FOXF1 promotes angiogenesis and accelerates bevacizumab resistance in colorectal cancer by transcriptionally activating VEGFA

Accepted Manuscript FOXF1 promotes angiogenesis and accelerates bevacizumab resistance in colorectal cancer by transcriptionally activating VEGFA Shuyang Wang, Zhiyuan Xiao, Zexuan Hong, Hongli Jiao, Shaowei Zhu, Yali Zhao, Jiaxin Bi, Junfeng Qiu, Dan Zhang, Junyu Yan, Lingjie Zhang, Chengmei Huang, Tingting Li, Li Liang, Wenting Liao, Yaping Ye, Yanqing Ding PII:

S0304-3835(18)30591-3

DOI:

10.1016/j.canlet.2018.09.026

Reference:

CAN 14071

To appear in:

Cancer Letters

Received Date: 12 July 2018 Revised Date:

17 September 2018

Accepted Date: 19 September 2018

Please cite this article as: S. Wang, Z. Xiao, Z. Hong, H. Jiao, S. Zhu, Y. Zhao, J. Bi, J. Qiu, D. Zhang, J. Yan, L. Zhang, C. Huang, T. Li, L. Liang, W. Liao, Y. Ye, Y. Ding, FOXF1 promotes angiogenesis and accelerates bevacizumab resistance in colorectal cancer by transcriptionally activating VEGFA, Cancer Letters (2018), doi: https://doi.org/10.1016/j.canlet.2018.09.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT FOXF1 promotes angiogenesis and accelerates bevacizumab resistance in colorectal cancer by transcriptionally activating VEGFA Shuyang Wang*1,2,3, Zhiyuan Xiao*1,2,3, Zexuan Hong*1,2,3, Hongli Jiao1,2,3, Shaowei Zhu1,2,3, Yali Zhao1,2,3, Jiaxin Bi1,2,3, Junfeng Qiu1,2,3, Dan Zhang1,2,3, Junyu Yan1,2,3,

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Liao#1,2,3, Yaping Ye#1,2,3, Yanqing Ding#1,2,3

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Lingjie Zhang1,2,3, Chengmei Huang1,2,3, Tingting Li1,2,3, Li Liang1,2,3, Wenting

Authors’ Affiliations:

Guangzhou, China;

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1. Department of Pathology, Nanfang Hospital, Southern Medical University,

2. Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China;

3. Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Guangzhou,

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China.

*These authors contributed equally to this work.

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Corresponding Authors:

Wen-Ting Liao: Department of Pathology, Nanfang Hospital and School of Basic Medical

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Sciences, Southern Medical University, Guangzhou 510515, China; Phone: 86 (20) 6164-8224; Fax: 86 (20) 6164-8224; E-mail: [email protected]

Ya-Ping Ye: Department of Pathology, Nanfang Hospital and School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China; Phone: 86 (20) 6164-2148; Fax: 86 (20) 6164-2148; E-mail: [email protected] Yan-Qing Ding: Department of Pathology, Nanfang Hospital and School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China; Phone: 86 (20) 6164-2148; Fax: 86 (20) 6164-2148; E-mail: [email protected]

ACCEPTED MANUSCRIPT

Abstract Forkhead box F1 (FOXF1) has been recently implicated in the progression and metastasis of lung cancer and breast cancer. However, the biological functions and

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underlying mechanisms by which FOXF1 regulates the progression of colorectal cancer (CRC) are largely unknown. As shown in our previous study, FOXF1 is

upregulated in 182 CRC tissues, and elevated FOXF1 expression is significantly

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associated with microvessel density and advanced TNM (T=primary tumour;

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N=regional lymph nodes; M=distant metastasis) stages. In this study, 43 CRC tissues collected from patients who underwent treatment with first-line standard chemotherapeutic regimens in combination with bevacizumab were used to explore the correlation between FOXF1 expression and resistance to bevacizumab. In addition,

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FOXF1 regulated angiogenesis by inducing the transcription of vascular endothelial growth factor A1 (VEGFA) in vitro and in vivo. Furthermore, upregulation of FOXF1 enhanced bevacizumab resistance in CRC, and inhibition of VEGFA attenuated

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angiogenesis and bevacizumab resistance in FOXF1-overexpressing CRC cells. These

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results suggest that FOXF1 plays critical roles in CRC angiogenesis and bevacizumab resistance by inducing VEGFA transcription and that FOXF1 represents a potentially new therapeutic strategy and biomarker for anti-angiogenic therapy against CRC.

