Med Oncol (2015)32:215 DOI 10.1007/s12032-015-0656-1
ORIGINAL PAPER
FBXW7 overexpression suppresses renal cancer cell proliferation and induces apoptosis Yu Fu1 • Youcheng Lin3 • Zhao Yang4 • Guosheng Yang1 • Gaoyuan Li1 • Yuejia Liu1 • Xi Tan2 • Yi Huang5 • Xun Wu3 • Yongqiang Wang5 • Hu Xiong8 • Meng Zhang5 • Lu Fang5 • Yukun Ge3 • Jun Zeng1 • Yangke Cai1 Jian Bai1 • Song Wu5,6,7
•
Received: 10 June 2015 / Accepted: 13 June 2015 Ó Springer Science+Business Media New York 2015
Abstract FBXW7 gene (F-box and WD-40 domain protein 7) is also named HCDC4 and is a significant tumor suppressor gene, which can regulate human cell cycle, proliferation and differentiation. In this study, we tend to investigate protein expression and related biological functions of FBXW7 gene. FBXW7 expression level in renal cell carcinoma (RCC) tissues is highly related to its clinical pathologic grade (P = 0.0094) and TNM phase (P = 0.0080) and is highly lower than in paracancerous normal tissues through immunohistochemistry study. FBXW7 high-expression patients have overall better prognosis than low-expression patients (P \ 0.001). After transfected with FBXW7 plasmid, the RCC cell lines ACHN and A704 showed a depressed proliferation activity and high proportion of apoptosis through CCK8, colony formation and flow cytometry assay studies. By Western blot analysis, expression of cell proliferation-activating
Yu Fu, Youcheng Lin, Zhao Yang and Guosheng Yang have contributed equally to this work.
protein c-Myc and c-Jun is downregulated in FBXW7 highexpression RCC compared with negative control. These data suggested that FBXW7 is a significant tumor suppressor gene in RCC. Keywords FBXW7 RCC Cell cycle Apoptosis Proliferation
Introduction As an important recognition factor of protein ubiquitination degradation, many downstream genes of FBXW7 are oncogenes, such as significant cyclin E, c-Myc, c-Jun and Notch, and are all degraded by FBXW7-induced protein ubiquitination [1–5]. Previous study revealed that FBXW7 mutation and deletion will cause accumulation of cancer cell proliferation-related protein, such as Myc and cyclin E [6]. FBXW7 can modulate cell cycle, cell proliferation and
& Guosheng Yang [email protected]
4
University of Chinese Academy of Sciences, Beijing 100049, China
& Song Wu [email protected]
5
National-Regional Key Technology Engineering Laboratory for Clinical Application of Cancer Genomics, Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China
6
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China
7
Luohu Hospital of Shenzhen University, Shenzhen Fifth People’s Hospital, Shenzhen 518035, China
8
The Second Hospital of Lanzhou University, Xian 710000, Shanxi Province, China
1
2
3
Department of Urology, University of South China of Collaboration Hospital, Guangdong No. 2 Provincial People’s Hospital, Guangzhou 510317, Guangdong Province, China Pathology Department, University of South China of Collaboration Hospital, Guangdong No. 2 Provincial People’s Hospital, Guangzhou 510095, Guangdong Province, China Southern Medical University, Guangzhou 510280, Guangdong Province, China
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differentiation through degrading these significant proteins in cell growth and differentiation process. Sahoko [7] and others discovered that FBXW7 is a significant defense factor in hematopoietic system, and FBXW7 deletion will cause hematopoietic cell hard to mature and acute lymphatic leukemia occurrence. Takehiko [8] found that P5 regulates FBXW7 transcription, which leads to FBXW7 expression depression and poor prognosis. Lwatukim demonstrated that FBXW7 expression obviously reduced t in colorectal tissues compared with normal tissues, which is a sign of poor prognosis [9]. In cervical cancer, FBXW7 mutation leads to its weak association with CDPS of cyclin E and induces unregulated cyclin E degradation, and this regulation deficiency will lead to abnormal cell cycle and tumorigenesis [10]. FBXW7 deletion in advanced glioma causes heteroploid increase in cell mitosis [11]. Related date indicated that FBXW7 mutation happens in 6 % human tumor; in cholangiocarcinoma, the FBXW7 mutation rate is even as high as 35 %. Thirty-one mutations occur in acute T cell lymphatic leukemia; 9 % and 6 % occurs in colon and stomach carcinoma. Sixty-three percent mutations occur at high-frequency mutational sites Arg465 and Arg479 [12]. However, little is known about FBXW7 biological function and clinical study significance in RCC. In this study, we tend to explore the correlation of FBXW7 protein expression level in RCC tissue and the clinicopathologic features. We investigated the effect of FBXW7 overexpression on proliferation, apoptosis and cell cycle of RCC cell lines ACHN and A704, to evaluate the influence of FBXW7 on growth and prognosis of RCC.
