Nagao et al. BMC Cancer (2018) 18:1114 https://doi.org/10.1186/s12885-018-5060-8 RESEARCHARTICLE Open Access Fatty acid binding protein 7 may be a marker and therapeutic targets in clear cell renal cell carcinoma Kazuhiro Nagao1,2*, Nachi Shinohara1, Frank Smit2, Mirjam de Weijert2, Sander Jannink2, Yuji Owada3, Peter Mulders2, Egbert Oosterwijk2 and Hideyasu Matsuyama1 Abstract Background: To identify potential therapeutic target in clear cell renal cell carcinoma (ccRCC), we performed a transcriptome analysis. Our analysis showed that fatty acid binding protein 7 (FABP7) has the highest mean differential overexpression in ccRCC compared to normal kidney. We aimed to investigate the significance of FABP7 in ccRCC. Methods: Immunohistochemical staining for 40 advanced ccRCC cases was performed to investigate correlation between clinicopathological parameters and FABP7. They were composed of 40–83 years old cases with 33 male, 22 cases with pT ≥ 3, 19 cases with M1, and 16 cases with grade 3. The effect of gene knockdown was analysed by a cell viability assay and invasion assay in FABP7-overexpressing cell lines (SKRC7 and SKRC10). Results: Our immunohistochemical analysis showed that higher FABP7 expression significantly correlated with distant metastasis and poor cancer-specific survival (CSS; both p < 0.05). Functional suppression of FABP7 significantly inhibited SKRC10 cell growth (p < 0.05) and resulted in a significant reduction of the invasive potential (p <0.01),butdidnot cause growth inhibition of SKRC7 cells. We found that The Cancer Genome Atlas Research Network (TCGA) database shows FABP6 and 7 as equally overexpressed in the FABP family. Functional suppression of fatty acid binding protein 6 (FABP6) resulted in significant growth inhibition of SKRC7 cells (p <0.005). Conclusions: Functional suppression of FABP7 significantly reduced cell viability and invasive potential in a ccRCC cell line. FABP7 may play a role in progression in some metastatic ccRCCs. The suppressed function may be compensated by another FABP family member. Keywords: Fatty acid binding protein 7, Clear cell renal cell carcinoma, Prognostic marker, Therapeutic target, Fatty acid binding protein 6 Background Clear cell RCC (ccRCC) is the most prevalent patho- Renal cell carcinoma (RCC) accounts for 3% of all cancer logical subtype of this cancer, accounting for 80% of all cases in adults, and it is the 11th most common cancer in cases. Recent advances in our understanding of the under- men and the 14th most common in women. Its worldwide lying genetic events leading to ccRCC have given rise to incidence and associated mortality in 2012 were ~ 337,000 treatment modalities targeting the vascular endothelial new cases and 143,000 deaths [1], both of which have growth factor (VEGF) signaling axis and its related path- increased steadily over the years [2, 3]. ways. Although VEGF tyrosine kinase inhibitors (VEGF-T- KIs) and mammalian target of rapamycin (mTOR) inhibitors significantly extend the progression free survival * Correspondence: [email protected] of patients with metastatic RCC [4], drug resistance invari- 1Department of Urology, Graduate School of Medicine, Yamaguchi ably occurs. University, 1-1-1, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan Recently, cabozantinib, an inhibitor of tyrosine kinases 2Department of Urology, Radboud University Medical Center, Nijmegen 267 Experimental Urology, Geert Grooteplein, 26-28, P.O. Box 9101, NL-6525, GA, including MET, VEGF receptor, and Anexelekto [5], and Nijmegen, The Netherlands nivolumab, an immune check point inhibitor targeting Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nagao et al. BMC Cancer (2018) 18:1114 Page 2 of 11 programmed cell death 1 on tumor-infiltrating lympho- Immunohistochemistry cytes [6], became available for clinical use. But it is still For immunohsitochemical analysis, we used the slides of necessary to identify new molecular targets for the treat- paraffin-embedded ccRCC and normal kidney samples ment of patients with metastatic ccRCC to improve the extracted from the same cases used in our transcriptome treatment outcome. To identify new therapeutic targets, we analysis. performed a transcriptome analysis of ccRCC and normal Slides of paraffin-embedded ccRCC samples were deparaf- kidney samples. Among the up-regulated genes in ccRCC, finized in xylene, followed by incubation in a graded series we focused on the five most differentially