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The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer
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Cancer Letters xxx (2016) 1e9
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Original Article
Q4 Q3
The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer Yue Wang a, 1, Runping Fang a, 1, Ming Cui b, Weiying Zhang a, Xiao Bai a, Huawei Wang a, Bowen Liu a, Xiaodong Zhang b, *, Lihong Ye a, ** a State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, People's Republic of China b State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 July 2016 Received in revised form 22 September 2016 Accepted 10 October 2016
The oncoprotein Yes-associated protein (YAP) in Hippo pathway plays crucial roles in the development of cancer. However, the mechanism of YAP regulation in cancer remains poorly understood. Here, we supposed that the oncoprotein hepatitis B X-interacting protein (HBXIP) might be involved in the modulation of YAP in liver cancer. Interestingly, our data showed that the expression levels of HBXIP were positively associated with those of YAP in clinical hepatocellular carcinoma (HCC) samples by immunohistochemistry (IHC) staining and real-time PCR assays. HBXIP was able to up-regulate YAP in hepatoma cells at the levels of promoter, mRNA and protein. Mechanistically, we identified that HBXIP up-regulated YAP through co-activating the transcription factor c-Myb in hepatoma cells. Functionally, silencing YAP abolished the proliferation of hepatoma cells mediated by HBXIP in vitro. Moreover, knockdown of YAP strongly blocked the HBXIP-enhanced tumor growth in mice. Thus, we conclude that HBXIP up-regulates YAP expression via activating transcription factor c-Myb to facilitate the growth of hepatoma cells. Our finding provides new insights into the mechanism of YAP regulation. Therapeutically, the oncoprotein HBXIP and YAP might serve as targets in liver cancer. © 2016 Published by Elsevier Ireland Ltd.
Keywords: HBXIP YAP c-Myb Proliferation Liver cancer
Introduction The dysregulation of Hippo pathway is wildly involved in the tumorigenesis [1e4], in which Yes-associated protein (YAP) is able to induce the expression of pro-proliferative and anti-apoptotic genes as a transcription co-activator in the complex of TEAD family [5e7]. CTGF, CYR61 and SOX2 serve as the downstream target genes of YAP [8,9]. A host of factors including cell density, extracellular matrix stiffness, G proteinecoupled receptors and leukemia inhibitory factor receptor can influence YAP activity through modulating the Hippo pathway. The heightened expression or genomic amplification of YAP is frequently observed in many cancers [10e15]. The highly expressed YAP is involved in multiple
* Corresponding author. Fax: þ86 22 23501385. ** Corresponding author. Fax: þ86 22 23501385. E-mail addresses: [email protected] (X. Zhang), [email protected] (L. Ye). 1 The authors contribute equally to the work.
process of liver disease including liver repair, regeneration, hepatocellular carcinoma (HCC) [16e18]. YAP can serve as an independent prognostic marker in HCC [19]. However, the mechanism of YAP regulation in liver cancer is poorly understood. Hepatitis B X-interacting protein (HBXIP), originally identified for interacting with hepatitis B virus X proteins, is a conserved ~18 kDa protein among mammalian species [20]. HBXIP acts as an oncoprotein in controlling cell proliferation, apoptosis and division [21e24]. HBXIP as a regulator component participates in amino acids-mediated mTORC1 activation [25]. Our group manifested that HBXIP could import into the nucleus of breast cancer cells, acting as a transcription co-activator of transcription factors such as Sp1, cAMP-response element binding protein (CREB), c-Myc and E2F1, to accelerate the progression of liver and breast cancer [26e30]. However, whether HBXIP is involved in the regulation of YAP in cancers is not well documented. The transcription factor c-Myb is encoded by the MYB protooncogene [31,32]. Overexpression of c-Myb contributes to malignant transformation by regulating genes participating in multiple
http://dx.doi.org/10.1016/j.canlet.2016.10.018 0304-3835/© 2016 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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processes of tumourigenesis, such as cell growth, angiogenesis and resistance to apoptosis [33e36]. Myb is able to affect tumor cell self-renewal through the Wnt signaling pathway [37]. Recent studies have shown that c-Myb plays an important role in the development of gastric cancer (GC) [38]. The target genes of c-Myb include SOX2, Bcl-2, Bax, c-Myc, and etc [39]. However, the role of c-Myb in the regulation of YAP remains unclear. In this study, we investigated the role of oncoprotein HBXIP in modulation of YAP in liver cancer. Interestingly, we found that HBXIP was able to up-regulate YAP through activating YAP promoter, involving co-activating c-Myb, to accelerate the growth of hepatoma cells. Our finding provides new insights into the mechanism by which HBXIP regulates YAP expression in the development of liver cancer. Materials and methods Patient samples Twenty-six hepatocellular carcinoma (HCC) tissue samples and their corresponding non-tumorous liver tissues were obtained from Tianjin First Center Hospital and Tianjin Tumor Hospital (Tianjin, China) after surgical resection. Written consents approving the use of tissue samples for research purposes were obtained from HCC patients. The study protocol was approved by the Institute Research Ethics Committee at Nankai University. The information of HCC patients was shown in Supplementary Table S1. Cell lines and cell culture The human hepatoma cell lines HepG2, HepG2-pcDNA3.1 (HepG2 cells stably transfected with pcDNA3.1) and HepG2-HBXIP (HepG2 cells stably transfected with pcDNA3.1-HBXIP) and human kidney epithelial (HEK) 293 T cells were maintained in Dulbecco's Modified Eagle's medium (Gibco, CA, USA). The human hepatoma H7402 cell line was cultured in RPMI-1640 medium (Gibco, CA, USA). All cell lines were supplemented with heat-inactivated 10% fetal bovine serum (FBS, Gibco, CA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin and grown at 5% CO2 and 37 C. Histological analysis and immunohistochemistry staining (IHC) Paraffin-embedded mouse liver tissue was cut into 5 mm sections, which were stained with hematoxylin-eosin for histological analysis. Immunohistochemical staining was performed as previously reported [30]. The primary antibodies of rabbit anti-HBXIP (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-YAP (Proteintech Group, USA) were used. The staining levels of HBXIP and YAP were classified into three groups using a modified scoring method based on the intensity of staining (0 ¼ negative; 1 ¼ low; 2 ¼ high) and the percentage of stained cells (0 ¼ 0% stained; 1 ¼ 1%e49% stained; 2 ¼ 50%e100% stained). A multiplied score (intensity score percentage score) lower than 1 was considered to be negative staining (), and 1e4 were considered to be positive staining (þ). Categorization of immunostaining intensity was performed by three independent observers. The information of the HCC patients and the staining level of HBXIP and YAP were listed in Supplementary Table S2. Construction of plasmids To construct the core region of YAP promoter, the region 354/þ115 and 354/ 62 of YAP were amplified by PCR from the pGL3-1536 and was inserted into the upstream of the pGL3-Basic vector (Promega, Madison, WI, USA) via KpnI and XhoI sites to generate YAPluc (354~þ115) and YAPluc (354~62). A mutant of YAPluc (354~62), which carried a substitution of three nucleotides (GGTT instead of wild-type AACT) within the binding site of c-Myb, was constructed by using overlapping extension PCR. According to the report [40], the 5'-flanking region of CTGF was amplified by PCR from the genomic DNA of HepG2 cells using specific primers and was cloned into the upstream of the pGL3-Basic vector via KpnI and HindⅢ sites. The resulting plasmid was sequenced and named CTGFluc. To construct the pcDNA3.1-c-Myb, the CDS of c-Myb were amplified by PCR and was inserted into the pcDNA3.1 (þ) vector (Invitrogen, CA, USA) via Hind III and Xho I sites. All primers used were listed in Supplementary Table S3. Chromatin immunoprecipitation (ChIP) ChIP assays were performed in HepG2-HBXIP cells transfected siRNA control (siControl) or c-Myb siRNA (si-c-Myb) according to the manufacturer's protocol (Epigentek Group Inc, Brooklyn, NY) as reported previously [28]. The fragment of YAP promoter including the c-Myb sites was amplified from the immunoprecipitated DNA samples with the specific primers. All primers are listed in Supplementary Table S3.
