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Transcription factor KLF9 suppresses the growth of hepatocellular carcinoma cells in vivo and positively regulates p53 expression
Cancer Letters 355 (2014) 25–33
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Transcription factor KLF9 suppresses the growth of hepatocellular carcinoma cells in vivo and positively regulates p53 expression Jiabin Sun a, Boshi Wang b, Yun Liu b, Li Zhang b, Aihui Ma b, Zhaojuan Yang b, Yuhua Ji a, Yongzhong Liu b,* a
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China b
A R T I C L E
I N F O
Article history: Received 18 July 2014 Received in revised form 5 September 2014 Accepted 5 September 2014 Keywords: Hepatocellular carcinoma KLF9 Apoptosis p53 protein Transcriptional regulation
A B S T R A C T
Krüppel-like factor 9 (KLF9) is known to be a tumor suppressor gene in colorectal tumors and glioblastoma; however, the functional status and significance of KLF9 in hepatocellular carcinoma (HCC) is unclear. We report here that KLF9 is downregulated in HCC tissues. Restoration of KLF9 significantly inhibited growth and caused apoptosis in SK-Hep1 and HepG2 cells. We found that KLF9 positively regulated p53 levels by directly binding to GC boxes within the proximal region of the p53 promoter. Moreover, in the presence of cycloheximide, KLF9 significantly increased p53 stability in HCC cells. Remarkably, ectopic expression of KLF9 was sufficient to delay the onset of tumors and to promote regression of the established tumors in vivo, suggesting that KLF9 plays a critical role in HCC development and that pharmacological or genetic activation of KLF9 may have potential in the treatment of HCC. © 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Krüppel-like factor 9 (KLF9), previously referred to as basic transcription element binding protein 1 (Bteb1), belongs to the mammalian Sp1/KLF family, which includes critical regulators of key cellular functions such as differentiation, proliferation, and development [1,2]. The members of this family are characterized by the presence of three conserved C2H2 zinc finger motifs in the carboxyl terminus, which recognize the consensus sequence CACCC and related GC-rich elements in promoters and enhancers. The N-terminal domains of these proteins, however, are quite variable and have different biological functions [3]. Based on their functional characteristics, the 17 human KLF proteins can be divided into three distinct groups by phylogenetic analysis of protein sequences. KLF9, along with KLF10, 11, 13, 14, and 16, belongs to subgroup 3, whose members most likely have repressor activity as they can interact with the common transcriptional co-repressor Sin3A [4,5]. As a thyroid hormone-induced immediate early gene, KLF9 participates in neural development in vertebrates [6]. In addition, KLF9 is also involved in the cellular response to progesterone and estrogen receptor activation [7,8] as well as in adipocyte differentiation [9]. Moreover, KLF9 in the human epidermis functions
Abbreviations: KLF, Krüppel-like factor; ZNF, Zinc finger domain; SID, Sin3A interaction domain; DOX, Doxycycline; CHX, Cycloheximide. * Corresponding author. Tel.: +86 21 3420 6283; fax: +86 21 3420 6283. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.canlet.2014.09.022 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.
as a circadian transcription factor that significantly affects keratinocyte proliferation and differentiation [10]. Recently, KLF9 has been reported to be down-regulated in human colorectal tumors, and the reduction of KLF9 expression is associated with tumor initiation and development [11]. Notably, KLF9 has been identified as a relatively unique differentiation-induced transcription factor in glioblastoma-derived neurospheres and can suppress glioblastoma stem cell maintenance [12]. Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide and causes approximately one million deaths annually [13]. Identification of causative genes and molecular pathways may improve strategies for the diagnosis, prevention and treatment of HCC. Specific KLF family members are now known to be involved in regulating the development of HCC. KLF6 status in HCC is controversial. On one hand, low expression of KLF6 mRNA was observed in 73% of HBV-associated HCCs, and reconstituting KLF6 in HepG2 cells decreased proliferation [14]. On the other hand, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression to trigger cell death [15]. KLF4 can induce the expression of the vitamin D receptor (VDR) by binding to its promoter, thereby sensitizing HCC cells to the antiproliferative effects of vitamin D3 (VD3) [16]. Most recently, KLF9 has been demonstrated to inhibit cellular proliferation and mobility and induce apoptosis in HepG2 cells [17]. In this study, we supply further evidence that the ectopic expression of KLF9 induces apoptosis in HCC cells and inhibits tumor growth in vitro and in vivo. Strikingly, the restoration of KLF9 caused significant remission of established tumors in mice. Moreover, the overexpression of KLF9 promoted the activation of p53, which may contribute to the tumor-suppressive function of KLF9.
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Material and methods
Statistical analysis
Tissue specimens and cell lines
Analysis of variance (ANOVA) and Student’s t tests were used to evaluate significant differences and variances. Quantitative data are described as the means ± SD. A P value less than 0.05 was considered significant.
Forty-five pairs of human HCC samples from Qi Dong Liver Cancer Institute in China were processed for RNA analysis. Patient information is listed in Table S1. The use of human material was approved by the institutional ethical review committee. A commercially available tissue microarray (TMA) containing 92 HCC pairs was used for immunohistochemical staining. The SK-Hep1, HepG2, Hep3B, PLC-PRF-5, SUN387, and THLE-2 cell lines were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). The Huh7 cell line was from Riken Cell Bank (Tsukuba Science City, Japan). The SMMC-7721 cell line was obtained from the cell bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The MHCC-97L and LM3 cell lines were provided by Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China). Xenograft model Male BALB/c nude mice (6–8 weeks of age) were injected s.c. with SK-Hep1 cells (5 × 106/0.1 mL saline) transfected with either pTRIPZ-wt-KLF9 (on the left flank) or empty vector (on the right flank) in groups one and two (n = 6 per group), and cells transfected with KLF9-ΔSID (on the left flank) or KLF9-ΔZNF (on the right flank) in group three (n = 6). Mice in group one were given normal water, groups two and three were administered daily doxycycline (DOX, 2 mg/mL) in the water. Tumor volumes were monitored every 2 days. To determine the effect of KLF9 on the growth of established tumors, the mice received single s.c. flank injections of Sk-Hep1 cells (5 × 106/ 0.1 mL saline) with either pTRIPZ-wt-KLF9 (n = 16) or empty vector (n = 16), and tumor growth was monitored. When the volume of the tumors reached 150–200 mm3, each group of mice was randomly divided into two groups (n = 8 per group). One group was given DOX (2 mg/mL) and the other given normal water. Tumor growth was monitored every 5 days. The tumor-bearing mice were sacrificed 40 days after inoculation, and the tumors were removed for further study. All experiments were subject to approval by the Institution of Animal Care and Use Committee (SHCI-11-020). Laboratory methods See Appendix S1 for detailed experimental procedures.