Materials and methods Tissue samples This research is supported by Ethics Committee of Guangdong No. 2 Provincial People’s Hospital (GD2H). All 105 pairs of samples were paraffin samples collected from the Department of Pathology, GD2H, dated from 2007 to 2012 (24 patients lost touch). All the histological diagnosis and pathologic grade are conducted by two independent senior pathology doctors from the Department of Pathology, GD2H. All the pathology did not receive any chemotherapy and radiotherapy before surgery. All the clinical information was collected from patient medical record. Immunohistochemistry All the processes are following the standard procedure. Surgical specimen was fixed in 10 % formalin and
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embedded in paraffin. Paraffin-embedded tissue block was sectioned at 4 lm thickness. The section slides were baked at 67 °C for 2 h, dewaxed in xylene, dehydrated in a gradient alcohol and boiled in 0.01 M citrate buffer (pH 6.0) in autoclave for antigen retrieval. The slides were incubated in 3 % H2O2 at 37 °C for 10 min to deactivate the endogenous peroxidase and incubated in normal goat serum at 37 °C for 10 min to prevent nonspecific binding. Primary antibody for FBXW7 (1:500, Abcam, USA) was added onto the slides and incubated at 4 °C overnight. The negative control was obtained with antibody diluents. The EliVision plus kit (Maixin, Fujian, China) detected the antibody binding. The slide was stained with DAB, washed in flowing water, re-stained with hematoxylin, dehydrated, cleared, dried and mounted. All the tumor sections were randomly reviewed by two independent observers. Five zones on each section were chosen for review; each zone was zoomed in 400-folds, contained 100 cells. Scores of FBXW7 immunostaining were marked by evaluation of staining strength and stained cell proportion on each section. FBXW7 staining strength was scored as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). Stained cell proportion was scored as 1 (1–5 %), 2 (5–25 %), 3 (25–50 %), 4 (50–75 %) and 5 (75–100 %). Each section final score was the multiple of the above two values, ranged from 0 to 15. Final score B4 was considered as FBXW7 low expression, and [4 was FBXW7 high expression. Cell culture and transfection with FBXW7 plasmid Human RCC cell lines AHCN and A704 were cultured using RPMI-1640 (Gibco, Invitrogen, NY, USA); PRMI medium was supplemented with 10 % fetal bovine serum (FBS) (Gibco, Invitrogen, NY, USA), 100 U/ml Penicillin (Gibco, Invitrogen, NY, USA) and 100 mg/ml streptomycin (Gibco, Invitrogen, NY, USA). Cells were cultivated at 37 °C in a 5 % CO2-humidified incubator. FBXW7 plasmid was purchased from Shanghai Genechem. FBXW7 plasmid was transfected into RCC cell lines using lipofectamine 2000. The transfection was performed following the manufacturer’s instruction. All the experiments were repeated for three times. Real-time PCR Total RNA was extracted with RNA isolation kit (Tiangen Biotech), and cDNA was synthesized using PrimeScript RT reagent kit (TakaraBio), following the manufacturer’s instruction. Real-time PCR quantified FBXW7 mRNA levels using a pair of qPCR primer sequences: forward, 50 AAGGGCAACAACGACG-30 ; reverse, 50 -AGGGAG CAATGAAATGAAGT-30 . The FBXW7 mRNA relative
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expression level referred to the housekeeping gene GAPDH using primer sequence: forward, 50 -CTGGGCT ACACTGAGCACC-30 ; reverse, 50 -AAGTGGTCGTT GAGGGCAATG-30 . All the experiments were repeated for three times.
Cell viability assays Twenty-four hours after transfection, cells were seeded in 96-well plates (3 9 103 cells/well), cultivated at 37 °C in a 5 % CO2-humidified incubator for 24 h. Ten microliters of Cell Counting Kit-8 kit (CCK-8; Dojindo, Kumamoto, Japan) was added into each well and incubated at 37 °C in a 5 % CO2-humidified incubator for 2 h. Spectrometer was used to measure 450-nm absorbance. All the experiments were repeated for three times.
