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Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells Xuquan Li 1, Tao Chen 1, Qiang Shi 1, Jian Li, Shilun Cai, Pinghong Zhou, Yunshi Zhong**, Liqing Yao* Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital of Fudan University, Shanghai 200032, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2015 Accepted 19 September 2015 Available online xxx
Angiopoietin-like 4 (ANGPTL4), a secretory glycoprotein, plays an important role in cancer metastasis. In the present study, we aim to investigate the roles and mechanisms of ANGPTL4 in the regulation of colorectal cancer metastasis. We found that expression level of ANGPTL4 was increased in colorectal cancer tissues, compared with that in normal tissues. Moreover, liver metastasis was significantly associated with higher expression of ANGPTL4. In vitro studies further showed that overexpression of ANGPTL4 enhanced cell migration, invasion and inhibited apoptosis. At the molecular level, ANGPTL4 overexpression resulted in an up-regulation of bone morphogenetic protein 7 (BMP7). Indeed, knockdown of BMP7 by small interfering RNA (siRNA) oligos reversed the roles of ANGPTL4 overexpression in HCT116 cells. Finally, in vivo studies further confirmed the metastatic roles of ANGPTL4 by inducing BMP7. Therefore, our study demonstrated that ANGPTL4 might promote metastasis and might inhibit apoptosis of colorectal cancer cells by up-regulation of BMP7. © 2015 Elsevier Inc. All rights reserved.
Keywords: ANGPTL4 BMP7 Metastasis Apoptosis Colorectal cancer
1. Introduction Colorectal cancer (CRC) has become one of the most common malignancy and leading cause of cancer-related death worldwide [1]. Liver metastasis is one of the leading causes of death in patients with colorectal cancer, but its mechanism remains poorly understood. Angiopoietin-like protein 4 (ANGPTL4), a secreted protein, exerts multifunctional roles in many aspects of tumorigenesis, such as motility, metastasis, apoptosis, angiogenesis, tumormicroenvironment, wound healing, vascular permeability and inflammation [2e7]. ANGPTL4 was up-regulated and could promote cancer occurrence and development in glioblastoma, breast cancer, oral Kaposi's sarcoma and clear cell renal-cell carcinoma [8e10]. ANGPTL4 can disrupt vascular endothelial cellecell
* Corresponding author. 180 Fenglin Road, Xuhui District, 200032 Shanghai, PR China. ** Corresponding author. 180 Fenglin Road, Xuhui District, 200032 Shanghai, PR China. E-mail addresses: [email protected] (Y. Zhong), yao.liqing@zs-hospital. sh.cn (L. Yao). 1 Equal contributors.
junctions and primes breast cancer cells for metastasis to the lungs by TGF-b/smad4 complex-induced expression [9]. The transcription factor c-Myc can bind to the promoter sequence on ANGPTL4 that markedly accelerates malignant glioma tumor growth [10]. However, ANGPTL4 could also inhibit metastasis in melanoma cancer, prostate cancer, and hepatocellular carcinoma [7,11,12]. In the human hepatocellular carcinoma (HCC), ANGPTL4 was hypoxiainducible factor 1a (HIF-1a) not HIF-2a directly up-regulated in hypoxia. ANGPTL4 promoted HUVECs cell transendothelial migration through the VCAM-1/integrin b1 axis [13]. Therefore, although the reason for the inconsistence remains largely unknown, the precise roles of ANGPTL4 might be cell or tissue-specific, which may rely on cellular microenvironments. ANGPTL4 is highly expressed in human CRC tissues. Upregulation of ANGPTL4 has been observed in CRC patients and may enhance cell invasion and migration [14]. However, to date, molecular mechanism study of ANGPTLl4 is relatively less, especially in colorectal cancer. Therefore, the expression and roles of ANGPTL4 in CRC metastasis remain need further studies. In the present study, we aimed to explore the expression and roles of ANGPTL4 in CRC metastasis. Moreover, we analyzed downstream of the molecular mechanisms of ANGPTL4 in CRC cell lines.
http://dx.doi.org/10.1016/j.bbrc.2015.09.104 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
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X. Li et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
2. Materials and methods 2.1. Human samples collection Fifty-four colon cancer tissues and 24 adjacent normal tissues were collected from routine therapeutic surgery, immediately snapfrozen in the nitrogen and stored at 80 C. The patient’ clinicopathological data were collected (Table 1). All patients were tested by computer tomography (CT) and B-type ultrasonic inspection before surgical operation. This project was approved by the institutional review board of the Zhongshan Hospital of Fudan University. 2.2. Cell and reagents The human CRC cell lines HCT116 and SW480 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Cells were grown in McCoy's 5A medium and DMEM (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (SigmaeAldrich,St. Louis, MO, USA) and maintained at 37 C in a humidified atmosphere with 5% CO2. Recombinant human BMP7 and ANGPTL4 were obtained from HumanZyme Company (USA) and Sion Biological Inc (China). 2.3. Lentivirus, siRNA and transfection HCT116 cells were transfected with Lentivirus (Genechem, Shanghai, China) containing empty vector or ANGPTL4. RNAimediated depletion of BMP7 was achieved by transfecting cells with small interfering RNA (siRNA) oligos targeting BMP7 or negative controls (GenePharma, Shanghai, China), using Lipofectamine 2000 reagents (Invitrogen,USA) according to the manufacturer's instructions. All the sequences of siRNA oligos were listed in the Supplementary Table S1. 2.4. Histology and immunohistochemistry All the tissues were fixed overnight in formalin solution, Table 1 Clinical characteristics of 54 CRC patients. Characteristics Age <60 60 Gender Male Female Tumor volume 5 >5 Invasion depth M S Differentiation Well Moderate Poor Lymph metastasis Yes No Liver metastasis Yes No Duke time AB CD
No.