Results KLF9 is downregulated in HCC First, the expression profile for the main subgroup 3 members, KLF9, KLF10, KLF11 and KLF13 were analyzed in 45 pairs of clinical specimens by real-time RT-PCR. The results showed that the mRNA levels of KLF9 and KLF11 were significantly down-regulated in HCC tissues when compared with adjacent noncancerous tissue, whereas there were no significant differences in the levels of KLF10 and KLF13 (Fig. 1A). Moreover, a subgroup of clinical human HCC samples (37%, 33/92) exhibited decreased KLF9 expression, as demonstrated by immunohistochemical staining in 92 pairs of HCC tissues (Fig. 1B). Consistently, we also found that mRNA levels of KLF9 were considerably decreased in human liver cancer cell lines compared with the immortalized liver cell line THLE-2 (Fig. 1C). Overexpression of KLF9 inhibits HCC cell proliferation and induces apoptosis The downregulation of KLF9 in liver cancer cell lines and primary HCC tissues suggests a suppressive function of this gene in HCC development. Therefore, we investigated the effects of ectopic KLF9 expression on two HCC cell lines, SK-Hep1 and HepG2. After 7 days of culture, cell growth and colony formation were significantly attenuated in KLF9-expressing cells (Fig. 2A, 2B and 2C). The expression
Fig. 1. KLF9 is downregulated in HCC tissues and liver cancer cell lines. (A) Scatter plots depicting the transcriptional levels of KLF9, KLF10, KLF11, and KLF13 in HCC tissues (T) and matched non-tumor tissues (N) (n = 45); GAPDH was used as an internal reference. (Black lines: median values, *** P < 0.001.) (B) Representative immunohistochemical staining of HCC samples with anti-KLF9 antibody. N: non-tumor, T: HCC tissue. Original magnification: 200×, 400×. (C) Histograms showing the relative expression of KLF9 in HCC-derived cell lines and the immortalized liver cell line THLE-2; GAPDH was used as an internal reference. The values indicate the means ± SD of three independent experiments.
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Fig. 2. KLF9 is a negative regulator of cell proliferation and induces apoptosis in HCC cells. SK-Hep1 and HepG2 cells transfected with Flag-tagged and Tet-induced KLF9 (pTRIPZ-KLF9) or empty vector were used. (A and B) The expression of exogenous (with Flag-specific antibody) and endogenous KLF9 was measured by western blot after treatment with DOX (0.5 μM) for 24 h. The cell growth rate was then determined using an MTT assay at the indicated time points. The results shown are the means ± SD of three independent experiments. ***P < 0.001. (C) Representative cell culture plates illustrating dense foci formation by the cells treated with or without DOX (0.5 μM) for 7 days. (D) The cells were double-stained with PI and 7-AAD, and cell cycle stages were analyzed by flow cytometry. The charts present 2D results of the cell distribution in the different cell cycle stages. The percentage of cells in G1, S and G2 phases are quantified in the histogram. The values indicate the means ± SD of three independent experiments. ***P < 0.001. (E and F) Cells were treated with DOX (0.5 μM) for the indicated times, and double-stained with Annexin-V and 7-AAD. The scattergrams show representative results after treatment for 48 h. The graph presents the percentage of Annexin-V-positive cells at the indicated time point. Each value is the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05.
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of exogenous KLF9 was marked with a Flag tag, while endogenous KLF9 could not be detected (Fig. 2A and 2B). These cells were further subjected to cell cycle and apoptosis assays. The results demonstrated that KLF9 caused a decrease in S-phase and an increase in G1-phase (Fig. 2D). Notably, the percentage of apoptotic cells was significantly increased from 9.01% to 39.54% (P < 0.01) in KLF9overexpressing SK-Hep1 cells after 48 h of DOX treatment (Fig. 2E). Similarly, the percentage of apoptotic cells was also increased from 8.2% to 13.58% in KLF9-overexpressing HepG2 cells (Fig. 2F). No meaningful differences were observed in the cells expressing empty vector. Collectively, these results suggest that KLF9 plays a significant role in inhibiting growth and promoting apoptosis in HCC cells. The tumor-suppressive function of KLF9 depends on its DNA-binding domain Some works have demonstrated that KLF10 and KLF11 act as transcriptional repressors through the Sin3 interaction domain in their amino-terminal regions, which forms a co-repressive complex with Sin3 proteins [18,19]. To structurally illuminate the contributions of the Sin3 interaction domain (SID) and DNA binding domain (zinc finger domain, ZNF) to the effects of KLF9, SK-Hep1 and HepG2 cells were transfected with KLF9 and the mutants without the SID domain (ΔSID) or ZNF domain (ΔZNF) (Fig. 3A). Compared with the effects of wt-KLF9, KLF9-ΔSID expression also robustly inhibited foci formation and triggered apoptosis, whereas the expression of
KLF9-ΔZNF had no such effects (Fig. 3B and 3C). Remarkably, the results were confirmed by in vivo experiments, in which the expression of wt and KLF9-ΔSID delayed the onset of tumors in mice. However, KLF9-ΔZNF expression had less significant effects, although it did result in subtle growth inhibition compared with the control mice (Fig. 3D). Therefore, these results suggest that the DNA-binding domain (ZNF) of KLF9 is essential for its tumor-suppressive role in HCC cells. KLF9 up-regulates p53 activity and enhances its stability To further explore the related molecular mechanisms underlying the pro-apoptotic effects of KLF9, the expression levels of selected apoptosis-related proteins were examined by western blot. The results demonstrated that overexpression of KLF9 significantly increased protein levels of p53 and cleaved caspase 3 and decreased the levels of β-Catenin and BCL-2 in DOX-treated cells at the indicated time points (Fig. 4A and 4B). Moreover, the expression of p53 at the mRNA level was also enhanced by KLF9 in SK-Hep1 and HepG2 cells (Fig. 4C and 4D). Further confirming the regulation of p53 by KLF9, we observed that expression of NOXA and PUMA, two genes downstream of p53, was also increased by KLF9 (Fig. S1A and S1B). Because ectopic expression of KLF9 dramatically increased protein levels of p53 in SK-Hep1 and HepG2 cells, we next explored whether the increase in p53 protein was attributable to protein stabilization or transcriptional enhancement. When treated with cycloheximide (CHX), the half-life of p53 protein in
Fig. 3. KLF9 inhibits proliferation and induces apoptosis in a ZNF domain-dependent manner both in vitro and in vivo. (A) Schematic representation of the two principal domains of KLF9 and the deletion mutants used [20]. (B) Representative dense foci formation by cells treated with or without DOX (0.5 μM) for 7 days. (C) The number of apoptotic cells was measured by flow cytometry after DOX (0.5 μM) treatment for the indicated time. Each value is the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05. (D) SK-Hep1 cells transfected with pTRIPZ-WT-KLF9, KLF9-ΔSID, KLF9-ΔZNF or empty vector were separately injected subcutaneously into the flanks of individual male BALB/c nude mice. Mice in group one (n = 6) and two (n = 6) were injected with cells containing empty vector (right flank) and WT-KLF9 (left flank); mice in group three (n = 6) were injected with cells containing KLF9-ΔSID (right flank) and KLF9-ΔZNF (left flank). Mice in group one received normal water and those in groups two and three received 2 mg/mL DOX in the drinking water beginning on the second day post-implantation. The tumor volumes were monitored and measured after treatment for 40 days. The scatter plots show the volumes for each group.