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Cell cycle assays Twenty-four hours after transfection, ACHN and A704 cells were collected. Cells were washed with PBS for three times, and cell cycle assay was performed using propidium iodide (PI) staining (KeyGEN, Nanjing, China). The assay was performed following the manufacturer’s instruction. All the experiments were repeated for three times. Cell apoptosis Cells were transfected for 24 h and then digested using trypsin without EDTA. Cell apoptosis assay was performed using PI Detection Kit (KeyGEN, Nanjing, China). Collected cells were washed with PBS for two times, resuspended with 500 ll of resuspension buffer and added with 5 ll of Annexin-KeyFour 488 and 5 ll PI. After mixing
Fig. 1 FBXW7 expression in RCC cancerous and paracancerous tissues. FBXW7 is primarily located in cell nucleus. FBXW7 is negatively (a) or weakly positively stained (b) in cancerous tissues (400X). c FBXW7 is moderately stained in paracancerous tissues (400X). d FBXW7 is strongly stained in paracancerous tissues (400X). FBXW7 showed strong staining differences between two tissues. e FBXW7 protein expression level increases in normal tissues. FBXW7 protein expression level is much higher than that in RCC tissues (n = 105, P \ 0.0001). At both ends of the box diagram are upper quartile and lower quartile, respectively, middle horizontal line is the median, and the connection line of both ends is maximum and minimum except for outliers
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Table 1 Correlation between FBXW7 expression and clinical pathologic characteristics of the patients with renal cell carcinoma (RCC)
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Characteristics
Number
Low expression of FBXW7
High expression of FBXW7
B50
76
21
55
[50
29
7
22
Male
59
17
42
Female
46
11
35
Age
P 0.7174
Gender
0.5732
Pathologic
0.0094
Stage I–II
82
17
65
III–IV
23
11
12
I
73
13
60
II
23
11
12
9
4
5
76
20
56
29
8
21
TNM
III–IV Tumor size B7 cm [7 cm
Fig. 2 Survival analysis of clear cell renal cell carcinoma patient (n = 81, 24 patients lost touch). a Kaplan–Meier method is recruited to analyze overall survival of FBXW7 lowexpression (n = 22) and highexpression (n = 59) groups of RCC patients. High-expression group has an obvious better survival than low-expression group (log-rank test, P \ 0.001). b TNM, phase I (P \ 0.001); c TNM, phase II (P = 0.030); d TNM, phases III–IV (P = 0.025). Analysis of survival in FBXW7 highexpression and low-expression RCC groups in phases I (b), II (c) and III–IV (d)
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0.0080
0.8953
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well, the sample reacted in dark for 5–15 min. The cells were detected using flow cytometry. The experiments were repeated for three times. Colony formation assay Cells were transfected for 24 h, seeded into 6-well plate (1000 cells/well) and incubated 37 °C in a 5 % CO2-humidified incubator. After 2 weeks, the cells were stained with gentian violet, and the wells with colony numbers larger than 50 were counted. The experiments were repeated for three times. Western blot Cells were transfected for 24 h, collected and washed with PBS for three times, then the cells were lysed with cell lysis buffer (RIPA, Beyotime Biotechnology), and the protein concentration of each sample was determined. Certain amount of SDS-PAGE gel loading buffer was added to total amount of 30 lg protein samples, boiled for 5 min and cooled down to room temperature. The protein samples were loaded into the well of SDS-PAGE gel, electrophoresed and transferred to PVDF membrane. The membrane was blocked with Western blocking buffer and incubated with rabbit against FBXW7 (1:500, Abcam, USA), c-Myc (1:500, Gene Tex, USA) and c-JUN (1:500,
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Abcam, USA) at 4 °C overnight. The membrane was washed and incubated with peroxisome-labeled secondary antibody at 4 °C for 1 h. ECL (Beyotime Biotechnology) was applied to detect the protein. Protein bands were analyzed using Bandscan 5.0. b-actin was used as a negative control of FBXW7. Statistic analysis We used Prism 5 software (GraphPad, San Diego, CA, USA) and SPSS 18.0 (SPSS, Chicago, IL, USA) for statistic analysis. All values are expressed as mean ± SD. v2 test was recruited to investigate the correlation of FBXW7 expression and clinical pathologic factors. All results were compared using Student’s t test. P value \ 0.05 was considered as a significant differentiation.
Results FBXW7 expression in RCC cancerous and paracancerous tissues and its correlation with clinical pathologic factors We have studied the FBXW7 expression in 105 pairs of RCC tissues by immunohistochemistry. FBXW7 is primarily expressed in nucleus of renal cell carcinoma cancerous and
Fig. 3 a RNA expression level in four kinds of RCC cell lines ACHN, A498, ACHN and A704. b Detection of FBXW7 protein expression level in four RCC cell lines using Western blot analysis. c Relative mRNA expression level in A704 and ACHN after transfected with FBXW7 plasmid. d Relative protein expression level in A704 and ACHN detected by Western blot after transfected with FBXW7 plasmid
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Fig. 4 FBXW7 overexpression induces cell apoptosis and cell cycle elongation and depresses cell proliferation. a CCK8 assay is performed using FBXW7 plasmid and control vectortransfected ACHN and A704 cells. OD value of FBXW7transfected cells is much lower than in control group, which indicates lower cell viability. b Colony formation assay is also performed using FBXW7 plasmid and control vectortransfected ACHN and A704 cells. Cell number of FBXW7transfected cell group is lower than that of control group