Low expression of High expression of P value ANGPTL4 n (%) ANGPTL4 n (%)
26 28
7 (26.9) 6 (21.4)
19 (73.1) 22 (78.6)
0.748
25 29
6 (24.0) 7 (24.1)
19 (76.0) 22 (75.9)
0.519
31 23
9 (29.0) 4 (17.4)
22 (71.0) 19 (82.6)
0.649
10 44
3 (30.0) 10 (22.7)
7 (70.0) 34 (77.3)
0.218
17 20 17
4 (23.5) 5 (25.0) 4 (23.5)
13 (76.5) 15 (75.0) 13 (76.5)
0.869
29 25
6 (20.7) 7 (28.0)
23 (79.3) 18 (72.0)
0.176
11 43
1 (9.1) 12 (27.9)
10 (90.9) 31 (72.1)
0.028*
25 29
7 (28.0) 6 (20.7)
18 (72.0) 23 (79.3)
0.176
dehydrated in ethanol, embedded in paraffin and sectioned at 5 mm. Immunohistochemical staining were performed according to a standard protocol. The staining intensity was rated according to a previous study [8]. The expression of the ANGPTL4 protein with an overall score of ‘/þ ’ was designated as ‘low,’ and with an overall score of ‘þþ/þþþ’ was designated as ‘high.’ The following primary antibodies were used: anti-ANGPTL4 antibody (diluted 1:50; Santa Cruz Biotechnology, CA, USA), anti-BMP7 antibody (diluted 1:50; Abgent, USA). 2.5. RNA isolation and quantitative real-time PCR Total RNA was extracted using TRIzol (Invitrogen, USA) and reversely transcribed to cDNA using the First-Strand Synthesis System (invitrogen, USA). Quantitative real-time PCR was performed as described using SYBR Green kit (Yeasen, China) in a 7500 Real-Time PCR system (Applied Biosystems), according to the manufacturer's instructions. Relative transcript quantities were calculated using the 2DDCt method with GAPDH as the endogenous reference gene. Primer sequences are listed in the Supplementary Table S2. 2.6. Western blotting Cells were harvested and lysed with RIPA (Thermo, USA) containing protease inhibitors (Invitrogen, USA). After centrifugation, proteins in the supernatants were quantified, separated by 8e12% SDS PAGE and transferred onto polyvinyl difluoride (PVDF) membrane. Proteins were incubated by primary antibody over night at 4 C and then incubated with secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. The antibody information is listed as follows: anti-ANGPTL4 (1:500, Santa Cruz Biotechnology, CA, USA), anti-BMP7 (1:1000, Santa Cruz Biotechnology, CA, USA), anti-GAPDH (1:1000, Abgent, USA), anti-Bcl-xl (1:1000, CST, USA), anti-Bax (1:1000, CST, USA), anti-Vimentin (1:1000, Gene Tax, Germany), anti-E-cadherin (1:1000, CST, USA). The proteins were visualized by an ECL chemiluminescence detection kit (Thermo scientific, USA). 2.7. Cell migration and invasion assay Cells (1.0 105) were subjected to Transwell with 8.0 mm polycarbonate membrane (Corning, USA) to analyze cell migration and invasion. After incubation at 37 C for 24 h, cells of inside Transwell were removed, the outside cells were dyed by Giemsa Stain (Nanjing JianCheng Bioengineering Institute, China)for 6e10 min and cleaned by PBS. All the migration and invasion assays were performed in triplicate in at least three independent experiments. 2.8. Flow cytometry
The significance of bold and asterisk stands for P value.
1 105 cells were added into 6-well plate, and cultured at 37 C 5% CO2 for 48 h. Cells were trypsinized without EDTA and washed twice with PBS. Subsequently, 50 mL of 1 binding buffer and 5 mL of 7-AAD (KeyGEN, China) were added into the pellet and incubated at room temperature (25 C) for 10 min in the dark. Then, 450 mL of 1 binding buffer and 1 mL of Annexin V-PE (KeyGEN, China) were added into the pellet and incubated at room temperature (25 C) for 10 min in the dark to measurement using FACS calibur flowcytometer and Cell Quest Pro software (BD Biosciences, USA). The data were analyzed using FlowJo 7.6 software and displayed in dot plot of 7-AAD (Y-axis) against Annexin V-PE (X-axis). 2.9. Liver metastasis model To examine whether ANGPTL4 expression in colon cancer cells
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
X. Li et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
affects metastasis, ANGPTL4-or empty vector-expressing HCT116 cells (1.0 105 cells/mouse) were injected into 6-week old female nude mice via intrasplenic injection. Mice were sacrificed 4 weeks after injection and examined for metastases development. Animal studies were carried out according to the Guideline for Animal Experiments, drawn up by the Committee for Ethics in Animal Experimentation of the National Cancer Center, which meet the ethical standards required by law and the guidelines about experimental animals in China.