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Fig. 4. KLF9 upregulates the expression of pro-apoptotic proteins. (A and B) The protein levels of Flag, β-Catenin, p53, BCL-2, cleaved Caspase 3, and α-Tubulin were determined by western blot at the indicated time points after DOX treatment in SK-Hep1 and HepG2 cells transfected with pTRIPZ-KLF9. (C) (D) The transcriptional level of p53 was examined at the indicated time points following KLF9 induction with DOX in SK-Hep1 and HepG2 cells. The values are the means ± SD of three independent experiments. *P < 0.05.
KLF9-overexpressing cells was significantly increased. The stability of p21 was also enhanced (Fig. 5A and 5B). These results suggest that KLF9 plays a role in the stabilization of p53 protein. We also found that the protein levels of p53 and p21 were not upregulated
by KLF9-ΔZNF (Fig. 5C and 5D). Consistently, KLF9-ΔZNF expression did not cause an increase in the p53 mRNA levels (Fig. 5E and 5F). The results suggest that the upregulation of p53 by KLF9 also occurred at the transcriptional level.
Fig. 5. KLF enhances p53 stability and promotes its activity. (A) (B) SK-Hep1 and HepG2 cells transfected with pTRIPZ-KLF9 or empty vector were treated with or without DOX (0.5 μM) for 24 h. The cells were then exposed to cycloheximide (CHX, 25 μg/mL) for the indicated times. The protein levels of p53, p21, Flag, and α-Tubulin were determined by western blot. (C) (D) The levels of Flag, p53, p21, and α-Tubulin were evaluated by western blot in SK-Hep1 and HepG2 cells transfected with WT or mutant KLF9 or empty vector with or without treatment with DOX (0.5 μM) for 24 h. (E) (F) The level of p53 was determined by RT-PCR in SK-Hep1 and HepG2 cells infected with WT or mutant KLF9 after treatment with DOX (0.5 μM) for 24 h. Values represent the means ± SD of three independent experiments. *P < 0.05.
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KLF9 binds the promoter of p53 and strengthens its transcriptional activity To further evaluate our hypothesis of transcriptional p53 upregulation by KLF9, we utilized luciferase reporter detection. As shown in Fig. 6A, the overexpression of KLF9 significantly increased the activity of the pGL3-p53 promoter in 293T, SK-Hep1 and HepG2 cells. To determine the efficiency of the three GC-box sequences within the p53 promoter, we performed site-specific mutations of these sites (from CACCC to AAAAA) and constructed four p53 promoter mutants: mut1, mut2, mut3, and mut all (Fig. 6B, lower panel). The results revealed that each of the GC-box mutations had deleterious effects on the transcriptional activity of p53. Indeed, the combinatory inactivation of these sites produced a more robust inhibition of luciferase activity than did any of the singlesite mutants (Fig. 6B). Furthermore, a ChIP assay was performed using Flag-specific mouse antibody and control (normal mouse IgG) from SK-Hep1 cells expressing KLF9 or empty vector following DOX treatment for 24 h. As shown in Fig. 6C, a clear enrichment of the proximal but not distal region of the p53 promoter was detected in the chromatin precipitated from cells expressing WT-KLF9. Collectively, our results demonstrate that KLF9 can increase p53 transcriptional activity by interacting with the proximal GC boxes of the p53 promoter. Inducible expression of KLF9 promotes regression of established HCC tumors Considering that KLF9 expression has robust inhibitory effects on the proliferation of HCC cells, we next explored whether KLF9 has therapeutic value in mice bearing established HCC tumors. Our results showed that all mice developed tumors after the subcutaneous inoculation of SK-Hep1 cells expressing empty vector in the
absence of inducible KLF9 expression. In the presence of DOXinduced KLF9 expression, however, established tumors in six of eight mice became entirely undetectable (Fig. 7A and 7B). To evaluate the levels of KLF9 induced in the tumor tissues, we performed immunohistochemical staining at days 0, 5 and 15 after DOX treatment. Marked induction of the KLF9 protein was observed with DOX in vivo and was mainly located in the nuclei of HCC cells. The expression patterns of Ki67, p53, and TUNEL were also detected in parallel. The results showed that at days 5 and 15 post-DOX treatment, the number of Ki67-positive cells was significantly decreased, whereas the number of TUNEL-positive cells was significantly increased in the presence of KLF9 expression (Fig. 7C). In addition, p53 expression was higher in the KLF9-expressing group than in the control group. The histograms present the quantification of positively stained cells (Fig. 7D). Overall, these results provide in vivo evidence that KLF9 expression can significantly attenuate the growth of established tumors by inhibiting proliferation and inducing p53 expression and apoptosis in situ, suggesting that induction of KLF9 may be an effective strategy to eradicate human HCC. Discussion In the present study, KLF9 was downregulated in the majority of HCC specimens, and exogenous KLF9 expression inhibited cell proliferation and induced apoptosis in cultured human HCC cells. A recent report also described an essential role for KLF9 in bortezomib- and LBH589-induced apoptosis in multiple myeloma cells [21]. Consistent with a recent report [17], KLF9 induced apoptosis in HCC cells. The authors of that report explained that programmed cell death protein 5 overexpression simulated the activation of KLF9 in HepG2 cells. In the present study, KLF9 overexpression increased the activation of p53. We have also
Fig. 6. KLF9 increases p53 transcriptional activity in HCC cells. (A) HEK 293T, SK-Hep1 and HepG2 cells were cotransfected with p53 luciferase reporter and pSIN-KLF9 or an empty vector for 48 h. The luciferase activity was subsequently assayed. Firefly-dependent luciferase emission was normalized to the Renilla signal. Values represent the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001. (B) Left, schematic representation of the pGL3-basic-p53 reporter construct, including the three predicted GC-rich KLF9 binding sites (CACCC). The proximal p53 promoter (region −651 to +50 base pairs) was cloned into the pGL3-basic luciferase vector, and the CACCC sites were individually or were combinatorially mutated to AAAAA in the luciferase vector. Hep3B cells (p53−/−) were transfected with p-SIN-KLF9 along with each of the promoter mutants: mutant1 (−273 to −267), mutant2 (−181 to −177), mutant3 (−128 to −124), or mutant all. After 48 h, the luciferase activity was analyzed. Values represent the means ± SD of three independent experiments. **P < 0.01, *P < 0.05. (C) The binding of KLF9 to the p53 promoter was evaluated by ChIP followed by RT-PCR analysis. Sk-Hep-1 cells transfected with KLF9-wt, ΔSID, ΔZNF and vector plasmids were treated with or without DOX for 24 h, then the cells were cross-linked, sonicated and subjected to chromatin immunoprecipitation with anti-Flag antibody beads or agarose-conjugated anti mouse IgG beads at 4 °C overnight. Isolated DNA was employed in quantitative PCR with GAPDH and p53 promoter-specific primers. All PCR signals were normalized to GAPDH-specific PCR signals, and the corresponding PCR signals obtained from reactions of DNA precipitated with IgG antibodies. Values represent the means ± SD of three independent experiments. **P < 0.01, *P < 0.05.
