Urologic Oncology: Seminars and Original Investigations 33 (2015) 113.e9–113.e17

Original article

Overexpression of FABP7 promotes cell growth and predicts poor prognosis of clear cell renal cell carcinoma Jiancheng Zhou, Ph.D.a,1, Zhuo Deng, Ph.D.b,1, Yule Chen, Ph.D.a, Yang Gao, Ph.D.a, Dapeng Wu, M.D.a, Guodong Zhu, Ph.D.a, Lei Li, Ph.D.a, Wenbin Song, M.D.a, Xinyang Wang, M.S.a, Kaijie Wu, Ph.D.a,*, Dalin He, M.D.a,* b

a Department of Urology, First Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an, China Department of Gynecology and Obstetrics, First Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an, China

Received 18 May 2014; received in revised form 31 July 2014; accepted 1 August 2014

Abstract Objectives: Renal cell carcinoma (RCC) is one of the most lethal urologic malignancies; however, the molecular events supporting RCC carcinogenesis remain poorly understood. The aim of the present study was to determine the differential expression of genes between normal kidney and clear cell RCC (ccRCC) samples and investigate the biological function of the most frequently altered gene in RCC cells. Materials and methods: The gene expression profiles of 60 ccRCC and matched normal kidney samples from The Cancer Genome Atlas were analyzed. The altered genes were subjected to functional annotation clustering and integrative pathway analysis. The expression of one of the most frequently altered gene, fatty acid–binding protein (FABP) 7, in ccRCC and matched normal kidney samples was verified by immunohistochemistry and the association between FABP7 level and patient survival was investigated. Furthermore, FABP7 DNA copy number alteration, methylation, and mutation status in ccRCC from The Cancer Genome Atlas were analyzed. Finally, FABP7overexpressing RCC cells were generated to determine the function of FABP7 in cell growth and the potential mechanisms of action. Results: FABP7 was significantly up-regulated in ccRCC, and the expression of FABP7 positively correlated with advanced clinical stage and poor survival of patients with ccRCC. FABP7 DNA copy number alteration was not frequently detected in ccRCC, and no mutation of FABP7 was found. FABP7 messenger RNA expression inversely correlated with its DNA methylation. Overexpression of FABP7 in RCC cells enhanced cell growth, clonogenicity, cell cycle progression and activated both extracellular-signal-regulated kinases (ERK) and signal transducer and activator of transcription 3 (Stat3) signaling. Conclusion: FABP7 is overexpressed in ccRCC and promotes cell growth by the activation of ERK and Stat3 signaling pathways. Evidence from the clinical observations and experimental data suggests that FABP7 is a novel prognostic marker and potential therapeutic target for ccRCC. r 2015 Elsevier Inc. All rights reserved.

Keywords: FABP7; Renal cell carcinoma; Gene profile; Prognostic factor; Cell growth

1. Introduction

This study was partially supported by the National Natural Science Foundation of China (NSFC 81202014 to K. Wu) and the National High Technology Research and Development Program of China (863 Program SS2014AA020607 to L. Li). 1 Contributed equally. * Corresponding authors. Tel.: þ86-29-85323661; fax: þ86-29-85323203. E-mail addresses: [email protected] (K. Wu), [email protected] (D. He). http://dx.doi.org/10.1016/j.urolonc.2014.08.001 1078-1439/r 2015 Elsevier Inc. All rights reserved.

Renal cell carcinoma (RCC) is by far the most lethal urologic malignancy, with deaths of more than 100,000 individuals per year worldwide [1]. It is a heterogeneous disease, which can be classified into various subtypes based on the morphological features, and clear cell RCC (ccRCC) represents the most common subtype, accounting for approximately 80% of cases [2]. RCC also shows molecular and genetic heterogeneity and complexity [3–5]. Wholegenome sequencing and integrated molecular analysis of

