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Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma Zhou Jiangqiao, Qiu Tao*, Chen Zhongbao, Ma Xiaoxiong, Zhang Long, Zou Jilin, Wang Tianyu Department of Organ Transplantation, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, 430060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 February 2019 Accepted 11 February 2019 Available online xxx
Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies. Anti-silencing function 1B histone chaperone (ASF1B) has been reported to be involved in various diseases. However, its role in ccRCC is largely unknown. In the present study, using genetic data and clinical information obtained from the TCGA data portal and GEO database, we found that ASF1B was highly expressed in ccRCC cancer tissue compared with normal tissue, and ASF1B expression was positively correlated with tumor stage, tumor grade and patient survival. The function of ASF1B in cell proliferation and migration was assessed by pathological and molecular analyses. The results showed that ASF1B overexpression significantly enhanced the proliferation and migration of 786-O cells and Caki-1 cells, while silencing ASF1B expression significantly inhibited the proliferation and migration. In addition, ASF1B overexpression enhanced cell proliferation by upregulating PCNA and downregulating P27 expression and promoted cell migration by upregulating MMP2 and MMP9. Furthermore, the phosphorylation levels of protein kinase B (AKT) and P-P70 S6K1 were significantly upregulated in the ASF1B overexpression group. More importantly, AKT inhibitor blocked the promotional effect of ASF1B on proliferation and migration. In summary, the present study demonstrated that ASF1B overexpression promoted tumor cell proliferation and migration, which was dependent on the AKT/P70 S6K1 pathway. © 2019 Elsevier Inc. All rights reserved.
Keywords: ASF1B Clear cell renal cell cancer Proliferation AKT
1. Introduction Worldwide, renal cell cancer is the ninth most common cancer [1]. Histologically, they are defined by clear cytoplasm, with nested clusters of cells surrounded by a dense endothelial network [2]. According to the guidelines, ccRCC can be effectively treated and even cured through surgery and targeted therapy [3], but over 90% of cancer-related deaths in individuals with ccRCC are caused by tumor relapse and metastasis [4]. With the development of bioinformatics, many more biomarkers have been found to predict the
Abbreviations: ccRCC, clear cell renal cell carcinoma; ASF1B, anti-silencing function 1B histone chaperone; AKT, protein kinase B; VHL, von Hippel-Lindau; OE, overexpression; PCNA, proliferating cell nuclear antigen. * Corresponding author.Department of Organ Transplantation, Renmin Hospital of Wuhan University, Wuhan University, ZiYang Road 99#, Wuhan, 430060, China. E-mail address: [email protected] (Q. Tao).
occurrence and development of ccRCC so that early intervention can be achieved [5]. Several novel recurrent mutations in histone modifying and chromatin remodeling genes have been identified in ccRCC. BAP1, PBRM1 and SETD2 could be molecular diagnostic markers and possible targets for personalized therapies in ccRCC [6]. Anti-silencing function 1 (ASF1) is a histone H3-H4 chaperone involved in DNA replication and repair as well as transcriptional regulation [7]. ASF1 regulates chromatin function and has been demonstrated to contribute to tumorigenesis [8]. ASF1 contains two subtypes, including ASF1B and ASF1A, which share a 70% sequence identity, but they are not functionally equivalent. Studies have shown that ASF1B is closely related to the development and progression of many tumors, including cervical cancer, breast cancer and prostate cancer [9,10]. However, the role and mechanism of ASF1B in RCC remains unclear. Therefore, we attempted to explore the role and mechanism of ASF1B in ccRCC.
https://doi.org/10.1016/j.bbrc.2019.02.060 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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2. Materials and methods 2.1. TCGA data analysis ccRCC mRNA-seq level3 data samples and clinical information were downloaded from the TCGA data portal. The R-packet DESeq2, which is based on the principle of negative binomial distribution, was used to standardize the expression quantity data and to analyze the differentially expressed genes between cancer samples and normal samples. The standardized data was used to visualize the expression level of ASF1B in ccRCC and adjacent noncancer tissues, and for grade classification, stage and survival correlation analysis. Clinical samples of ccRCC were divided into 2 groups according to the expression level of ASF1B. Then, the log-rank test algorithm was used to generate the survival curve, and the followup data obtained from the ccRCC clinical information were visualized using R package survival analysis. Correspondingly, the correlation between ASF1B expression and tumor grade and stage was calculated by T-test, and the data were visualized using R package ggplot2. The correlation between the expression of ASF1B mRNA and 2 genes (PCNA, P27) related to proliferation in the TCGA KIRC was determined using the Pearson correlation coefficient.
culture dishes for target cell infection. After cell passage, the cells were screened with 4 ml/mL puromycin for both cells at indicated time. After screening, ASF1B expression was detected. 2.6. CCK8 assay Cell viability was examined using a CCK-8 kit according to the manufacturer's protocol. 786-O and Caki-1 cells were inoculated in 96-well plates at a density of 3000 cells/well. After the cells adhered to the wells, 10 ml CCK8 reagent (Dojindo, 44786) and 90 ml DMEM were added to the cells. The cells were further cultured for 0 h, 24 h, 48 h, 72 h, 96 h or 120 h, respectively. Then, the absorbance at 450 nm was measured at the indicated time. 2.7. BrdU experiment 786-O and Caki-1 cells were inoculated in 96-well plates at a density of 3000 cells per well. After 24 h and 48 h, BrdU solution (Roche, Cat. No. 11647229001) was added followed by a 6 h incubation. The standard procedure was performed according to the kit's instructions. Finally, the absorbance was measured at a wavelength of 370 nm.