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Fig. 7. KLF9 expression abolishes the growth of established tumors. (A) SK-Hep1 cells transfected with pTRIPZ-WT-KLF9 and empty vector were separately injected subcutaneously into the right flanks of individual male BALB/c nude mice. All mice were provided with normal water until the tumor size reached approximately 150– 200 mm3. The mice from each group were then randomly divided into two groups per cell line. One group was provided with normal water (n = 8), and the other received 2 mg/mL DOX in the drinking water (n = 8). The graph presents the growth of the tumors in the mice at the end of the experiment after treatment for 40 days. (B) The tumor volume was measured every 5 days from day 20 after inoculation until the end of the experiment. (C) The tumors were collected at days 5 and 15 after DOX treatment and analyzed by immunohistochemical staining with anti-KLF9 anti-p53, Ki67 and TUNEL. All photographs are at the same magnification. Original magnification: 400×. (D) Histograms showing the quantification of the numbers of positively stained cells at the indicated day (derived from the experiments described in (C)). For each staining, 10 separate fields at 400× magnification were recorded. Each value represents the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05.
reported that KLF9 induces apoptosis and suppresses the proliferation of prostate cancer cells by inhibiting the activation of AKT [22]. These findings indicate that KLF9 plays critical roles in the development and progression of human cancers. More importantly, we found KLF9 expression not only delayed the onset of tumors but also efficiently promoted the regression of established tumors.
KLF9 belongs to subgroup 3 of the KLF family, members of which are likely to have repressor activity because they contain SID domains that interact with the common transcriptional co-repressor Sin3A. Structurally, KLF family members are highly conserved in their C-termini, and the ZNF domains in this region are usually responsible for the DNA-binding activity or protein-protein interactions.
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Indeed, we found that deletion of the ZNF domain almost entirely attenuated KLF9-mediated transcriptional activation of p53. The N-terminal regions of the KLFs vary considerably and are usually responsible for transcriptional activation or repression [23]. The Sin3interacting-domain (SID) is found in the N-terminal of KLF9. In general, the SID has been characterized as a domain with transcriptional repression activity [20]. We found that the examined activity of SID deletion mutant was comparable with intact KLF9, suggesting that the SID domain-mediated function, if any involved in gene silencing, was dispensable for KLF9-mediated p53 activation and suppression of cell proliferation. Another notable finding of this study is that KLF9 efficiently increases p53 activity. p53 is a very labile protein, with a half-life as short as a few minutes. Defects in the ability to stabilize p53 are likely to contribute to malignancy development, and restoration of this activity represents an extremely attractive possibility for tumor therapy [24,25]. Our results indicate that the protein levels of p53 were clearly increased by KLF9; this increase may be attributable to enhanced stability of p53 at the posttranslational level and increased transcriptional activity. Consistent with the observation that cells with ectopic KLF9 expression displayed an increase in the proportion of apoptotic cells, we observed that the p53 downstream targets p21, Puma and Noxa [26] were significantly activated by KLF9. These proteins have long been appreciated to be the mediators of p53-induced apoptosis. In addition, the pro-survival proteins Bcl-2 was down-regulated in cells expressing exogenous KLF9. In the present study, we also showed the coincidence of p53 activation and β-catenin inhibition in the context of KLF9 induction. Hyperactivation of the WNT/β-catenin signaling contributes to the development and pathogenesis of HCC. Consistently, it has been shown that p53 activation negatively regulates β-catenin expression [27]; and the accumulation of wild-type β-catenin is linked with the inactivation of p53 in HCC cells [28]. Obviously, further exploration of the details of how KLF9 regulates the stabilization of p53 will be helpful in the understanding of the roles of KLF9 in cancer development. Notably, in addition to p53, other pathways contribute to KLF9mediated apoptosis. A recent study [29] has demonstrated that KLF9 is associated with the transcriptional regulation of several genes related to ROS metabolism, and KLF9 thereby facilitates oxidative stress and subsequent cell death. These findings indicate that KLF9 activation may contribute to cell-autonomous limitation to inhibit cell transformation and tumor genesis in hepatocytes, especially in the presence of stress, such as HBV replication, DNA damage, and liver injury. Induction of KLF9 expression may provide a strategy for cancer treatment. KLF9 is required for bortezomib-induced apoptosis in myeloma cells [21]. Interestingly, a phase II trial of bortezomib has been performed to evaluate this drug as a mono-therapy in patients with unresectable HCC [30], and bortezomib can sensitize HCC cells to several cell death-inducing reagents, such as TRAIL, mapatumumab, lexatumumab, and CS-1008 [31,32], suggesting that reagents that induce KLF9 expression may have clinical potential as therapy for HCC patients. In summary, our study suggests that the development of specific activators of KLF9 may be a useful approach for the clinical treatment of HCC. Conflict of interest The authors have no conflict of interest. Acknowledgments This work was supported by grants from The National Science Foundation of China (Grant No. 81201542 and No. 81301716), and Natural Science Foundation of Shanghai (No. 12ZR1430100). Key
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Liu, The fate of Kruppel-like factor 9-positive hepatic carcinoma cells may be determined by the programmed cell death protein 5, Int. J. Oncol. 44 (2014) 153–160. [18] V. Ellenrieder, A. Buck, A. Harth, K. Jungert, M. Buchholz, G. Adler, et al., KLF11 mediates a critical mechanism in TGF-beta signaling that is inactivated by Erk-MAPK in pancreatic cancer cells, Gastroenterology 127 (2004) 607–620. [19] Y. Xiong, P.A. Svingen, O.O. Sarmento, T.C. Smyrk, M. Dave, S. Khanna, et al., Differential coupling of KLF10 to Sin3-HDAC and PCAF regulates the inducibility of the FOXP3 gene, Am. J. Physiol. Regul. Integr. Comp. Physiol. 307 (2014) R608–R620. [20] J.S. Zhang, M.C. Moncrieffe, J. Kaczynski, V. Ellenrieder, F.G. Prendergast, R. Urrutia, A conserved alpha-helical motif mediates the interaction of Sp1-like transcriptional repressors with the corepressor mSin3A, Mol. Cell. Biol. 21 (2001) 5041–5049. [21] S. Mannava, D. Zhuang, J.R. Nair, R. Bansal, J.A. Wawrzyniak, S.N. 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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Transcription factor KLF9 suppresses the growth of hepatocellular carcinoma cells in vivo and positively regulates p53 expression Jiabin Sun a, Boshi Wang b, Yun Liu b, Li Zhang b, Aihui Ma b, Zhaojuan Yang b, Yuhua Ji a, Yongzhong Liu b,* a