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compared with those in normal tissues (P < 0.001) (Fig. 1B). There were 11 cases (19.6%) showing a higher level of ANGPTL4 expression in tumor tissues. Moreover, the higher expression of ANGPTL4 was significantly associated with liver metastasis (P ¼ 0.028) (Table 1). 3.2. ANGPTL4 up-regulates BMP7 expression
3. Results
To test whether ANGPTL4 could regulate BMP7, HCT116 cells were transfected with lentivirus containing empty vector or ANGPTL4 (A4-OVER) (Fig. 1C and E). As expected, ANGPTL4 overexpression led to an increased mRNA and protein expression of BMP7 (Fig. 1DeE). However, treatment of HCT116 cells with recombinant BMP7 (25 ng/ml) proteins did not affect ANGPTL4 expression (Fig. 1E). BMP7 expression were increased gradually when HCT116 and SW480 were co-cultured with recombinant human-ANGPTL4 protein from 150 ng/ml to 450 ng/ml for 60 h (Fig. 1F), suggesting that ANGPTL4 may be an up-stream regulator of BMP7 in CRC cells.
3.1. Up-regulation of ANGPTL4 in CRC tissues
3.3. ANGPTL4 inhibits cell apoptosis in HCT116 cells
To investigate the roles of ANGPTL4 in CRC pathogenesis, its protein expression was analyzed by immunohistochemistry using 54 CRC and 24 adjacent normal specimens. As shown in Fig. 1A, expression levels of ANGPTL4 are significantly up-regulated in CRC tissues,
We found that the apoptotic rate of HCT116 cells overexpressing ANGPTL4 (about 3.79%) was reduced compared to the control group (about 11.23%) (Fig. 2A). mRNA and protein levels of Bax, a proapoptotic marker [15], were significantly reduced in HCT116 cells
2.10. Statistical analysis Data were expressed as mean ± SD of at least three independent experiments, and statistical analysis for single comparison was performed using the SPSS 17.0. The criterion for statistical significance was *p < 0.05, **p < 0.01, ***P < 0.001.
Fig. 1. Up-regulation of ANGPTL4 in CRC tissues and ANGPTL4 regulates BMP7 expression in HCT116 cells. (A) Representative immunohistochemistry staining of ANGPTL4 in cancer with metastasis (Metastasis cancer), cancer without metastasis (No metastasis cancer), and adjacent normal tissues (Normal). (B) The average immunostaining scores of ANGPTL4 expression in 11 cancer with metastasis, 43 cancer without metastasis and 24 normal tissues. (CeD) Relative mRNA levels of ANGPTL4 (C) and BMP7 (D) in HCT116 cells transfected with lentivirus expressing ANGPTL4 (A4-OVER) or empty vector (Con). (E) Representative protein levels of ANGPTL4 and BMP7 in HCT116 cells transfected with A4-OVER or treated with recombinant BMP7 protein. (F) BMP7 expression were increased gradually when HCT116 and SW480 were co-cultured with different concentration of recombinant humanANGPTL4 protein for 60 h. *p < 0.05, ***p < 0.001.
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
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Fig. 2. ANGPTL4 inhibits cell apoptosis by inducing BMP7. (A) Apoptosis was determined in HCT116 cells. Cells were transfected with A4-OVER or empty vector. (B) Transfection efficiency was tested after transfecting siRNA oligos targeting BMP7 (B7RNAi) in HCT116 cells with overexpressed ANGPTL4. (C) Apoptosis was determined in HCT116 cells. Cells were transfected with A4-OVER plus siRNA oligos targeting BMP7 (B7RNAi). (DeF) Relative mRNA (DeE) and representative protein (F) levels of Bax and BCL-xL in HCT116 cells. *p < 0.05, ***p < 0.001.
with ANGPTL4 overexpression (Fig. 2D and F). Besides, expression levels of Bcl-xl, an apoptotic inhibitor [15], were increased (Fig. 2D and F). To confirm the relationship between ANGPTL4 and BMP7, HCT116 cells were administrated with siRNA oligos targeting BMP7 (B7RNAi) or negative control (A4(OE)-Con), after transfection of ANGPTL4 (Fig. 2B). As expected, knockdown of BMP7 significantly reversed the anti-apoptotic roles of ANGPTL4 (Fig. 2C). Besides, the changes in the mRNA and protein levels of Bax and Bcl-xl were attenuated by B7RNAi (Fig. 2EeF).
affect the metastatic potential and growth of the cancer cells in vivo. For this purpose, HCT116 cells were stably transfected with ANGPTL4 or empty vector and equally were introduced into the nude mice via intrasplenic injection. After the inoculation of tumor cells, mice were sacrificed for the examination of metastatic growth in the liver. As a result, more tumor nodules were found in the livers from A4-OVER group than control group (Fig. 4AeB), with much higher expression of ANGPTL4 (Fig. 4C) and BMP7 (Fig. 4D), suggesting that ANGPTL4 could also promote tumor metastasis by BMP7 in vivo.
3.4. ANGPTL4 promotes migration and invasion of HCT116 cells
4. Discussion
Moreover, we found that overexpression of ANGPTL4 significantly enhanced cell migration and invasion abilities (Fig. 3A). In agreement, knockdown of BMP7 significantly blocked the roles of ANGPTL4 to promote cell migration and invasion (Fig. 3B). At the molecular level, expression levels of E-cadherin were significantly reduced, while Vimentin were increased in HCT116 cells with ANGPTL4 overexpression (Fig. 3C and E). However, these changes were largely blocked by B7RNAi (Fig. 3DeE).