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this tumor have identified many alterations in protooncogenes and tumor suppressor genes such as VHL [4], MET [6], PTEN [7], TSC1/2 [8], BAP1 [9], PBRM1 [10], and PIK3CA [3], which can lead to the dysregulated signal networks associated with RCC tumor growth, survival, angiogenesis, and metabolism [2,11,12]. By analysis of the differential expression of genes between normal kidney and ccRCC samples, we identified that fatty acid–binding protein (FABP) 7, a small cytoplasmic protein plays crucial roles in cellular fatty acid uptake, transport, and metabolism [13,14] and is the most significantly up-regulated gene in ccRCC. It is well known that cancer cells alter their metabolism to suit their needs for uncontrolled proliferation [15,16]. One of the metabolic changes frequently found in cancer cells is their increased fatty acid synthesis, which is most clearly demonstrated in ccRCC [17]. To address the hypothesis that FABP7 is associated with ccRCC progression, we performed a retrospective analysis using 2 independent cohorts with ccRCC and demonstrated that increased FABP7 expression was indeed associated with poor prognosis in patients with ccRCC. In addition, further biological function studies indicated that FABP7 was important for RCC cell proliferation and cell cycle progression by activation of extracellularsignal-regulated kinases (ERK) and signal transducer and activator of transcription 3 (Stat3) signaling pathways. These suggest that FABP7 may be a novel prognostic marker and potential therapeutic target for ccRCC.

2. Materials and methods 2.1. Patients and clinical database In this retrospective analysis, we assessed 77 primary ccRCC and matched normal tissues in a tissue microarray (TMA) from radical or partial nephrectomy between July 2006 and February 2008. All the patients had no systemic treatment or targeted therapy at the time of sample collection. Detailed patient information, including age at diagnosis, sex, and tumor stage and grade, are listed in Supplementary Table S1. Follow-up data regarding recurrence and survival have been collected from patient records. Date of last follow-up in this cohort was December 2013 and median follow-up for survivors was 75 months (range: 67–82). We also used another independent cohort from The Cancer Genome Atlas (TCGA), which was a publically available, open-access, and provided multidimensional genomic and clinical data (update to February 4, 2014). This cohort contained 491 patients with ccRCC in which overall survival data were available for 462 patients and tumor-free survival data were available for 381 patients. Median follow-up for survivors was 47 months (range: 1–122). Detailed patient information is listed in Supplementary Table S1. In this cohort, FABP7 messenger

RNA (mRNA) data for 491 patients, DNA copy number data for 411 patients, mutation data for 475 patients, and methylation data for 295 patients were available for analysis. Both FABP7 mRNA and methylation data were available for 290 patients. Importantly, among the 491 patients, mRNA data were available for 60 paired tumor and matched normal samples. Gene methylation, DNA copy number, and mRNA expression data were retrieved using the TCGA data portal [18]. Gene mutation data were retrieved from the Sanger Institute Catalogue Of Somatic Mutations In Cancer [19]. Gene methylation assay was performed using HumanMethylation450 BeadChip arrays, and mRNA expression was determined using the Illumina HiSeq RNA Sequencing (RNA-Seq) platform. Platforms and methods for DNA copy number alteration (CNA) assay and gene mutation assay have been described previously [18,19]. 2.2. Tissue samples and immunohistochemistry staining This human RCC TMA (HKid-CRC180Sur-01; Shanghai Outdo Biotech, Co) was constructed with formalin-fixed, paraffin-embedded RCC tissues and their corresponding normal kidney tissues. The TMA sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. Sections were blocked with goat serum, incubated with anti-FABP7 (Abcam, Cambridge, 1:100 dilutions) primary antibody, and developed with 3,30 -diaminobenzidine chromogen followed by counterstaining with hematoxylin. An H-score was assigned to each tissue based on the product of staining intensity (0, no staining; 1, weak; 2, moderate; and 3, strong) and percentage of stained cells (0-0%, 1-1% to 30%, 2-31% to 70%, and 3-71% to 100%). Expression of FABP7 in each tissue was considered as either negative (H-score o2) or positive (H-score Z2). 2.3. Cell culture Human RCC 786-0 cell was maintained in RPMI1640 medium (Gibco, Santa Clara, CA) containing 10% fetal bovine serum (FBS), ACHN was maintained in Dulbecco modified Eagle medium (Gibco) containing 10% FBS. FABP7-overexpressing stable cell lines were maintained in appropriate medium supplemented with blasticidin. All the cells were cultured in a humidified incubator containing 5% CO2 at 371C. 2.4. Plasmid constructs and transfection FABP7 plasmid was obtained from the DNASU Plasmid Repository. Transfections were performed using Lipofectamine LTX (Invitrogen) and PLUS according to the manufacturer's instructions. To generate stable sublines, 48 hours after the transfection, cells were selected with 5 μg/ml blasticidin for 14 days.

