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BRG1 promotes VEGF-A expression and angiogenesis in human colorectal cancer cells
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BRG1 promotes VEGF-A expression and angiogenesis in human colorectal cancer cells Jingqin Lan, Haijie Li, Xuelai Luo, Junbo Hu, Guihua Wang
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Cancer Research Institute, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Keywords: BRG1 VEGF Angiogenesis Colorectal cancer
Angiogenesis plays an important role in tumor growth and progression in solid tumors. Vascular endothelial growth factor (VEGF) is one of the most critical and specific factors that stimulate both physiological and pathological angiogenesis. Here, we report a novel role of BRG1, the core subunit of SWI/SNF family complexes, in angiogenesis. In this study, we demonstrate that BRG1 is overexpressed in colorectal cancer and decreased expression of BRG1 not only blocks cell proliferation but remarkably inhibits the ability of HUVECs to form capillary-like structures. Moreover, our study shows that BRG1 can regulate the expression of VEGF-A by interacting with HIF-1α. Furthermore, we find VEGF-A is overexpressed in colorectal cancer and is positively correlated with BRG1 expression. Taken together, our study demonstrated that BRG1 can promote VEGF-A expression and angiogenesis in colorectal cancer and BRG1 may be a novel drug target for the treatment of colorectal cancer.
1. Introduction Colorectal cancer(CRC) is the third most common cancer and the fourth most common cancer cause of death globally, accounting for roughly 1.2 million new cases and 600 000 deaths per year [1]. Although great progress has been made in the diagnosis and treatment of colorectal cancer, the outcome for the patients is still pessimistic [2]. Therefore, better strategies of treatment will ultimately require understanding of the molecular mechanisms of the initiation and progression of CRC. Angiogenesis is the formation of new blood vessels from pre-existing vasculature and plays an important role in tumor growth and progression. Normally, angiogenesis is tightly controlled by a balance of angiogenic and anti-angiogenic factors [3]. Among the many angiogenic factors, vascular endothelial growth factor (VEGF) is one of the most critical and specific factors that stimulate both physiological and pathological angiogenesis. VEGF is a large family of growth factors that includes VEGF-A, B, C, D and placental growth factor (PLGF). These family members differ in their expression pattern, receptor specificity and biological functions [4]. VEGF-A, which is often referred to as VEGF, has been studied more than the other members of this family and is a critical factor that regulates angiogenesis. Overexpression of VEGF is associated with progression of and poor prognoses for several tumors, including prostate [5] and breast cancer [6], as well as hepatocellular carcinoma [7].
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VEGF expression is induced by growth factors, oncogenes and hypoxia. The key regulator of VEGF expression in response to hypoxia is hypoxia-inducible factor-1 (HIF-1) [8]. HIF-1 activates VEGF transcription by binding to the hypoxia response element (HRE) in the VEGF promoter. Treatment with a VEGF antagonist significantly attenuated angiogenesis and tumor progress and this treatment has been listed in NCCN guidelines as a molecular therapy for colorectal cancer [9]. Mammalian switch/sucrose nonfermentable (SWI/SNF) ATP-dependent chromatin remodeling enzymes disrupt Histone-DNA contacts on the nucleosome using the energy released by ATP hydrolysis [10]. These structural alterations result in increased or decreased chromatin accessibility for the binding of regulatory proteins that modulate transcription. SWI/SNF complex has two catalytic subunits, including BRG1 or BRM and 9–12 associated factors, which have tissue context specificity [11]. As the core subunit of SWI/SNF family complexes, BRG1 has been linked to progenitor cell proliferation, differentiation and survival in a variety of organs [12], the central nervous system [13] and T cells [14]. Previously, BRG1 was reported as a tumor suppressor [15]. BRG1 knockdown promoted tumor metastasis in CRC via the miR550/RNF43 pathway, indicating that BRG1 is a potential tumor suppressor [16]. However, the role of BRG1 in cell proliferation remains controversial. BRG1 is upregulated and correlated with tumor growth in gastric cancer [17]. Significant overexpression of BRG1 was observed in carcinoma compare to adjacent normal colon tissue [18]. Angiogenesis, as a crucial process to tumor growth, has been widely
Corresponding author. E-mail address: [email protected] (G. Wang).
http://dx.doi.org/10.1016/j.yexcr.2017.09.013 Received 14 July 2017; Received in revised form 30 August 2017; Accepted 8 September 2017 0014-4827/ © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Lan, J., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.09.013
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2.7. Chromatin immunoprecipitation(ChIP) assay
investigated. However, the functional role of BRG1 in angiogenesis and its molecular mechanism remain to be elucidated. In this study, we use human colorectal cancer cells to provide a comprehensive analysis of the role played by BRG1 in angiogenesis in vivo and vitro study. Our study demonstrates that BRG1 can promote angiogenesis in CRC via its regulation of VEGF. The further understanding of the role of BRG1 in angiogenesis may promote the development of therapeutic strategies for CRC.
ChIP assay were performed in accordance with the manufacturer's protocols (EpiQuik Chromatin Immunoprecipitation Kit, Epigentek Group Inc.). To examine changes in BRG1-binding activity at the VEGFA promoter, ChIP assays were conducted with the anti-BRG1 antibody (49360, Cell Signaling Technology). The primer sequences for ChIP are listed in Supplement Table 1.
2. Materials and methods
2.8. Mouse tumor xenograft
2.1. Cell culture The CRC cell line SW48 were obtained from the Type Culture Collection Cell Bank (Beijing, China). Cells were cultured at 37 °C，5% CO2 in DMEM with 10% fetal bovine serum (Thermo Fisher Scientific, Shanghai, China).
Four weeks old, female, nude mice were housed in sterile filtercapped cages. A total of 1 × 106 Sh-BRG1 or Sh-Con SW48 cells in 200ul Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were injected subcutaneously into nude mice. Measurement of tumor volume started from their visual appearance and operated every 3 days. After 2 weeks, all mice were sacrificed and tumors were collected and weighed.
2.2. Clinical samples
2.9. Statistical analysis
This study was approved by the Huazhong University of Science and Technology Research Ethics Committee. Tumor tissues and adjacent normal colorectal tissues were collected from primary resections of colorectal tumors from the same patient and kept at −80 °C until used.