paracancerous tissues (Fig. 1), and cancerous staining is obviously lower than in paracancerous normal tissues. FBXW7 protein expression level is much higher than that in RCC tissues (Fig. 1E, n = 105, P \ 0.0001). About all of the 105 RCC samples, 77 cases have strong positive staining, and 28 cases have week positive staining. We have analyzed the correlation of FBXW7 overexpression and clinical pathologic factors. As Table 1 describes, FBXW7 protein expression upregulation is highly correlated with RCC clinical pathologic grading (P = 0.0094). Tumor-nodemetastasis (TNM) stage also has a strong correlation with FBXW7 expression upregulation (P = 0.0080). However, age, gender and tumor size (small:\7 cm, big:[7 cm) have no correlations with FBXW7 protein expression level. All the above contents are concluded in Table 1. Survival analysis FBXW7 expression downregulation effect on RCC patient survival was analyzed using Kaplan–Meier and the log-
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rank tests. Five-year survival rate of FBXW7 low-expression patients is much lower than that of FBXW7 highexpression patients (Fig. 2a, P \ 0.001). Overall survival time of FBXW7 expression upregulation patients is longer than that of FBXW7 expression depression patients. All the patients were grouped into three groups (I, II, III–IV) based on TNM staging; the survival rate of FBXW7 high-expression and low-expression patients was compared within one group. The result of group I was shown in Fig. 2b (P \ 0.001), group II in Fig. 2c (P = 0.030) and III–IV result in Fig. 2d (P = 0.025). FBXW7 overexpression represses cell proliferation and induces apoptosis RNA extract from RCC cell lines ACHN, A704, A498 and 786-0 was reverse-transcribed into cDNA, and real-time PCR was performed to get the relative FBXW7 RNA expression level of the four cell lines; the result is 786-0[A498[A704[ACHN (Fig. 3a). From Western blot
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Fig. 5 a FBXW7-transfected cell has a prolonged G0/G1 phase. b FBXW7-transfected group has a higher percentage of early and late apoptotic cells
result, FBXW7 protein expression level was consistent with real-time PCR result (Fig. 3b). To further investigate the FBXW7 regulation to RCC cell lines, the cell lines A704 and ACHN were chosen as research
targets. We use FBXW7-transfected ACHN and A704 to perform CCK8 and colony formation assay to study FBXW7 regulation effect on RCC cell line proliferation, apoptosis and cell cycle. After FBXW7 transfection, real-time PCR and
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Western blot analysis were performed to detect FBXW7 RNA (Fig. 3c) as well as FBXW7 and its downstream target protein c-Myc and c-Jun protein relative expression level (Fig. 3d) to demonstrate the effectiveness of FBXW7 plasmid transfection. FBXW7 expression upregulation increased ACHN and A704 cell apoptosis and repressed cell proliferation, as shown in CCK8 analysis (Fig. 4a, vector vs plasmid, A704: 1.327 ± 0.036 vs 1.035 ± 0.042, P \ 0.05; ACHN: 1.403 ± 0.024 vs 1.166 ± 0.029, P \ 0.05) and colony formation assay (Fig. 4b vector vs plasmid, ACHN: 567 ± 19 vs 412 ± 21, P \ 0.05; A704: 279 ± 13 vs 144 ± 7, P \ 0.05). Figure 5a shows cell cycle changes detected by PI method using flow cytometry. FBXW7 highexpression ACHN and A704 showed a high ratio of G0/G1 phase than in control group (vector vs plasmid, ACHN: 51.93 vs 63.25 %, P \ 0.05; A704: 60.31 vs 68.32 %, P \ 0.05). Figure 5b shows that FBXW7 high-expression ACHN and A704 have a higher percentage of early and late apoptotic cells than in control group (vector vs plasmid, ACHN: 5.1 vs 8.5 %, P \ 0.05; A704 14.1 vs 20.4 %, P \ 0.05).
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expressed FBXW7 in RCC cell lines can induce ubiquitination and degradation of c-Myc and c-Jun protein and suppress tumor cell growth. Furthermore, mTOR is also the target protein of FBXW7 and can be degraded by ubiquitination [19, 20]. mTOR is a protein kinase, which induces cell growth and division through regulating protein synthesis and autophagy [21]. mTOR cell signaling pathway mainly involves classic PI3K/Akt/MTOR signaling pathway, and this pathway is repressed after mTOR protein degraded by FBXW7, which elongates cell G1 phase and suppresses cell proliferation. In summary, this research elucidated that FBXW7 expression level is largely correlated with RCC clinical pathologic grade and TNM phase. FBXW7 high-expression patient has a better overall prognosis than those with FBXW7 low expression. FBXW7 high-expression RCC cell lines ACHN and A704 have a decreased proliferation activity, elongated G1 phase and induced apoptosis, which may be modulated through FBXW7-regulated c-Myc, c-Jun and mTOR. Generally speaking, FBXW7 is a significant tumor suppressor gene in RCC, and molecular mechanism of FBXW7 gene needs to be further studied.
Conclusion Conflict of interest
FBXW7 high expression in RCC is highly related to its pathologic grade and clinical stage. FBXW7 high-expression patient has a good overall survival than those with FBXW7 low expression. In RCC cell lines, FBXW7 overexpression repressed cell proliferation and cell cycle and increased cell apoptosis. FBXW7 downstream protein c-Jun and c-Myc expression also decreased after FBXW7 transfection. The immunohistochemistry and cell experiment data support each other. Pervious literatures revealed FBXW7 functions in other cancers. FBXW7 expression downregulation induced liver, stomach and breast cancer tumorigenesis [13–15]. Our study result coordinates with previous research, FBXW7 is an important tumor suppressor gene, and little is known about FBXW7 tumor suppression mechanism. At molecular biology level, c-Myc oncogene transcription product c-Myc protein is highly related to cell cycle regulation, DNA polymerase activity and cell proliferation and differentiation [16]. At cell stationary and terminal differentiation stage, c-Myc transcription is depressed and the mRNA level is decreased; at normal proliferation stage, c-Myc transcription is transiently activated and is depressed before protein translation. Phosphorylation-activated c-Jun induces cell proliferation, differentiation and carcinogenesis [17]. It also regulates downstream target gene through AP-1, degrades extra cellular matrix, and increases tumor migration and invasion activity, metastatic cell vascularization and cell cycle abnormality, which activate tumor cell migration and invasion [18]. Therefore, highly
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None.
