CYP1B1 promotes tumorigenesis via altered expression of CDC20 and DAPK1 genes in renal cell carcinoma

Normal kidney sample and tissue microarray

Fresh frozen normal kidney tissue was purchased from BioServe (Beltsville, MD, USA). In total, 96 primary RCC comprised of 31 specimens from tissue microarray (TMA) KD951, 40 specimens from TMA KD485 (both acquired from US Biomax, Rockville, MD, USA), and 25 specimens from TMA CT565907 (ORIGENE, Rockville, MD, USA) were evaluated. Also, twenty-five normal kidney specimens were obtained from these 3 TMAs. Median patient age at surgery was 57 years old. Of the 96 RCC specimens, 83 were clear cell carcinoma and the remaining 13 specimens were non-clear cell carcinoma (3 papillary carcinoma, 3 chromophobe carcinoma, 3 sarcomatoid carcinoma, 2 granular carcinoma and 2 collecting duct carcinoma). Forty-seven (49.0 %) patients had Fuhrman grade 1 or 2, 15 (15.6 %) were grade 3 or 4, and the remaining 34 (35.4 %) were unknown. Forty (41.7 %) patients were stage I, 16 (16.7 %) patients stage II, 7 (7.3 %) stage III, 2 (2.1 %) stage IV, and the remaining 31 (32.2 %) were unknown.

Cell lines and reagents

Renal cancer cell lines, Caki-1, Caki-2, A498, ACHN, 786-O and 769-P were obtained from the American Type Culture Collection (Manassas, VA, USA). McCoy’s 5A, MEM Eagle’s BSS (EMEM), RPMI 1640, Opti-MEM and penicillin/streptomycin mixture were obtained from the UCSF Cell Culture Facility (San Francisco, CA, USA). Fetal bovine serum (FBS) was a product of Atlanta Biologicals (Lawrenceville, GA, USA).

Cell culture

Caki-1 and Caki-2 cells were cultured in McCoy’s 5A, and A498 and ACHN cells were maintained in EMEM medium while 786-O and 769-P cells were cultured in RPMI 1640 medium. All culture medium contained 10 % FBS and 100 μg/ml penicillin/streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere composed of 5 % CO₂ and 95 % air.

Knockdown of CYP1B1 in Caki-1 and 769-P cells

Oligonucleotides siRNA against human CYP1B1 and mismatch control oligonucleotides were purchased from Life technologies. For inhibition of CYP1B1, 5 μl of siRNA oligonucleotides (siRNA-CYP1B1 or siRNA-control) and 5 μl of lipofectamine RNAiMAX reagent (Invitrogen-Life Technologies Inc., Carlsbad, CA, USA) were separately diluted with 250 μl of Opti-MEM (Gibco, Carlsbad, CA, USA). Cells were then transfected with lipofectamine + siRNA-CYP1B1, lipofectamine + siRNA-control, or left untransfected. Transfection was terminated after 5 h by aspirating the transfection medium and adding fresh RPMI 1640 containing 10 % FBS. Non-adherent cells were washed off and the remaining cells were incubated at 37 °C.

RNA extraction and quantitative RT-PCR

Total RNA was extracted from cultured cells using RNeasy Mini Kit (Qiagen) and was converted into cDNA by using an iScript^TM cDNA Synthesis Kit (BIO-RAD) according to the manufacturer’s instruction. To assess gene expression, cDNA was amplified with the TaqMan® Gene Expression Assays and TaqMan® Fast Universal PCR Master Mix using the 7500 Fast Real-Time PCR System. The target genes and their Assay ID were as follows: CYP1B1 (HSpp16383_m1), DKC1 (Hs00154737_m1), OCLN (Hs00170162_m1), FASLG (Hs00181225_m1), MKI67 (Hs01032443_m1), LPL (Hs00173425_m1), CA9 (Hs00154208_m1), CDC20 (Hs00426680_mH), TP73 (Hs00390315_m1), BCL2A1 (Hs00187845_m1), LTA (Hs04188773_g1), DAPK1 (Hs00234489_m1), BIRC5 (Hs04194391_sH) and GAPDH (Hs03929097_g1). The relative level was calculated by the comparative C_t method (ΔΔC_t) using the 7500 Fast System Sequence Detection Software (Applied Biosystems).