2.2. GEO data analysis
2.8. Wound healing test
Data on 10 paired tumor tissue samples were downloaded from the GSE102101, a GEO database, and sequence reads were mapped to the human genome (hg38) using hisat2. Transcripts were quantified using stringtie according to the RefSeq annotation and were normalized; differential expression analysis was performed using DESeq2.
The 786-O and Caki-1 cells were inoculated into 6-well plates at a density of 3.5 105 cells per well (Thermo, Cat. No. 140675). After the cells were incubated overnight, confluent monolayers for wounding were yielded. The original culture medium was replaced with DMEM supplemented with mitomycin. Then, the cells were cultured for 12 h. Wounds were generated using a pipette tip, and photographs were immediately obtained (time zero) and at the indicated times for 786-O cells and Caki-1 cells. The migration index was then measured according to the following formula: migration index ¼ (width of the wound at 0 h-width of the wound at 24 or 48 h) *100/width of the wound at time zero.
2.3. Cells and reagents All the reagents information is in Supplementary Table 1. The human ccRCC cell line 786-O and Caki-1 cell line were cultured in standard procedure at 37 C in a humidified atmosphere containing 5% CO2. 2.4. Lentivirus construction The overexpression vector of the ASF1B gene with flag tag was designed and constructed. The pHAGE-3xFlag carrier was used as the carrier vector. The knockdown vector of the ASF1B gene was designed and constructed based on the pLKO.1 carrier vector. Primers were designed via software on the NCBI website (Supplementary Table 2). After the amplification of the target gene fragment by over-nested PCR, the vector was digested and purified. The target gene fragment was ligated to the vector to obtain the recombinant vector, which was then transferred into Escherichia coli. Finally, the positive clones were screened for expansion culture. 2.5. Lentivirus packaging and target cell infection The 293T cell line was used as packaging cells. The package plasmid pMD2-G (addgene, Plasmid #12259 pMD2-G), Pspax2 (addgene, asmid #12260 Pspax2) and PEI transfection Reagent (sigma, # GF95977287) were mixed with the control vector Plasmid Flag and target gene vector Plasmid ASF1B for 15 min, respectively. The virus was collected after 48 h, centrifuged at 12000 rpm for 3 min, and then collected through a 0.45 mm pore filter. The ASF1B shRNA lentivirus were produced as the procedure abovementioned in supplemental material. The viruses collected above were added to 786-O and Caki-1 cell
2.9. Transwell migration assay The 786-O cells were resuspended and plated at a density of 3 104 cells/well in the upper compartment of a Transwell chamber (Corning, REF:3421). The cells were cultured for 2 h and were washed twice with PBS. Next, 4% paraformaldehyde solution was used to fix the cells at room temperature for 15 min, which was followed by staining with 600 ml 0.1% crystal violet for 2 h at 37 C. Images were acquired under a microscope. The same experiment was performed for the Caki-1 cells, which were cultured for 20 h. The number of positively stained cells reflected the cell migration ability. 2.10. Western blot The proteins were extracted from the cells according to standard protocols. Briefly, the protein samples were separated on 12.5% SDS-PAGE and then transferred to a nitrocellulose membrane. We blocked the membrane with 5% nonfat dry milk in TBS-T buffer and incubated the membrane overnight at 4 C with the primary antibody. After the blots were rinsed extensively with TBS-T buffer, they were incubated with the secondary antibodies for 1 h. A Bio-Rad Chemi Doc XRS þ gel imaging system was used to detect the target bands. 2.11. Statistical analysis Statistical differences among more than 2 groups were
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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compared using one-way ANOVA, followed by Bonferroni analysis (for data meeting homogeneity of variance) or Tamhane's T2 analysis (for data demonstrating heteroscedasticity). Statistical differences between two groups were compared with a two-tailed Student's t-test. P < 0.05 was considered significant. 3. Results 3.1. ASF1B is highly expressed in ccRCC and is correlated with tumor stage, grade and patient survival To determine whether ASF1B is related to ccRCC tumorigenesis, we first analyzed information from the database of the TCGA data portal. The results showed that ASF1B expression was significantly higher in ccRCC specimens than in normal tissues (Fig. 1A). Furthermore, an independent analysis of the data from the GEO database (GSE102101) also demonstrated that ASF1B expression was highly expressed in tumor tissue compared with normal tissue
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(Fig. 1B). Then, we performed a subgroup analysis according to the stage and grade of the tumor and found that higher ASF1B expression was closely related to both tumor stage and grade (Fig. 1C and D). As the stage and grade increased, the expression of ASF1B was also gradually increased. In addition, we examined the survival rate of the patients and found that the expression level of ASF1B was closely related to the survival rate of the patients (Fig. 1E). According to these results, ASF1B may be a predictive marker of tumor progression and play an important roles in progress of ccRCC. 3.2. ASF1B overexpression promotes ccRCC cell proliferation Subsequently, we explored the role of ASF1B in ccRCC in vitro. We used two renal cell carcinoma cell lines, 786-O and Caki-1, which exhibit different potencies with respect to invasion and metastasis. The overexpressed stable cell lines were generated using a lentivirus, and the successful overexpression of ASF1B in