Cancer metastasis is a complicated process involving several sequential steps, including intravasation into the blood, survival in the circulation systems, extravasation into a new organ, initiation and maintenance of growth [16,17]. ANGPTL4 plays multiple important roles in the occurrence and progression of different cancer. However, the molecular events of ANGPTL4 that are directly involved in the colorectal cancer metastatic process remains poorly understood. In the present study, our data showed that ANGPTL4 could be a novel and important regulator of cell metastasis in CRC. These are supported by multiple lines of evidence. ANGPTL4 expression was up-regulated in CRC tissues and higher expression of ANGPTL4 was significantly associated with liver metastasis. Overexpression of
3.5. Overexpression of ANGPTL4 in colorectal cancer cells potently promote metastatic tumor growth in the liver We next examined whether ANGPTL4 overexpression could
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
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Fig. 3. ANGPTL4 promotes cell migration and invasion by inducing BMP7. (AeB) Cell migration and invasion abilities were analyzed HCT116 cells. Cells were transfected with A4OVER or A4-OVER plus siRNA oligos targeting BMP7 (B7RNAi). (CeD) Relative mRNA levels of Vimentin and E-cadherin in HCT116 cells. (E) Representative protein levels of Vimentin and E-cadherin were determined by western blot in HCT116 cells.
ANGPTL4 could promote HCT116 migration and invasion in vitro and enhance cancer metastasis in the nude mice. ANGPTL4 can also restrain E-cadherin and enhance Vimentin expression, two markers in cancer cell metastasis. Recently, ANGPTL4 was shown to promote the metastatic process by increasing vascular permeability in cancer cells [2]. Besides, Nakayama T et al. reported that ANGPTL4 plays a role in the progression of human colorectal cancer, especially venous invasion and distant metastasis [18]. Meanwhile, ANGPTL4 could inhibit HCT116 apoptosis by down-regulation of Bax and upregulation Bcl-xl expression, in the absence of other external stimuli. Our results were consistent with previous observations, which showed that ANGPTL4 might be a potential therapeutic target for preventing cancer metastasis. However, further studies, such as ANGPTL4 whole-body knockout or colon cell-specific knockout mice, are still needed to determine the precise roles of ANGPTL4 in cancer metastasis. BMP7, a member of the TGF-b superfamily, regulates cell differentiation, proliferation, and apoptosis, by activation of BMP receptors and intracellular SMADs signaling. Previous studies have shown that BMP7 is overexpressed in several types of human cancers, including breast cancer [19], hepatocellular carcinoma [20], esophageal squamous cell carcinoma (ESCC) [21] and colorectal cancer [22]. Importantly, BMP7 could affect proliferation, migration, and invasion of breast cancer cells [19]. In addition, expression of BMP7 in CRC correlates with parameters of pathological aggressiveness such as liver metastasis and poor prognosis [22].
Our results suggested that, at the molecular level, ANGPTL4 was a positive regulator of BMP7 in CRC cells. Indeed, knockdown of BMP7 significantly attenuated the roles of ANGPTL4 in the regulation of cell apoptosis, migration and invasion. Besides, BMP7 expression were increased in liver tissues from the spleen-liver metastasis models. It has been shown that BMP7 protects prostate cancer cells from stress-induced apoptosis via both Smad and c-Jun NH2-terminal kinase pathways [23]. Besides, BMP7 expression was associated with bone metastasis in breast carcinomas [24]. Therefore, our data also proposed a molecular determinant for the aberrant expression of BMP7 in cancer tissues. Interestingly, ANGPTL4 was shown to bind integrin b1 and activate the FAK-SrcPAK1 signaling pathway, which is important for cell migration [25]. Whether these complex signaling by ANGPTL4 is involved in the regulation of BMP7 is still under investigation. Recent studies have demonstrated that several secreted cytokines are involved in the regulation of cancer metastasis. For instance, periostin could promote metastatic growth of colon cancer through activation of PI3K/AKT signaling pathway [26]. Besides, compared to primary tumor cell lines, metastatic CRC cell lines expressed higher expression of many cytokines [27]. Therefore, together with ANGPTL4 and BMP7, these factors might provide the implications for intervention targets in the metastatic CRC setting. In conclusion, our data revealed that ANGPTL4 not only inhibits cell apoptosis but also promotes migration, invasion and metastasis
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
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Fig. 4. ANGPTL4 enhances liver metastasis by inducing BMP7 in nude mouse. (A) The results showing that injection of ANGPTL4-producing HCT116 cells into spleen led to the formation of large metastases in the liver. (B) The numbers of metastatic tumors in the liver isolated from 7 animals survived the procedure with injection of the ANGPTL4overexpressing HCT116 cells and from 7 mice injected with the control cells. (C) Representative immunohistochemistry staining of ANGPTL4 in tumors from two groups of mice. (D) Representative immunohistochemistry staining of BMP7 in tumors from two groups of mice.
in colorectal cancer. Moreover, higher expression of ANGPTL4 accelerated CRC progression by up-regulation of BMP7. Taken together, our data suggest that ANGPTL4 might be an important prognostic target for CRC patients. Conflict of interest The authors have no conflict of interest. Acknowledgments The project was supported by the Research Fund for the Doctoral Project (20120071110061), The Youth Fund for Affiliated Zhongshan Hospital of Fudan University (2014ZSQN33), Shanghai Natural Fund (15ZR1406800, No. 20144Y0195, 13411950800), Open Fund of Key Laboratory of Carcinogenesis and Cancer Invasion, Fudan University, Ministry of Education (KLCCI2014-6), Shanghai Engineering and Research Center of Diagnostic and Theraputic Endoscopy (laboratory-009) and National Science Foundation of China (81502523, 81502000). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.09.104. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.104. References [1] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int. J. Cancer 127 (2010) 2893e2917.