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ORIGINAL PAPER
FBXW7 overexpression suppresses renal cancer cell proliferation and induces apoptosis Yu Fu1 • Youcheng Lin3 • Zhao Yang4 • Guosheng Yang1 • Gaoyuan Li1 • Yuejia Liu1 • Xi Tan2 • Yi Huang5 • Xun Wu3 • Yongqiang Wang5 • Hu Xiong8 • Meng Zhang5 • Lu Fang5 • Yukun Ge3 • Jun Zeng1 • Yangke Cai1 Jian Bai1 • Song Wu5,6,7
•
Received: 10 June 2015 / Accepted: 13 June 2015 Ó Springer Science+Business Media New York 2015
Abstract FBXW7 gene (F-box and WD-40 domain protein 7) is also named HCDC4 and is a significant tumor suppressor gene, which can regulate human cell cycle, proliferation and differentiation. In this study, we tend to investigate protein expression and related biological functions of FBXW7 gene. FBXW7 expression level in renal cell carcinoma (RCC) tissues is highly related to its clinical pathologic grade (P = 0.0094) and TNM phase (P = 0.0080) and is highly lower than in paracancerous normal tissues through immunohistochemistry study. FBXW7 high-expression patients have overall better prognosis than low-expression patients (P \ 0.001). After transfected with FBXW7 plasmid, the RCC cell lines ACHN and A704 showed a depressed proliferation activity and high proportion of apoptosis through CCK8, colony formation and flow cytometry assay studies. By Western blot analysis, expression of cell proliferation-activating
Yu Fu, Youcheng Lin, Zhao Yang and Guosheng Yang have contributed equally to this work.
protein c-Myc and c-Jun is downregulated in FBXW7 highexpression RCC compared with negative control. These data suggested that FBXW7 is a significant tumor suppressor gene in RCC. Keywords FBXW7 RCC Cell cycle Apoptosis Proliferation
Introduction As an important recognition factor of protein ubiquitination degradation, many downstream genes of FBXW7 are oncogenes, such as significant cyclin E, c-Myc, c-Jun and Notch, and are all degraded by FBXW7-induced protein ubiquitination [1–5]. Previous study revealed that FBXW7 mutation and deletion will cause accumulation of cancer cell proliferation-related protein, such as Myc and cyclin E [6]. FBXW7 can modulate cell cycle, cell proliferation and
& Guosheng Yang [email protected]
4
University of Chinese Academy of Sciences, Beijing 100049, China
& Song Wu [email protected]
5
National-Regional Key Technology Engineering Laboratory for Clinical Application of Cancer Genomics, Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China
6
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China
7
Luohu Hospital of Shenzhen University, Shenzhen Fifth People’s Hospital, Shenzhen 518035, China
8
The Second Hospital of Lanzhou University, Xian 710000, Shanxi Province, China
1
2
3
Department of Urology, University of South China of Collaboration Hospital, Guangdong No. 2 Provincial People’s Hospital, Guangzhou 510317, Guangdong Province, China Pathology Department, University of South China of Collaboration Hospital, Guangdong No. 2 Provincial People’s Hospital, Guangzhou 510095, Guangdong Province, China Southern Medical University, Guangzhou 510280, Guangdong Province, China
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differentiation through degrading these significant proteins in cell growth and differentiation process. Sahoko [7] and others discovered that FBXW7 is a significant defense factor in hematopoietic system, and FBXW7 deletion will cause hematopoietic cell hard to mature and acute lymphatic leukemia occurrence. Takehiko [8] found that P5 regulates FBXW7 transcription, which leads to FBXW7 expression depression and poor prognosis. Lwatukim demonstrated that FBXW7 expression obviously reduced t in colorectal tissues compared with normal tissues, which is a sign of poor prognosis [9]. In cervical cancer, FBXW7 mutation leads to its weak association with CDPS of cyclin E and induces unregulated cyclin E degradation, and this regulation deficiency will lead to abnormal cell cycle and tumorigenesis [10]. FBXW7 deletion in advanced glioma causes heteroploid increase in cell mitosis [11]. Related date indicated that FBXW7 mutation happens in 6 % human tumor; in cholangiocarcinoma, the FBXW7 mutation rate is even as high as 35 %. Thirty-one mutations occur in acute T cell lymphatic leukemia; 9 % and 6 % occurs in colon and stomach carcinoma. Sixty-three percent mutations occur at high-frequency mutational sites Arg465 and Arg479 [12]. However, little is known about FBXW7 biological function and clinical study significance in RCC. In this study, we tend to explore the correlation of FBXW7 protein expression level in RCC tissue and the clinicopathologic features. We investigated the effect of FBXW7 overexpression on proliferation, apoptosis and cell cycle of RCC cell lines ACHN and A704, to evaluate the influence of FBXW7 on growth and prognosis of RCC.