Analysis of cell proliferation HepG2 or H7402 cells were seeded into 96-well plates (1000 cells/well) for 24 h before transfection. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay or 5-ethynyl-20 -deoxyuridine (EdU) incorporation assay was performed to access the proliferation of cells as described previously. For colony formation assay, 48 h after transfection, about 500 cells were placed into 6-well plates and maintained in complete medium for 2 weeks. Colonies were fixed with methanol and stained with methylene blue. Tumor xenograft in mice BALB/c athymic nude mice (Experiment Animal Center of Peking, China) were housed and treated according to guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. HepG2-pcDNA3.1 or HepG2-HBXIP cells with the transfection of si-Control or si-YAP were harvested and re-suspended at 2 107 per ml with sterile phosphate-buffered saline. Groups of 4week-old female BALB/c athymic nude mice (each group, n ¼ 6) were subcutaneously injected at the shoulder with 0.2 ml of the cell suspensions. Tumor growth was measured after 10 days from injection and then every 5 days. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L W2) 0.5. After 30 days, the mice were sacrificed, and the tumors were excised and measured. Statistical analysis Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (±SD) using a Student's t test for independent groups and was assumed for *P < 0.05 and **P < 0.01; and ***P < 0.005. Pearson's correlation coefficient was used to determine the correlation between YAP mRNA levels and HBXIP mRNA levels in HCC tissues.
Results The expression of HBXIP is positively associated with that of YAP in clinical HCC tissues Our laboratory has reported that the oncoprotein HBXIP contributes to the progression of cancer through acting as a coactivator of transcription factors [26e30]. Lots of studies have proven that the highly expressed YAP plays a crucial role in the development of cancer [10e15]. Accordingly, we supposed that HBXIP might be involved in the regulation of Hippo-YAP signaling. Immunohistochemistry (IHC) staining analysis exhibited that the positive rate of HBXIP was 100% (40/40) in HCC tissues, in which the positive rate of YAP was 77.5% (31/40) in HBXIP-positive HCC tissues using tissue microarray (Fig. 1A). Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression levels of HBXIP were positively related to those of YAP in 26 cases of clinical HCC tissues (r ¼ 0.6178, P < 0.001, Pearson's correlation, Fig. 1B). Meanwhile, we verified the correlation between YAP and HBXIP using 6 paired HCC tissues by Western blot analysis (Fig. 1C), These results suggest that the expression of HBXIP is positively associated with that of YAP in clinical HCC tissues. In addition, we evaluated the relationship between HBXIP and CTGF, a target gene of YAP, in clinical HCC tissues. Our data indicated that the expression levels of HBXIP were positively correlated with those of CTGF in 26 cases (Fig. 1D). Thus, we conclude that HBXIP is positively associated with YAP in clinical HCC tissues. HBXIP up-regulates YAP expression in hepatoma cells Next, we tried to evaluate the effect of HBXIP on YAP in hepatoma cells. Interestingly, we showed that HBXIP was capable of upregulating YAP at the levels of mRNA and protein in hepatoma HepG2 and H7402 cells in a dose-dependent manner (Fig. 2A and Supplementary Fig. S1A), and silencing HBXIP by siRNA significantly decreased YAP at the levels of mRNA and protein in HepG2HBXIP cells in a dose-dependent manner (Fig. 2B), suggesting that HBXIP is able to up-regulate YAP in hepatoma cells. Interestingly, we observed that HBXIP enhanced the expression and nuclear
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Fig. 1. The expression of HBXIP is positively associated with that of YAP in clinical HCC tissues. (A) The expression levels of HBXIP and YAP were examined by IHC staining analysis in clinical HCC tissues and normal tissues. (B) The correlation between HBXIP mRNA levels and YAP mRNA levels was detected by qRT-PCR analysis in 26 cases of clinical HCC samples. (C) The correlation between HBXIP protein levels and YAP protein levels was examined by Western blot analysis in 6 paired HCC clinical tissues. T, tumor; N, normal tissue. (D) The correlation between HBXIP mRNA levels and CTGF mRNA levels was detected by qRT-PCR analysis in 26 cases of clinical HCC samples. Statistically significant differences are indicated: ***P < 0.001; Student's t test.