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Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells Xuquan Li 1, Tao Chen 1, Qiang Shi 1, Jian Li, Shilun Cai, Pinghong Zhou, Yunshi Zhong**, Liqing Yao* Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital of Fudan University, Shanghai 200032, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 September 2015 Accepted 19 September 2015 Available online xxx
Angiopoietin-like 4 (ANGPTL4), a secretory glycoprotein, plays an important role in cancer metastasis. In the present study, we aim to investigate the roles and mechanisms of ANGPTL4 in the regulation of colorectal cancer metastasis. We found that expression level of ANGPTL4 was increased in colorectal cancer tissues, compared with that in normal tissues. Moreover, liver metastasis was significantly associated with higher expression of ANGPTL4. In vitro studies further showed that overexpression of ANGPTL4 enhanced cell migration, invasion and inhibited apoptosis. At the molecular level, ANGPTL4 overexpression resulted in an up-regulation of bone morphogenetic protein 7 (BMP7). Indeed, knockdown of BMP7 by small interfering RNA (siRNA) oligos reversed the roles of ANGPTL4 overexpression in HCT116 cells. Finally, in vivo studies further confirmed the metastatic roles of ANGPTL4 by inducing BMP7. Therefore, our study demonstrated that ANGPTL4 might promote metastasis and might inhibit apoptosis of colorectal cancer cells by up-regulation of BMP7. © 2015 Elsevier Inc. All rights reserved.
Keywords: ANGPTL4 BMP7 Metastasis Apoptosis Colorectal cancer
1. Introduction Colorectal cancer (CRC) has become one of the most common malignancy and leading cause of cancer-related death worldwide [1]. Liver metastasis is one of the leading causes of death in patients with colorectal cancer, but its mechanism remains poorly understood. Angiopoietin-like protein 4 (ANGPTL4), a secreted protein, exerts multifunctional roles in many aspects of tumorigenesis, such as motility, metastasis, apoptosis, angiogenesis, tumormicroenvironment, wound healing, vascular permeability and inflammation [2e7]. ANGPTL4 was up-regulated and could promote cancer occurrence and development in glioblastoma, breast cancer, oral Kaposi's sarcoma and clear cell renal-cell carcinoma [8e10]. ANGPTL4 can disrupt vascular endothelial cellecell
* Corresponding author. 180 Fenglin Road, Xuhui District, 200032 Shanghai, PR China. ** Corresponding author. 180 Fenglin Road, Xuhui District, 200032 Shanghai, PR China. E-mail addresses: [email protected] (Y. Zhong), yao.liqing@zs-hospital. sh.cn (L. Yao). 1 Equal contributors.
junctions and primes breast cancer cells for metastasis to the lungs by TGF-b/smad4 complex-induced expression [9]. The transcription factor c-Myc can bind to the promoter sequence on ANGPTL4 that markedly accelerates malignant glioma tumor growth [10]. However, ANGPTL4 could also inhibit metastasis in melanoma cancer, prostate cancer, and hepatocellular carcinoma [7,11,12]. In the human hepatocellular carcinoma (HCC), ANGPTL4 was hypoxiainducible factor 1a (HIF-1a) not HIF-2a directly up-regulated in hypoxia. ANGPTL4 promoted HUVECs cell transendothelial migration through the VCAM-1/integrin b1 axis [13]. Therefore, although the reason for the inconsistence remains largely unknown, the precise roles of ANGPTL4 might be cell or tissue-specific, which may rely on cellular microenvironments. ANGPTL4 is highly expressed in human CRC tissues. Upregulation of ANGPTL4 has been observed in CRC patients and may enhance cell invasion and migration [14]. However, to date, molecular mechanism study of ANGPTLl4 is relatively less, especially in colorectal cancer. Therefore, the expression and roles of ANGPTL4 in CRC metastasis remain need further studies. In the present study, we aimed to explore the expression and roles of ANGPTL4 in CRC metastasis. Moreover, we analyzed downstream of the molecular mechanisms of ANGPTL4 in CRC cell lines.
http://dx.doi.org/10.1016/j.bbrc.2015.09.104 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
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X. Li et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
2. Materials and methods 2.1. Human samples collection Fifty-four colon cancer tissues and 24 adjacent normal tissues were collected from routine therapeutic surgery, immediately snapfrozen in the nitrogen and stored at 80 C. The patient’ clinicopathological data were collected (Table 1). All patients were tested by computer tomography (CT) and B-type ultrasonic inspection before surgical operation. This project was approved by the institutional review board of the Zhongshan Hospital of Fudan University. 2.2. Cell and reagents The human CRC cell lines HCT116 and SW480 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Cells were grown in McCoy's 5A medium and DMEM (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (SigmaeAldrich,St. Louis, MO, USA) and maintained at 37 C in a humidified atmosphere with 5% CO2. Recombinant human BMP7 and ANGPTL4 were obtained from HumanZyme Company (USA) and Sion Biological Inc (China). 2.3. Lentivirus, siRNA and transfection HCT116 cells were transfected with Lentivirus (Genechem, Shanghai, China) containing empty vector or ANGPTL4. RNAimediated depletion of BMP7 was achieved by transfecting cells with small interfering RNA (siRNA) oligos targeting BMP7 or negative controls (GenePharma, Shanghai, China), using Lipofectamine 2000 reagents (Invitrogen,USA) according to the manufacturer's instructions. All the sequences of siRNA oligos were listed in the Supplementary Table S1. 2.4. Histology and immunohistochemistry All the tissues were fixed overnight in formalin solution, Table 1 Clinical characteristics of 54 CRC patients. Characteristics Age <60 60 Gender Male Female Tumor volume 5 >5 Invasion depth M S Differentiation Well Moderate Poor Lymph metastasis Yes No Liver metastasis Yes No Duke time AB CD
No.