Materials and methods Tissue samples This research is supported by Ethics Committee of Guangdong No. 2 Provincial People’s Hospital (GD2H). All 105 pairs of samples were paraffin samples collected from the Department of Pathology, GD2H, dated from 2007 to 2012 (24 patients lost touch). All the histological diagnosis and pathologic grade are conducted by two independent senior pathology doctors from the Department of Pathology, GD2H. All the pathology did not receive any chemotherapy and radiotherapy before surgery. All the clinical information was collected from patient medical record. Immunohistochemistry All the processes are following the standard procedure. Surgical specimen was fixed in 10 % formalin and
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embedded in paraffin. Paraffin-embedded tissue block was sectioned at 4 lm thickness. The section slides were baked at 67 °C for 2 h, dewaxed in xylene, dehydrated in a gradient alcohol and boiled in 0.01 M citrate buffer (pH 6.0) in autoclave for antigen retrieval. The slides were incubated in 3 % H2O2 at 37 °C for 10 min to deactivate the endogenous peroxidase and incubated in normal goat serum at 37 °C for 10 min to prevent nonspecific binding. Primary antibody for FBXW7 (1:500, Abcam, USA) was added onto the slides and incubated at 4 °C overnight. The negative control was obtained with antibody diluents. The EliVision plus kit (Maixin, Fujian, China) detected the antibody binding. The slide was stained with DAB, washed in flowing water, re-stained with hematoxylin, dehydrated, cleared, dried and mounted. All the tumor sections were randomly reviewed by two independent observers. Five zones on each section were chosen for review; each zone was zoomed in 400-folds, contained 100 cells. Scores of FBXW7 immunostaining were marked by evaluation of staining strength and stained cell proportion on each section. FBXW7 staining strength was scored as 0 (no staining), 1 (weak), 2 (moderate) and 3 (strong). Stained cell proportion was scored as 1 (1–5 %), 2 (5–25 %), 3 (25–50 %), 4 (50–75 %) and 5 (75–100 %). Each section final score was the multiple of the above two values, ranged from 0 to 15. Final score B4 was considered as FBXW7 low expression, and [4 was FBXW7 high expression. Cell culture and transfection with FBXW7 plasmid Human RCC cell lines AHCN and A704 were cultured using RPMI-1640 (Gibco, Invitrogen, NY, USA); PRMI medium was supplemented with 10 % fetal bovine serum (FBS) (Gibco, Invitrogen, NY, USA), 100 U/ml Penicillin (Gibco, Invitrogen, NY, USA) and 100 mg/ml streptomycin (Gibco, Invitrogen, NY, USA). Cells were cultivated at 37 °C in a 5 % CO2-humidified incubator. FBXW7 plasmid was purchased from Shanghai Genechem. FBXW7 plasmid was transfected into RCC cell lines using lipofectamine 2000. The transfection was performed following the manufacturer’s instruction. All the experiments were repeated for three times. Real-time PCR Total RNA was extracted with RNA isolation kit (Tiangen Biotech), and cDNA was synthesized using PrimeScript RT reagent kit (TakaraBio), following the manufacturer’s instruction. Real-time PCR quantified FBXW7 mRNA levels using a pair of qPCR primer sequences: forward, 50 AAGGGCAACAACGACG-30 ; reverse, 50 -AGGGAG CAATGAAATGAAGT-30 . The FBXW7 mRNA relative
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expression level referred to the housekeeping gene GAPDH using primer sequence: forward, 50 -CTGGGCT ACACTGAGCACC-30 ; reverse, 50 -AAGTGGTCGTT GAGGGCAATG-30 . All the experiments were repeated for three times.
Cell viability assays Twenty-four hours after transfection, cells were seeded in 96-well plates (3 9 103 cells/well), cultivated at 37 °C in a 5 % CO2-humidified incubator for 24 h. Ten microliters of Cell Counting Kit-8 kit (CCK-8; Dojindo, Kumamoto, Japan) was added into each well and incubated at 37 °C in a 5 % CO2-humidified incubator for 2 h. Spectrometer was used to measure 450-nm absorbance. All the experiments were repeated for three times.