Statistical analysis was performed using SPSS 19.0 software and GraphPad Prism 5.0. Data were presented as mean ± S.D. and clinical samples were analyzed by paired t-test. The Pearson r correlation test was used for the correlation analysis. Statistical significance was considered at P < 0.05.
2.3. Cell transfection and lentiviral system
3. Results
The coding sequence of BRG1 was cloned into the expression vector pcDNA 3.1. The transfection of colorectal cancer cells was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and the expression levels of BRG1 were quantified 48 h after transfection. Lentivirus-expression BRG1-targeted short hairpin RNA(Sh-BRG1) and control lentivirus (Sh-Con) was previously constructed in our laboratory.
3.1. BRG1 is overexpressed in CRC Firstly, we performed the NCBI Gene Expression Omnibus(GEO) dataset parameters to investigate the expression of BRG1 in normal tissues and tumors. The analysis result showed that BRG1 mRNA levels were significantly higher in colorectal tumor samples than normal tissues (P < 0.05, Fig. 1A, B and C). To validate the results, we measured BRG1 mRNA and protein expression in 12 primary colorectal tumor samples and corresponding adjacent normal colorectal tissues. The result showed that BRG1 was significantly overexpressed in colorectal tumors compared with normal tissues (Fig. 1D and E). The IHC results were also consistent with the previous results (Fig. 1F). CM from shBRG1 SW48 inhibits the proliferation and tube formation of HUVECs.
2.4. Western bolt analysis and real-time PCR assay Western bolt analysis was conducted as previously described [19]. Rabbit anti-BRG1 (ab110640) and rabbit anti-VEGFA were purchased from Abcam. Mouse anti-HIF-1α (BD610958) was purchased from BD. Mouse anti-GAPDH (sc-47724) was purchased from Santa Cruz. For real-time PCR, total mRNA was extracted using TRIzol regent (Invitrogen, Carlsbad, CA, USA) and reverse transcription was performed with PrimeScript RT Master Mix (Takara Biotechnology, Dalian, China). Real-time PCR assay was performed on ABI Prism 7300 Real-time system (Applied Biosystems, Foster City). The primer sequences for real-time PCR are listed in Supplement Table 1.
3.2. CM from sh-BRG1 SW48 inhibits the proliferation and tube formation of HUVECs The sustain proliferation and angiogenesis of endothelial cells are the fundamental trait of cancer cells [22]. To further investigate the function of BRG1 in the proliferation and tube formation of HUVECs, we produced a BRG1 knockdown cell model by transfecting CRC cells with BRG1 shRNA (Sh-BRG1). As shown in Supplementary Figure 1A and B, the treatment of SW48 cells with Sh-BRG1 led to a significant knockdown of BRG1 in both mRNA and protein levels. We next analyzed the proliferation by BrdU cell proliferation assay and Cell Count Kit-8 assay in HUVECs, which were incubated with CM collected from SW48 Sh-Con and SW48 Sh-BRG1 for 48 h. The result showed that the CM of BRG1 knockdown SW48 cells significantly inhibited HUVECs proliferation both in normoxia and hypoxia (Fig. 2A and B, Supplementary Figure 2A). Cell cycle analysis by flow cytometry detection was also performed, and the results revealed that the CM of BRG1 knockdown SW48 cells led to cell cycle arrest at G2-M phase both in normoxia and hypoxia (Figure C and Supplementary Figure 2B). Furthermore, the knockdown of BRG1 reduced the ability of HUVECs to form capillary-like structures (Fig. 2D), as indicated by the reduced number of branching points (Fig. 2E). Taken together, CM of BRG1 knockdown SW48 cells inhibited the proliferation and tube formation of HUVECs.
2.5. Cell proliferation and cell cycle analysis Cell proliferation was conducted by BrdU incorporation assay and CCK8 assay. The methods of Brdu incorporation and CCK8 assay was performed as previously described [20,21]. For cell cycle analysis, cells were fixed in 80% ethanol overnight at −20 °C, washed with PBS and then stained with propidium iodide and RNase. DNA content was measured by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). 2.6. Tube formation assay 100ul growth factor-reduced Matrigel was loaded into pre-chilled 96-well tissue plates and plates were placed at 37 ℃ for 30 min. HUVECs (4 × 104 cells) were added gently into each well and cultured in conditioned media (CM) on top of this gel for 12 h. For evaluating the tube formation, branching points per field were counted. 2
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Fig. 1. BRG1 is overexpressed in CRC (A-C) GEO cohorts (GSE5 5261(A), GSE 9689(B), GSE 20916 (C)were used to calculate the expression of BRG1 in colorectal cancer and normal colorectal tissues. (D) Quantitative real-time PCR of BRG1 was performed using 12 pairs of colorectal cancer tissues and corresponding colorectal normal tissues (P=0.0089). (E) Western blot analysis of BRG1 protein expression in 12 pairs of colorectal tissues. (F) Representative images of immunohistochemical (IHC) staining (using anti-BRG1 antibody) of colorectal cancers and corresponding colorectal normal tissues were shown. 40× scale bars, 50 µm, 100 × scale bars, 20 µm, 200× scale bars, 20 µm.
Fig. 2. Knockdown of BRG1 inhibits the proliferation and tube formation of HUVECs induced by CM from SW48 cells (A) HUVECs DNA synthesis was determined by BrdU incorporation assay treated with CM from SW48 cells which were infected with Sh-Con or Sh-BRG1 lentivirus cultured under normoxic or hypoxic conditions. (B) HUVECs proliferation was determined by CCK8 assay treated with CM from SW48 cells which were infected with Sh-Con or Sh-BRG1 lentivirus cultured under normoxic or hypoxic conditions. (C) HUVECs cell cycle distribution analysis measured by propodium iodide staining treated with CM from SW48 cells which were infected with Sh-Con or Sh-BRG1 lentivirus cultured under normoxic or hypoxic conditions. (D-E) Tube formed of HUVECs were detected by tube formation treated with CMs from SW48 cells treated with Sh-Con or Sh-BRG1 under normoxic and hypoxic conditions. The ability of tube formation was quantified by calculating branch points per field. Scale bars, 50 µm.