Fig. 1. ASF1B is closely related to the occurrence and development of ccRCC. (A,B)The mRNA expression of ASF1B in normal kidney tissue and ccRCC according to the gene sequencing results from the TCGA data analysis and GEO data analysis (GSE102101). (C) ASF1B mRNA expression in ccRCC tissues at different tumor stages. (D) ASF1B mRNA expression in ccRCC tissues of different tumor grades. (E) Kaplan-Meier curve for ccRCC patients with low and high expression of ASF1B.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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these two cell lines was confirmed by qPCR and Western blot (Fig. 2AeD). A CCK8 assay was then performed to evaluate cell viability. As shown in Fig. 2E and F, ASF1B overexpression significantly promoted the viability of the two cell lines compared with the negative control. To further investigate the direct effect of ASF1B on the proliferation of ccRCC cells, we added BrdU to ASF1B-
overexpressing and control cells to label proliferating cells. The results showed that ASF1B overexpression significantly increased the uptake of BrdU in both cell lines, which indicates that ASF1B promotes the proliferation of ccRCC cells (Fig. 2G and H). PCNA and P27 play an important role in tumorigenesis. PCNA is considered a pro-proliferative marker, while P27 is considered an anti-
Fig. 2. ASF1B overexpression promotes the proliferation of ccRCC cells. (A-D) mRNA and WB result of ASF1B in ctrl and OE groups of 786-O cells(A,B) and Caki-1 cell(C,D). (E,F) The relative proliferation of 786-O cells and Caki-1 in the ctrl and OE groups. (G,H) The OD370 value in the ctrl and OE groups of 786-O cells and Caki-1 cells. (I,J) The correlation between ASF1B expression and the pro-proliferative marker PCNA and anti-proliferative marker P27 in the TCGA KIRC was determined using the Pearson correlation coefficient. (K,L) Western blot analysis of PCNA and P27 expression in the ctrl and OE groups of 786-O cells and Caki-1 cells. *p < 0.05, **<0.01.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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proliferative marker [11,12]. Based on the correlation analysis between the expression of ASF1B mRNA and these two genes in the TCGA data portal, we found that ASF1B expression was positively correlated with PCNA and negatively correlated with P27 expression (Fig. 2I and J). Furthermore, we examined the expression of PCNA and P27 in ASF1B-overexpressing and control cells. We found that ASF1B overexpression led to an increased expression of PCNA protein, whereas the expression of P27 protein was decreased in both cells (Fig. 2K and L). Taken together, these results demonstrate that ASF1B promotes the proliferation of ccRCC.
3.3. ASF1B overexpression promotes cell migration We then performed a series of assays to explore whether ASF1B overexpression affects ccRCC cell migration. Wound healing assays revealed that ASF1B overexpression accelerated migration of both 786-O and Caki-1 cells (Fig. 3A and B). Given that these two types of tumor cells have different migration capacities, ASF1B overexpression promoted the enhancement of 786-O cell migration in the early stage (8.5 h, 12 h), while ASF1B overexpression in the late stage of culture (20 h, 40 h) still increased the migration capacity of Caki-1 cells. To further confirm the effect of ASF1B on the migration of tumor cells, we also used a transwell experiment to evaluate cell migration. Consistent with the abovementioned results, ASF1B overexpression significantly promoted cell migration (Fig. 3C and D). At the molecular level, MMP2 and MMP2 are classical markers of cell migration in tumor [13]. We also found that ASF1B overexpression upregulated the expression of MMP2 and MMP9 in both cell lines (Fig. 3E and F). Collectively, these results confirm the promoting effect of ASF1B on the migration of ccRCC cells.
3.3.1. Silencing of ASF1B inhibit cell proliferation and migration To further validate the function of ASF1B deficiency in ccRCC cell, the ASF1B shRNA lentivirus was constructed to infect the Caki1 cell and 786-O cell. The two shRNA vectors were successfully to silence ASF1B expression in 786-O cell and Caki-1 cell (Supplementary Figs. 1A and 1D). CCK8 and Transwell experiments were employed to detect the effect of ASF1B deficiency on cell proliferation and migration. The result showed that ASF1B silencing significantly inhibits the 786-O cell and Caki-1 cell proliferation and migration (Supplementary 1B,C,E,F). Collectively, these results proved the function of ASF1B in the development of ccRCC from the reverse side.
3.4. ASF1B promotes cancer cell growth and migration through AKT/P70 S6K1 signaling pathway The PI3K/AKT signal transduction pathway plays a crucial role in the occurrence and development of malignant tumors [14]. To decipher the potential underlying mechanism by which ASF1B regulates tumor cell growth, we detected activation of the PI3K/ AKT signaling pathway in ASF1B-overexpressing and control cells. The western blot results showed that ASF1B overexpression increased the level of phosphorylated p70 S6K1 and AKT compared with control cells in both cell lines (Fig. 4A and B). More importantly, when AKT inhibitors were added to the overexpressing cells of ASF1B, the ability of ASF1B to promote proliferation and migration was significantly reduced, which suggested that ASF1B regulated proliferation and migration in ccRCC through AKT signaling pathway (Fig. 4CeF). All these results demonstrate that the AKT signaling pathway is involved in the function of ASF1B in the regulation of ccRCC cell proliferation and migration.
