Fatty acid binding protein 7 may be a marker and therapeutic targets in clear cell renal cell carcinoma

Transcriptome analysis

Total RNA was extracted from 60 ccRCC samples (49 cases without metastasis, and 11 cases with synchronous metastasis at nephrectomy), and 20 corresponding normal kidney samples using the TRIzol Reagent (Invitrogen®). The tumor samples contained > 70% tumor cells as estimated by microscopy. Gene expression analysis was performed on the Genechip Human Gene 1.0 ST Array (Affymetrix®), according to the manufacturers’ instructions. The expression profiles were analyzed using an unsupervised hierarchical average linkage-clustering algorithm. The analysis was ethically approved at our institution (Radboud University Medical Center, Nijmegen).

Antibodies

The following primary antibodies were used: anti-CAIX monoclonal antibody M75 (HB-11128, ATCC), rabbit anti-NDUFA4L2 (Proteintech®), anti-PHD3 monoclonal antibody (Novus Biologicals®), rabbit anti-MCT4 (Millipore®), rabbit anti-FABP7 polyclonal antibody (produced by a co-author, YO [8]), and a mouse anti-Stat3 monoclonal antibody (Abcam®). Primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin antibody) (DAKO®) and HRP-conjugated swine anti-rabbit immunoglobulin antibody (DAKO®) (secondary antibodies).

Immunohistochemistry

For immunohsitochemical analysis, we used the slides of paraffin-embedded ccRCC and normal kidney samples extracted from the same cases used in our transcriptome analysis.

Slides of paraffin-embedded ccRCC samples were deparaffinized in xylene, followed by incubation in a graded series of ethanol (100, 70 and 50%) and re-hydration in PBS. The slides were immersed in 3% H₂O₂ for 30 min at room temperature to block endogenous peroxidase activity. After a PBS wash, the slides were immersed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven for 10 min at 100 °C. After cooling to room temperature, the slides were blocked in 20% normal goat serum for 10 min. The tissue slices were then incubated with the mouse anti-CAIX monoclonal M75 antibody (1600 dilution), rabbit anti-NDUFA4L2 polyclonal antibody (12,000 dilution), mouse anti-PHD3 monoclonal antibody (1200 dilution), rabbit anti-MCT4 polyclonal antibody (1200), or the rabbit anti-FABP7 polyclonal antibody (11000) for 90 min at room temperature, washed in PBS, and then incubated with the appropriate secondary antibody for 1 h at room temperature (1100 dilution). After washing with PBS, the slides were incubated in a PowerVision DAB solution (40 μl of Solution A + 40 μl of Solution B + 5 μl of Tween 20 brought up to 1 ml with PBS, ImmunoLogic®) for 10 min at room temperature, washed again in tap water, counterstained with hematoxylin, and mounted in Permount (Fisher Scientific®).

Patients’ characteristics in the cases analyzed by FABP7 immunohistochemical staining

We retrospectively reviewed the clinical data of 40 patients with metastatic ccRCC who had undergone nephrectomy, followed by systemic therapy using either a cytokine, VEGF-TKIs, or mTOR inhibitors in Yamaguchi University Hospital. The analysis was ethically approved at our institution (Yamaguchi University Hospital).

Evaluation of FABP7 expression status in ccRCC specimens

We scored the FABP7 staining index based on the staining intensity (Fig. 2a, low: 1, intermediate: 2, high: 3, and nuclear staining: 4 points) and the number of the cells as calculated by the formula: (staining intensity 1, 2, 3, or 4) × cell counts per 500 cells in each sample of ccRCC specimen. The mean value of the index was 11 in all the cases analyzed. We defined the case with more than mean value of the index as high FABP7 expression case, and also defined the case with less than mean value of the index as low FABP7 expression case.

RNA extraction and real-time quantitative RT-PCR

Total RNA was extracted from the cultured cells using the TRIzol Reagent (Invitrogen®). RNA was used for reverse transcription with SuperScript II RNase Hˉ Reverse Transcriptase (Invitrogen®). The cDNA synthesis was performed using 2 μg of RNA with a mix of reverse transcriptase, 5× first-strand buffer, DTT, dNTPs, and random hexamers.

HPRT mRNA expression served as an internal control. All the measurements were repeated at least twice to confirm reproducibility. The expression of the target mRNA was quantified relative to that of the HPRT mRNA and untreated controls were used as a reference according to the model described by Pfaffl [9].

Cell culture

Human ccRCC-derived cell lines (SKRC1, SKRC7, SKRC10, SKRC12, SKRC17, SKRC59, and CaKi1) were cultured in the RPMI 1640 medium supplemented with 10% of fetal calf serum and ʟ-glutamate (Gibco®) in a humidified atmosphere containing 5% of CO₂ at 37 °C.