PCR assay of apoptosis and cancer pathways

cDNAs were evaluated for genes using the RT² Profiler^TM PCR Array PAHS-012ZC (Human Apoptosis PCR Array) and the RT² Profiler^TM PCR Array PAHS-033ZC (Human Cancer Pathway Finder^TM ) on the ABI Fast 7900 real-time PCR system with RT² Real-time SYBR Green PCR master mix according to the manufacturer’s protocol.

MTS assay

Cells were plated in triplicate in 96-well microplates at a density of 3 × 10³ cells per well. The number of viable cells was determined by adding 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-based CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) to each well and measuring the absorbance at 490 nm on SPECTRA MAX 190 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Migration and invasion assay

Cell migration was evaluated by a wound-healing assay. Cells were plated in six-well dishes and the cell monolayers were scraped using a P-20 micropipette tip. Wound closure was monitored and the percent closure was measured. A cell invasion assay was carried out using modified Boyden Chambers consisting of transwell-precoated Matrigel membrane filter inserts with eight micro pores in 24-well tissue culture plates (BD Biosciences, Bedford, MA, USA). Cells were re-suspended in culture medium without FBS and placed in the upper chamber in triplicate. After 48 h incubation at 37 °C, cells migrating through the membrane were stained. The results were expressed as invaded cells quantified at OD 560 nm.

Apoptosis assay

Fluorescence-activated cell-sorting (FACS) analysis for apoptosis was done 48 h post-transfection, using an annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining system obtained from BD Biosciences (San Diego, CA, USA) and a Cell Lab Quanta^TM SC MPL (Beckman Coulter, Fullerton, CA, USA). Cells were stained with annexin V-FITC only (early apoptotic) or both annexin V-FITC and 7-AAD (late apoptotic) and considered to be total apoptotic cell fractions.

Western analyses

Normal kidney tissue and whole cell extracts were prepared using radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Pierce) according to the manufacturer’s instructions. Total cell protein (15–20 μg) was used for Western blotting. Samples were run on the polyacrylamide gels and then transferred to PVDF membranes. The membranes were immersed in 3 % skim milk in antibody against CYP1B1 (#ab32649, Abcam, Cambridge, MA, USA), CDC20 (#4823, Cell Signaling Technology), DAPK1 (#3008, Cell Signaling Technology) and GAPDH (#sc-32233, Santa Cruz) overnight at 4 °C. Blots were washed in TBS containing 0.1 % Tween20 and labeled with horseradish peroxidase conjugated secondary anti-rabbit antibody (Cell Signaling Technology). Specific complexes were visualized with an enhanced chemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK) using the Chemidoc imaging system (Bio Rad, CA, USA). Protein expression levels were expressed relative to GAPDH.