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4. Discussion ccRCC is the most common subtype of renal cell carcinoma [1]. As of now, the molecular mechanism of ccRCC has been closely related to apoptosis, autophagy, inflammation, hypoxia, metabolism and immune imbalance [15e18]. Based on these findings, many new drugs and methods to treat renal cancer have been proposed [19,20]. However, the long-term survival of metastatic renal carcinoma is not high, and the pathogenesis of mechanism of tumor metastasis have not been fully elucidated. Chromatin structures are maintained through the assembly and disassembly of nucleosomes, which is catalyzed by histone chaperones. When chromatin is not regulated appropriately due to dysfunction of chromatin remodeling factors, many diseases occur, including several types of cancers [21,22]. Facilitates Chromatin Transcription (FACT) is involved in DNA repair, replication and transcription by eviction of histones or loosening their contact with DNA, while the previous study showed that FACT can promotes proliferative switch of G0 cancer cells [23]. ASF1 is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation. ASF1 regulates chromatin functions and has been demonstrated to contribute to tumorigenesis [24]. The previous study showed that ASF1a overexpression is associated with increased proliferation of cancer cells, whereas ASF1a inhibition induces DNA damage and subsequent up-regulation of p53-p21cip1 expression, thereby triggering cellular senescence [25]. In this study, we explored tumor markers based on information from the TCGA data portal and GEO database. The results showed that ASF1B was highly expressed in tumor tissues and was correlated with clinical stage, pathological degree and patient overall survival (Fig. 1). Abnormal cell proliferation is an important marker of tumor development. CCK-8 assay and BrdU incorporation detection are the conventional proliferation assays to evaluate cell proliferation. PCNA and p27 can often serve as an indicator to evaluate cell proliferation status [26,27]. According to these methods, our results showed that ASF1B overexpression promoted ccRCC cell proliferation and was positively correlated with PCNA and negatively correlated with P27 expression in tumor cell lines (Fig. 2). These findings indicate that ASF1B may be a new marker that can predict ccRCC progression. A previous study also demonstrated that ASF1B markedly enhances the proliferation of breast cancer [9], which is consistent with our result. The migration potential of ccRCC cell is the main factor that leads to the decrease in the survival rate of metastasis patients [28]. Using a wound healing assay and Transwell chamber experiments, we also demonstrated that ASF1B overexpression promoted tumor cell migration (Fig. 3AeD). MMP2 and MM9 are two typical representatives of the MMP family and are closely related to the invasion and metastasis of many tumors [13]. The results showed that overexpression ASF1B also serve as a reminder that ccRCC metastasis is related to MMP2 and MMP9 expression (Fig. 3E and F). In addition, silencing ASF1B expression can inhibit the proliferation and migration ccRCC cells (Supplementary Fig. 1). In addition, when cells are stimulated by extracellular signals, PI3K activates AKT, which then activates its downstream factor mTOR and p70S6K1. Previous studies of ASF1B has shown that the knockout of ASF1B could affect the apoptosis of prostate cancer cells by inhibiting the PI3K/AKT pathway [8]. In this experiment, we detected the protein activity of AKT and its downstream p70S6K1, and found that ASF1B overexpression could promote the activation of AKT pathway (Fig. 4A and B), which was consistent with the mechanism of action in prostate cancer. More importantly, when AKT inhibitors were added to the overexpressed cells of ASF1B, the
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Fig. 3. ASF1B overexpression promotes ccRCC cell migration. (A,B) Wound healing assay result for 786-O cells (A) and Caki-1 cells (B). The histogram shows cell mobility. (C,D) Image of Transwell assay result for 786-O cells (C) and Caki-1 cells (D). The histogram shows the number of migrated cells. (E,F) MMP2 and MMP9 protein expression in ctrl and OE groups of 786-O cells (E) and Caki-1 cells (F). **p < 0.01.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Fig. 4. AKT/P70 S6K1 signaling pathway is involved in the promotional effect of ASF1B on proliferation and migration of ccRCC. (AeB) Western blot analysis of AKT/P70 S6K1 signaling pathway molecules (AKT, p-AKT, p70, p-p70) in ctrl and OE groups of 786-O cells (A) and Caki-1 cells (B). **p < 0.01. (C-D) CCK8 results showed the relative proliferation of 786-O cells (C) and Caki-1 cells (D) in the ctrl and OE groups with or without AKT inhibitor MK-2206.*p < 0.05,**p < 0.01 vs. the ctrl group; ##p < 0.05 vs. the OE group. (E-F) Transwell experiments showed the migration capacity of 786-O cells (E) and Caki-1 cells (F) in the ctrl and OE groups with or without AKT inhibitor MK-2206. **p < 0.01.
ability of ASF1B to promote proliferation and migration was significantly reduced (Fig. 4CeF). Therefore, ASF1B can affect cell proliferation and migration through the AKT pathway. In conclusion, our results demonstrated that ASF1B was highly
expressed in ccRCC and ASF1B significantly induced cell proliferation and increased tumor cell migration. AKT/P70 S6K1 is the key regulatory pathway.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Disclosures The authors have no conflicts of interest.