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Fig. 1. Differential gene expression in ccRCC and matched normal kidney tissues. (A) Heat map showing the top 100 genes differentially expressed between ccRCC (n ¼ 60) and matched normal kidney samples (n ¼ 60) (TCGA, RNA-Seq). (B) Relative FABP7 mRNA expression level in the individual tumors compared with the corresponding normal tissues (TCGA, RNA-Seq). (Color version of figure is available online.)

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Fig. 2. FABP7 is associated with poor prognosis in patients with ccRCC. (A) Representative IHC staining of FABP7 in ccRCC and matched normal samples. (B) The results of Fisher exact test of FABP7 expression in ccRCC and normal kidney samples. (C) The results of the Student t-test comparing tumor volume in FABP7-positive and FABP7-negative patients. (D) Representative IHC staining of FABP7 in low or high clinical stage of ccRCC. (E) The results of the Kaplan-Meier analysis (log-rank test) showing overall survival of FABP7-positive (n ¼ 47) and FABP7-negative (n ¼ 30) patients with ccRCC (TMA cohort). (F) The results of the Kaplan-Meier analysis (log-rank test) showing overall and tumor-free survival of FABP7-positive and FABP7-negative patients with ccRCC (TCGA cohort, optimum cut-point determined by X-tile). All scale bar ¼ 100 μm. (Color version of figure is available online.)

2.5. Western blot Western blot was performed as described previously [20]. In brief, cell lysates were harvested, and proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with appropriate primary antibodies overnight and horseradish peroxidase– conjugated secondary antibodies for 1 hour. Signals were detected using chemiluminescence (Pierce, Rockford, IL).

analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche, Indianapolis, IN). 2.7. Cell cycle assay Cells were cultured in 10% FBS medium overnight and then changed to 1% FBS medium for another 24 hours. After that, they were harvested and washed with PBS 3 times, then fixed in 96% ethanol overnight, washed, stained with 50-μg/ml propidium iodide, and examined with a fluorescence-activated cell-sorting flow cytometer.

2.6. In vitro cell proliferation assay 2.8. Colony-formation assay Cells were resuspended in appropriate medium containing 10% FBS and plated in 96-well plates at a concentration of 1,500 cells/well. For FBS stimulation or PD98059 and S3I-201 treatment, cells were pretreated in FBS-free medium for 6 hours and then changed to the indicated treatment. After the indicated time of treatment, cell viability was

A total of 1,000 cells were plated in a 6-well plate in triplicate and cultured in complete medium in a humidified incubator containing 5% CO2 at 371C. Cells were fed with fresh medium weekly for 3 weeks. Colonies were fixed with 4% paraformaldehyde for 10 minutes, and stained with

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0.5% crystal violet for 5 minutes. Colonies were photographed and visible colonies were counted. 2.9. Statistical analysis Initially, the alteration in genes was determined by analyzing the mRNA expression in 60 ccRCC and matched normal samples from TCGA (cutoff, fold changes of gene expression Z10 and P o 0.0001), Student two-tailed t-test was used to compare the differences between 2 groups. The X-tile algorithm was used to generate an optimal cutoff point to dichotomize FABP7 mRNA expression as positive and negative using the Monte Carlo method, with P o 0.05, because they were molecules with no established cutoff points for their expression in RCC. The Kaplan-Meier method was used to analyze patient survival, and the long-rank test was used to assess the differences between groups. Overall survival was defined as the interval from the date of initial surgery to the date of death or last contact (censored). Tumor-free survival was determined by the time between initial surgery and first recurrence or last contact (censored). The Cox proportional hazards regression model was performed for multivariate analysis to study the contribution of various potential prognostic parameters to overall survival of patients. All the data from in vitro assay were presented as the mean ⫾ standard error of the mean from 3 independent experiments, Student two-tailed t-test was used to compare the differences between the 2 groups. The Pearson correlation coefficient was used to test the association between different variables. All statistical analyses were performed using GraphPad Prism and SPSS16.0 software. 3. Results 3.1. FABP7 is overexpressed in patients with RCC As shown in a scheme of methodology in Supplementary Fig. S1, we firstly analyzed differential gene expression in 60 paired ccRCC tissues and matched normal tissues (RNASeq) from TCGA. The altered genes were submitted to The Database for annotation, visualization, and integrated discovery for functional annotation clustering and integrative pathway analysis (Supplementary Tables S2 and S3). Within the most significantly altered genes (fold changes Z10 and P o 0.0001), we found that FABP7, a gene involved in fatty acid metabolism and associated with many types of cancers [21–23], was significantly up-regulated in ccRCC tissues compared with normal tissues (Fig. 1A). In the 60 paired tumor and matched normal tissues, 57/60 (95%) tumor tissues showed increased FABP7 mRNA expression when compared with normal tissues (Fig. 1B, P o 0.0001). To verify the RNA-Seq data, we examined FABP7 expression in 77 ccRCC tissues and matched normal tissues in

