [6] Keefe SM, Nathanson KL and Rathmell WK. prognostic factor for OS. The molecular biology of renal cell carcinoma. Semin Oncol 2013; 40: 421-428. To the best of our knowledge, this is the [7] Pavlakis E, Chiotaki R and Chalepakis G. The first evidence that FRAS1, FREM1 and FREM2 role of Fras1/Frem proteins in the structure mRNA levels were downregulated in KIRC tis- and function of basement membrane. Int J Bio- sues than normal tissues and significantly dif- chem Cell Biol 2011; 43: 487-495. ferent in histologic grade, pathologic stage and [8] Nayak SS, Salian S, Shukla A, Mathew M and pathologic T (all P < 0.001). The univariate and Girisha KM. Variable presentation of Fraser multivariate Cox regression analysis confirmed syndrome in two fetuses and a novel mutation that FREM2 could be an independent prognos- in FRAS1. Congenit Anom (Kyoto) 2017; 57: tic factor for OS in renal clear cell carcinoma. 83-85. These results manifest that FRAS1/FREM1/2 [9] Saleem AA and Siddiqui SN. Fraser syndrome. may act as novel roles in renal clear cell carci- J Coll Physicians Surg Pak 2015; 25 Suppl 2: noma. Moreover, the molecular mechanism by S124-126. which FRAS1/FREM1/2 was decreased in KIRC [10] Alazami AM, Shaheen R, Alzahrani F, Snape K, Saggar A, Brinkmann B, Bavi P, Al-Gazali LI and also should be explored in the future works. Alkuraya FS. FREM1 mutations cause bifid Conclusion nose, renal agenesis, and anorectal malforma- tions syndrome. Am J Hum Genet 2009; 85: In all, the present study revealed FRAS1/ 414-418. [11] Beck TF, Veenma D, Shchelochkov OA, Yu Z, FREM1/2 expression were positively associat- Kim BJ, Zaveri HP, van Bever Y, Choi S, Douben ed with advanced stage, high histologic grade H, Bertin TK, Patel PI, Lee B, Tibboel D, de and FREM2 could be an independent predictor Klein A, Stockton DW, Justice MJ and Scott DA. of poor survival in renal clear cell carcinoma. Deficiency of FRAS1-related extracellular ma- However, the molecular mechanisms of three trix 1 (FREM1) causes congenital diaphrag- genes impacting on KIRC was largely unclear. matic hernia in humans and mice. Hum Mol Therefore, further studies are needed to vali- Genet 2013; 22: 1026-1038. date our analysis and explore the molecular [12] Pitera JE, Scambler PJ and Woolf AS. Fras1, a mechanisms in KIRC. basement membrane-associated protein mutated in Fraser syndrome, mediates both the Disclosure of conflict of interest initiation of the mammalian kidney and the integrity of renal glomeruli. Hum Mol Genet None. 2008; 17: 3953-64. [13] Petrou P, Makrygiannis AK and Chalepakis G. Address correspondence to: Dr. Zhixian Yu, De- The Fras1/Frem family of extracellular matrix partment of Urology, The First Affiliated Hospital proteins: structure, function, and association

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