import of YAP in HepG2-HBXIP cells by immunofluorescent staining (Fig. 2C). It has been reported that the phosphorylation of YAP causes cytoplasmic retention, and results in inactivating its gene regulation ability [41]. Western blot analysis showed that overexpressed HBXIP decreased the phosphorylation levels of YAP in hepatoma cells (Supplementary Fig. S1B), further supporting that HBXIP is able to increase the nuclear import of YAP via inhibiting YAP phosphorylation. We have reported that HBXIP as a coactivator regulates the gene transcription through interacting with transcription factors [26e30]. Accordingly, we verified the effect of HBXIP on the promoter region of YAP (1420/þ115, pGL3-
1536) [42]. Luciferase reporter gene assays demonstrated that HBXIP was able to increase the activities of pGL3-1536 in a dosedependent manner in HepG2 and H7402 cells (Fig. 2D and Supplementary Fig. S1C), whereas the activities of pGL3-1536 were decreased by the treatment with HBXIP siRNA in HepG2-HBXIP cells (Fig. 2E), suggesting that HBXIP up-regulates the expression of YAP at the transcription level in hepatoma cells. We further validated the effect of HBXIP on YAP using the Gal4-TEAD1 system. Interestingly, we observed that the co-transfection of HBXIP and Gal4-TEAD1 increased the luciferase activities, while cotransfection of si-HBXIP and Gal4-TEAD1 decreased the luciferase
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Fig. 2. HBXIP up-regulates YAP expression in hepatoma cells. (A, B) The effect of HBXIP (or HBXIP siRNA) on the expression of YAP was measured by RT-PCR analysis and Western blot analysis at the levels of mRNA and protein in HepG2 (or HepG2-HBXIP) cells, respectively. (C) The effect of HBXIP on the expression and localization of YAP was detected by laser confocal microscope in HepG2-HBXIP cells. Scale bar, 20 mm. (D, E) The effect of HBXIP (or HBXIP siRNA) on the activities of YAP promoter was examined by luciferase reporter gene assays in HepG2 (or HepG2-HBXIP) cells, respectively. (F) The effect of HBXIP (or HBXIP siRNA) on the activities of YAP was examined by Gal4-TEAD1 reporter system. Each experiment was repeated at least three times. Statistically significant differences are indicated: *P < 0.05, **P < 0.01; Student's t test.
activities (Fig. 2F), suggesting that the HBXIP is able to stimulate activities of YAP in the cells. It has been reported that CTGF is a classic downstream gene of YAP [40]. To further validate that HBXIP up-regulates YAP, we examined the effect of HBXIP on CTGF in hepatoma cells. As expected, we observed that HBXIP was able to up-regulate CTGF at the levels of mRNA, protein and promoter in HepG2 and H7402 cells in a dose-dependent manner, whereas silencing of HBXIP by HBXIP siRNA led to the down-regulation of CTGF in HepG2-HBXIP cells in a dose-dependent manner (Supplementary Fig. S1D-H), supporting that HBXIP up-regulates YAP in the cells. Thus, we conclude that HBXIP is able to upregulate the expression of YAP in hepatoma cells.
HBXIP stimulates YAP promoter through co-activation of transcription factor c-Myb We have reported that the core region of YAP promoter locates at the region 354/þ115 in hepatoma cells [42]. To identify the mechanism by which HBXIP up-regulates YAP, we further minimized the core region of YAP promoter. Luciferase reporter gene assays revealed that pGL3-293 (354/62) exhibited the maximum luciferase activities and pGL3-Basic (61/þ115) exhibited the minimum luciferase activities (Fig. 3A). We further observed that HBXIP was able to increase the activities of pGL3-293 in HepG2 and H7402 cells in a dose-dependent manner (Fig. 3B and
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Fig. 3. HBXIP stimulates YAP promoter through co-activation of transcription factor c-Myb. (A) The activities of different fragments of YAP promoter (pGL3-1536, pGL3-470 or pGL3293) were measured by luciferase reporter gene assays in HepG2 cells. (B) The activities of pGL3-293 (354~62) were measured by luciferase reporter gene assays in HepG2 cells transfected with pcDNA3.1-HBXIP. (C) Schematic diagram depicting the potential transcription factors in the promoter region 354~62 and 61~þ115 of YAP by bioinformatics. (D) The activities of pGL3-293 (354~62) were measured by luciferase reporter gene assays in HepG2 cells transfected with pcDNA3.1-HBXIP and si-c-Myb. (E) Schematic diagram depicting the YAP promoter with c-Myb binding site was shown. The effect of HBXIP on the activities of YAP promoter of wild type (WT) or the mutant (MUT) (pGL3-293) was examined by luciferase reporter gene assays in HepG2 and 293 T cells. (F) The interaction of HBXIP with the promoter region of YAP was examined by ChIP assays in HepG2-HBXIP cells. (G) The interaction of HBXIP with c-Myb was examined by co-IP assays in HepG2-HBXIP cells. (H) The interaction between HBXIP and c-Myb binding element in the region of YAP promoter was examined by EMSA. Each experiment was repeated at least three times. Statistically significant differences are indicated: *P < 0.05, **P < 0.01, Student's t test.
Supplementary Fig. S2D-H), suggesting that the region 354/62 of YAP promoter might be responsible for the activation of YAP promoter mediated by HBXIP. To identify the mechanism by which HBXIP activates YAP, we analyzed the region of 354/62 of YAP promoter with the highest luciferase activities using PROMO [43,44] (http://alggen.lsi.upc.es/cgi-bin/promov3/promo/ promoinit.cgi?dirDB¼TF8.3), with a parameter 95% similarity. Interestingly, we observed that there were 25 potential transcription factors in the region, in which 8 transcription factors, including c-Myb, NF-AT2, c-Ets-2, Sp3, c-Ets-1, Elk-1, IRF-1, and NF-AT1, showed the RE query value less than 0.01 (Supplementary Table S4). Moreover, we analyzed the region of 61/þ115 of YAP promoter with the lowest luciferase activities. The data showed that there were 4 transcription factors, including c-Ets-2, c-Ets-1, Elk-1, and IRF-1, which were same with above transcription factors (Fig. 3C), suggesting that these 4 transcription factors can be excluded in activation of YAP promoter. It has been reported that overexpression of c-Myb contributes to malignant transformation by regulating genes participating in multiple aspects of
tumourigenesis [33e36]. Thus, we hypothesized that c-Myb as an oncogenic transcription factor might be involved in the activation of YAP promoter induced by HBXIP. As expected, the treatment with c-Myb siRNA could abrogate the augment of YAP promoter activities induced by HBXIP in HepG2 cells (Fig. 3D). Furthermore, luciferase reporter gene assays showed that HBXIP failed to work when the c-Myb binding site was mutated in HepG2, 293 T and H7402 cells (Fig. 3E and Supplementary Fig. S2B), suggesting that cMyb is required for the activation of YAP mediated by HBXIP in the cells. Moreover, Western blot analysis showed that the overexpression of c-Myb was able to up-regulate YAP in HepG2 cells (Supplementary Fig. S2C), supporting that the c-Myb as a transcriptional factor contributes to the up-regulation of YAP in the cells. Moreover, ChIP assays indicated that HBXIP was able to bind to the YAP promoter, but it was undetectable when the cells were treated with c-Myb siRNA (Fig. 3F). Co-immunoprecipitation assays validated that the endogenous c-Myb could interact with HBXIP in HepG2-HBXIP cells (Fig. 3G). Then, we performed electrophoresis mobility shift assays (EMSA) using the probe of DNA sequence
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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(193/155) from YAP promoter containing the binding site of cMyb. Our data revealed that HBXIP could bind to the c-Myb consensus sequence in YAP promoter, whereas the interaction could be abolished when the cold competitor, anti-c-Myb antibody or anti-HBXIP antibody was added into nuclear extracts of HepG2HBXIP cells (Fig. 3H), suggesting that HBXIP is able to bind to the core region of YAP promoter through interacting with the transcription factor c-Myb. According to the reports that CTGF, CYR61 and SOX2 were members of the YAP target genes [8,9], we evaluated the effect of c-Myb on HBXIP-induced expression of these genes in hepatoma cells. Interestingly, c-Myb siRNA was able to block the up-regulation of YAP, CTGF, CYR61 and SOX2 at mRNA level when HBXIP was over-expressed in HepG2 cells in a dosedependent manner (Supplementary Fig. 2D). Additionally,
overexpressed HBXIP did not mRNA levels (Supplementary HBXIP activates YAP promoter tion factor c-Myb in hepatoma
affect the expression of c-Myb at Fig. 2D). Thus, we conclude that through co-activating of transcripcells.