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Acknowledgements
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This study was supported by the National Natural Science Foundation of China (No. 81870067, No. 81400753).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.060.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.060
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Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma Zhou Jiangqiao, Qiu Tao*, Chen Zhongbao, Ma Xiaoxiong, Zhang Long, Zou Jilin, Wang Tianyu Department of Organ Transplantation, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, 430060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 February 2019 Accepted 11 February 2019 Available online xxx
Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies. Anti-silencing function 1B histone chaperone (ASF1B) has been reported to be involved in various diseases. However, its role in ccRCC is largely unknown. In the present study, using genetic data and clinical information obtained from the TCGA data portal and GEO database, we found that ASF1B was highly expressed in ccRCC cancer tissue compared with normal tissue, and ASF1B expression was positively correlated with tumor stage, tumor grade and patient survival. The function of ASF1B in cell proliferation and migration was assessed by pathological and molecular analyses. The results showed that ASF1B overexpression significantly enhanced the proliferation and migration of 786-O cells and Caki-1 cells, while silencing ASF1B expression significantly inhibited the proliferation and migration. In addition, ASF1B overexpression enhanced cell proliferation by upregulating PCNA and downregulating P27 expression and promoted cell migration by upregulating MMP2 and MMP9. Furthermore, the phosphorylation levels of protein kinase B (AKT) and P-P70 S6K1 were significantly upregulated in the ASF1B overexpression group. More importantly, AKT inhibitor blocked the promotional effect of ASF1B on proliferation and migration. In summary, the present study demonstrated that ASF1B overexpression promoted tumor cell proliferation and migration, which was dependent on the AKT/P70 S6K1 pathway. © 2019 Elsevier Inc. All rights reserved.
Keywords: ASF1B Clear cell renal cell cancer Proliferation AKT
1. Introduction Worldwide, renal cell cancer is the ninth most common cancer [1]. Histologically, they are defined by clear cytoplasm, with nested clusters of cells surrounded by a dense endothelial network [2]. According to the guidelines, ccRCC can be effectively treated and even cured through surgery and targeted therapy [3], but over 90% of cancer-related deaths in individuals with ccRCC are caused by tumor relapse and metastasis [4]. With the development of bioinformatics, many more biomarkers have been found to predict the
Abbreviations: ccRCC, clear cell renal cell carcinoma; ASF1B, anti-silencing function 1B histone chaperone; AKT, protein kinase B; VHL, von Hippel-Lindau; OE, overexpression; PCNA, proliferating cell nuclear antigen. * Corresponding author.Department of Organ Transplantation, Renmin Hospital of Wuhan University, Wuhan University, ZiYang Road 99#, Wuhan, 430060, China. E-mail address: [email protected] (Q. Tao).
occurrence and development of ccRCC so that early intervention can be achieved [5]. Several novel recurrent mutations in histone modifying and chromatin remodeling genes have been identified in ccRCC. BAP1, PBRM1 and SETD2 could be molecular diagnostic markers and possible targets for personalized therapies in ccRCC [6]. Anti-silencing function 1 (ASF1) is a histone H3-H4 chaperone involved in DNA replication and repair as well as transcriptional regulation [7]. ASF1 regulates chromatin function and has been demonstrated to contribute to tumorigenesis [8]. ASF1 contains two subtypes, including ASF1B and ASF1A, which share a 70% sequence identity, but they are not functionally equivalent. Studies have shown that ASF1B is closely related to the development and progression of many tumors, including cervical cancer, breast cancer and prostate cancer [9,10]. However, the role and mechanism of ASF1B in RCC remains unclear. Therefore, we attempted to explore the role and mechanism of ASF1B in ccRCC.
https://doi.org/10.1016/j.bbrc.2019.02.060 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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2. Materials and methods 2.1. TCGA data analysis ccRCC mRNA-seq level3 data samples and clinical information were downloaded from the TCGA data portal. The R-packet DESeq2, which is based on the principle of negative binomial distribution, was used to standardize the expression quantity data and to analyze the differentially expressed genes between cancer samples and normal samples. The standardized data was used to visualize the expression level of ASF1B in ccRCC and adjacent noncancer tissues, and for grade classification, stage and survival correlation analysis. Clinical samples of ccRCC were divided into 2 groups according to the expression level of ASF1B. Then, the log-rank test algorithm was used to generate the survival curve, and the followup data obtained from the ccRCC clinical information were visualized using R package survival analysis. Correspondingly, the correlation between ASF1B expression and tumor grade and stage was calculated by T-test, and the data were visualized using R package ggplot2. The correlation between the expression of ASF1B mRNA and 2 genes (PCNA, P27) related to proliferation in the TCGA KIRC was determined using the Pearson correlation coefficient.
culture dishes for target cell infection. After cell passage, the cells were screened with 4 ml/mL puromycin for both cells at indicated time. After screening, ASF1B expression was detected. 2.6. CCK8 assay Cell viability was examined using a CCK-8 kit according to the manufacturer's protocol. 786-O and Caki-1 cells were inoculated in 96-well plates at a density of 3000 cells/well. After the cells adhered to the wells, 10 ml CCK8 reagent (Dojindo, 44786) and 90 ml DMEM were added to the cells. The cells were further cultured for 0 h, 24 h, 48 h, 72 h, 96 h or 120 h, respectively. Then, the absorbance at 450 nm was measured at the indicated time. 2.7. BrdU experiment 786-O and Caki-1 cells were inoculated in 96-well plates at a density of 3000 cells per well. After 24 h and 48 h, BrdU solution (Roche, Cat. No. 11647229001) was added followed by a 6 h incubation. The standard procedure was performed according to the kit's instructions. Finally, the absorbance was measured at a wavelength of 370 nm.