Western blot analysis

Protein expression levels were determined by western blot analysis. In brief, cells were lysed in a buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mmol/l NaCl, 0.1% SDS, 5 mmol/l EDTA, 1% of Triton X-100 (Sigma-Aldrich®), and 1 tablet of a protease inhibitor cocktail (cOmplete Mini, Roche®) per 10 mL of the buffer. The lysates were centrifuged at 13,200 rpm for 15 min at 4 °C, and the supernatants were collected to determine protein concentration using the BSA protein assay reagent. Total protein (10 or 20 μg) was separated by SDS-PAGE in a 10% gel and transferred to a filter using a semidry blotter. After blocking with 1% skimmed milk powder dissolved in PBS, the blots were incubated with the appropriate primary and secondary antibodies: the rabbit anti-FABP7 polyclonal antibody (1,5000) and mouse anti-Stat3 monoclonal antibody (110000). Detection was performed using the Amersham ECL Plus Western Blotting Detection Reagents kit (Amersham®), and protein expression levels were quantified in the ImageJ software (NIH, USA).

The knockdown with small interfering RNA (siRNA)

A commercially available mixture of 4 single-stranded 19-bp siRNAs (ON-TARGETplus SMARTpool, Invitrogen®) was used to transfect SKRC7 and SKRC10 cells according to the manufacturer’s instructions. The sequences of the siRNAs were 5’CAACGGUAAUUAUCAGUCA3’, 5’GUCAGAACUUUGAUGAGUA3’, 5’GAACACGGAGAUUAGUUUC3’, and 5’GAUGAUAGAAACUGUAAGU3’ for FABP7, 5’UCGGAGGCGUGACCUAUGA3’, 5’CCUCAGAGAUCGUGGGUGA3’, 5’GUGAGAAGAAUUAUGAUGA3’, and 5’GCAAGGAAAGCAACAUACA3’ for FABP6.

The final concentration of siRNA for transfection was 33 nmol/L. A scrambled siRNA served as a negative control (Invitrogen®). Transfection was carried out using Lipofectamine 2000 (Invitrogen®).

Cell viability assay

Cell viability was tested by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

SKRC7 and SKRC10 cells transfected with scrambled siRNA, FABP7 siRNA, or FABP6 siRNA were seeded in triplicate in 96-well plates at 4000 cells per well and cultured at 37 °C and 5% CO₂ for up to 7 days. On the day of measurement, 30 μL of MTT Thiazolyl Blue (5 mg/mL: Sigma-Aldrich®) was added into each well, and the cells were incubated for an additional 4 h. Subsequently, 100 μL of DMSO was added, and the plate was shaken for 5 min at room temperature to dissolve the formazan crystals. Finally, optical density (OD) at 595 nm was measured (3550 Microplate Reader, Bio-rad®).

Cell invasion assay

This assay was conducted using a BD BioCoat Matrigel Invasion Chamber (BD-Biosciences®).

SKRC10 cells were harvested 48 h after transfection at 37 °C. The transfected cells were re-suspended in serum-free Dulbecco’s modified Eagle’s medium and then added to the upper chamber at a density of 2 × 10⁵ cells/well. After 24 h of incubation at 37 °C, cells migrating through the membrane were stained. The results are expressed as invading cells quantified at OD 560 nm.

Statistical analysis

Student’s t test was used for statistical analysis using commercially available software (JMP version 4, SAS). A different with a p-value less than 0.05 was considered significant.

Transcriptome analysis

This analysis revealed numerous genes that were over-expressed in ccRCC samples compared to normal kidney tissues (Table 1). Five genes (PHD3, FABP7, CAIX, NDUFA4L2, and MCT4) were expressed approximately 10-fold higher in ccRCC specimens compared to normal kidney samples (Fig. 1a). Higher expression levels of these genes were also observed in The Cancer Genome Atlas Research Network (TCGA) database. The mRNA expression levels of PHD3, CAIX, NDUFA4L2, and MCT4 were equally high in most ccRCC samples, and did not depend on cancer progression. In contrast, the levels of FABP7 mRNA varied. Among the overexpressed genes, FABP7 had the highest mean expression level at the mRNA level, and the expression levels varied depending on the ccRCC cases.

Gene Symbol	Ref Seq	LSMean (NK)	LSMean (RCC)	Fold-Change (RCC/NK)
FABP7	NM_001446	3.20163	9.76575	94.6231
NDUFA4L2	NM_020142	5.26123	10.5235	38.3803
CP	NM_000096	5.94544	10.8426	29.7987
HIG2	NM_013332	4.40522	9.16447	27.0818
ANGPTL4	NM_139314	3.92434	8.28295	20.5151
CA9	NM_001216	4.36817	8.66135	19.6054
ANGPT2	NM_001147	4.81109	8.97052	17.8695
EGLN3	NM_022073	5.49587	9.45616	15.5656
C3	NM_000064	7.32719	11.2612	15.2848
AHNAK2	NM_138420	3.73036	7.61734	14.7944
NETO2	NM_018092	4.68377	8.51052	14.1895
ENO2	NM_001975	4.74515	8.47351	13.254
NNMT	NM_006169	5.63127	9.29046	12.6335
LOX	NM_002317	6.23912	9.89302	12.5873
CYP2J2	NM_000775	4.48354	8.08306	12.1217
TNFAIP6	NM_007115	3.21484	6.78895	11.9101
PNMA2	NM_007257	4.42318	7.86148	10.8401
RRM2	NM_001034	3.95228	7.36327	10.6368
VWF	NM_000552	5.54317	8.90743	10.2978
CXCL10	NM_001565	4.73448	8.09726	10.2872
NPTX2	NM_002523	4.07029	7.42967	10.263
TMEM45A	NM_018004	3.75004	7.07615	10.029
CXCL11	NM_005409	3.39841	6.6673	9.63905
SLC16A3	NM_001042422	4.92069	8.18727	9.62364
IGFBP3	NM_001013398	7.18489	10.4391	9.54115
ENPP3	NM_005021	6.77557	9.96963	9.15181
DNAH11	NM_003777	3.34526	6.53492	9.12395
SCD	NM_005063	6.71436	9.89784	9.08496
CXCL9	NM_002416	4.06238	7.23311	9.00504
FCGR3A	NM_000569	4.98514	8.13677	8.88659
BHLHE41	NM_030762	5.29995	8.40999	8.63406
HK2	NM_000189	4.30467	7.39848	8.53744
PCSK6	NM_002570	4.33254	7.34167	8.05077
GBP5	NM_052942	4.17666	7.1851	8.04697
ESM1	NM_007036	6.14498	9.1468	8.0101
PDK1	NM_002610	6.19808	9.19637	7.99055