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Fig. 3. BRG1 can regulate the expression of VEGF-A by interacting with HIF-1α (A) Western blot detected BRG1, HIF-1α and VEGF-A expression levels in SW48 Sh-Con and Sh-BRG1 under normoxic and hypoxic conditions. (B) BRG1 and VEGF-A mRNA level in SW48 cells treated with Sh-con and Sh-BRG1 under normoxic (up) and hypoxic (down) conditions. (C) Western blot detected BRG1, HIF-1α and VEGF-A expression levels in SW48 Ad-GFP and Ad-BRG1 cells under normoxic and hypoxic conditions. (D) VEGF-a protein level in CM collected from SW48s treated with Sh-con and Sh-BRG1 under normoxic or hypoxic conditions were measured by ELISA kits. (E) Co-immunoprecipitation analysis in SW48 cells using anti-BRG1 antibodies and anti-mouse IgG antibodies or anti HIF-α antibodies and anti-mouse IgG under hypoxia condition, respectively. (F) Chromatin immunoprecipitation (ChIP) analyses of BRG1 binding on the promoters of VEGF-A under hypoxia condition. (G)ChIP assay and real-time PCR analysis showing the effect of HIF-1α on the VEGF-A promoter after the downregulation of BRG1 under hypoxia condition. P＜0.01.
3.3. BRG1 can regulate the expression of VEGF-A by interacting with HIF1α
3.4. Knockdown of BRG1 inhibits the proliferation and angiogenesis in mouse xenograft models
Given that VEGF-A is the most critical factor which stimulates both physiological and pathological angiogenesis, we detected the effect of Brg1 on the expression of VEGF-A to explore the regulation of Brg1 on angiogenesis. We found that Brg1 knockdown inhibited the expression of VEGF-A both in protein and mRNA levels in normoxia and hypoxia (Fig. 3A -B). The overexpression of BRG1 promoted the expression of VEGF-A in protein levels under normoxic and hypoxic conditions (Fig. 3C). To further demonstrate the role of BRG1 on VEGF-A expression, we transfected Ad-BRG1 in SW48-sh-BRG1 cells and diminished VEGF-A expression was recovered (Supplementary Figure 3A). The ELISA results further confirmed the relationship between Brg1 and VEGF-A. Our data showed that Brg1 knockdown significantly inhibited the expression of VEGF-A (Fig. 3D). To further clarify the mechanisms of the regulation of BRG1 on VEGF-A, we investigated the relationship between BRG1 and HIF-1α, which is the key regulator of the expression of VEGF-A. Our results showed that BRG1 interacted with HIF-1α detected by immunoprecipitation assay (Fig. 3E). It has been shown that HIF-1α regulates VEGF transcription by binding to the HRE in the VEGF promoter by ChIP assay. In our study, we found that VEGF promoter was occupied by BRG1 (Fig. 3F). Interestingly, knockdown BRG1 inhibited the function of HIF-1α to VEGF-A promoter(Fig. 3G). These results indicated that BRG1 binded with VEGF-A promoter to regulate the expression of VEGF-A by interacted with HIF-1α. To further investigate the role of HIF-1α on BRG1-regulated VEGF-A expression, we performed ‘rescue’ assay to show that the increases of VEGF-A induced by BRG1 overexpression were abrogated in the presence of HIF-1α inhibitor YC-1 (Supplementary Figure 3B).
To further evaluate the role of BRG1 in proliferation and angiogenesis in vivo, we generated stably BRG1-depleted cell lines by transducing SW48 cells with BRG1 shRNA (Sh-BRG1) and negative control shRNA (Sh-Con) lentivirus. Then the Sh-BRG1 cells and Sh-Con cells were subcutaneously injected into nude mice. We calculated the tumor volume every 4 days and the volumes were plotted into a tumor growth curve (Fig. 4A). After the mice were executed, we observed that Sh-BRG1 groups were significantly depressed in tumor volume (Fig. 4B) and weight (Fig. 4C) compared with Sh-Con groups. IHC analysis showed decreased expression of BRG1 and VEGF in the Sh-BRG1 group compared with the Sh-Con group. Moreover, CD34 antibody were used to measure tumor angiogenesis [23]. The expression of CD34 in tumors were obviously reduced in Sh-BRG1 group. These results revealed that knockdown of BRG1 inhibits CRC proliferation and angiogenesis in vivo.
3.5. BRG1 and VEGF expression are significantly correlated in colorectal tissues To investigate the expression of BRG1 and VEGF in clinic colorectal tissues, we performed q RT-PCR and Western Blot analysis to evaluate the expression of BRG1 and VEGF in the 12 pairs colorectal samples mentioned above. We found that VEGF was significantly overexpressed in colorectal tumor samples compared with normal tissues (Fig. 5A and B). Next we used the Pearson r correlation test to investigate the association between BRG1 and VEGF expression. We observed a significantly positive correlation between BRG1 and VEGF at mRNA levels (Fig. 5C). These results were also confirmed by IHC (Fig. 5D).
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Fig. 4. Knockdown of BRG1 inhibits the proliferation and angiogenesis in mouse xenograft models (A) Stable BRG1 knockdown SW48 cells and control cells were injected subcutaneously into nude mice (n = 7 for each group). The tumors were collected and shown. (B) Tumor development was measured as methods and material, and tumor volume was shown. (C) Tumor weight of each group was measured(Sh-Con vs Sh-BRG1 = 0.4457 ± 0.03993 vs 0.1114 + 0.008571). (D) representative images of IHC staining of indicated protein in tumors xenograft are shown. BRG1 scale bars, 50 µm, VEGF-A scale bars, 20 µm, CD34 scale bars, 200 µm.