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TMA by immunohistochemistry staining. Consistent with the mRNA expression data, the proportion of FABP7positive tissues in ccRCC was higher than normal tissues (Fig. 2A). Overall, FABP7 was positively expressed in 47/ 77 (61%) tumor tissues, but only in 15/77 (19%) normal tissues (P o 0.0001) (Fig. 2B). To further clarify the mechanisms leading to the dysregulation of FABP7 in ccRCC, we retrieved FABP7 DNA CNAs and methylation data in patients with ccRCC from cBioPortal for Cancer Genomics and mutation data from Sanger Institute Catalogue Of Somatic Mutations In Cancer. Only 1/411 (0.2%) of patients were found to harbor FABP7 homozygous deletion, whereas most patients exhibited either heterozygous deletion, 2 alleles present, or low-level gene amplification event. No patient showed high-level of gene amplification (Supplementary Fig. S2A). These data indicated that the up-regulation of FABP7 in ccRCC was not because of CNA. Notably, the Pearson correlation analysis of FABP7 DNA methylation and mRNA expression in those 290 patients indicated an inverse correlation between FABP7 mRNA expression and methylation level (r ¼ 0.67, P o 0.0001) (Supplementary Fig. S2B), indicating that DNA methylation might be important for the regulation of FABP7 expression in ccRCC. Furthermore, the FABP7 mutation data showed that no FABP7 mutation was detected in RCC samples, although FABP7 mutation were detected in few samples of stomach, endometrium, large intestine, liver, lung and ovary cancers (Supplementary Table S4).

3.2. Overexpression of FABP7 is associated with poor prognosis in ccRCC We further investigated the correlation between FABP7 expression and clinicopathologic features of 77 patients with ccRCC in TMA cohort (Table 1). FABP7-positive patients showed higher tumor volume than FABP7-negative patients (Fig. 2C). Patients with advanced clinical stage were frequently identified with FABP7-positive expression (Table 1 and Fig. 2D). Univariate survival analysis (Fig. 2E) and multivariate survival analysis (Table 2) demonstrated that FABP7-positive expression was associated with poor overall survival in patients. In addition, we investigated the TCGA cohort, in which FABP7 mRNA expression data were available for 491 patients with ccRCC, overall survival information was available for 462 patients, and tumor-free survival information was available for 381 patients. The data showed that FABP7-positive patients exhibited higher tumor pathologic stage, advanced clinical stage, and poor tumor-free survival; however, there was no significant correlation between FABP7 expression and overall survival of patients in this cohort (Supplementary Table S5, Supplementary Fig. S3, and Fig. 2F).

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Table 1 Relationship between FABP7 protein expression and clinicopathologic feature in 77 ccRCC n

P value

FABP7

Table 2 Multivariate Cox regression analysis on the contribution of various potential prognostic parameters to overall survival of 77 patients with ccRCC P value

Negative

Positive

30 (39%)

47 (61%)

HR

95.0% CI for HR Lower

Total

77

Sex M F

0.351 46 31

20 (43%) 10 (32%)

26 (57%) 21 (68%)

Age, y o60 Z60

33 44

13 (39%) 17 (39%)

20 (61%) 27 (61%)

Grade 1 2 3 4

29 23 18 7

9 10 8 3

(31%) (43%) (44%) (43%)

20 13 10 4

(69%) (57%) (56%) (57%)

Pathological stage T1 44 T2 23 T3 8 T4 2

18 10 2 0

(41%) (43%) (25%) (0%)

26 13 6 2

(59%) (57%) (75%) (100%)

Lymph nodes N0 N1

75 2

29 (39%) 1 (50%)

46 (61%) 1 (50%)

Metastasis M0 M1

75 2

30 (40%) 0 (0%)

45 (60%) 2 (100%)

Clinical stage I II III IV

40 22 11 4

19 9 2 0

21 13 9 4

1.000

0.744

Sex Age Furman grade Pathological stage (T) Lymph nodes (N) Metastasis (M) Clinical stage FABP7 protein expression

0.930 0.177 0.334 0.494 0.985 0.985 0.002 0.019

0.964 1.023 1.302 0.798 0.000 0.000 2.249 3.807

0.432 0.990 0.762 0.418 0.000 0.000 1.330 1.246

Upper 2.155 1.058 2.225 1.523

3.803 11.632

HR ¼ hazard ratio.