Low expression of High expression of P value ANGPTL4 n (%) ANGPTL4 n (%)
26 28
7 (26.9) 6 (21.4)
19 (73.1) 22 (78.6)
0.748
25 29
6 (24.0) 7 (24.1)
19 (76.0) 22 (75.9)
0.519
31 23
9 (29.0) 4 (17.4)
22 (71.0) 19 (82.6)
0.649
10 44
3 (30.0) 10 (22.7)
7 (70.0) 34 (77.3)
0.218
17 20 17
4 (23.5) 5 (25.0) 4 (23.5)
13 (76.5) 15 (75.0) 13 (76.5)
0.869
29 25
6 (20.7) 7 (28.0)
23 (79.3) 18 (72.0)
0.176
11 43
1 (9.1) 12 (27.9)
10 (90.9) 31 (72.1)
0.028*
25 29
7 (28.0) 6 (20.7)
18 (72.0) 23 (79.3)
0.176
dehydrated in ethanol, embedded in paraffin and sectioned at 5 mm. Immunohistochemical staining were performed according to a standard protocol. The staining intensity was rated according to a previous study [8]. The expression of the ANGPTL4 protein with an overall score of ‘/þ ’ was designated as ‘low,’ and with an overall score of ‘þþ/þþþ’ was designated as ‘high.’ The following primary antibodies were used: anti-ANGPTL4 antibody (diluted 1:50; Santa Cruz Biotechnology, CA, USA), anti-BMP7 antibody (diluted 1:50; Abgent, USA). 2.5. RNA isolation and quantitative real-time PCR Total RNA was extracted using TRIzol (Invitrogen, USA) and reversely transcribed to cDNA using the First-Strand Synthesis System (invitrogen, USA). Quantitative real-time PCR was performed as described using SYBR Green kit (Yeasen, China) in a 7500 Real-Time PCR system (Applied Biosystems), according to the manufacturer's instructions. Relative transcript quantities were calculated using the 2DDCt method with GAPDH as the endogenous reference gene. Primer sequences are listed in the Supplementary Table S2. 2.6. Western blotting Cells were harvested and lysed with RIPA (Thermo, USA) containing protease inhibitors (Invitrogen, USA). After centrifugation, proteins in the supernatants were quantified, separated by 8e12% SDS PAGE and transferred onto polyvinyl difluoride (PVDF) membrane. Proteins were incubated by primary antibody over night at 4 C and then incubated with secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. The antibody information is listed as follows: anti-ANGPTL4 (1:500, Santa Cruz Biotechnology, CA, USA), anti-BMP7 (1:1000, Santa Cruz Biotechnology, CA, USA), anti-GAPDH (1:1000, Abgent, USA), anti-Bcl-xl (1:1000, CST, USA), anti-Bax (1:1000, CST, USA), anti-Vimentin (1:1000, Gene Tax, Germany), anti-E-cadherin (1:1000, CST, USA). The proteins were visualized by an ECL chemiluminescence detection kit (Thermo scientific, USA). 2.7. Cell migration and invasion assay Cells (1.0 105) were subjected to Transwell with 8.0 mm polycarbonate membrane (Corning, USA) to analyze cell migration and invasion. After incubation at 37 C for 24 h, cells of inside Transwell were removed, the outside cells were dyed by Giemsa Stain (Nanjing JianCheng Bioengineering Institute, China)for 6e10 min and cleaned by PBS. All the migration and invasion assays were performed in triplicate in at least three independent experiments. 2.8. Flow cytometry
The significance of bold and asterisk stands for P value.
1 105 cells were added into 6-well plate, and cultured at 37 C 5% CO2 for 48 h. Cells were trypsinized without EDTA and washed twice with PBS. Subsequently, 50 mL of 1 binding buffer and 5 mL of 7-AAD (KeyGEN, China) were added into the pellet and incubated at room temperature (25 C) for 10 min in the dark. Then, 450 mL of 1 binding buffer and 1 mL of Annexin V-PE (KeyGEN, China) were added into the pellet and incubated at room temperature (25 C) for 10 min in the dark to measurement using FACS calibur flowcytometer and Cell Quest Pro software (BD Biosciences, USA). The data were analyzed using FlowJo 7.6 software and displayed in dot plot of 7-AAD (Y-axis) against Annexin V-PE (X-axis). 2.9. Liver metastasis model To examine whether ANGPTL4 expression in colon cancer cells
Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104
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affects metastasis, ANGPTL4-or empty vector-expressing HCT116 cells (1.0 105 cells/mouse) were injected into 6-week old female nude mice via intrasplenic injection. Mice were sacrificed 4 weeks after injection and examined for metastases development. Animal studies were carried out according to the Guideline for Animal Experiments, drawn up by the Committee for Ethics in Animal Experimentation of the National Cancer Center, which meet the ethical standards required by law and the guidelines about experimental animals in China.