4. Discussion
impact vascular development in midgestation embryo by promoting Wnt signaling and venous specification. The deletion of BRG1 from embryonic blood vessels results in yolk sac vascular remodeling defects [25]. Another study shows that BRG1 is requited for vascular integrity in infant mice [26]. However, these results were drawn from study in endothelial cells in embryology and showed no effect on postnatal angiogenesis. Meanwhile, the primary effect of the BRG1 in tumor cells on angiogenesis remains unclear. In our study, we showed that knockdown
Mammalian SWI/SNF adenosine ATP-dependent chromatin remodeling complexes promotes or represses transcription of genes by increasing or decreasing accessibility of DNA to large transcriptional machinery at specific loci [24]. BRG1, as the specific ATPase of SWI/ SNF chromatin-remodeling complex, is correlated with embryonic vascular development in previous study. It has been shown that BRG1
Fig. 5. BRG1 and VEGF expression are significantly correlated in colorectal tissues (A) Analysis of VEGF-A and BRG1 mRNA expression in colorectal tissues (including 12 colorectal tumor tissues and 12 corresponding colorectal normal tissues) are shown in box-whisker plot. Student's t-test (P = 0.0034) (B) Pearson r correlation was used to measure the relationship between BRG1 and VEGF-A mRNA levels, r = 0.8948, p < 0.0001. (C) BRG1 and VEGF-A protein levels in colorectal tissues (12 paired samples) were determined by western blot. (D) Representative images of IHC staining of CRC tumor tissues and corresponding colorectal normal tissues with anti-BRG1, anti-VEGF-A and anti-CD34 antibodies, 100 × scale bars, 50 µm, 200 × scale bars, 20 µm.
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Fig. 6. A hypothetical representation of the regulatory pathway underlying BRG1-induced angiogenesis.
functions as a general modulator of VEGF in CRC. We propose a model that suggests BRG1 bind to the HRE in the VEGE promoter to regulation the expression of VEGF to promote angiogenesis in a complex with HIF1α (Fig. 6). These findings provide evidence that BRG1 can be used as anti-angiogenic therapy for treating colorectal cancer.
of BRG1 inhibited the HUVEC capillary tube formation (Fig. 2), indicating that BRG1 played an important role in angiogenesis in CRC. Meanwhile, we investigated the expression of BRG1 in GEO datasets and clinical colorectal tissues and we observed BRG1 was overexpressed in colorectal tumor compared normal tissues (Fig. 1). These results indicating that BRG1 is closely correlated with tumor progression in CRC. Our results were not completely consistent with previous study about BRG1 in angiogenesis in endothelial cells [26]. The different cell type may contribute to explain the specific influence of BRG1 in angiogenesis. Although the correlation between BRG1 and vascular development was previously reported, our study is the first to systematically investigated the effect of BRG1 in angiogenesis in CRC. Tumor growth require angiogenesis when the tumor reaches 1–2 mm in diameter. Inhibition of angiogenesis specifically suppressed tumor growth without affecting the normal mature vessels in human body [27]. The angiogenic process is balanced by various positive (such as VEGF) and negative (such as thrombospondin-1) regulatory molecules of endothelial proliferation and migration [9]. VEGF is critical for both normal and tumor angiogenesis. Treatment with a VEGF antagonist significantly attenuated angiogenesis and tumor growth [28]. In our study, we observed that BRG1 knockdown inhibited the expression of VEGF in normoxic and hypoxic condition (Fig. 3). HIF-1 is a key factor in carcinogenesis, angiogenesis, tumor growth and can be induced by hypoxia. HIF-1α is often upregulated in human cancer to regulate VEGF expression by binding to the hypoxia responsive element of VEGF promoter [29]. Kenneth has reported that modulation of SWI/ SNF levels results in pronounced changes in hypoxia-inducible factor1α expression in osteosarcoma which stands in contrast to our results and Johnny A. Sena has reported that BRG1 could bind with HIF-1α. These previous data revealed that the role of BRG1 is complicated and changeable in different cancer types [30,31]. Our study showed that HIF-1α expression is not impacted by BRG1 in SW48 CRC cell line and BRG1 could interact with HIF-1α by the IP assay (Fig. 3). Furthermore, we observed that the hypoxia response element (HRE) in the VEGF promoter was occupied by BRG1 by the ChIP assays. These results indicating that BRG1 could bind to VEGF promoter to regulate the transcription of VEGF in a complex with HIF-1α. Another finding of this study is the overexpression of BRG1 in colorectal cancer and the promotion of BRG1 in the proliferation of colorectal cancer cells. The role of BRG1 in tumor progression was contradictor in previous study. For example, studies using BRG1 knock-out mouse models have shown that the loss of BRG1 increased susceptibility to both mammary gland and lung tumourigenesis [32,33]. These studies suggested that BRG1 acted as a tumor suppressor. However, BRG1 is upregulated and promoted tumor proliferation in gastric cancer and contributed to acute myeloid leukemia maintenance [17,34]. The cell-type-specific influence of BRG1 on VEGF expression may help explain the varying effects of BRG1 on tumor promotion or inhibition in different tumor types. In our study, we showed the expression of BRG1 in CRC and the role of BRG1 played in proliferation and angiogenesis of CRC. The mouse xenograft models further confirmed the promotion of BRG1 in colorectal cancer progression (Fig. 4). Taken together, this study provides significant evidence that BRG1
Author contribution Guihua Wang designed this project. Jingqin Lan, Haijie Li, Xuelai Luon and Junbo Hu performed the experiments. Conflict of interest There is no conflict of interest. Compliance with ethical standards Ethical approval Research involving animals: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Research Involving human participants: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Acknowledgements This work is supported by the National Natural Science Foundation of China (81570525, 81572725). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2017.09.013. References [1] H. Brenner, M. Kloor, C.P. Pox, Colorectal cancer, Lancet 383 (2014) 1490–1502. [2] M.G. Fakih, Metastatic colorectal cancer: current state and future directions, J. Clin. Oncol. 33 (2015) 1809–1824. [3] D. Hanahan, J. Folkman, Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis, Cell 86 (1996) 353–364. [4] H.L. Goel, A.M. Mercurio, VEGF targets the tumour cell, Nat. Rev. Cancer 13 (2013) 871–882. [5] C. Kwak, R.J. Jin, C. Lee, M.S. Park, S.E. Lee, Thrombospondin-1, vascular endothelial growth factor expression and their relationship with p53 status in prostate cancer and benign prostatic hyperplasia, BJU Int. 89 (2002) 303–309. [6] M. Toi, K. Inada, H. Suzuki, T. Tominaga, Tumor angiogenesis in breast cancer: its importance as a prognostic indicator and the association with vascular endothelial growth factor expression, Breast Cancer Res. Treat. 36 (1995) 193–204. [7] O.N. El-Assal, A. Yamanoi, Y. Soda, M. Yamaguchi, M. Igarashi, A. Yamamoto, T. Nabika, N. Nagasue, Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver, Hepatology 27 (1998) 1554–1562. [8] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003)
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
BRG1 promotes VEGF-A expression and angiogenesis in human colorectal cancer cells Jingqin Lan, Haijie Li, Xuelai Luo, Junbo Hu, Guihua Wang
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Cancer Research Institute, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
A R T I C L E I N F O
A B S T R A C T
Keywords: BRG1 VEGF Angiogenesis Colorectal cancer
Angiogenesis plays an important role in tumor growth and progression in solid tumors. Vascular endothelial growth factor (VEGF) is one of the most critical and specific factors that stimulate both physiological and pathological angiogenesis. Here, we report a novel role of BRG1, the core subunit of SWI/SNF family complexes, in angiogenesis. In this study, we demonstrate that BRG1 is overexpressed in colorectal cancer and decreased expression of BRG1 not only blocks cell proliferation but remarkably inhibits the ability of HUVECs to form capillary-like structures. Moreover, our study shows that BRG1 can regulate the expression of VEGF-A by interacting with HIF-1α. Furthermore, we find VEGF-A is overexpressed in colorectal cancer and is positively correlated with BRG1 expression. Taken together, our study demonstrated that BRG1 can promote VEGF-A expression and angiogenesis in colorectal cancer and BRG1 may be a novel drug target for the treatment of colorectal cancer.