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China b
A R T I C L E
I N F O
Article history: Received 18 July 2014 Received in revised form 5 September 2014 Accepted 5 September 2014 Keywords: Hepatocellular carcinoma KLF9 Apoptosis p53 protein Transcriptional regulation
A B S T R A C T
Krüppel-like factor 9 (KLF9) is known to be a tumor suppressor gene in colorectal tumors and glioblastoma; however, the functional status and significance of KLF9 in hepatocellular carcinoma (HCC) is unclear. We report here that KLF9 is downregulated in HCC tissues. Restoration of KLF9 significantly inhibited growth and caused apoptosis in SK-Hep1 and HepG2 cells. We found that KLF9 positively regulated p53 levels by directly binding to GC boxes within the proximal region of the p53 promoter. Moreover, in the presence of cycloheximide, KLF9 significantly increased p53 stability in HCC cells. Remarkably, ectopic expression of KLF9 was sufficient to delay the onset of tumors and to promote regression of the established tumors in vivo, suggesting that KLF9 plays a critical role in HCC development and that pharmacological or genetic activation of KLF9 may have potential in the treatment of HCC. © 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction Krüppel-like factor 9 (KLF9), previously referred to as basic transcription element binding protein 1 (Bteb1), belongs to the mammalian Sp1/KLF family, which includes critical regulators of key cellular functions such as differentiation, proliferation, and development [1,2]. The members of this family are characterized by the presence of three conserved C2H2 zinc finger motifs in the carboxyl terminus, which recognize the consensus sequence CACCC and related GC-rich elements in promoters and enhancers. The N-terminal domains of these proteins, however, are quite variable and have different biological functions [3]. Based on their functional characteristics, the 17 human KLF proteins can be divided into three distinct groups by phylogenetic analysis of protein sequences. KLF9, along with KLF10, 11, 13, 14, and 16, belongs to subgroup 3, whose members most likely have repressor activity as they can interact with the common transcriptional co-repressor Sin3A [4,5]. As a thyroid hormone-induced immediate early gene, KLF9 participates in neural development in vertebrates [6]. In addition, KLF9 is also involved in the cellular response to progesterone and estrogen receptor activation [7,8] as well as in adipocyte differentiation [9]. Moreover, KLF9 in the human epidermis functions
Abbreviations: KLF, Krüppel-like factor; ZNF, Zinc finger domain; SID, Sin3A interaction domain; DOX, Doxycycline; CHX, Cycloheximide. * Corresponding author. Tel.: +86 21 3420 6283; fax: +86 21 3420 6283. E-mail address: [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.canlet.2014.09.022 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.
as a circadian transcription factor that significantly affects keratinocyte proliferation and differentiation [10]. Recently, KLF9 has been reported to be down-regulated in human colorectal tumors, and the reduction of KLF9 expression is associated with tumor initiation and development [11]. Notably, KLF9 has been identified as a relatively unique differentiation-induced transcription factor in glioblastoma-derived neurospheres and can suppress glioblastoma stem cell maintenance [12]. Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide and causes approximately one million deaths annually [13]. Identification of causative genes and molecular pathways may improve strategies for the diagnosis, prevention and treatment of HCC. Specific KLF family members are now known to be involved in regulating the development of HCC. KLF6 status in HCC is controversial. On one hand, low expression of KLF6 mRNA was observed in 73% of HBV-associated HCCs, and reconstituting KLF6 in HepG2 cells decreased proliferation [14]. On the other hand, KLF6 silencing caused p53 upregulation and inhibited Bcl-xL expression to trigger cell death [15]. KLF4 can induce the expression of the vitamin D receptor (VDR) by binding to its promoter, thereby sensitizing HCC cells to the antiproliferative effects of vitamin D3 (VD3) [16]. Most recently, KLF9 has been demonstrated to inhibit cellular proliferation and mobility and induce apoptosis in HepG2 cells [17]. In this study, we supply further evidence that the ectopic expression of KLF9 induces apoptosis in HCC cells and inhibits tumor growth in vitro and in vivo. Strikingly, the restoration of KLF9 caused significant remission of established tumors in mice. Moreover, the overexpression of KLF9 promoted the activation of p53, which may contribute to the tumor-suppressive function of KLF9.
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Material and methods
Statistical analysis
Tissue specimens and cell lines
Analysis of variance (ANOVA) and Student’s t tests were used to evaluate significant differences and variances. Quantitative data are described as the means ± SD. A P value less than 0.05 was considered significant.
Forty-five pairs of human HCC samples from Qi Dong Liver Cancer Institute in China were processed for RNA analysis. Patient information is listed in Table S1. The use of human material was approved by the institutional ethical review committee. A commercially available tissue microarray (TMA) containing 92 HCC pairs was used for immunohistochemical staining. The SK-Hep1, HepG2, Hep3B, PLC-PRF-5, SUN387, and THLE-2 cell lines were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). The Huh7 cell line was from Riken Cell Bank (Tsukuba Science City, Japan). The SMMC-7721 cell line was obtained from the cell bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The MHCC-97L and LM3 cell lines were provided by Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China). Xenograft model Male BALB/c nude mice (6–8 weeks of age) were injected s.c. with SK-Hep1 cells (5 × 106/0.1 mL saline) transfected with either pTRIPZ-wt-KLF9 (on the left flank) or empty vector (on the right flank) in groups one and two (n = 6 per group), and cells transfected with KLF9-ΔSID (on the left flank) or KLF9-ΔZNF (on the right flank) in group three (n = 6). Mice in group one were given normal water, groups two and three were administered daily doxycycline (DOX, 2 mg/mL) in the water. Tumor volumes were monitored every 2 days. To determine the effect of KLF9 on the growth of established tumors, the mice received single s.c. flank injections of Sk-Hep1 cells (5 × 106/ 0.1 mL saline) with either pTRIPZ-wt-KLF9 (n = 16) or empty vector (n = 16), and tumor growth was monitored. When the volume of the tumors reached 150–200 mm3, each group of mice was randomly divided into two groups (n = 8 per group). One group was given DOX (2 mg/mL) and the other given normal water. Tumor growth was monitored every 5 days. The tumor-bearing mice were sacrificed 40 days after inoculation, and the tumors were removed for further study. All experiments were subject to approval by the Institution of Animal Care and Use Committee (SHCI-11-020). Laboratory methods See Appendix S1 for detailed experimental procedures.