Immunohistochemistry

Immunohistochemical staining was used to determine whether the differential mRNA expression of these genes was reflected in differential protein expression in the ccRCC and normal kidney samples. Homogenous staining was observed in ccRCC samples for PHD3, CAIX, NDUFA4L2, and MCT4, while the staining level of FABP7 varied among the cases. Representative cases of differential immunohistochemical staining for PHD3, CAIX, NDUFA4L2, and MCT4 in ccRCC and normal kidney samples are shown in Fig. 1b. CAIX was not expressed in normal kidneys. PHD3 expression was weak in all epithelial cells of the kidney, whereas MCT4 and NDUFA4L2 expression was restricted to the distal tubuli.

FABP7 immunohistochemical staining as a prognostic marker

We retrospectively reviewed the clinical data of 40 patients with metastatic ccRCC who had undergone nephrectomy, followed by systemic therapy using either a cytokine, VEGF-TKIs, or mTOR inhibitors in Yamaguchi University Hospital. The analysis was ethically approved at our institution (Yamaguchi University Hospital).

To evaluate the efficacy of FABP7 as a biomarker, we retrospectively reviewed the clinical data of 40 patients with metastatic ccRCC who had undergone nephrectomy, followed by systemic therapy using either a cytokine, VEGF-TKIs, or mTOR inhibitors in Yamaguchi University Hospital. The expression pattern of FABP7 was consistent in the cytoplasm, while nuclear expression varied. The expression of FABP7 was frequently observed in the peripheral lesion of the tumor with viable cancer cell, while was not in the central lesion with necrotic tissue. The expression level of FABP7 depended on the cases in agreement with transcriptome analysis. Distribution status of the FABP7 staining index among the 40 ccRCC cases is shown in Fig. 2b. We classified the cases into those with higher or lower expression of FABP7 based on the FABP7 staining index.

Functional effects of a FABP7 knockdown

The mRNA and protein expression levels of FABP7 were evaluated in 7 ccRCC-derived cell lines: SKRC1, SKRC7, SKRC10, SKRC12, SKRC17, SKRC59, and Caki1. The mRNA levels are shown in Fig. 3a. SKRC1, SKRC7, and SKRC10 showed higher expression levels of FABP7 compared to the other cell lines.

We transfected FABP7- siRNA into SKRC10 cells to knock down the expression of FABP7. This transfection resulted in a > 90% reduction of mRNA expression for 7 days as compared to scramble transfection (Fig. 3b), and the protein level was also reduced (Fig. 3c). Interestingly, FABP7 knockdown reduced the expression of Stat3 (Fig. 3c).

We evaluated the effect of the FABP7 knockdown on SKRC10 cells by the MTT assay. Functional suppression of FABP7 resulted in significant growth inhibition of SKRC10 cells at 7 days after transfection (p < 0.05, Fig. 4a). A Matrigel invasion assay indicated a significant reduction in the invasive potential by the functional suppression of FABP7 (p < 0.01, Fig. 4b).

FABP6 expression

Although higher expression of FABP7 was observed in SKRC7 cells than in SKRC10 cells, suppression of FABP7 in SKRC7 cells did not affect the cell viability as shown in Fig. 5b. Therefore, we analysed relative mRNA expression of FABP6, a member of the FABP family, in ccRCC cell lines and showed high expression of FABP6 mRNA in SKRC7 cells, while SKRC10 cells had low expression of FABP6 mRNA (Fig. 5a).

We evaluated the effect of the FABP6 knockdown on SKRC7 cells by the MTT assay. Functional suppression of FABP6 resulted in significant growth inhibition of SKRC10 cells at 7 days after transfection (p < 0.05, Fig. 5b), while combined knock down of FABP7 and FABP6 did not inhibit the cell growth more than knock down of FABP6 alone. In SKRC7 cells, FABP6 seemed to compensate the role of FABP7 in cell proliferation.