Immunohistochemical analyses

Immunostaining of CYP1B1, CDC20 and DAPK1 were performed on TMA slides using UltraVision Detection System (Thermo Scientific) according to the manufacture’s instruction, and 2 TMA slides were evaluated for each antibody. After 12 h incubation with rabbit polyclonal antibody for CYP1B1 (1:1500, #ab32649, Abcam), CDC20 (1:100, #ab86104, Abcam) and DAPK1 (1:250, #ab109382, Abcam), 3, 3'-diaminobenzidine (DAB) was added as chromogen followed by counterstaining with hematoxylin. The degree of immunostaining was evaluated by two independent observers who were blind to the clinical data of the TMAs. For CYP1B1, cytoplasmic expression was analyzed by the intensity of positive cells using Image J software (http://rsb.info.nih.gov/ij) and was ranked on an overall scale from 0 to 3; with 0 indicating the absence of staining; 1, weak staining; 2, moderate staining; and 3, strong staining [12]. CDC20 and DAPK were scored by multiplication of the percentage of positively stained tumor cells and staining intensity according to previous studies [17, 18, 28]. Briefly, the percentage of positive cells was scored as: 0, 0 %; 1+, 1–10 %; 2+, 11–50 %; or 3+, 51–100 %. The intensity of nuclear staining and/or cytoplasmic staining was scored as follows: grade 0, negative; 1+, weakly positive; 2+, moderately positive; or 3+, strongly positive. The two scores were then multiplied to calculate the final score (range from 0 to 9).

Statistical analysis

Values are presented as the mean ± standard error mean based on results obtained from at least three independent experiments. All data were analyzed by the StatView version 5 statistical software (SAS Institute, Inc., Cary, NC). The relationship between two variables and the numerical values were analyzed using the two-tailed unpaired Student’s t-test. Chi-square test was used for analyzing the correlation between clinicopathologic parameters and CYP1B1 expression. A p-value of less than 0.05 was considered to be statistically significant.

Ethics statement

All samples obtained with informed consent according to US federal law were purchased commercially and at the laboratory in San Francisco, specimens and de-identified patient data were used for analysis. This study was approved by the Clinical Research Office of the San Francisco Veterans Affairs Medical Center and the Institutional Review Board (study number 10–03240) of the University of California at San Francisco.

CYP1B1 is up-regulated in RCC cell lines and RCC tissues

To determine CYP1B1 protein expression, Western analysis was performed using normal kidney tissue, Caki-1, Caki-2, A498, ACHN, 786-O, and 769-P cells. CYP1B1 protein expression was significantly up-regulated in most RCC cell lines compared with normal kidney (Fig. 1a). Next, RCC TMA consisting of 96 cases of primary RCC was immunostained with CYP1B1 antibody. While CYP1B1 expression was weak or not detected in most of the normal kidney tissues, the majority of RCC samples showed moderate or strong CYP1B1 immunoreactivity with an average staining score of 1.84 ± 0.10 (versus 1.16 ± 0.21 in normal kidney tissues) (p < 0.01; Fig. 1b).

The clinicopathological status of the 96 RCC samples is shown in Table 1. RCC were classified into 2 groups according to CYP1B1 staining score; High (2–3) and Low (0–1). Increased expression of CYP1B1 was correlated with high grade or high stage disease, with a trend toward statistical significance (P = 0.0875, P = 0.0692, respectively). However, there were no significant differences for age, gender, histological type, lymph node and systematic metastasis between the groups.

Attenuation of CYP1B1 expression inhibits renal cancer cell viability, migration, and invasion

CYP1B1 levels were increased in RCC and thus, the functional significance of this gene were explored. This was done by examining whether reduction of CYP1B1 expression has an effect on cell viability, migration, or invasion properties of RCC cell lines. After transfection with two different CYP1B1 siRNAs, significant reduction of CYP1B1 mRNA and proteins were detected in both Caki-1 and 769-P cells (Fig. 2a). Cell proliferation (Fig. 2b) and wound healing assays (Fig. 2c) demonstrated significant inhibition in CYP1B1 transfectants in both Caki-1 and 769-P cells compared to the control siRNA transfectants. Matrigel invasion assay also showed that the number of invaded cells was significantly decreased in CYP1B1 transfectants compared with their control counterparts after 24 h (Fig. 2d). These results suggest that CYP1B1 plays an important role in RCC progression.