2.2. GEO data analysis
2.8. Wound healing test
Data on 10 paired tumor tissue samples were downloaded from the GSE102101, a GEO database, and sequence reads were mapped to the human genome (hg38) using hisat2. Transcripts were quantified using stringtie according to the RefSeq annotation and were normalized; differential expression analysis was performed using DESeq2.
The 786-O and Caki-1 cells were inoculated into 6-well plates at a density of 3.5 105 cells per well (Thermo, Cat. No. 140675). After the cells were incubated overnight, confluent monolayers for wounding were yielded. The original culture medium was replaced with DMEM supplemented with mitomycin. Then, the cells were cultured for 12 h. Wounds were generated using a pipette tip, and photographs were immediately obtained (time zero) and at the indicated times for 786-O cells and Caki-1 cells. The migration index was then measured according to the following formula: migration index ¼ (width of the wound at 0 h-width of the wound at 24 or 48 h) *100/width of the wound at time zero.
2.3. Cells and reagents All the reagents information is in Supplementary Table 1. The human ccRCC cell line 786-O and Caki-1 cell line were cultured in standard procedure at 37 C in a humidified atmosphere containing 5% CO2. 2.4. Lentivirus construction The overexpression vector of the ASF1B gene with flag tag was designed and constructed. The pHAGE-3xFlag carrier was used as the carrier vector. The knockdown vector of the ASF1B gene was designed and constructed based on the pLKO.1 carrier vector. Primers were designed via software on the NCBI website (Supplementary Table 2). After the amplification of the target gene fragment by over-nested PCR, the vector was digested and purified. The target gene fragment was ligated to the vector to obtain the recombinant vector, which was then transferred into Escherichia coli. Finally, the positive clones were screened for expansion culture. 2.5. Lentivirus packaging and target cell infection The 293T cell line was used as packaging cells. The package plasmid pMD2-G (addgene, Plasmid #12259 pMD2-G), Pspax2 (addgene, asmid #12260 Pspax2) and PEI transfection Reagent (sigma, # GF95977287) were mixed with the control vector Plasmid Flag and target gene vector Plasmid ASF1B for 15 min, respectively. The virus was collected after 48 h, centrifuged at 12000 rpm for 3 min, and then collected through a 0.45 mm pore filter. The ASF1B shRNA lentivirus were produced as the procedure abovementioned in supplemental material. The viruses collected above were added to 786-O and Caki-1 cell
2.9. Transwell migration assay The 786-O cells were resuspended and plated at a density of 3 104 cells/well in the upper compartment of a Transwell chamber (Corning, REF:3421). The cells were cultured for 2 h and were washed twice with PBS. Next, 4% paraformaldehyde solution was used to fix the cells at room temperature for 15 min, which was followed by staining with 600 ml 0.1% crystal violet for 2 h at 37 C. Images were acquired under a microscope. The same experiment was performed for the Caki-1 cells, which were cultured for 20 h. The number of positively stained cells reflected the cell migration ability. 2.10. Western blot The proteins were extracted from the cells according to standard protocols. Briefly, the protein samples were separated on 12.5% SDS-PAGE and then transferred to a nitrocellulose membrane. We blocked the membrane with 5% nonfat dry milk in TBS-T buffer and incubated the membrane overnight at 4 C with the primary antibody. After the blots were rinsed extensively with TBS-T buffer, they were incubated with the secondary antibodies for 1 h. A Bio-Rad Chemi Doc XRS þ gel imaging system was used to detect the target bands. 2.11. Statistical analysis Statistical differences among more than 2 groups were
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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compared using one-way ANOVA, followed by Bonferroni analysis (for data meeting homogeneity of variance) or Tamhane's T2 analysis (for data demonstrating heteroscedasticity). Statistical differences between two groups were compared with a two-tailed Student's t-test. P < 0.05 was considered significant. 3. Results 3.1. ASF1B is highly expressed in ccRCC and is correlated with tumor stage, grade and patient survival To determine whether ASF1B is related to ccRCC tumorigenesis, we first analyzed information from the database of the TCGA data portal. The results showed that ASF1B expression was significantly higher in ccRCC specimens than in normal tissues (Fig. 1A). Furthermore, an independent analysis of the data from the GEO database (GSE102101) also demonstrated that ASF1B expression was highly expressed in tumor tissue compared with normal tissue
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(Fig. 1B). Then, we performed a subgroup analysis according to the stage and grade of the tumor and found that higher ASF1B expression was closely related to both tumor stage and grade (Fig. 1C and D). As the stage and grade increased, the expression of ASF1B was also gradually increased. In addition, we examined the survival rate of the patients and found that the expression level of ASF1B was closely related to the survival rate of the patients (Fig. 1E). According to these results, ASF1B may be a predictive marker of tumor progression and play an important roles in progress of ccRCC. 3.2. ASF1B overexpression promotes ccRCC cell proliferation Subsequently, we explored the role of ASF1B in ccRCC in vitro. We used two renal cell carcinoma cell lines, 786-O and Caki-1, which exhibit different potencies with respect to invasion and metastasis. The overexpressed stable cell lines were generated using a lentivirus, and the successful overexpression of ASF1B in