HBXIP contributes to the growth of hepatoma cells in vitro and in vivo through YAP To better understand the role of HBXIP-induced expression of YAP in the promotion of proliferation of hepatoma cells, we performed MTT assays, EdU incorporation assays and colony formation assays. Interestingly, our data revealed that YAP knockdown was able to block the HBXIP-enhanced proliferation of hepatoma cells (Fig. 4AeC). Additionally, we found that the silencing of c-Myb by
Fig. 4. HBXIP contributes to the proliferation of hepatoma cells in vitro through YAP. (AeC) The effect of HBXIP (or HBXIP plus YAP siRNA) on the proliferation of HepG2 cells was detected by MTT assays, EdU incorporation assays and colony formation assays, respectively. Each experiment was repeated at least three times. Statistically significant differences are indicated: **P < 0.01; Student's t test.
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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siRNA could restrict the HBXIP-induced proliferation of the cells (Supplementary Fig. S2E). Next, we used nude mouse models to verify our above findings in vivo. As expected, YAP siRNA remarkably abolished the HBXIP-promoted growth of hepatoma cells in mice (Fig. 5AeC), suggesting that YAP is implicated in the accelerated-proliferation of hepatoma cells induced by HBXIP. Meanwhile, the expression levels of HBXIP, YAP and CTGF in each group of tumor samples from mice were validated by Western blot analysis (Fig. 5D). Taken together, we conclude that HBXIP contributes to the growth of hepatoma cells in vitro and in vivo through YAP.
Discussion Hippo signaling plays pivotal roles in cell proliferation and organ size, and mounting studies have reported that this pathway is involved in the development of numerous cancers [1e4,45]. Our laboratory has reported that HBXIP, as an oncoprotein and a transcription co-activator, contributes to the development of breast cancer and liver cancer [26e30]. However, the effect of HBXIP on YAP in hepatocarcinogenesis is poorly understood. In this study, we investigated the role of HBXIP in regulation of YAP in HCC.
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To address the effect of HBXIP on YAP, we evaluated the relationship between HBXIP and YAP in clinical HCC tissues. Our data showed that both HBXIP and YAP were over-expressed in HCC tissues. Particularly, the expression levels of HBXIP were positively associated with those of YAP and its target gene, CTGF, in clinical HCC samples. It suggests that HBXIP is associated with YAP in the development of liver cancer. Next, our data revealed that HBXIP was able to up-regulate the expression of YAP in hepatoma cells. Meanwhile, we examined the expression of CTGF in hepatoma cells. Our data showed that HBXIP up-regulated the mRNA, protein and promoter levels of CTGF. It indicates that HBXIP is implicated in the modulation of YAP in liver cancer. Given that HBXIP acts as an oncogenic transcription co-activator to promote the development of cancer [26e30], we speculated that HBXIP might be involved in the regulation of YAP by stimulating its promoter. To address our hypothesis, we minimized the core region of YAP promoter. c-Myb is important for the continued proliferation of leukemia cells and c-Myb silencing is able to inhibit the growth of prostate cancer cells [46,47]. In addition, c-Myb mediates inflammatory reaction against oxidative stress in breast cancer [48]. Based on the bioinformatics analysis, we found that transcription factor c-Myb might be involved in the activation of YAP promoter mediated by HBXIP. As expected, our data showed that HBXIP was
Fig. 5. HBXIP contributes to the growth of hepatoma cells in vivo through YAP. (A, B) The growth curves and the average weights of tumors from nude mice transplanted with HepG2-pcDNA3.1 cells were shown. (C) The photographs of dissected tumors from nude mice were shown. (D) The expression levels of HBXIP, YAP and CTGF were examined by Western blot analysis in tumor tissues from mice. (E) A model shows the mechanism by which HBXIP up-regulates YAP by c-Myb in hepatoma cells. Statistically significant differences are indicated: **P < 0.01; ***P < 0.001; Student's t test.
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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able to interact with c-Myb to activate the promoter of YAP. It suggests that c-Myb is essential for the up-regulation of YAP induced by HBXIP. In terms of function, our data showed that HBXIP was able to facilitate the growth of hepatoma cells, whereas YAP siRNA significantly blocked the effect of HBXIP in vitro and in vivo. It suggests that HBXIP-mediated YAP expression contributes to hepatocarcinogenesis. Our finding is consistent with that YAP is a key factor in carcinogenesis [5e7,10e15]. Thus, we conclude that oncoprotein HBXIP up-regulates YAP through activating transcription factor c-Myb to promote the growth of liver cancer. Taken together, in this study we report a novel function of HBXIP in regulation of YAP through transcription factor c-Myb in HCC. We summarized a model showing the mechanism by which HBXIP upregulates YAP through activating c-Myb in hepatoma cells (Fig. 5E). Our finding provides new insights into the mechanism of HippoYAP signaling regulation mediated by HBXIP in hepatocarcinogenesis. Therapeutically, HBXIP and YAP may serve as new targets in liver cancer. Acknowledgements This work was supported by the grants of the National Basic Research Program of China (973 Program, No. 2015CB553905, 2015CB553703), the National Natural Scientific Foundation of China (Nos. 81372186, 81272218, 31470756, 31670771, 31670769), and Tianjin Natural Scientific Foundation (No. 14JCZDJC32800). Conflict of interest The authors declare that they have no competing interests.
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2016.10.018.