1. Introduction Colorectal cancer(CRC) is the third most common cancer and the fourth most common cancer cause of death globally, accounting for roughly 1.2 million new cases and 600 000 deaths per year [1]. Although great progress has been made in the diagnosis and treatment of colorectal cancer, the outcome for the patients is still pessimistic [2]. Therefore, better strategies of treatment will ultimately require understanding of the molecular mechanisms of the initiation and progression of CRC. Angiogenesis is the formation of new blood vessels from pre-existing vasculature and plays an important role in tumor growth and progression. Normally, angiogenesis is tightly controlled by a balance of angiogenic and anti-angiogenic factors [3]. Among the many angiogenic factors, vascular endothelial growth factor (VEGF) is one of the most critical and specific factors that stimulate both physiological and pathological angiogenesis. VEGF is a large family of growth factors that includes VEGF-A, B, C, D and placental growth factor (PLGF). These family members differ in their expression pattern, receptor specificity and biological functions [4]. VEGF-A, which is often referred to as VEGF, has been studied more than the other members of this family and is a critical factor that regulates angiogenesis. Overexpression of VEGF is associated with progression of and poor prognoses for several tumors, including prostate [5] and breast cancer [6], as well as hepatocellular carcinoma [7].
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VEGF expression is induced by growth factors, oncogenes and hypoxia. The key regulator of VEGF expression in response to hypoxia is hypoxia-inducible factor-1 (HIF-1) [8]. HIF-1 activates VEGF transcription by binding to the hypoxia response element (HRE) in the VEGF promoter. Treatment with a VEGF antagonist significantly attenuated angiogenesis and tumor progress and this treatment has been listed in NCCN guidelines as a molecular therapy for colorectal cancer [9]. Mammalian switch/sucrose nonfermentable (SWI/SNF) ATP-dependent chromatin remodeling enzymes disrupt Histone-DNA contacts on the nucleosome using the energy released by ATP hydrolysis [10]. These structural alterations result in increased or decreased chromatin accessibility for the binding of regulatory proteins that modulate transcription. SWI/SNF complex has two catalytic subunits, including BRG1 or BRM and 9–12 associated factors, which have tissue context specificity [11]. As the core subunit of SWI/SNF family complexes, BRG1 has been linked to progenitor cell proliferation, differentiation and survival in a variety of organs [12], the central nervous system [13] and T cells [14]. Previously, BRG1 was reported as a tumor suppressor [15]. BRG1 knockdown promoted tumor metastasis in CRC via the miR550/RNF43 pathway, indicating that BRG1 is a potential tumor suppressor [16]. However, the role of BRG1 in cell proliferation remains controversial. BRG1 is upregulated and correlated with tumor growth in gastric cancer [17]. Significant overexpression of BRG1 was observed in carcinoma compare to adjacent normal colon tissue [18]. Angiogenesis, as a crucial process to tumor growth, has been widely
Corresponding author. E-mail address: [email protected] (G. Wang).
http://dx.doi.org/10.1016/j.yexcr.2017.09.013 Received 14 July 2017; Received in revised form 30 August 2017; Accepted 8 September 2017 0014-4827/ © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Lan, J., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.09.013
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2.7. Chromatin immunoprecipitation(ChIP) assay
investigated. However, the functional role of BRG1 in angiogenesis and its molecular mechanism remain to be elucidated. In this study, we use human colorectal cancer cells to provide a comprehensive analysis of the role played by BRG1 in angiogenesis in vivo and vitro study. Our study demonstrates that BRG1 can promote angiogenesis in CRC via its regulation of VEGF. The further understanding of the role of BRG1 in angiogenesis may promote the development of therapeutic strategies for CRC.
ChIP assay were performed in accordance with the manufacturer's protocols (EpiQuik Chromatin Immunoprecipitation Kit, Epigentek Group Inc.). To examine changes in BRG1-binding activity at the VEGFA promoter, ChIP assays were conducted with the anti-BRG1 antibody (49360, Cell Signaling Technology). The primer sequences for ChIP are listed in Supplement Table 1.
2. Materials and methods
2.8. Mouse tumor xenograft
2.1. Cell culture The CRC cell line SW48 were obtained from the Type Culture Collection Cell Bank (Beijing, China). Cells were cultured at 37 °C，5% CO2 in DMEM with 10% fetal bovine serum (Thermo Fisher Scientific, Shanghai, China).
Four weeks old, female, nude mice were housed in sterile filtercapped cages. A total of 1 × 106 Sh-BRG1 or Sh-Con SW48 cells in 200ul Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were injected subcutaneously into nude mice. Measurement of tumor volume started from their visual appearance and operated every 3 days. After 2 weeks, all mice were sacrificed and tumors were collected and weighed.
2.2. Clinical samples
2.9. Statistical analysis
This study was approved by the Huazhong University of Science and Technology Research Ethics Committee. Tumor tissues and adjacent normal colorectal tissues were collected from primary resections of colorectal tumors from the same patient and kept at −80 °C until used.