Fig. 1. ASF1B is closely related to the occurrence and development of ccRCC. (A,B)The mRNA expression of ASF1B in normal kidney tissue and ccRCC according to the gene sequencing results from the TCGA data analysis and GEO data analysis (GSE102101). (C) ASF1B mRNA expression in ccRCC tissues at different tumor stages. (D) ASF1B mRNA expression in ccRCC tissues of different tumor grades. (E) Kaplan-Meier curve for ccRCC patients with low and high expression of ASF1B.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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these two cell lines was confirmed by qPCR and Western blot (Fig. 2AeD). A CCK8 assay was then performed to evaluate cell viability. As shown in Fig. 2E and F, ASF1B overexpression significantly promoted the viability of the two cell lines compared with the negative control. To further investigate the direct effect of ASF1B on the proliferation of ccRCC cells, we added BrdU to ASF1B-
overexpressing and control cells to label proliferating cells. The results showed that ASF1B overexpression significantly increased the uptake of BrdU in both cell lines, which indicates that ASF1B promotes the proliferation of ccRCC cells (Fig. 2G and H). PCNA and P27 play an important role in tumorigenesis. PCNA is considered a pro-proliferative marker, while P27 is considered an anti-
Fig. 2. ASF1B overexpression promotes the proliferation of ccRCC cells. (A-D) mRNA and WB result of ASF1B in ctrl and OE groups of 786-O cells(A,B) and Caki-1 cell(C,D). (E,F) The relative proliferation of 786-O cells and Caki-1 in the ctrl and OE groups. (G,H) The OD370 value in the ctrl and OE groups of 786-O cells and Caki-1 cells. (I,J) The correlation between ASF1B expression and the pro-proliferative marker PCNA and anti-proliferative marker P27 in the TCGA KIRC was determined using the Pearson correlation coefficient. (K,L) Western blot analysis of PCNA and P27 expression in the ctrl and OE groups of 786-O cells and Caki-1 cells. *p < 0.05, **<0.01.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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proliferative marker [11,12]. Based on the correlation analysis between the expression of ASF1B mRNA and these two genes in the TCGA data portal, we found that ASF1B expression was positively correlated with PCNA and negatively correlated with P27 expression (Fig. 2I and J). Furthermore, we examined the expression of PCNA and P27 in ASF1B-overexpressing and control cells. We found that ASF1B overexpression led to an increased expression of PCNA protein, whereas the expression of P27 protein was decreased in both cells (Fig. 2K and L). Taken together, these results demonstrate that ASF1B promotes the proliferation of ccRCC.
3.3. ASF1B overexpression promotes cell migration We then performed a series of assays to explore whether ASF1B overexpression affects ccRCC cell migration. Wound healing assays revealed that ASF1B overexpression accelerated migration of both 786-O and Caki-1 cells (Fig. 3A and B). Given that these two types of tumor cells have different migration capacities, ASF1B overexpression promoted the enhancement of 786-O cell migration in the early stage (8.5 h, 12 h), while ASF1B overexpression in the late stage of culture (20 h, 40 h) still increased the migration capacity of Caki-1 cells. To further confirm the effect of ASF1B on the migration of tumor cells, we also used a transwell experiment to evaluate cell migration. Consistent with the abovementioned results, ASF1B overexpression significantly promoted cell migration (Fig. 3C and D). At the molecular level, MMP2 and MMP2 are classical markers of cell migration in tumor [13]. We also found that ASF1B overexpression upregulated the expression of MMP2 and MMP9 in both cell lines (Fig. 3E and F). Collectively, these results confirm the promoting effect of ASF1B on the migration of ccRCC cells.
3.3.1. Silencing of ASF1B inhibit cell proliferation and migration To further validate the function of ASF1B deficiency in ccRCC cell, the ASF1B shRNA lentivirus was constructed to infect the Caki1 cell and 786-O cell. The two shRNA vectors were successfully to silence ASF1B expression in 786-O cell and Caki-1 cell (Supplementary Figs. 1A and 1D). CCK8 and Transwell experiments were employed to detect the effect of ASF1B deficiency on cell proliferation and migration. The result showed that ASF1B silencing significantly inhibits the 786-O cell and Caki-1 cell proliferation and migration (Supplementary 1B,C,E,F). Collectively, these results proved the function of ASF1B in the development of ccRCC from the reverse side.
3.4. ASF1B promotes cancer cell growth and migration through AKT/P70 S6K1 signaling pathway The PI3K/AKT signal transduction pathway plays a crucial role in the occurrence and development of malignant tumors [14]. To decipher the potential underlying mechanism by which ASF1B regulates tumor cell growth, we detected activation of the PI3K/ AKT signaling pathway in ASF1B-overexpressing and control cells. The western blot results showed that ASF1B overexpression increased the level of phosphorylated p70 S6K1 and AKT compared with control cells in both cell lines (Fig. 4A and B). More importantly, when AKT inhibitors were added to the overexpressing cells of ASF1B, the ability of ASF1B to promote proliferation and migration was significantly reduced, which suggested that ASF1B regulated proliferation and migration in ccRCC through AKT signaling pathway (Fig. 4CeF). All these results demonstrate that the AKT signaling pathway is involved in the function of ASF1B in the regulation of ccRCC cell proliferation and migration.