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Original Article
Q4 Q3
The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer Yue Wang a, 1, Runping Fang a, 1, Ming Cui b, Weiying Zhang a, Xiao Bai a, Huawei Wang a, Bowen Liu a, Xiaodong Zhang b, *, Lihong Ye a, ** a State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, People's Republic of China b State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 July 2016 Received in revised form 22 September 2016 Accepted 10 October 2016
The oncoprotein Yes-associated protein (YAP) in Hippo pathway plays crucial roles in the development of cancer. However, the mechanism of YAP regulation in cancer remains poorly understood. Here, we supposed that the oncoprotein hepatitis B X-interacting protein (HBXIP) might be involved in the modulation of YAP in liver cancer. Interestingly, our data showed that the expression levels of HBXIP were positively associated with those of YAP in clinical hepatocellular carcinoma (HCC) samples by immunohistochemistry (IHC) staining and real-time PCR assays. HBXIP was able to up-regulate YAP in hepatoma cells at the levels of promoter, mRNA and protein. Mechanistically, we identified that HBXIP up-regulated YAP through co-activating the transcription factor c-Myb in hepatoma cells. Functionally, silencing YAP abolished the proliferation of hepatoma cells mediated by HBXIP in vitro. Moreover, knockdown of YAP strongly blocked the HBXIP-enhanced tumor growth in mice. Thus, we conclude that HBXIP up-regulates YAP expression via activating transcription factor c-Myb to facilitate the growth of hepatoma cells. Our finding provides new insights into the mechanism of YAP regulation. Therapeutically, the oncoprotein HBXIP and YAP might serve as targets in liver cancer. © 2016 Published by Elsevier Ireland Ltd.
Keywords: HBXIP YAP c-Myb Proliferation Liver cancer
Introduction The dysregulation of Hippo pathway is wildly involved in the tumorigenesis [1e4], in which Yes-associated protein (YAP) is able to induce the expression of pro-proliferative and anti-apoptotic genes as a transcription co-activator in the complex of TEAD family [5e7]. CTGF, CYR61 and SOX2 serve as the downstream target genes of YAP [8,9]. A host of factors including cell density, extracellular matrix stiffness, G proteinecoupled receptors and leukemia inhibitory factor receptor can influence YAP activity through modulating the Hippo pathway. The heightened expression or genomic amplification of YAP is frequently observed in many cancers [10e15]. The highly expressed YAP is involved in multiple
* Corresponding author. Fax: þ86 22 23501385. ** Corresponding author. Fax: þ86 22 23501385. E-mail addresses: [email protected] (X. Zhang), [email protected] (L. Ye). 1 The authors contribute equally to the work.
process of liver disease including liver repair, regeneration, hepatocellular carcinoma (HCC) [16e18]. YAP can serve as an independent prognostic marker in HCC [19]. However, the mechanism of YAP regulation in liver cancer is poorly understood. Hepatitis B X-interacting protein (HBXIP), originally identified for interacting with hepatitis B virus X proteins, is a conserved ~18 kDa protein among mammalian species [20]. HBXIP acts as an oncoprotein in controlling cell proliferation, apoptosis and division [21e24]. HBXIP as a regulator component participates in amino acids-mediated mTORC1 activation [25]. Our group manifested that HBXIP could import into the nucleus of breast cancer cells, acting as a transcription co-activator of transcription factors such as Sp1, cAMP-response element binding protein (CREB), c-Myc and E2F1, to accelerate the progression of liver and breast cancer [26e30]. However, whether HBXIP is involved in the regulation of YAP in cancers is not well documented. The transcription factor c-Myb is encoded by the MYB protooncogene [31,32]. Overexpression of c-Myb contributes to malignant transformation by regulating genes participating in multiple
http://dx.doi.org/10.1016/j.canlet.2016.10.018 0304-3835/© 2016 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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processes of tumourigenesis, such as cell growth, angiogenesis and resistance to apoptosis [33e36]. Myb is able to affect tumor cell self-renewal through the Wnt signaling pathway [37]. Recent studies have shown that c-Myb plays an important role in the development of gastric cancer (GC) [38]. The target genes of c-Myb include SOX2, Bcl-2, Bax, c-Myc, and etc [39]. However, the role of c-Myb in the regulation of YAP remains unclear. In this study, we investigated the role of oncoprotein HBXIP in modulation of YAP in liver cancer. Interestingly, we found that HBXIP was able to up-regulate YAP through activating YAP promoter, involving co-activating c-Myb, to accelerate the growth of hepatoma cells. Our finding provides new insights into the mechanism by which HBXIP regulates YAP expression in the development of liver cancer. Materials and methods Patient samples Twenty-six hepatocellular carcinoma (HCC) tissue samples and their corresponding non-tumorous liver tissues were obtained from Tianjin First Center Hospital and Tianjin Tumor Hospital (Tianjin, China) after surgical resection. Written consents approving the use of tissue samples for research purposes were obtained from HCC patients. The study protocol was approved by the Institute Research Ethics Committee at Nankai University. The information of HCC patients was shown in Supplementary Table S1. Cell lines and cell culture The human hepatoma cell lines HepG2, HepG2-pcDNA3.1 (HepG2 cells stably transfected with pcDNA3.1) and HepG2-HBXIP (HepG2 cells stably transfected with pcDNA3.1-HBXIP) and human kidney epithelial (HEK) 293 T cells were maintained in Dulbecco's Modified Eagle's medium (Gibco, CA, USA). The human hepatoma H7402 cell line was cultured in RPMI-1640 medium (Gibco, CA, USA). All cell lines were supplemented with heat-inactivated 10% fetal bovine serum (FBS, Gibco, CA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin and grown at 5% CO2 and 37 C. Histological analysis and immunohistochemistry staining (IHC) Paraffin-embedded mouse liver tissue was cut into 5 mm sections, which were stained with hematoxylin-eosin for histological analysis. Immunohistochemical staining was performed as previously reported [30]. The primary antibodies of rabbit anti-HBXIP (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-YAP (Proteintech Group, USA) were used. The staining levels of HBXIP and YAP were classified into three groups using a modified scoring method based on the intensity of staining (0 ¼ negative; 1 ¼ low; 2 ¼ high) and the percentage of stained cells (0 ¼ 0% stained; 1 ¼ 1%e49% stained; 2 ¼ 50%e100% stained). A multiplied score (intensity score percentage score) lower than 1 was considered to be negative staining (), and 1e4 were considered to be positive staining (þ). Categorization of immunostaining intensity was performed by three independent observers. The information of the HCC patients and the staining level of HBXIP and YAP were listed in Supplementary Table S2. Construction of plasmids To construct the core region of YAP promoter, the region 354/þ115 and 354/ 62 of YAP were amplified by PCR from the pGL3-1536 and was inserted into the upstream of the pGL3-Basic vector (Promega, Madison, WI, USA) via KpnI and XhoI sites to generate YAPluc (354~þ115) and YAPluc (354~62). A mutant of YAPluc (354~62), which carried a substitution of three nucleotides (GGTT instead of wild-type AACT) within the binding site of c-Myb, was constructed by using overlapping extension PCR. According to the report [40], the 5'-flanking region of CTGF was amplified by PCR from the genomic DNA of HepG2 cells using specific primers and was cloned into the upstream of the pGL3-Basic vector via KpnI and HindⅢ sites. The resulting plasmid was sequenced and named CTGFluc. To construct the pcDNA3.1-c-Myb, the CDS of c-Myb were amplified by PCR and was inserted into the pcDNA3.1 (þ) vector (Invitrogen, CA, USA) via Hind III and Xho I sites. All primers used were listed in Supplementary Table S3. Chromatin immunoprecipitation (ChIP) ChIP assays were performed in HepG2-HBXIP cells transfected siRNA control (siControl) or c-Myb siRNA (si-c-Myb) according to the manufacturer's protocol (Epigentek Group Inc, Brooklyn, NY) as reported previously [28]. The fragment of YAP promoter including the c-Myb sites was amplified from the immunoprecipitated DNA samples with the specific primers. All primers are listed in Supplementary Table S3.