Statistical analysis was performed using SPSS 19.0 software and GraphPad Prism 5.0. Data were presented as mean ± S.D. and clinical samples were analyzed by paired t-test. The Pearson r correlation test was used for the correlation analysis. Statistical significance was considered at P < 0.05.
2.3. Cell transfection and lentiviral system
3. Results
The coding sequence of BRG1 was cloned into the expression vector pcDNA 3.1. The transfection of colorectal cancer cells was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and the expression levels of BRG1 were quantified 48 h after transfection. Lentivirus-expression BRG1-targeted short hairpin RNA(Sh-BRG1) and control lentivirus (Sh-Con) was previously constructed in our laboratory.
3.1. BRG1 is overexpressed in CRC Firstly, we performed the NCBI Gene Expression Omnibus(GEO) dataset parameters to investigate the expression of BRG1 in normal tissues and tumors. The analysis result showed that BRG1 mRNA levels were significantly higher in colorectal tumor samples than normal tissues (P < 0.05, Fig. 1A, B and C). To validate the results, we measured BRG1 mRNA and protein expression in 12 primary colorectal tumor samples and corresponding adjacent normal colorectal tissues. The result showed that BRG1 was significantly overexpressed in colorectal tumors compared with normal tissues (Fig. 1D and E). The IHC results were also consistent with the previous results (Fig. 1F). CM from shBRG1 SW48 inhibits the proliferation and tube formation of HUVECs.
2.4. Western bolt analysis and real-time PCR assay Western bolt analysis was conducted as previously described [19]. Rabbit anti-BRG1 (ab110640) and rabbit anti-VEGFA were purchased from Abcam. Mouse anti-HIF-1α (BD610958) was purchased from BD. Mouse anti-GAPDH (sc-47724) was purchased from Santa Cruz. For real-time PCR, total mRNA was extracted using TRIzol regent (Invitrogen, Carlsbad, CA, USA) and reverse transcription was performed with PrimeScript RT Master Mix (Takara Biotechnology, Dalian, China). Real-time PCR assay was performed on ABI Prism 7300 Real-time system (Applied Biosystems, Foster City). The primer sequences for real-time PCR are listed in Supplement Table 1.
3.2. CM from sh-BRG1 SW48 inhibits the proliferation and tube formation of HUVECs The sustain proliferation and angiogenesis of endothelial cells are the fundamental trait of cancer cells [22]. To further investigate the function of BRG1 in the proliferation and tube formation of HUVECs, we produced a BRG1 knockdown cell model by transfecting CRC cells with BRG1 shRNA (Sh-BRG1). As shown in Supplementary Figure 1A and B, the treatment of SW48 cells with Sh-BRG1 led to a significant knockdown of BRG1 in both mRNA and protein levels. We next analyzed the proliferation by BrdU cell proliferation assay and Cell Count Kit-8 assay in HUVECs, which were incubated with CM collected from SW48 Sh-Con and SW48 Sh-BRG1 for 48 h. The result showed that the CM of BRG1 knockdown SW48 cells significantly inhibited HUVECs proliferation both in normoxia and hypoxia (Fig. 2A and B, Supplementary Figure 2A). Cell cycle analysis by flow cytometry detection was also performed, and the results revealed that the CM of BRG1 knockdown SW48 cells led to cell cycle arrest at G2-M phase both in normoxia and hypoxia (Figure C and Supplementary Figure 2B). Furthermore, the knockdown of BRG1 reduced the ability of HUVECs to form capillary-like structures (Fig. 2D), as indicated by the reduced number of branching points (Fig. 2E). Taken together, CM of BRG1 knockdown SW48 cells inhibited the proliferation and tube formation of HUVECs.
2.5. Cell proliferation and cell cycle analysis Cell proliferation was conducted by BrdU incorporation assay and CCK8 assay. The methods of Brdu incorporation and CCK8 assay was performed as previously described [20,21]. For cell cycle analysis, cells were fixed in 80% ethanol overnight at −20 °C, washed with PBS and then stained with propidium iodide and RNase. DNA content was measured by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). 2.6. Tube formation assay 100ul growth factor-reduced Matrigel was loaded into pre-chilled 96-well tissue plates and plates were placed at 37 ℃ for 30 min. HUVECs (4 × 104 cells) were added gently into each well and cultured in conditioned media (CM) on top of this gel for 12 h. For evaluating the tube formation, branching points per field were counted. 2
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Fig. 1. BRG1 is overexpressed in CRC (A-C) GEO cohorts (GSE5 5261(A), GSE 9689(B), GSE 20916 (C)were used to calculate the expression of BRG1 in colorectal cancer and normal colorectal tissues. (D) Quantitative real-time PCR of BRG1 was performed using 12 pairs of colorectal cancer tissues and corresponding colorectal normal tissues (P=0.0089). (E) Western blot analysis of BRG1 protein expression in 12 pairs of colorectal tissues. (F) Representative images of immunohistochemical (IHC) staining (using anti-BRG1 antibody) of colorectal cancers and corresponding colorectal normal tissues were shown. 40× scale bars, 50 µm, 100 × scale bars, 20 µm, 200× scale bars, 20 µm.
Fig. 2. Knockdown of BRG1 inhibits the proliferation and tube formation of HUVECs induced by CM from SW48 cells (A) HUVECs DNA synthesis was determined by BrdU incorporation assay treated with CM from SW48 cells which were infected with Sh-Con or Sh-BRG1 lentivirus cultured under normoxic or hypoxic conditions. (B) HUVECs proliferation was determined by CCK8 assay treated with CM from SW48 cells which were infected with Sh-Con or Sh-BRG1 lentivirus cultured under normoxic or hypoxic conditions. (C) HUVECs cell cycle distribution analysis measured by propodium iodide staining treated with CM from SW48 cells which were infected with Sh-Con or Sh-BRG1 lentivirus cultured under normoxic or hypoxic conditions. (D-E) Tube formed of HUVECs were detected by tube formation treated with CMs from SW48 cells treated with Sh-Con or Sh-BRG1 under normoxic and hypoxic conditions. The ability of tube formation was quantified by calculating branch points per field. Scale bars, 50 µm.