3.4. Overexpression of FABP7 in RCC activates ERK and Stat3 signaling 0.532

1.000

0.252

(40%) (56%) (13%) (0%)

(60%) (44%) (88%) (100%)

0.121 I–II vs. III–IV: 0.023

3.3. FABP7 promotes RCC cell growth in vitro To determine the biological function of FABP7 in RCC, stable FABP7-overexpressing (FABP7) and vector control (VC) cells from 786-0 and ACHN were established (Fig. 3A). Although there was no significance in cell growth rate between FABP7-overexpressing and VC cells under nutrition-abundant condition (i.e., 10% FBS), FABP7-overexpressing cells exhibited higher growth rate than VC cells when cultured in low-nutrition condition (i.e., 1% FBS) (Fig. 3B). In addition, FABP7-overexpressing cells became more sensitive to FBS stimulation after a period of FBS starvation, resulting in an increased cell growth with the supplement of 10% FBS (Fig. 3C). Additionally, FABP7-overexpressing cells exhibited enhanced clonogenicity compared with VC cells in vitro (Fig. 3D) and enhanced cell cycle progression by promoting G1/S transition (Fig. 3E). Taken together, these data suggest that FABP7 plays a critical role in promoting RCC cell growth.

To dissect the mechanisms of FABP7 in regulation of RCC cell growth, we examined the activities of ERK and Stat3 signaling pathways, which have been reported to be activated in RCC [24,25]. We observed increased phosphorylated ERK (p-ERK) and Stat3 (p-Stat3) in FABP7-overexpressing 786-0 cells under low-serum cultured condition (Fig. 4A). More interestingly, although FBS stimulation could increase p-ERK and p-Stat3 expression and cell growth in both FABP7-overexpressing and VC cells, levels of p-ERK and p-Stat3 expression and cell viability were consistently higher in FABP7-overexpressing cells than in VC cells. When cells were treated with specific ERK inhibitor PD98059 (PD) or Stat3 inhibitor S3I-201 (S3I), the expression of p-ERK and p-Stat3 dramatically decreased in both FABP7-overexpressing cells and VC cells, and no significant difference exited between the FABP7-overexpressing and VC cells according to p-ERK and p-Stat3 expression levels and cell viability (Fig. 4B and C). Collectively, these data indicate that FABP7 promotes cell growth in RCC cells via activating both ERK and Stat3 signaling.

4. Discussion RCC is characterized by substantial genetic heterogeneity and distinctive histological features and clinical phenotypes. Systemic genome screenings of this tumor have identified many alterations in proto-oncogenes and antioncogenes, leading to the dysregulation of signal transduction that underlies abnormal cell growth and metabolism [3–5]. Germline and somatic mutation [26], deletion [27], or epigenetic silencing of VHL [28] in ccRCC leads to stabilization of hypoxia-inducible factors, which are involved in the carcinogenesis, pathological angiogenesis, glucose metabolism, and progression of ccRCC [29,30]. More recently, whole-genome/exome sequencing has also identified frequent alterations in ccRCC, such as in BAP1 [9],

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Fig. 3. FABP7 promotes RCC cell growth in vitro. (A) The results of a Western blot analysis showing FABP7 expression in 786-0 and ACHN cells stably transfected with FABP7 plasmids (FABP7) and control vectors (VC). Actin was used as loading control. (B) 786-0 VC and FBAP7-overexpressing cells, ACHN VC and FABP7-overexpressing cells were cultured in 10% FBS medium or 1% FBS medium, cell viabilities were analyzed by MTT assay at the indicated time point. *P o 0.05. (C) VC and FABP7-overexpressing cells were pretreated with FBS-free medium for 4 hours and then cultured in either FBSfree medium () or 10% FBS medium (þ) for the indicated time, cell viabilities were analyzed by MTT assay. *P o 0.05. (D) Colony-formation assay of VC and FBAP7-overexpressing cells. Quantification of colony number is shown. *P o 0.05. (E) VC and FABP7-overexpressing cells were pretreated with FBSfree medium for 4 hours and then treated with 10% FBS medium for 12 hours followed by FASC analyzing cell cycles. Percentage of cells in different cell cycle phase is shown.