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compared with those in normal tissues (P < 0.001) (Fig. 1B). There were 11 cases (19.6%) showing a higher level of ANGPTL4 expression in tumor tissues. Moreover, the higher expression of ANGPTL4 was significantly associated with liver metastasis (P ¼ 0.028) (Table 1). 3.2. ANGPTL4 up-regulates BMP7 expression
3. Results
To test whether ANGPTL4 could regulate BMP7, HCT116 cells were transfected with lentivirus containing empty vector or ANGPTL4 (A4-OVER) (Fig. 1C and E). As expected, ANGPTL4 overexpression led to an increased mRNA and protein expression of BMP7 (Fig. 1DeE). However, treatment of HCT116 cells with recombinant BMP7 (25 ng/ml) proteins did not affect ANGPTL4 expression (Fig. 1E). BMP7 expression were increased gradually when HCT116 and SW480 were co-cultured with recombinant human-ANGPTL4 protein from 150 ng/ml to 450 ng/ml for 60 h (Fig. 1F), suggesting that ANGPTL4 may be an up-stream regulator of BMP7 in CRC cells.
3.1. Up-regulation of ANGPTL4 in CRC tissues
3.3. ANGPTL4 inhibits cell apoptosis in HCT116 cells
To investigate the roles of ANGPTL4 in CRC pathogenesis, its protein expression was analyzed by immunohistochemistry using 54 CRC and 24 adjacent normal specimens. As shown in Fig. 1A, expression levels of ANGPTL4 are significantly up-regulated in CRC tissues,
We found that the apoptotic rate of HCT116 cells overexpressing ANGPTL4 (about 3.79%) was reduced compared to the control group (about 11.23%) (Fig. 2A). mRNA and protein levels of Bax, a proapoptotic marker [15], were significantly reduced in HCT116 cells
2.10. Statistical analysis Data were expressed as mean ± SD of at least three independent experiments, and statistical analysis for single comparison was performed using the SPSS 17.0. The criterion for statistical significance was *p < 0.05, **p < 0.01, ***P < 0.001.
Fig. 1. Up-regulation of ANGPTL4 in CRC tissues and ANGPTL4 regulates BMP7 expression in HCT116 cells. (A) Representative immunohistochemistry staining of ANGPTL4 in cancer with metastasis (Metastasis cancer), cancer without metastasis (No metastasis cancer), and adjacent normal tissues (Normal). (B) The average immunostaining scores of ANGPTL4 expression in 11 cancer with metastasis, 43 cancer without metastasis and 24 normal tissues. (CeD) Relative mRNA levels of ANGPTL4 (C) and BMP7 (D) in HCT116 cells transfected with lentivirus expressing ANGPTL4 (A4-OVER) or empty vector (Con). (E) Representative protein levels of ANGPTL4 and BMP7 in HCT116 cells transfected with A4-OVER or treated with recombinant BMP7 protein. (F) BMP7 expression were increased gradually when HCT116 and SW480 were co-cultured with different concentration of recombinant humanANGPTL4 protein for 60 h. *p < 0.05, ***p < 0.001.
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Fig. 2. ANGPTL4 inhibits cell apoptosis by inducing BMP7. (A) Apoptosis was determined in HCT116 cells. Cells were transfected with A4-OVER or empty vector. (B) Transfection efficiency was tested after transfecting siRNA oligos targeting BMP7 (B7RNAi) in HCT116 cells with overexpressed ANGPTL4. (C) Apoptosis was determined in HCT116 cells. Cells were transfected with A4-OVER plus siRNA oligos targeting BMP7 (B7RNAi). (DeF) Relative mRNA (DeE) and representative protein (F) levels of Bax and BCL-xL in HCT116 cells. *p < 0.05, ***p < 0.001.
with ANGPTL4 overexpression (Fig. 2D and F). Besides, expression levels of Bcl-xl, an apoptotic inhibitor [15], were increased (Fig. 2D and F). To confirm the relationship between ANGPTL4 and BMP7, HCT116 cells were administrated with siRNA oligos targeting BMP7 (B7RNAi) or negative control (A4(OE)-Con), after transfection of ANGPTL4 (Fig. 2B). As expected, knockdown of BMP7 significantly reversed the anti-apoptotic roles of ANGPTL4 (Fig. 2C). Besides, the changes in the mRNA and protein levels of Bax and Bcl-xl were attenuated by B7RNAi (Fig. 2EeF).
affect the metastatic potential and growth of the cancer cells in vivo. For this purpose, HCT116 cells were stably transfected with ANGPTL4 or empty vector and equally were introduced into the nude mice via intrasplenic injection. After the inoculation of tumor cells, mice were sacrificed for the examination of metastatic growth in the liver. As a result, more tumor nodules were found in the livers from A4-OVER group than control group (Fig. 4AeB), with much higher expression of ANGPTL4 (Fig. 4C) and BMP7 (Fig. 4D), suggesting that ANGPTL4 could also promote tumor metastasis by BMP7 in vivo.
3.4. ANGPTL4 promotes migration and invasion of HCT116 cells
4. Discussion
Moreover, we found that overexpression of ANGPTL4 significantly enhanced cell migration and invasion abilities (Fig. 3A). In agreement, knockdown of BMP7 significantly blocked the roles of ANGPTL4 to promote cell migration and invasion (Fig. 3B). At the molecular level, expression levels of E-cadherin were significantly reduced, while Vimentin were increased in HCT116 cells with ANGPTL4 overexpression (Fig. 3C and E). However, these changes were largely blocked by B7RNAi (Fig. 3DeE).