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Fig. 3. BRG1 can regulate the expression of VEGF-A by interacting with HIF-1α (A) Western blot detected BRG1, HIF-1α and VEGF-A expression levels in SW48 Sh-Con and Sh-BRG1 under normoxic and hypoxic conditions. (B) BRG1 and VEGF-A mRNA level in SW48 cells treated with Sh-con and Sh-BRG1 under normoxic (up) and hypoxic (down) conditions. (C) Western blot detected BRG1, HIF-1α and VEGF-A expression levels in SW48 Ad-GFP and Ad-BRG1 cells under normoxic and hypoxic conditions. (D) VEGF-a protein level in CM collected from SW48s treated with Sh-con and Sh-BRG1 under normoxic or hypoxic conditions were measured by ELISA kits. (E) Co-immunoprecipitation analysis in SW48 cells using anti-BRG1 antibodies and anti-mouse IgG antibodies or anti HIF-α antibodies and anti-mouse IgG under hypoxia condition, respectively. (F) Chromatin immunoprecipitation (ChIP) analyses of BRG1 binding on the promoters of VEGF-A under hypoxia condition. (G)ChIP assay and real-time PCR analysis showing the effect of HIF-1α on the VEGF-A promoter after the downregulation of BRG1 under hypoxia condition. P＜0.01.
3.3. BRG1 can regulate the expression of VEGF-A by interacting with HIF1α
3.4. Knockdown of BRG1 inhibits the proliferation and angiogenesis in mouse xenograft models
Given that VEGF-A is the most critical factor which stimulates both physiological and pathological angiogenesis, we detected the effect of Brg1 on the expression of VEGF-A to explore the regulation of Brg1 on angiogenesis. We found that Brg1 knockdown inhibited the expression of VEGF-A both in protein and mRNA levels in normoxia and hypoxia (Fig. 3A -B). The overexpression of BRG1 promoted the expression of VEGF-A in protein levels under normoxic and hypoxic conditions (Fig. 3C). To further demonstrate the role of BRG1 on VEGF-A expression, we transfected Ad-BRG1 in SW48-sh-BRG1 cells and diminished VEGF-A expression was recovered (Supplementary Figure 3A). The ELISA results further confirmed the relationship between Brg1 and VEGF-A. Our data showed that Brg1 knockdown significantly inhibited the expression of VEGF-A (Fig. 3D). To further clarify the mechanisms of the regulation of BRG1 on VEGF-A, we investigated the relationship between BRG1 and HIF-1α, which is the key regulator of the expression of VEGF-A. Our results showed that BRG1 interacted with HIF-1α detected by immunoprecipitation assay (Fig. 3E). It has been shown that HIF-1α regulates VEGF transcription by binding to the HRE in the VEGF promoter by ChIP assay. In our study, we found that VEGF promoter was occupied by BRG1 (Fig. 3F). Interestingly, knockdown BRG1 inhibited the function of HIF-1α to VEGF-A promoter(Fig. 3G). These results indicated that BRG1 binded with VEGF-A promoter to regulate the expression of VEGF-A by interacted with HIF-1α. To further investigate the role of HIF-1α on BRG1-regulated VEGF-A expression, we performed ‘rescue’ assay to show that the increases of VEGF-A induced by BRG1 overexpression were abrogated in the presence of HIF-1α inhibitor YC-1 (Supplementary Figure 3B).
To further evaluate the role of BRG1 in proliferation and angiogenesis in vivo, we generated stably BRG1-depleted cell lines by transducing SW48 cells with BRG1 shRNA (Sh-BRG1) and negative control shRNA (Sh-Con) lentivirus. Then the Sh-BRG1 cells and Sh-Con cells were subcutaneously injected into nude mice. We calculated the tumor volume every 4 days and the volumes were plotted into a tumor growth curve (Fig. 4A). After the mice were executed, we observed that Sh-BRG1 groups were significantly depressed in tumor volume (Fig. 4B) and weight (Fig. 4C) compared with Sh-Con groups. IHC analysis showed decreased expression of BRG1 and VEGF in the Sh-BRG1 group compared with the Sh-Con group. Moreover, CD34 antibody were used to measure tumor angiogenesis [23]. The expression of CD34 in tumors were obviously reduced in Sh-BRG1 group. These results revealed that knockdown of BRG1 inhibits CRC proliferation and angiogenesis in vivo.
3.5. BRG1 and VEGF expression are significantly correlated in colorectal tissues To investigate the expression of BRG1 and VEGF in clinic colorectal tissues, we performed q RT-PCR and Western Blot analysis to evaluate the expression of BRG1 and VEGF in the 12 pairs colorectal samples mentioned above. We found that VEGF was significantly overexpressed in colorectal tumor samples compared with normal tissues (Fig. 5A and B). Next we used the Pearson r correlation test to investigate the association between BRG1 and VEGF expression. We observed a significantly positive correlation between BRG1 and VEGF at mRNA levels (Fig. 5C). These results were also confirmed by IHC (Fig. 5D).
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Fig. 4. Knockdown of BRG1 inhibits the proliferation and angiogenesis in mouse xenograft models (A) Stable BRG1 knockdown SW48 cells and control cells were injected subcutaneously into nude mice (n = 7 for each group). The tumors were collected and shown. (B) Tumor development was measured as methods and material, and tumor volume was shown. (C) Tumor weight of each group was measured(Sh-Con vs Sh-BRG1 = 0.4457 ± 0.03993 vs 0.1114 + 0.008571). (D) representative images of IHC staining of indicated protein in tumors xenograft are shown. BRG1 scale bars, 50 µm, VEGF-A scale bars, 20 µm, CD34 scale bars, 200 µm.