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Cell cycle assays Twenty-four hours after transfection, ACHN and A704 cells were collected. Cells were washed with PBS for three times, and cell cycle assay was performed using propidium iodide (PI) staining (KeyGEN, Nanjing, China). The assay was performed following the manufacturer’s instruction. All the experiments were repeated for three times. Cell apoptosis Cells were transfected for 24 h and then digested using trypsin without EDTA. Cell apoptosis assay was performed using PI Detection Kit (KeyGEN, Nanjing, China). Collected cells were washed with PBS for two times, resuspended with 500 ll of resuspension buffer and added with 5 ll of Annexin-KeyFour 488 and 5 ll PI. After mixing
Fig. 1 FBXW7 expression in RCC cancerous and paracancerous tissues. FBXW7 is primarily located in cell nucleus. FBXW7 is negatively (a) or weakly positively stained (b) in cancerous tissues (400X). c FBXW7 is moderately stained in paracancerous tissues (400X). d FBXW7 is strongly stained in paracancerous tissues (400X). FBXW7 showed strong staining differences between two tissues. e FBXW7 protein expression level increases in normal tissues. FBXW7 protein expression level is much higher than that in RCC tissues (n = 105, P \ 0.0001). At both ends of the box diagram are upper quartile and lower quartile, respectively, middle horizontal line is the median, and the connection line of both ends is maximum and minimum except for outliers
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Table 1 Correlation between FBXW7 expression and clinical pathologic characteristics of the patients with renal cell carcinoma (RCC)
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Characteristics
Number
Low expression of FBXW7
High expression of FBXW7
B50
76
21
55
[50
29
7
22
Male
59
17
42
Female
46
11
35
Age
P 0.7174
Gender
0.5732
Pathologic
0.0094
Stage I–II
82
17
65
III–IV
23
11
12
I
73
13
60
II
23
11
12
9
4
5
76
20
56
29
8
21
TNM
III–IV Tumor size B7 cm [7 cm
Fig. 2 Survival analysis of clear cell renal cell carcinoma patient (n = 81, 24 patients lost touch). a Kaplan–Meier method is recruited to analyze overall survival of FBXW7 lowexpression (n = 22) and highexpression (n = 59) groups of RCC patients. High-expression group has an obvious better survival than low-expression group (log-rank test, P \ 0.001). b TNM, phase I (P \ 0.001); c TNM, phase II (P = 0.030); d TNM, phases III–IV (P = 0.025). Analysis of survival in FBXW7 highexpression and low-expression RCC groups in phases I (b), II (c) and III–IV (d)
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0.0080
0.8953
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well, the sample reacted in dark for 5–15 min. The cells were detected using flow cytometry. The experiments were repeated for three times. Colony formation assay Cells were transfected for 24 h, seeded into 6-well plate (1000 cells/well) and incubated 37 °C in a 5 % CO2-humidified incubator. After 2 weeks, the cells were stained with gentian violet, and the wells with colony numbers larger than 50 were counted. The experiments were repeated for three times. Western blot Cells were transfected for 24 h, collected and washed with PBS for three times, then the cells were lysed with cell lysis buffer (RIPA, Beyotime Biotechnology), and the protein concentration of each sample was determined. Certain amount of SDS-PAGE gel loading buffer was added to total amount of 30 lg protein samples, boiled for 5 min and cooled down to room temperature. The protein samples were loaded into the well of SDS-PAGE gel, electrophoresed and transferred to PVDF membrane. The membrane was blocked with Western blocking buffer and incubated with rabbit against FBXW7 (1:500, Abcam, USA), c-Myc (1:500, Gene Tex, USA) and c-JUN (1:500,
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Abcam, USA) at 4 °C overnight. The membrane was washed and incubated with peroxisome-labeled secondary antibody at 4 °C for 1 h. ECL (Beyotime Biotechnology) was applied to detect the protein. Protein bands were analyzed using Bandscan 5.0. b-actin was used as a negative control of FBXW7. Statistic analysis We used Prism 5 software (GraphPad, San Diego, CA, USA) and SPSS 18.0 (SPSS, Chicago, IL, USA) for statistic analysis. All values are expressed as mean ± SD. v2 test was recruited to investigate the correlation of FBXW7 expression and clinical pathologic factors. All results were compared using Student’s t test. P value \ 0.05 was considered as a significant differentiation.
Results FBXW7 expression in RCC cancerous and paracancerous tissues and its correlation with clinical pathologic factors We have studied the FBXW7 expression in 105 pairs of RCC tissues by immunohistochemistry. FBXW7 is primarily expressed in nucleus of renal cell carcinoma cancerous and
Fig. 3 a RNA expression level in four kinds of RCC cell lines ACHN, A498, ACHN and A704. b Detection of FBXW7 protein expression level in four RCC cell lines using Western blot analysis. c Relative mRNA expression level in A704 and ACHN after transfected with FBXW7 plasmid. d Relative protein expression level in A704 and ACHN detected by Western blot after transfected with FBXW7 plasmid
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Fig. 4 FBXW7 overexpression induces cell apoptosis and cell cycle elongation and depresses cell proliferation. a CCK8 assay is performed using FBXW7 plasmid and control vectortransfected ACHN and A704 cells. OD value of FBXW7transfected cells is much lower than in control group, which indicates lower cell viability. b Colony formation assay is also performed using FBXW7 plasmid and control vectortransfected ACHN and A704 cells. Cell number of FBXW7transfected cell group is lower than that of control group