Results KLF9 is downregulated in HCC First, the expression profile for the main subgroup 3 members, KLF9, KLF10, KLF11 and KLF13 were analyzed in 45 pairs of clinical specimens by real-time RT-PCR. The results showed that the mRNA levels of KLF9 and KLF11 were significantly down-regulated in HCC tissues when compared with adjacent noncancerous tissue, whereas there were no significant differences in the levels of KLF10 and KLF13 (Fig. 1A). Moreover, a subgroup of clinical human HCC samples (37%, 33/92) exhibited decreased KLF9 expression, as demonstrated by immunohistochemical staining in 92 pairs of HCC tissues (Fig. 1B). Consistently, we also found that mRNA levels of KLF9 were considerably decreased in human liver cancer cell lines compared with the immortalized liver cell line THLE-2 (Fig. 1C). Overexpression of KLF9 inhibits HCC cell proliferation and induces apoptosis The downregulation of KLF9 in liver cancer cell lines and primary HCC tissues suggests a suppressive function of this gene in HCC development. Therefore, we investigated the effects of ectopic KLF9 expression on two HCC cell lines, SK-Hep1 and HepG2. After 7 days of culture, cell growth and colony formation were significantly attenuated in KLF9-expressing cells (Fig. 2A, 2B and 2C). The expression
Fig. 1. KLF9 is downregulated in HCC tissues and liver cancer cell lines. (A) Scatter plots depicting the transcriptional levels of KLF9, KLF10, KLF11, and KLF13 in HCC tissues (T) and matched non-tumor tissues (N) (n = 45); GAPDH was used as an internal reference. (Black lines: median values, *** P < 0.001.) (B) Representative immunohistochemical staining of HCC samples with anti-KLF9 antibody. N: non-tumor, T: HCC tissue. Original magnification: 200×, 400×. (C) Histograms showing the relative expression of KLF9 in HCC-derived cell lines and the immortalized liver cell line THLE-2; GAPDH was used as an internal reference. The values indicate the means ± SD of three independent experiments.
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Fig. 2. KLF9 is a negative regulator of cell proliferation and induces apoptosis in HCC cells. SK-Hep1 and HepG2 cells transfected with Flag-tagged and Tet-induced KLF9 (pTRIPZ-KLF9) or empty vector were used. (A and B) The expression of exogenous (with Flag-specific antibody) and endogenous KLF9 was measured by western blot after treatment with DOX (0.5 μM) for 24 h. The cell growth rate was then determined using an MTT assay at the indicated time points. The results shown are the means ± SD of three independent experiments. ***P < 0.001. (C) Representative cell culture plates illustrating dense foci formation by the cells treated with or without DOX (0.5 μM) for 7 days. (D) The cells were double-stained with PI and 7-AAD, and cell cycle stages were analyzed by flow cytometry. The charts present 2D results of the cell distribution in the different cell cycle stages. The percentage of cells in G1, S and G2 phases are quantified in the histogram. The values indicate the means ± SD of three independent experiments. ***P < 0.001. (E and F) Cells were treated with DOX (0.5 μM) for the indicated times, and double-stained with Annexin-V and 7-AAD. The scattergrams show representative results after treatment for 48 h. The graph presents the percentage of Annexin-V-positive cells at the indicated time point. Each value is the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05.
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of exogenous KLF9 was marked with a Flag tag, while endogenous KLF9 could not be detected (Fig. 2A and 2B). These cells were further subjected to cell cycle and apoptosis assays. The results demonstrated that KLF9 caused a decrease in S-phase and an increase in G1-phase (Fig. 2D). Notably, the percentage of apoptotic cells was significantly increased from 9.01% to 39.54% (P < 0.01) in KLF9overexpressing SK-Hep1 cells after 48 h of DOX treatment (Fig. 2E). Similarly, the percentage of apoptotic cells was also increased from 8.2% to 13.58% in KLF9-overexpressing HepG2 cells (Fig. 2F). No meaningful differences were observed in the cells expressing empty vector. Collectively, these results suggest that KLF9 plays a significant role in inhibiting growth and promoting apoptosis in HCC cells. The tumor-suppressive function of KLF9 depends on its DNA-binding domain Some works have demonstrated that KLF10 and KLF11 act as transcriptional repressors through the Sin3 interaction domain in their amino-terminal regions, which forms a co-repressive complex with Sin3 proteins [18,19]. To structurally illuminate the contributions of the Sin3 interaction domain (SID) and DNA binding domain (zinc finger domain, ZNF) to the effects of KLF9, SK-Hep1 and HepG2 cells were transfected with KLF9 and the mutants without the SID domain (ΔSID) or ZNF domain (ΔZNF) (Fig. 3A). Compared with the effects of wt-KLF9, KLF9-ΔSID expression also robustly inhibited foci formation and triggered apoptosis, whereas the expression of
KLF9-ΔZNF had no such effects (Fig. 3B and 3C). Remarkably, the results were confirmed by in vivo experiments, in which the expression of wt and KLF9-ΔSID delayed the onset of tumors in mice. However, KLF9-ΔZNF expression had less significant effects, although it did result in subtle growth inhibition compared with the control mice (Fig. 3D). Therefore, these results suggest that the DNA-binding domain (ZNF) of KLF9 is essential for its tumor-suppressive role in HCC cells. KLF9 up-regulates p53 activity and enhances its stability To further explore the related molecular mechanisms underlying the pro-apoptotic effects of KLF9, the expression levels of selected apoptosis-related proteins were examined by western blot. The results demonstrated that overexpression of KLF9 significantly increased protein levels of p53 and cleaved caspase 3 and decreased the levels of β-Catenin and BCL-2 in DOX-treated cells at the indicated time points (Fig. 4A and 4B). Moreover, the expression of p53 at the mRNA level was also enhanced by KLF9 in SK-Hep1 and HepG2 cells (Fig. 4C and 4D). Further confirming the regulation of p53 by KLF9, we observed that expression of NOXA and PUMA, two genes downstream of p53, was also increased by KLF9 (Fig. S1A and S1B). Because ectopic expression of KLF9 dramatically increased protein levels of p53 in SK-Hep1 and HepG2 cells, we next explored whether the increase in p53 protein was attributable to protein stabilization or transcriptional enhancement. When treated with cycloheximide (CHX), the half-life of p53 protein in
Fig. 3. KLF9 inhibits proliferation and induces apoptosis in a ZNF domain-dependent manner both in vitro and in vivo. (A) Schematic representation of the two principal domains of KLF9 and the deletion mutants used [20]. (B) Representative dense foci formation by cells treated with or without DOX (0.5 μM) for 7 days. (C) The number of apoptotic cells was measured by flow cytometry after DOX (0.5 μM) treatment for the indicated time. Each value is the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05. (D) SK-Hep1 cells transfected with pTRIPZ-WT-KLF9, KLF9-ΔSID, KLF9-ΔZNF or empty vector were separately injected subcutaneously into the flanks of individual male BALB/c nude mice. Mice in group one (n = 6) and two (n = 6) were injected with cells containing empty vector (right flank) and WT-KLF9 (left flank); mice in group three (n = 6) were injected with cells containing KLF9-ΔSID (right flank) and KLF9-ΔZNF (left flank). Mice in group one received normal water and those in groups two and three received 2 mg/mL DOX in the drinking water beginning on the second day post-implantation. The tumor volumes were monitored and measured after treatment for 40 days. The scatter plots show the volumes for each group.