Analysis of cell proliferation HepG2 or H7402 cells were seeded into 96-well plates (1000 cells/well) for 24 h before transfection. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay or 5-ethynyl-20 -deoxyuridine (EdU) incorporation assay was performed to access the proliferation of cells as described previously. For colony formation assay, 48 h after transfection, about 500 cells were placed into 6-well plates and maintained in complete medium for 2 weeks. Colonies were fixed with methanol and stained with methylene blue. Tumor xenograft in mice BALB/c athymic nude mice (Experiment Animal Center of Peking, China) were housed and treated according to guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. HepG2-pcDNA3.1 or HepG2-HBXIP cells with the transfection of si-Control or si-YAP were harvested and re-suspended at 2 107 per ml with sterile phosphate-buffered saline. Groups of 4week-old female BALB/c athymic nude mice (each group, n ¼ 6) were subcutaneously injected at the shoulder with 0.2 ml of the cell suspensions. Tumor growth was measured after 10 days from injection and then every 5 days. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L W2) 0.5. After 30 days, the mice were sacrificed, and the tumors were excised and measured. Statistical analysis Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (±SD) using a Student's t test for independent groups and was assumed for *P < 0.05 and **P < 0.01; and ***P < 0.005. Pearson's correlation coefficient was used to determine the correlation between YAP mRNA levels and HBXIP mRNA levels in HCC tissues.
Results The expression of HBXIP is positively associated with that of YAP in clinical HCC tissues Our laboratory has reported that the oncoprotein HBXIP contributes to the progression of cancer through acting as a coactivator of transcription factors [26e30]. Lots of studies have proven that the highly expressed YAP plays a crucial role in the development of cancer [10e15]. Accordingly, we supposed that HBXIP might be involved in the regulation of Hippo-YAP signaling. Immunohistochemistry (IHC) staining analysis exhibited that the positive rate of HBXIP was 100% (40/40) in HCC tissues, in which the positive rate of YAP was 77.5% (31/40) in HBXIP-positive HCC tissues using tissue microarray (Fig. 1A). Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression levels of HBXIP were positively related to those of YAP in 26 cases of clinical HCC tissues (r ¼ 0.6178, P < 0.001, Pearson's correlation, Fig. 1B). Meanwhile, we verified the correlation between YAP and HBXIP using 6 paired HCC tissues by Western blot analysis (Fig. 1C), These results suggest that the expression of HBXIP is positively associated with that of YAP in clinical HCC tissues. In addition, we evaluated the relationship between HBXIP and CTGF, a target gene of YAP, in clinical HCC tissues. Our data indicated that the expression levels of HBXIP were positively correlated with those of CTGF in 26 cases (Fig. 1D). Thus, we conclude that HBXIP is positively associated with YAP in clinical HCC tissues. HBXIP up-regulates YAP expression in hepatoma cells Next, we tried to evaluate the effect of HBXIP on YAP in hepatoma cells. Interestingly, we showed that HBXIP was capable of upregulating YAP at the levels of mRNA and protein in hepatoma HepG2 and H7402 cells in a dose-dependent manner (Fig. 2A and Supplementary Fig. S1A), and silencing HBXIP by siRNA significantly decreased YAP at the levels of mRNA and protein in HepG2HBXIP cells in a dose-dependent manner (Fig. 2B), suggesting that HBXIP is able to up-regulate YAP in hepatoma cells. Interestingly, we observed that HBXIP enhanced the expression and nuclear
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Fig. 1. The expression of HBXIP is positively associated with that of YAP in clinical HCC tissues. (A) The expression levels of HBXIP and YAP were examined by IHC staining analysis in clinical HCC tissues and normal tissues. (B) The correlation between HBXIP mRNA levels and YAP mRNA levels was detected by qRT-PCR analysis in 26 cases of clinical HCC samples. (C) The correlation between HBXIP protein levels and YAP protein levels was examined by Western blot analysis in 6 paired HCC clinical tissues. T, tumor; N, normal tissue. (D) The correlation between HBXIP mRNA levels and CTGF mRNA levels was detected by qRT-PCR analysis in 26 cases of clinical HCC samples. Statistically significant differences are indicated: ***P < 0.001; Student's t test.