Cancer metastasis is a complicated process involving several sequential steps, including intravasation into the blood, survival in the circulation systems, extravasation into a new organ, initiation and maintenance of growth [16,17]. ANGPTL4 plays multiple important roles in the occurrence and progression of different cancer. However, the molecular events of ANGPTL4 that are directly involved in the colorectal cancer metastatic process remains poorly understood. In the present study, our data showed that ANGPTL4 could be a novel and important regulator of cell metastasis in CRC. These are supported by multiple lines of evidence. ANGPTL4 expression was up-regulated in CRC tissues and higher expression of ANGPTL4 was significantly associated with liver metastasis. Overexpression of
3.5. Overexpression of ANGPTL4 in colorectal cancer cells potently promote metastatic tumor growth in the liver We next examined whether ANGPTL4 overexpression could
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Fig. 3. ANGPTL4 promotes cell migration and invasion by inducing BMP7. (AeB) Cell migration and invasion abilities were analyzed HCT116 cells. Cells were transfected with A4OVER or A4-OVER plus siRNA oligos targeting BMP7 (B7RNAi). (CeD) Relative mRNA levels of Vimentin and E-cadherin in HCT116 cells. (E) Representative protein levels of Vimentin and E-cadherin were determined by western blot in HCT116 cells.
ANGPTL4 could promote HCT116 migration and invasion in vitro and enhance cancer metastasis in the nude mice. ANGPTL4 can also restrain E-cadherin and enhance Vimentin expression, two markers in cancer cell metastasis. Recently, ANGPTL4 was shown to promote the metastatic process by increasing vascular permeability in cancer cells [2]. Besides, Nakayama T et al. reported that ANGPTL4 plays a role in the progression of human colorectal cancer, especially venous invasion and distant metastasis [18]. Meanwhile, ANGPTL4 could inhibit HCT116 apoptosis by down-regulation of Bax and upregulation Bcl-xl expression, in the absence of other external stimuli. Our results were consistent with previous observations, which showed that ANGPTL4 might be a potential therapeutic target for preventing cancer metastasis. However, further studies, such as ANGPTL4 whole-body knockout or colon cell-specific knockout mice, are still needed to determine the precise roles of ANGPTL4 in cancer metastasis. BMP7, a member of the TGF-b superfamily, regulates cell differentiation, proliferation, and apoptosis, by activation of BMP receptors and intracellular SMADs signaling. Previous studies have shown that BMP7 is overexpressed in several types of human cancers, including breast cancer [19], hepatocellular carcinoma [20], esophageal squamous cell carcinoma (ESCC) [21] and colorectal cancer [22]. Importantly, BMP7 could affect proliferation, migration, and invasion of breast cancer cells [19]. In addition, expression of BMP7 in CRC correlates with parameters of pathological aggressiveness such as liver metastasis and poor prognosis [22].
Our results suggested that, at the molecular level, ANGPTL4 was a positive regulator of BMP7 in CRC cells. Indeed, knockdown of BMP7 significantly attenuated the roles of ANGPTL4 in the regulation of cell apoptosis, migration and invasion. Besides, BMP7 expression were increased in liver tissues from the spleen-liver metastasis models. It has been shown that BMP7 protects prostate cancer cells from stress-induced apoptosis via both Smad and c-Jun NH2-terminal kinase pathways [23]. Besides, BMP7 expression was associated with bone metastasis in breast carcinomas [24]. Therefore, our data also proposed a molecular determinant for the aberrant expression of BMP7 in cancer tissues. Interestingly, ANGPTL4 was shown to bind integrin b1 and activate the FAK-SrcPAK1 signaling pathway, which is important for cell migration [25]. Whether these complex signaling by ANGPTL4 is involved in the regulation of BMP7 is still under investigation. Recent studies have demonstrated that several secreted cytokines are involved in the regulation of cancer metastasis. For instance, periostin could promote metastatic growth of colon cancer through activation of PI3K/AKT signaling pathway [26]. Besides, compared to primary tumor cell lines, metastatic CRC cell lines expressed higher expression of many cytokines [27]. Therefore, together with ANGPTL4 and BMP7, these factors might provide the implications for intervention targets in the metastatic CRC setting. In conclusion, our data revealed that ANGPTL4 not only inhibits cell apoptosis but also promotes migration, invasion and metastasis
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Fig. 4. ANGPTL4 enhances liver metastasis by inducing BMP7 in nude mouse. (A) The results showing that injection of ANGPTL4-producing HCT116 cells into spleen led to the formation of large metastases in the liver. (B) The numbers of metastatic tumors in the liver isolated from 7 animals survived the procedure with injection of the ANGPTL4overexpressing HCT116 cells and from 7 mice injected with the control cells. (C) Representative immunohistochemistry staining of ANGPTL4 in tumors from two groups of mice. (D) Representative immunohistochemistry staining of BMP7 in tumors from two groups of mice.
in colorectal cancer. Moreover, higher expression of ANGPTL4 accelerated CRC progression by up-regulation of BMP7. Taken together, our data suggest that ANGPTL4 might be an important prognostic target for CRC patients. Conflict of interest The authors have no conflict of interest. Acknowledgments The project was supported by the Research Fund for the Doctoral Project (20120071110061), The Youth Fund for Affiliated Zhongshan Hospital of Fudan University (2014ZSQN33), Shanghai Natural Fund (15ZR1406800, No. 20144Y0195, 13411950800), Open Fund of Key Laboratory of Carcinogenesis and Cancer Invasion, Fudan University, Ministry of Education (KLCCI2014-6), Shanghai Engineering and Research Center of Diagnostic and Theraputic Endoscopy (laboratory-009) and National Science Foundation of China (81502523, 81502000). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.09.104. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.104. References [1] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int. J. Cancer 127 (2010) 2893e2917.
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Please cite this article in press as: X. Li, et al., Angiopoietin-like 4 enhances metastasis and inhibits apoptosis via inducing bone morphogenetic protein 7 in colorectal cancer cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.09.104