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4. Discussion ccRCC is the most common subtype of renal cell carcinoma [1]. As of now, the molecular mechanism of ccRCC has been closely related to apoptosis, autophagy, inflammation, hypoxia, metabolism and immune imbalance [15e18]. Based on these findings, many new drugs and methods to treat renal cancer have been proposed [19,20]. However, the long-term survival of metastatic renal carcinoma is not high, and the pathogenesis of mechanism of tumor metastasis have not been fully elucidated. Chromatin structures are maintained through the assembly and disassembly of nucleosomes, which is catalyzed by histone chaperones. When chromatin is not regulated appropriately due to dysfunction of chromatin remodeling factors, many diseases occur, including several types of cancers [21,22]. Facilitates Chromatin Transcription (FACT) is involved in DNA repair, replication and transcription by eviction of histones or loosening their contact with DNA, while the previous study showed that FACT can promotes proliferative switch of G0 cancer cells [23]. ASF1 is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation. ASF1 regulates chromatin functions and has been demonstrated to contribute to tumorigenesis [24]. The previous study showed that ASF1a overexpression is associated with increased proliferation of cancer cells, whereas ASF1a inhibition induces DNA damage and subsequent up-regulation of p53-p21cip1 expression, thereby triggering cellular senescence [25]. In this study, we explored tumor markers based on information from the TCGA data portal and GEO database. The results showed that ASF1B was highly expressed in tumor tissues and was correlated with clinical stage, pathological degree and patient overall survival (Fig. 1). Abnormal cell proliferation is an important marker of tumor development. CCK-8 assay and BrdU incorporation detection are the conventional proliferation assays to evaluate cell proliferation. PCNA and p27 can often serve as an indicator to evaluate cell proliferation status [26,27]. According to these methods, our results showed that ASF1B overexpression promoted ccRCC cell proliferation and was positively correlated with PCNA and negatively correlated with P27 expression in tumor cell lines (Fig. 2). These findings indicate that ASF1B may be a new marker that can predict ccRCC progression. A previous study also demonstrated that ASF1B markedly enhances the proliferation of breast cancer [9], which is consistent with our result. The migration potential of ccRCC cell is the main factor that leads to the decrease in the survival rate of metastasis patients [28]. Using a wound healing assay and Transwell chamber experiments, we also demonstrated that ASF1B overexpression promoted tumor cell migration (Fig. 3AeD). MMP2 and MM9 are two typical representatives of the MMP family and are closely related to the invasion and metastasis of many tumors [13]. The results showed that overexpression ASF1B also serve as a reminder that ccRCC metastasis is related to MMP2 and MMP9 expression (Fig. 3E and F). In addition, silencing ASF1B expression can inhibit the proliferation and migration ccRCC cells (Supplementary Fig. 1). In addition, when cells are stimulated by extracellular signals, PI3K activates AKT, which then activates its downstream factor mTOR and p70S6K1. Previous studies of ASF1B has shown that the knockout of ASF1B could affect the apoptosis of prostate cancer cells by inhibiting the PI3K/AKT pathway [8]. In this experiment, we detected the protein activity of AKT and its downstream p70S6K1, and found that ASF1B overexpression could promote the activation of AKT pathway (Fig. 4A and B), which was consistent with the mechanism of action in prostate cancer. More importantly, when AKT inhibitors were added to the overexpressed cells of ASF1B, the
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Fig. 3. ASF1B overexpression promotes ccRCC cell migration. (A,B) Wound healing assay result for 786-O cells (A) and Caki-1 cells (B). The histogram shows cell mobility. (C,D) Image of Transwell assay result for 786-O cells (C) and Caki-1 cells (D). The histogram shows the number of migrated cells. (E,F) MMP2 and MMP9 protein expression in ctrl and OE groups of 786-O cells (E) and Caki-1 cells (F). **p < 0.01.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Fig. 4. AKT/P70 S6K1 signaling pathway is involved in the promotional effect of ASF1B on proliferation and migration of ccRCC. (AeB) Western blot analysis of AKT/P70 S6K1 signaling pathway molecules (AKT, p-AKT, p70, p-p70) in ctrl and OE groups of 786-O cells (A) and Caki-1 cells (B). **p < 0.01. (C-D) CCK8 results showed the relative proliferation of 786-O cells (C) and Caki-1 cells (D) in the ctrl and OE groups with or without AKT inhibitor MK-2206.*p < 0.05,**p < 0.01 vs. the ctrl group; ##p < 0.05 vs. the OE group. (E-F) Transwell experiments showed the migration capacity of 786-O cells (E) and Caki-1 cells (F) in the ctrl and OE groups with or without AKT inhibitor MK-2206. **p < 0.01.
ability of ASF1B to promote proliferation and migration was significantly reduced (Fig. 4CeF). Therefore, ASF1B can affect cell proliferation and migration through the AKT pathway. In conclusion, our results demonstrated that ASF1B was highly
expressed in ccRCC and ASF1B significantly induced cell proliferation and increased tumor cell migration. AKT/P70 S6K1 is the key regulatory pathway.
Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060
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Disclosures The authors have no conflicts of interest.
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Acknowledgements
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This study was supported by the National Natural Science Foundation of China (No. 81870067, No. 81400753).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.060.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.02.060
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Please cite this article as: Z. Jiangqiao et al., Anti-silencing function 1B histone chaperone promotes cell proliferation and migration via activation of the AKT pathway in clear cell renal cell carcinoma, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.02.060