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Fig. 4. KLF9 upregulates the expression of pro-apoptotic proteins. (A and B) The protein levels of Flag, β-Catenin, p53, BCL-2, cleaved Caspase 3, and α-Tubulin were determined by western blot at the indicated time points after DOX treatment in SK-Hep1 and HepG2 cells transfected with pTRIPZ-KLF9. (C) (D) The transcriptional level of p53 was examined at the indicated time points following KLF9 induction with DOX in SK-Hep1 and HepG2 cells. The values are the means ± SD of three independent experiments. *P < 0.05.
KLF9-overexpressing cells was significantly increased. The stability of p21 was also enhanced (Fig. 5A and 5B). These results suggest that KLF9 plays a role in the stabilization of p53 protein. We also found that the protein levels of p53 and p21 were not upregulated
by KLF9-ΔZNF (Fig. 5C and 5D). Consistently, KLF9-ΔZNF expression did not cause an increase in the p53 mRNA levels (Fig. 5E and 5F). The results suggest that the upregulation of p53 by KLF9 also occurred at the transcriptional level.
Fig. 5. KLF enhances p53 stability and promotes its activity. (A) (B) SK-Hep1 and HepG2 cells transfected with pTRIPZ-KLF9 or empty vector were treated with or without DOX (0.5 μM) for 24 h. The cells were then exposed to cycloheximide (CHX, 25 μg/mL) for the indicated times. The protein levels of p53, p21, Flag, and α-Tubulin were determined by western blot. (C) (D) The levels of Flag, p53, p21, and α-Tubulin were evaluated by western blot in SK-Hep1 and HepG2 cells transfected with WT or mutant KLF9 or empty vector with or without treatment with DOX (0.5 μM) for 24 h. (E) (F) The level of p53 was determined by RT-PCR in SK-Hep1 and HepG2 cells infected with WT or mutant KLF9 after treatment with DOX (0.5 μM) for 24 h. Values represent the means ± SD of three independent experiments. *P < 0.05.
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KLF9 binds the promoter of p53 and strengthens its transcriptional activity To further evaluate our hypothesis of transcriptional p53 upregulation by KLF9, we utilized luciferase reporter detection. As shown in Fig. 6A, the overexpression of KLF9 significantly increased the activity of the pGL3-p53 promoter in 293T, SK-Hep1 and HepG2 cells. To determine the efficiency of the three GC-box sequences within the p53 promoter, we performed site-specific mutations of these sites (from CACCC to AAAAA) and constructed four p53 promoter mutants: mut1, mut2, mut3, and mut all (Fig. 6B, lower panel). The results revealed that each of the GC-box mutations had deleterious effects on the transcriptional activity of p53. Indeed, the combinatory inactivation of these sites produced a more robust inhibition of luciferase activity than did any of the singlesite mutants (Fig. 6B). Furthermore, a ChIP assay was performed using Flag-specific mouse antibody and control (normal mouse IgG) from SK-Hep1 cells expressing KLF9 or empty vector following DOX treatment for 24 h. As shown in Fig. 6C, a clear enrichment of the proximal but not distal region of the p53 promoter was detected in the chromatin precipitated from cells expressing WT-KLF9. Collectively, our results demonstrate that KLF9 can increase p53 transcriptional activity by interacting with the proximal GC boxes of the p53 promoter. Inducible expression of KLF9 promotes regression of established HCC tumors Considering that KLF9 expression has robust inhibitory effects on the proliferation of HCC cells, we next explored whether KLF9 has therapeutic value in mice bearing established HCC tumors. Our results showed that all mice developed tumors after the subcutaneous inoculation of SK-Hep1 cells expressing empty vector in the
absence of inducible KLF9 expression. In the presence of DOXinduced KLF9 expression, however, established tumors in six of eight mice became entirely undetectable (Fig. 7A and 7B). To evaluate the levels of KLF9 induced in the tumor tissues, we performed immunohistochemical staining at days 0, 5 and 15 after DOX treatment. Marked induction of the KLF9 protein was observed with DOX in vivo and was mainly located in the nuclei of HCC cells. The expression patterns of Ki67, p53, and TUNEL were also detected in parallel. The results showed that at days 5 and 15 post-DOX treatment, the number of Ki67-positive cells was significantly decreased, whereas the number of TUNEL-positive cells was significantly increased in the presence of KLF9 expression (Fig. 7C). In addition, p53 expression was higher in the KLF9-expressing group than in the control group. The histograms present the quantification of positively stained cells (Fig. 7D). Overall, these results provide in vivo evidence that KLF9 expression can significantly attenuate the growth of established tumors by inhibiting proliferation and inducing p53 expression and apoptosis in situ, suggesting that induction of KLF9 may be an effective strategy to eradicate human HCC. Discussion In the present study, KLF9 was downregulated in the majority of HCC specimens, and exogenous KLF9 expression inhibited cell proliferation and induced apoptosis in cultured human HCC cells. A recent report also described an essential role for KLF9 in bortezomib- and LBH589-induced apoptosis in multiple myeloma cells [21]. Consistent with a recent report [17], KLF9 induced apoptosis in HCC cells. The authors of that report explained that programmed cell death protein 5 overexpression simulated the activation of KLF9 in HepG2 cells. In the present study, KLF9 overexpression increased the activation of p53. We have also
Fig. 6. KLF9 increases p53 transcriptional activity in HCC cells. (A) HEK 293T, SK-Hep1 and HepG2 cells were cotransfected with p53 luciferase reporter and pSIN-KLF9 or an empty vector for 48 h. The luciferase activity was subsequently assayed. Firefly-dependent luciferase emission was normalized to the Renilla signal. Values represent the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001. (B) Left, schematic representation of the pGL3-basic-p53 reporter construct, including the three predicted GC-rich KLF9 binding sites (CACCC). The proximal p53 promoter (region −651 to +50 base pairs) was cloned into the pGL3-basic luciferase vector, and the CACCC sites were individually or were combinatorially mutated to AAAAA in the luciferase vector. Hep3B cells (p53−/−) were transfected with p-SIN-KLF9 along with each of the promoter mutants: mutant1 (−273 to −267), mutant2 (−181 to −177), mutant3 (−128 to −124), or mutant all. After 48 h, the luciferase activity was analyzed. Values represent the means ± SD of three independent experiments. **P < 0.01, *P < 0.05. (C) The binding of KLF9 to the p53 promoter was evaluated by ChIP followed by RT-PCR analysis. Sk-Hep-1 cells transfected with KLF9-wt, ΔSID, ΔZNF and vector plasmids were treated with or without DOX for 24 h, then the cells were cross-linked, sonicated and subjected to chromatin immunoprecipitation with anti-Flag antibody beads or agarose-conjugated anti mouse IgG beads at 4 °C overnight. Isolated DNA was employed in quantitative PCR with GAPDH and p53 promoter-specific primers. All PCR signals were normalized to GAPDH-specific PCR signals, and the corresponding PCR signals obtained from reactions of DNA precipitated with IgG antibodies. Values represent the means ± SD of three independent experiments. **P < 0.01, *P < 0.05.