4. Discussion
impact vascular development in midgestation embryo by promoting Wnt signaling and venous specification. The deletion of BRG1 from embryonic blood vessels results in yolk sac vascular remodeling defects [25]. Another study shows that BRG1 is requited for vascular integrity in infant mice [26]. However, these results were drawn from study in endothelial cells in embryology and showed no effect on postnatal angiogenesis. Meanwhile, the primary effect of the BRG1 in tumor cells on angiogenesis remains unclear. In our study, we showed that knockdown
Mammalian SWI/SNF adenosine ATP-dependent chromatin remodeling complexes promotes or represses transcription of genes by increasing or decreasing accessibility of DNA to large transcriptional machinery at specific loci [24]. BRG1, as the specific ATPase of SWI/ SNF chromatin-remodeling complex, is correlated with embryonic vascular development in previous study. It has been shown that BRG1
Fig. 5. BRG1 and VEGF expression are significantly correlated in colorectal tissues (A) Analysis of VEGF-A and BRG1 mRNA expression in colorectal tissues (including 12 colorectal tumor tissues and 12 corresponding colorectal normal tissues) are shown in box-whisker plot. Student's t-test (P = 0.0034) (B) Pearson r correlation was used to measure the relationship between BRG1 and VEGF-A mRNA levels, r = 0.8948, p < 0.0001. (C) BRG1 and VEGF-A protein levels in colorectal tissues (12 paired samples) were determined by western blot. (D) Representative images of IHC staining of CRC tumor tissues and corresponding colorectal normal tissues with anti-BRG1, anti-VEGF-A and anti-CD34 antibodies, 100 × scale bars, 50 µm, 200 × scale bars, 20 µm.
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Fig. 6. A hypothetical representation of the regulatory pathway underlying BRG1-induced angiogenesis.
functions as a general modulator of VEGF in CRC. We propose a model that suggests BRG1 bind to the HRE in the VEGE promoter to regulation the expression of VEGF to promote angiogenesis in a complex with HIF1α (Fig. 6). These findings provide evidence that BRG1 can be used as anti-angiogenic therapy for treating colorectal cancer.
of BRG1 inhibited the HUVEC capillary tube formation (Fig. 2), indicating that BRG1 played an important role in angiogenesis in CRC. Meanwhile, we investigated the expression of BRG1 in GEO datasets and clinical colorectal tissues and we observed BRG1 was overexpressed in colorectal tumor compared normal tissues (Fig. 1). These results indicating that BRG1 is closely correlated with tumor progression in CRC. Our results were not completely consistent with previous study about BRG1 in angiogenesis in endothelial cells [26]. The different cell type may contribute to explain the specific influence of BRG1 in angiogenesis. Although the correlation between BRG1 and vascular development was previously reported, our study is the first to systematically investigated the effect of BRG1 in angiogenesis in CRC. Tumor growth require angiogenesis when the tumor reaches 1–2 mm in diameter. Inhibition of angiogenesis specifically suppressed tumor growth without affecting the normal mature vessels in human body [27]. The angiogenic process is balanced by various positive (such as VEGF) and negative (such as thrombospondin-1) regulatory molecules of endothelial proliferation and migration [9]. VEGF is critical for both normal and tumor angiogenesis. Treatment with a VEGF antagonist significantly attenuated angiogenesis and tumor growth [28]. In our study, we observed that BRG1 knockdown inhibited the expression of VEGF in normoxic and hypoxic condition (Fig. 3). HIF-1 is a key factor in carcinogenesis, angiogenesis, tumor growth and can be induced by hypoxia. HIF-1α is often upregulated in human cancer to regulate VEGF expression by binding to the hypoxia responsive element of VEGF promoter [29]. Kenneth has reported that modulation of SWI/ SNF levels results in pronounced changes in hypoxia-inducible factor1α expression in osteosarcoma which stands in contrast to our results and Johnny A. Sena has reported that BRG1 could bind with HIF-1α. These previous data revealed that the role of BRG1 is complicated and changeable in different cancer types [30,31]. Our study showed that HIF-1α expression is not impacted by BRG1 in SW48 CRC cell line and BRG1 could interact with HIF-1α by the IP assay (Fig. 3). Furthermore, we observed that the hypoxia response element (HRE) in the VEGF promoter was occupied by BRG1 by the ChIP assays. These results indicating that BRG1 could bind to VEGF promoter to regulate the transcription of VEGF in a complex with HIF-1α. Another finding of this study is the overexpression of BRG1 in colorectal cancer and the promotion of BRG1 in the proliferation of colorectal cancer cells. The role of BRG1 in tumor progression was contradictor in previous study. For example, studies using BRG1 knock-out mouse models have shown that the loss of BRG1 increased susceptibility to both mammary gland and lung tumourigenesis [32,33]. These studies suggested that BRG1 acted as a tumor suppressor. However, BRG1 is upregulated and promoted tumor proliferation in gastric cancer and contributed to acute myeloid leukemia maintenance [17,34]. The cell-type-specific influence of BRG1 on VEGF expression may help explain the varying effects of BRG1 on tumor promotion or inhibition in different tumor types. In our study, we showed the expression of BRG1 in CRC and the role of BRG1 played in proliferation and angiogenesis of CRC. The mouse xenograft models further confirmed the promotion of BRG1 in colorectal cancer progression (Fig. 4). Taken together, this study provides significant evidence that BRG1
Author contribution Guihua Wang designed this project. Jingqin Lan, Haijie Li, Xuelai Luon and Junbo Hu performed the experiments. Conflict of interest There is no conflict of interest. Compliance with ethical standards Ethical approval Research involving animals: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Research Involving human participants: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Acknowledgements This work is supported by the National Natural Science Foundation of China (81570525, 81572725). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2017.09.013. References [1] H. Brenner, M. Kloor, C.P. Pox, Colorectal cancer, Lancet 383 (2014) 1490–1502. [2] M.G. Fakih, Metastatic colorectal cancer: current state and future directions, J. Clin. Oncol. 33 (2015) 1809–1824. [3] D. Hanahan, J. Folkman, Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis, Cell 86 (1996) 353–364. [4] H.L. Goel, A.M. Mercurio, VEGF targets the tumour cell, Nat. Rev. Cancer 13 (2013) 871–882. [5] C. Kwak, R.J. Jin, C. Lee, M.S. Park, S.E. Lee, Thrombospondin-1, vascular endothelial growth factor expression and their relationship with p53 status in prostate cancer and benign prostatic hyperplasia, BJU Int. 89 (2002) 303–309. [6] M. Toi, K. Inada, H. Suzuki, T. Tominaga, Tumor angiogenesis in breast cancer: its importance as a prognostic indicator and the association with vascular endothelial growth factor expression, Breast Cancer Res. Treat. 36 (1995) 193–204. [7] O.N. El-Assal, A. Yamanoi, Y. Soda, M. Yamaguchi, M. Igarashi, A. Yamamoto, T. Nabika, N. Nagasue, Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver, Hepatology 27 (1998) 1554–1562. [8] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003)
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