import of YAP in HepG2-HBXIP cells by immunofluorescent staining (Fig. 2C). It has been reported that the phosphorylation of YAP causes cytoplasmic retention, and results in inactivating its gene regulation ability [41]. Western blot analysis showed that overexpressed HBXIP decreased the phosphorylation levels of YAP in hepatoma cells (Supplementary Fig. S1B), further supporting that HBXIP is able to increase the nuclear import of YAP via inhibiting YAP phosphorylation. We have reported that HBXIP as a coactivator regulates the gene transcription through interacting with transcription factors [26e30]. Accordingly, we verified the effect of HBXIP on the promoter region of YAP (1420/þ115, pGL3-
1536) [42]. Luciferase reporter gene assays demonstrated that HBXIP was able to increase the activities of pGL3-1536 in a dosedependent manner in HepG2 and H7402 cells (Fig. 2D and Supplementary Fig. S1C), whereas the activities of pGL3-1536 were decreased by the treatment with HBXIP siRNA in HepG2-HBXIP cells (Fig. 2E), suggesting that HBXIP up-regulates the expression of YAP at the transcription level in hepatoma cells. We further validated the effect of HBXIP on YAP using the Gal4-TEAD1 system. Interestingly, we observed that the co-transfection of HBXIP and Gal4-TEAD1 increased the luciferase activities, while cotransfection of si-HBXIP and Gal4-TEAD1 decreased the luciferase
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Fig. 2. HBXIP up-regulates YAP expression in hepatoma cells. (A, B) The effect of HBXIP (or HBXIP siRNA) on the expression of YAP was measured by RT-PCR analysis and Western blot analysis at the levels of mRNA and protein in HepG2 (or HepG2-HBXIP) cells, respectively. (C) The effect of HBXIP on the expression and localization of YAP was detected by laser confocal microscope in HepG2-HBXIP cells. Scale bar, 20 mm. (D, E) The effect of HBXIP (or HBXIP siRNA) on the activities of YAP promoter was examined by luciferase reporter gene assays in HepG2 (or HepG2-HBXIP) cells, respectively. (F) The effect of HBXIP (or HBXIP siRNA) on the activities of YAP was examined by Gal4-TEAD1 reporter system. Each experiment was repeated at least three times. Statistically significant differences are indicated: *P < 0.05, **P < 0.01; Student's t test.
activities (Fig. 2F), suggesting that the HBXIP is able to stimulate activities of YAP in the cells. It has been reported that CTGF is a classic downstream gene of YAP [40]. To further validate that HBXIP up-regulates YAP, we examined the effect of HBXIP on CTGF in hepatoma cells. As expected, we observed that HBXIP was able to up-regulate CTGF at the levels of mRNA, protein and promoter in HepG2 and H7402 cells in a dose-dependent manner, whereas silencing of HBXIP by HBXIP siRNA led to the down-regulation of CTGF in HepG2-HBXIP cells in a dose-dependent manner (Supplementary Fig. S1D-H), supporting that HBXIP up-regulates YAP in the cells. Thus, we conclude that HBXIP is able to upregulate the expression of YAP in hepatoma cells.
HBXIP stimulates YAP promoter through co-activation of transcription factor c-Myb We have reported that the core region of YAP promoter locates at the region 354/þ115 in hepatoma cells [42]. To identify the mechanism by which HBXIP up-regulates YAP, we further minimized the core region of YAP promoter. Luciferase reporter gene assays revealed that pGL3-293 (354/62) exhibited the maximum luciferase activities and pGL3-Basic (61/þ115) exhibited the minimum luciferase activities (Fig. 3A). We further observed that HBXIP was able to increase the activities of pGL3-293 in HepG2 and H7402 cells in a dose-dependent manner (Fig. 3B and
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Fig. 3. HBXIP stimulates YAP promoter through co-activation of transcription factor c-Myb. (A) The activities of different fragments of YAP promoter (pGL3-1536, pGL3-470 or pGL3293) were measured by luciferase reporter gene assays in HepG2 cells. (B) The activities of pGL3-293 (354~62) were measured by luciferase reporter gene assays in HepG2 cells transfected with pcDNA3.1-HBXIP. (C) Schematic diagram depicting the potential transcription factors in the promoter region 354~62 and 61~þ115 of YAP by bioinformatics. (D) The activities of pGL3-293 (354~62) were measured by luciferase reporter gene assays in HepG2 cells transfected with pcDNA3.1-HBXIP and si-c-Myb. (E) Schematic diagram depicting the YAP promoter with c-Myb binding site was shown. The effect of HBXIP on the activities of YAP promoter of wild type (WT) or the mutant (MUT) (pGL3-293) was examined by luciferase reporter gene assays in HepG2 and 293 T cells. (F) The interaction of HBXIP with the promoter region of YAP was examined by ChIP assays in HepG2-HBXIP cells. (G) The interaction of HBXIP with c-Myb was examined by co-IP assays in HepG2-HBXIP cells. (H) The interaction between HBXIP and c-Myb binding element in the region of YAP promoter was examined by EMSA. Each experiment was repeated at least three times. Statistically significant differences are indicated: *P < 0.05, **P < 0.01, Student's t test.
Supplementary Fig. S2D-H), suggesting that the region 354/62 of YAP promoter might be responsible for the activation of YAP promoter mediated by HBXIP. To identify the mechanism by which HBXIP activates YAP, we analyzed the region of 354/62 of YAP promoter with the highest luciferase activities using PROMO [43,44] (http://alggen.lsi.upc.es/cgi-bin/promov3/promo/ promoinit.cgi?dirDB¼TF8.3), with a parameter 95% similarity. Interestingly, we observed that there were 25 potential transcription factors in the region, in which 8 transcription factors, including c-Myb, NF-AT2, c-Ets-2, Sp3, c-Ets-1, Elk-1, IRF-1, and NF-AT1, showed the RE query value less than 0.01 (Supplementary Table S4). Moreover, we analyzed the region of 61/þ115 of YAP promoter with the lowest luciferase activities. The data showed that there were 4 transcription factors, including c-Ets-2, c-Ets-1, Elk-1, and IRF-1, which were same with above transcription factors (Fig. 3C), suggesting that these 4 transcription factors can be excluded in activation of YAP promoter. It has been reported that overexpression of c-Myb contributes to malignant transformation by regulating genes participating in multiple aspects of
tumourigenesis [33e36]. Thus, we hypothesized that c-Myb as an oncogenic transcription factor might be involved in the activation of YAP promoter induced by HBXIP. As expected, the treatment with c-Myb siRNA could abrogate the augment of YAP promoter activities induced by HBXIP in HepG2 cells (Fig. 3D). Furthermore, luciferase reporter gene assays showed that HBXIP failed to work when the c-Myb binding site was mutated in HepG2, 293 T and H7402 cells (Fig. 3E and Supplementary Fig. S2B), suggesting that cMyb is required for the activation of YAP mediated by HBXIP in the cells. Moreover, Western blot analysis showed that the overexpression of c-Myb was able to up-regulate YAP in HepG2 cells (Supplementary Fig. S2C), supporting that the c-Myb as a transcriptional factor contributes to the up-regulation of YAP in the cells. Moreover, ChIP assays indicated that HBXIP was able to bind to the YAP promoter, but it was undetectable when the cells were treated with c-Myb siRNA (Fig. 3F). Co-immunoprecipitation assays validated that the endogenous c-Myb could interact with HBXIP in HepG2-HBXIP cells (Fig. 3G). Then, we performed electrophoresis mobility shift assays (EMSA) using the probe of DNA sequence
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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(193/155) from YAP promoter containing the binding site of cMyb. Our data revealed that HBXIP could bind to the c-Myb consensus sequence in YAP promoter, whereas the interaction could be abolished when the cold competitor, anti-c-Myb antibody or anti-HBXIP antibody was added into nuclear extracts of HepG2HBXIP cells (Fig. 3H), suggesting that HBXIP is able to bind to the core region of YAP promoter through interacting with the transcription factor c-Myb. According to the reports that CTGF, CYR61 and SOX2 were members of the YAP target genes [8,9], we evaluated the effect of c-Myb on HBXIP-induced expression of these genes in hepatoma cells. Interestingly, c-Myb siRNA was able to block the up-regulation of YAP, CTGF, CYR61 and SOX2 at mRNA level when HBXIP was over-expressed in HepG2 cells in a dosedependent manner (Supplementary Fig. 2D). Additionally,
overexpressed HBXIP did not mRNA levels (Supplementary HBXIP activates YAP promoter tion factor c-Myb in hepatoma
affect the expression of c-Myb at Fig. 2D). Thus, we conclude that through co-activating of transcripcells.