paracancerous tissues (Fig. 1), and cancerous staining is obviously lower than in paracancerous normal tissues. FBXW7 protein expression level is much higher than that in RCC tissues (Fig. 1E, n = 105, P \ 0.0001). About all of the 105 RCC samples, 77 cases have strong positive staining, and 28 cases have week positive staining. We have analyzed the correlation of FBXW7 overexpression and clinical pathologic factors. As Table 1 describes, FBXW7 protein expression upregulation is highly correlated with RCC clinical pathologic grading (P = 0.0094). Tumor-nodemetastasis (TNM) stage also has a strong correlation with FBXW7 expression upregulation (P = 0.0080). However, age, gender and tumor size (small:\7 cm, big:[7 cm) have no correlations with FBXW7 protein expression level. All the above contents are concluded in Table 1. Survival analysis FBXW7 expression downregulation effect on RCC patient survival was analyzed using Kaplan–Meier and the log-
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rank tests. Five-year survival rate of FBXW7 low-expression patients is much lower than that of FBXW7 highexpression patients (Fig. 2a, P \ 0.001). Overall survival time of FBXW7 expression upregulation patients is longer than that of FBXW7 expression depression patients. All the patients were grouped into three groups (I, II, III–IV) based on TNM staging; the survival rate of FBXW7 high-expression and low-expression patients was compared within one group. The result of group I was shown in Fig. 2b (P \ 0.001), group II in Fig. 2c (P = 0.030) and III–IV result in Fig. 2d (P = 0.025). FBXW7 overexpression represses cell proliferation and induces apoptosis RNA extract from RCC cell lines ACHN, A704, A498 and 786-0 was reverse-transcribed into cDNA, and real-time PCR was performed to get the relative FBXW7 RNA expression level of the four cell lines; the result is 786-0[A498[A704[ACHN (Fig. 3a). From Western blot
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Fig. 5 a FBXW7-transfected cell has a prolonged G0/G1 phase. b FBXW7-transfected group has a higher percentage of early and late apoptotic cells
result, FBXW7 protein expression level was consistent with real-time PCR result (Fig. 3b). To further investigate the FBXW7 regulation to RCC cell lines, the cell lines A704 and ACHN were chosen as research
targets. We use FBXW7-transfected ACHN and A704 to perform CCK8 and colony formation assay to study FBXW7 regulation effect on RCC cell line proliferation, apoptosis and cell cycle. After FBXW7 transfection, real-time PCR and
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Western blot analysis were performed to detect FBXW7 RNA (Fig. 3c) as well as FBXW7 and its downstream target protein c-Myc and c-Jun protein relative expression level (Fig. 3d) to demonstrate the effectiveness of FBXW7 plasmid transfection. FBXW7 expression upregulation increased ACHN and A704 cell apoptosis and repressed cell proliferation, as shown in CCK8 analysis (Fig. 4a, vector vs plasmid, A704: 1.327 ± 0.036 vs 1.035 ± 0.042, P \ 0.05; ACHN: 1.403 ± 0.024 vs 1.166 ± 0.029, P \ 0.05) and colony formation assay (Fig. 4b vector vs plasmid, ACHN: 567 ± 19 vs 412 ± 21, P \ 0.05; A704: 279 ± 13 vs 144 ± 7, P \ 0.05). Figure 5a shows cell cycle changes detected by PI method using flow cytometry. FBXW7 highexpression ACHN and A704 showed a high ratio of G0/G1 phase than in control group (vector vs plasmid, ACHN: 51.93 vs 63.25 %, P \ 0.05; A704: 60.31 vs 68.32 %, P \ 0.05). Figure 5b shows that FBXW7 high-expression ACHN and A704 have a higher percentage of early and late apoptotic cells than in control group (vector vs plasmid, ACHN: 5.1 vs 8.5 %, P \ 0.05; A704 14.1 vs 20.4 %, P \ 0.05).
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expressed FBXW7 in RCC cell lines can induce ubiquitination and degradation of c-Myc and c-Jun protein and suppress tumor cell growth. Furthermore, mTOR is also the target protein of FBXW7 and can be degraded by ubiquitination [19, 20]. mTOR is a protein kinase, which induces cell growth and division through regulating protein synthesis and autophagy [21]. mTOR cell signaling pathway mainly involves classic PI3K/Akt/MTOR signaling pathway, and this pathway is repressed after mTOR protein degraded by FBXW7, which elongates cell G1 phase and suppresses cell proliferation. In summary, this research elucidated that FBXW7 expression level is largely correlated with RCC clinical pathologic grade and TNM phase. FBXW7 high-expression patient has a better overall prognosis than those with FBXW7 low expression. FBXW7 high-expression RCC cell lines ACHN and A704 have a decreased proliferation activity, elongated G1 phase and induced apoptosis, which may be modulated through FBXW7-regulated c-Myc, c-Jun and mTOR. Generally speaking, FBXW7 is a significant tumor suppressor gene in RCC, and molecular mechanism of FBXW7 gene needs to be further studied.
Conclusion Conflict of interest
FBXW7 high expression in RCC is highly related to its pathologic grade and clinical stage. FBXW7 high-expression patient has a good overall survival than those with FBXW7 low expression. In RCC cell lines, FBXW7 overexpression repressed cell proliferation and cell cycle and increased cell apoptosis. FBXW7 downstream protein c-Jun and c-Myc expression also decreased after FBXW7 transfection. The immunohistochemistry and cell experiment data support each other. Pervious literatures revealed FBXW7 functions in other cancers. FBXW7 expression downregulation induced liver, stomach and breast cancer tumorigenesis [13–15]. Our study result coordinates with previous research, FBXW7 is an important tumor suppressor gene, and little is known about FBXW7 tumor suppression mechanism. At molecular biology level, c-Myc oncogene transcription product c-Myc protein is highly related to cell cycle regulation, DNA polymerase activity and cell proliferation and differentiation [16]. At cell stationary and terminal differentiation stage, c-Myc transcription is depressed and the mRNA level is decreased; at normal proliferation stage, c-Myc transcription is transiently activated and is depressed before protein translation. Phosphorylation-activated c-Jun induces cell proliferation, differentiation and carcinogenesis [17]. It also regulates downstream target gene through AP-1, degrades extra cellular matrix, and increases tumor migration and invasion activity, metastatic cell vascularization and cell cycle abnormality, which activate tumor cell migration and invasion [18]. Therefore, highly
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None.
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