expressed genes; of ethanol (100, 70 and 50%) and re-hydration in PBS. The prolyl hydroxylase 3 (PHD3), fatty acid binding protein 7 slides were immersed in 3% H2O2 for 30 min at room (FABP7), carbonic anhydrase IX (CAIX), NADH dehydro- temperature to block endogenous peroxidase activity. After genase 1 alpha subcomplex, 4-like 2 (NDUFA4L2), and a PBS wash, the slides were immersed in 10 mM citrate buf- monocarboxylate transporter 4 (MCT4). fer (pH 6.0) and heated in a microwave oven for 10 min at FABP7 is known to be upregulated in brain tissue, and 100 °C. After cooling to room temperature, the slides were its function is well studied in glioblastoma cell lines. Zhou blocked in 20% normal goat serum for 10 min. The tissue et al. reported that FABP7 is significantly up-regulated in slices were then incubated with the mouse anti-CAIX ccRCC and that the expression of FABP7 positively corre- monoclonal M75 antibody (1600 dilution), rabbit anti-NDU- lates with advanced clinical stage and poor survival of pa- FA4L2 polyclonal antibody (12,000 dilution), mouse tients with ccRCC [7]. Overexpression of FABP7 in RCC anti-PHD3 monoclonal antibody (1200 dilution), rabbit cells enhances cell growth and cell cycle progression, anti-MCT4 polyclonal antibody (1200), or the rabbit which activates both extracellular-signal-regulated kinases anti-FABP7 polyclonal antibody (11000) for 90 min at room (ERK) and signal transducer and activator of transcription temperature, washed in PBS, and then incubated with the 3 (Stat3) signaling. appropriate secondary antibody for 1 h at room temperature To the best of our knowledge, this is the first report (1100 dilution). After washing with PBS, the slides were about the complementary role of fatty acid binding pro- incubated in a PowerVision DAB solution (40 μlof tein 6 (FABP6) for FABP7 in ccRCC. Solution A + 40 μlofSolutionB+5μl of Tween 20 brought up to 1 ml with PBS, ImmunoLogic®) for 10 min at room temperature, washed again in tap water, Methods counterstained with hematoxylin, and mounted in Per- Transcriptome analysis mount (Fisher Scientific®). Total RNA was extracted from 60 ccRCC samples (49 cases without metastasis, and 11 cases with synchronous metasta- sis at nephrectomy), and 20 corresponding normal kidney Patients’ characteristics in the cases analyzed by FABP7 samples using the TRIzol Reagent (Invitrogen®). The tumor immunohistochemical staining samples contained > 70% tumor cells as estimated by We retrospectively reviewed the clinical data of 40 microscopy. Gene expression analysis was performed on the patients with metastatic ccRCC who had undergone Genechip Human Gene 1.0 ST Array (Affymetrix®), accord- nephrectomy, followed by systemic therapy using either ing to the manufacturers’ instructions. The expression a cytokine, VEGF-TKIs, or mTOR inhibitors in Yama- profiles were analyzed using an unsupervised hierarchical guchi University Hospital. The analysis was ethically average linkage-clustering algorithm. The analysis was approved at our institution (Yamaguchi University ethically approved at our institution (Radboud University Hospital). Medical Center, Nijmegen). Antibodies Evaluation of FABP7 expression status in ccRCC The following primary antibodies were used: anti-CAIX specimens monoclonal antibody M75 (HB-11128, ATCC), rabbit We scored the FABP7 staining index based on the staining anti-NDUFA4L2 (Proteintech®), anti-PHD3 monoclonal intensity (Fig. 2a, low: 1, intermediate: 2, high: 3, and antibody (Novus Biologicals®), rabbit anti-MCT4 (Milli- nuclear staining: 4 points) and the number of the cells as pore®), rabbit anti-FABP7 polyclonal antibody (produced calculated by the formula: (staining intensity 1, 2, 3, or by a co-author, YO [8]), and a mouse anti-Stat3 mono- 4) × cell counts per 500 cells in each sample of ccRCC clonal antibody (Abcam®). Primary antibodies were de- specimen. The mean value of the index was 11 in all the tected using a horseradish peroxidase (HRP)-conjugated cases analyzed. We defined the case with more than mean rabbit anti-mouse immunoglobulin antibody) (DAKO®) value of the index as high FABP7 expression case, and also and HRP-conjugated swine anti-rabbit immunoglobulin defined the case with less than mean value of the index as antibody (DAKO®) (secondary antibodies). low FABP7 expression case. Nagao et al. BMC Cancer (2018) 18:1114 Page 3 of 11 RNA extraction and real-time quantitative RT-PCR Reverse primer sequence: 5’GTCTCTTGCTCACGCG