PBRM1 [10], SETD2, and PIK3CA [5], which are associated with ubiquitin-mediated proteolysis, chromatin remodeling, histone methylation, and protein kinase activation, respectively. In our study, by analyzing the RNA-Seq data of ccRCC and matched normal samples from TCGA, we found that the categories of altered genes mainly included polymorphism (e.g., BMP1 and KLF10), membrane (e.g., ATP2B2 and TNFSF2), glycoprotein (e.g., COL14A1 and GPR31), alternative splicing (e.g., SKIL and SF1), transmembrane (e.g., VDR and SMAD7), and signal (e.g.,

WISP1 and IL8) (Supplementary Table S2). Pathway annotation showed that the alterations mainly included cytokine-cytokine receptor interaction, neuroactive ligandreceptor interaction, cell adhesion molecules, and chemokine signaling pathways (Supplementary Table S3). All these indicate the genetic complexity of ccRCC. Cancer cells alter their metabolism to suit their needs for uncontrolled proliferation [15,16]. One of the metabolic changes frequently found in cancer cells is increased fatty acid synthesis, which is most clearly demonstrated in

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Fig. 4. FABP7 activates ERK and Stat3 signaling. (A) Western blot showing total and phosphorylated ERK and Stat3 expression in 786-0 VC and FABP7overexpressing cells cultured in 10% FBS and 1% FBS medium. 786-0 VC and FABP7-overexpressing cells were pretreated with FBS-free medium for 4 hours and then supplied with 10% FBS, 10% FBS plus 20-μM ERK inhibitor PD98059 (PD), or 10% FBS plus 50-μM Stat3 inhibitor S3I-201 (S3I) for 12 hours. Cells supplied with DMSO were used as control. Western blot analysis (B) was used to detect the expression of ERK and Stat3 expression and MTT assay (C) was used to determine the cell growth after treatment. *P o 0.05; ns, not significant.

ccRCC [17]. For example, it is reported that FAF1 and Ubxd7 [31] are involved in fatty acid metabolism of ccRCC. Here, we noticed that one of the FABPs gene family, FABP7, which plays key roles in cellular fatty acid uptake, transport, and metabolism [13,14], was significantly up-regulated in ccRCC. Retrospective analysis demonstrated that FABP7 was a predictive biomarker for patients with ccRCC who had advanced clinical stage and poor survival. Recently, the associations between FABP7 and other cancers have also been studied. FABP7 enhances the motility of glioblastoma cells [32] and promotes the cell proliferation and invasion of malignant melanoma [22,23]. FABP7 is also preliminarily reported to be a biomarker for RCC [33]; however, the mechanisms leading to the dysregulation of FABP7 expression and its biological function in RCC remain unknown. We demonstrated that FABP7 methylation and not DNA amplification or mutation may contribute to its up-regulation in ccRCC, which is

consistent with the results of a promoter activity assay in another study [34]. Functionally, FABP7 acts as a proto-oncogene in RCC. Overexpression of FABP7 promotes RCC cell proliferation by the mechanisms of activating both ERK and Stat3 signaling pathways. Now, only about 30% of advanced or metastatic patients with RCC show response to targeted therapies and all patients will eventually develop resistance [35]. Commercial therapeutic agents combined with other molecular approaches may provide alternative strategies for RCC treatment. Owing to the critical roles of ERK and Stat3 pathway in cancer, small molecular agents specifically targeting these 2 pathways are now under clinical trials for advanced cancer treatment (NCT01378377 and NCT00 955812). In the present study, we unveil that FABP7 can activate both ERK and Stat3 signaling. In addition, given the well-known roles of ERK [24] and Stat3 [25] pathways in the regulation of RCC growth and progression,

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direct-targeting FABP7 may provide another potential strategy to combat RCC. 5. Conclusion This study unveiled that FABP7 was significantly upregulated in ccRCC and was a potential prognostic marker for patients with advanced clinical stage and poor survival. Functionally, overexpression of FABP7 promoted cell growth by activating both ERK and Stat3 signaling. Thus, FABP7 may be a novel potential therapeutic target for ccRCC. However, further studies on the effects of FABP7 on tumor growth in vivo and its functional role in fatty acid metabolism of ccRCC are needed. Appendix A. Supplemental materials Supplementary material cited in this article is available online at http://dx.doi.org/10.1016/j.urolonc.2014.08.001.

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Overexpression of FABP7 promotes cell growth and predicts poor prognosis of clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most lethal urologic malignancies; however, the molecular events supporting RCC carcinogenesis remain poorly ...
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