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Fig. 7. KLF9 expression abolishes the growth of established tumors. (A) SK-Hep1 cells transfected with pTRIPZ-WT-KLF9 and empty vector were separately injected subcutaneously into the right flanks of individual male BALB/c nude mice. All mice were provided with normal water until the tumor size reached approximately 150– 200 mm3. The mice from each group were then randomly divided into two groups per cell line. One group was provided with normal water (n = 8), and the other received 2 mg/mL DOX in the drinking water (n = 8). The graph presents the growth of the tumors in the mice at the end of the experiment after treatment for 40 days. (B) The tumor volume was measured every 5 days from day 20 after inoculation until the end of the experiment. (C) The tumors were collected at days 5 and 15 after DOX treatment and analyzed by immunohistochemical staining with anti-KLF9 anti-p53, Ki67 and TUNEL. All photographs are at the same magnification. Original magnification: 400×. (D) Histograms showing the quantification of the numbers of positively stained cells at the indicated day (derived from the experiments described in (C)). For each staining, 10 separate fields at 400× magnification were recorded. Each value represents the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05.
reported that KLF9 induces apoptosis and suppresses the proliferation of prostate cancer cells by inhibiting the activation of AKT [22]. These findings indicate that KLF9 plays critical roles in the development and progression of human cancers. More importantly, we found KLF9 expression not only delayed the onset of tumors but also efficiently promoted the regression of established tumors.
KLF9 belongs to subgroup 3 of the KLF family, members of which are likely to have repressor activity because they contain SID domains that interact with the common transcriptional co-repressor Sin3A. Structurally, KLF family members are highly conserved in their C-termini, and the ZNF domains in this region are usually responsible for the DNA-binding activity or protein-protein interactions.
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Indeed, we found that deletion of the ZNF domain almost entirely attenuated KLF9-mediated transcriptional activation of p53. The N-terminal regions of the KLFs vary considerably and are usually responsible for transcriptional activation or repression [23]. The Sin3interacting-domain (SID) is found in the N-terminal of KLF9. In general, the SID has been characterized as a domain with transcriptional repression activity [20]. We found that the examined activity of SID deletion mutant was comparable with intact KLF9, suggesting that the SID domain-mediated function, if any involved in gene silencing, was dispensable for KLF9-mediated p53 activation and suppression of cell proliferation. Another notable finding of this study is that KLF9 efficiently increases p53 activity. p53 is a very labile protein, with a half-life as short as a few minutes. Defects in the ability to stabilize p53 are likely to contribute to malignancy development, and restoration of this activity represents an extremely attractive possibility for tumor therapy [24,25]. Our results indicate that the protein levels of p53 were clearly increased by KLF9; this increase may be attributable to enhanced stability of p53 at the posttranslational level and increased transcriptional activity. Consistent with the observation that cells with ectopic KLF9 expression displayed an increase in the proportion of apoptotic cells, we observed that the p53 downstream targets p21, Puma and Noxa [26] were significantly activated by KLF9. These proteins have long been appreciated to be the mediators of p53-induced apoptosis. In addition, the pro-survival proteins Bcl-2 was down-regulated in cells expressing exogenous KLF9. In the present study, we also showed the coincidence of p53 activation and β-catenin inhibition in the context of KLF9 induction. Hyperactivation of the WNT/β-catenin signaling contributes to the development and pathogenesis of HCC. Consistently, it has been shown that p53 activation negatively regulates β-catenin expression [27]; and the accumulation of wild-type β-catenin is linked with the inactivation of p53 in HCC cells [28]. Obviously, further exploration of the details of how KLF9 regulates the stabilization of p53 will be helpful in the understanding of the roles of KLF9 in cancer development. Notably, in addition to p53, other pathways contribute to KLF9mediated apoptosis. A recent study [29] has demonstrated that KLF9 is associated with the transcriptional regulation of several genes related to ROS metabolism, and KLF9 thereby facilitates oxidative stress and subsequent cell death. These findings indicate that KLF9 activation may contribute to cell-autonomous limitation to inhibit cell transformation and tumor genesis in hepatocytes, especially in the presence of stress, such as HBV replication, DNA damage, and liver injury. Induction of KLF9 expression may provide a strategy for cancer treatment. KLF9 is required for bortezomib-induced apoptosis in myeloma cells [21]. Interestingly, a phase II trial of bortezomib has been performed to evaluate this drug as a mono-therapy in patients with unresectable HCC [30], and bortezomib can sensitize HCC cells to several cell death-inducing reagents, such as TRAIL, mapatumumab, lexatumumab, and CS-1008 [31,32], suggesting that reagents that induce KLF9 expression may have clinical potential as therapy for HCC patients. In summary, our study suggests that the development of specific activators of KLF9 may be a useful approach for the clinical treatment of HCC. Conflict of interest The authors have no conflict of interest. Acknowledgments This work was supported by grants from The National Science Foundation of China (Grant No. 81201542 and No. 81301716), and Natural Science Foundation of Shanghai (No. 12ZR1430100). Key
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