HBXIP contributes to the growth of hepatoma cells in vitro and in vivo through YAP To better understand the role of HBXIP-induced expression of YAP in the promotion of proliferation of hepatoma cells, we performed MTT assays, EdU incorporation assays and colony formation assays. Interestingly, our data revealed that YAP knockdown was able to block the HBXIP-enhanced proliferation of hepatoma cells (Fig. 4AeC). Additionally, we found that the silencing of c-Myb by
Fig. 4. HBXIP contributes to the proliferation of hepatoma cells in vitro through YAP. (AeC) The effect of HBXIP (or HBXIP plus YAP siRNA) on the proliferation of HepG2 cells was detected by MTT assays, EdU incorporation assays and colony formation assays, respectively. Each experiment was repeated at least three times. Statistically significant differences are indicated: **P < 0.01; Student's t test.
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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siRNA could restrict the HBXIP-induced proliferation of the cells (Supplementary Fig. S2E). Next, we used nude mouse models to verify our above findings in vivo. As expected, YAP siRNA remarkably abolished the HBXIP-promoted growth of hepatoma cells in mice (Fig. 5AeC), suggesting that YAP is implicated in the accelerated-proliferation of hepatoma cells induced by HBXIP. Meanwhile, the expression levels of HBXIP, YAP and CTGF in each group of tumor samples from mice were validated by Western blot analysis (Fig. 5D). Taken together, we conclude that HBXIP contributes to the growth of hepatoma cells in vitro and in vivo through YAP.
Discussion Hippo signaling plays pivotal roles in cell proliferation and organ size, and mounting studies have reported that this pathway is involved in the development of numerous cancers [1e4,45]. Our laboratory has reported that HBXIP, as an oncoprotein and a transcription co-activator, contributes to the development of breast cancer and liver cancer [26e30]. However, the effect of HBXIP on YAP in hepatocarcinogenesis is poorly understood. In this study, we investigated the role of HBXIP in regulation of YAP in HCC.
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To address the effect of HBXIP on YAP, we evaluated the relationship between HBXIP and YAP in clinical HCC tissues. Our data showed that both HBXIP and YAP were over-expressed in HCC tissues. Particularly, the expression levels of HBXIP were positively associated with those of YAP and its target gene, CTGF, in clinical HCC samples. It suggests that HBXIP is associated with YAP in the development of liver cancer. Next, our data revealed that HBXIP was able to up-regulate the expression of YAP in hepatoma cells. Meanwhile, we examined the expression of CTGF in hepatoma cells. Our data showed that HBXIP up-regulated the mRNA, protein and promoter levels of CTGF. It indicates that HBXIP is implicated in the modulation of YAP in liver cancer. Given that HBXIP acts as an oncogenic transcription co-activator to promote the development of cancer [26e30], we speculated that HBXIP might be involved in the regulation of YAP by stimulating its promoter. To address our hypothesis, we minimized the core region of YAP promoter. c-Myb is important for the continued proliferation of leukemia cells and c-Myb silencing is able to inhibit the growth of prostate cancer cells [46,47]. In addition, c-Myb mediates inflammatory reaction against oxidative stress in breast cancer [48]. Based on the bioinformatics analysis, we found that transcription factor c-Myb might be involved in the activation of YAP promoter mediated by HBXIP. As expected, our data showed that HBXIP was
Fig. 5. HBXIP contributes to the growth of hepatoma cells in vivo through YAP. (A, B) The growth curves and the average weights of tumors from nude mice transplanted with HepG2-pcDNA3.1 cells were shown. (C) The photographs of dissected tumors from nude mice were shown. (D) The expression levels of HBXIP, YAP and CTGF were examined by Western blot analysis in tumor tissues from mice. (E) A model shows the mechanism by which HBXIP up-regulates YAP by c-Myb in hepatoma cells. Statistically significant differences are indicated: **P < 0.01; ***P < 0.001; Student's t test.
Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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able to interact with c-Myb to activate the promoter of YAP. It suggests that c-Myb is essential for the up-regulation of YAP induced by HBXIP. In terms of function, our data showed that HBXIP was able to facilitate the growth of hepatoma cells, whereas YAP siRNA significantly blocked the effect of HBXIP in vitro and in vivo. It suggests that HBXIP-mediated YAP expression contributes to hepatocarcinogenesis. Our finding is consistent with that YAP is a key factor in carcinogenesis [5e7,10e15]. Thus, we conclude that oncoprotein HBXIP up-regulates YAP through activating transcription factor c-Myb to promote the growth of liver cancer. Taken together, in this study we report a novel function of HBXIP in regulation of YAP through transcription factor c-Myb in HCC. We summarized a model showing the mechanism by which HBXIP upregulates YAP through activating c-Myb in hepatoma cells (Fig. 5E). Our finding provides new insights into the mechanism of HippoYAP signaling regulation mediated by HBXIP in hepatocarcinogenesis. Therapeutically, HBXIP and YAP may serve as new targets in liver cancer. Acknowledgements This work was supported by the grants of the National Basic Research Program of China (973 Program, No. 2015CB553905, 2015CB553703), the National Natural Scientific Foundation of China (Nos. 81372186, 81272218, 31470756, 31670771, 31670769), and Tianjin Natural Scientific Foundation (No. 14JCZDJC32800). Conflict of interest The authors declare that they have no competing interests.
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2016.10.018.
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Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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Please cite this article in press as: Y. Wang, et al., The oncoprotein HBXIP up-regulates YAP through activation of transcription factor c-Myb to promote growth of liver cancer, Cancer Letters (2016), http://dx.doi.org/10.1016/j.canlet.2016.10.018
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