CYP1B1 influences cellular apoptosis in RCC cells

Since attenuation of CYP1B1 expression significantly inhibited cell growth and progression of RCC cells, we hypothesized that its expression may induce apoptosis. Apoptosis was examined in control siRNA-treated cells or CYP1B1 siRNA-treated cells. Results of apoptosis assay in Caki-1 and 769-P cells done 48 h post-transfection are shown in Fig. 3. In Caki-1 cells, the apoptotic and early apoptotic fractions (upper right and lower right in the quadrant images, respectively) were significantly greater in CYP1B1 -depleted cells (2.88 % + 5.23 %) compared to control cells (0.33 % + 1.36 %) (p < 0.05; Fig. 3a). In contrast, these differences were not seen in 769-P cells (Fig. 3b).

CYP1B1 regulates the expression of CDC20 and DAPK1 in RCC cell lines

To further understand the precise mechanism of the anti-tumor effect on RCC cells induced by CYP1B1 knockdown, we looked for changes in gene expression in CYP1B1 siRNA-treated and NS siRNA-treated control cells using the Human Cancer Pathway RT² Profiler^TM PCR Array and the Human Apoptosis RT² Profiler^TM PCR Array. From the cancer pathway-related genes analyzed, eight were down-regulated ~ 2- to 5- fold after CYP1B1 depletion and among them, CDC20 was the greatest (Table 2). To verify array data, we performed real-time PCR using Taqman probes. Consistently, a robust decrease of CDC20 mRNA expression was detected in CYP1B1 siRNA-treated Caki-1 (6.1- fold) and 769-P (2.2- fold) (Fig. 4a). However, a significant decrease of DKC1 (Dyskeratosis congenita1), OCLN (Occuludin), and MIK67 (Antigen identified by monoclonal antibody Ki-67) expression were seen in only Caki-1 cells (Fig. 4a). We also examined the protein expression of CDC20 to identify the association with their mRNA expression levels. As shown by Western blotting, CDC20 protein levels were significantly decreased in CYP1B1 siRNA-treated cells compared with NS siRNA-treated control cells (Fig. 4c). These results indicate that CDC20 is potentially regulated by CYP1B1 in RCC.

Table 2 displays genes that were up-regulated and down-regulated 2 or greater due to CYP1B1 knockdown in apoptosis-related gene array analyses. Among 4 up-regulated genes, only DAPK1 was found to be significantly increased in both Caki-1 (1.6- fold) and 769-P (1.3- fold) cells by real-time PCR (Fig. 4b). As shown in Fig. 4c, an increase in DAPK1 protein was also found in both CYP1B1 siRNA-treated RCC cells. Though TP73 (Tumor protein p73) was observed to be most increased by array analyses, its up-regulation was not confirmed in Caki-1 by real-time PCR (Fig. 4b). Among 15 down-regulated genes, BIRC5 (Baculoviral IAP repeat containing 5) was observed to be the lowest. However, a significant decrease of BIRC5 mRNA expression was detected only in CYP1B1 siRNA-treated Caki-1 (Fig. 4b). Thus, these data indicate that the attenuation of CYP1B1 expression caused induction of DAPK1 and could lead to apoptosis in RCC.

CYP1B1 expression is associated with CDC20 and DAPK1 expression in clinical samples

To validate the correlation between CYP1B1 expression and CDC20 or DAPK1 expression, we performed immunohistochemical studies using clinical samples. Case specimens were grouped as Low or High CYP1B1 expression categories as previously determined (Table 1). Representative CDC20 and DAPK1 immunostaining pattern are shown in Fig. 5a. RCC samples showed a higher level of CDC20 expression in comparison with normal kidney specimens (Fig. 5b). In addition, the High CYP1B1 group in RCC showed higher CDC20 expression than Low CYP1B1 group, though the difference did not reach statistical significance (Fig. 5a, b). As for DAPK1, strong cytoplasmic staining was more common in the normal kidney samples than in the RCC tissues (Fig. 5a, b). Moreover, RCC samples with Low CYP1B1 expression had a significantly higher level of DAPK1 expression as compared to those with High CYP1B1 expression (p < 0.01; Fig. 5a, b).