Keywords: FOXF1; Angiogenesis; Colorectal Cancer; VEGFA; Bevacizumab.

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Introduction Colorectal cancer (CRC) is one of the most common malignant solid tumours worldwide [1], and it develops through a complex biological process involving

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multiple steps, stages and genes. Angiogenesis is one of the biological steps that occurs during the progression and metastasis of CRC and functions as a rate-limiting step in tumour growth [2]. Anti-angiogenic therapy has become an important and

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common therapeutic strategy for multiple tumour types, including CRC [3].

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Forkhead box F1 (FOXF1) belongs to the FOX transcription factor family and contains a highly conserved DNA binding region (DBD) [4]. FOXF1 acts as a critical regulator of embryonic angiogenesis [5]. Mutation, deficiency or inactivation of FOXF1 has been shown to cause various developmental abnormalities and lesions,

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such as abnormal alveolar capillary development [6, 7] and pulmonary oedema [8]. FOXF1 expression is required for lung regeneration after injury [9]. FOXF1 also has been reported to participate in the progression and metastasis of many tumours.

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FOXF1 was recently reported to accelerate the growth and maintenance of

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gastrointestinal stromal tumours by regulating the expression of KIT and ETV1 [10]. FOXF1 promotes the progression of prostate tumours by inducing ERK5 expression [11] and increasing the invasive phenotype of breast cancer by upregulating lysyl oxidase (LOX) [12, 13]. High expression of FOXF1 in lung cancer fibroblasts is negatively correlated with the survival of patients with lung cancer and facilitates the expression of HGF [14, 15]. On the other hand, upregulation of FOXF1 expression reduces aggressive behaviours in hepatocellular cancer and is negatively correlated

ACCEPTED MANUSCRIPT with a poorer prognosis [16]. Based on these findings, FOXF1 may play an important and complicated functional role in the progression of malignancies. However, the clinicopathological expression pattern and functional role of FOXF1 in CRC remain

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unknown. As shown in our previous study FOXF1 expression is upregulated in CRC tissues; nevertheless, the functional role of FOXF1 expression in the progression of CRC requires further exploration.

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Angiogenesis plays an essential role in the proliferation and metastasis of CRC

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cells [17]. Generally, a dynamic balance exists between pro-angiogenic and anti-angiogenic processes in normal tissues. However, tumour cells tend to equilibrate towards the production of pro-angiogenic factors to promote angiogenesis by recruiting cells from the tumour microenvironment to sustain and promote tumour

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growth [18]. The strength of the angiogenic response depends on the activated pathway and level of pro-angiogenic signals [19]. This process requires the participation of multiple angiogenesis regulators, such as vascular endothelial growth

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factor (VEGF) [20], fibroblast growth factor (FGF) [21], endothelial growth factor

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(EGF) [22] and transforming growth factor (TGF) [23]. The identification of the regulatory mechanisms of these different components has resulted in the development of effective anti-angiogenic therapies for patients with CRC. Bevacizumab is the first and most commonly used anti-angiogenic drug that specifically targets VEGFA [24]. Bevacizumab combined with chemotherapy is the first-line treatment option for metastatic colorectal cancer, breast cancer, renal cell carcinoma, and advanced non-small cell lung cancer [25]. Even with its significant effect and wide range of

ACCEPTED MANUSCRIPT applications, bevacizumab may lose its therapeutic effect due to drug resistance problems. As shown in our previous study, high expression of FOXF1 is strongly correlated with the microvessel density, and patients with CRC displaying

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bevacizumab resistance exhibit increased expression of FOXF1, indicating that FOXF1 may influence angiogenesis and the resistance to bevacizumab in patients with CRC.

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Here, we probed the expression patterns and functional role of FOXF1 in CRC

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angiogenesis and bevacizumab resistance and investigated the underlying mechanism by which FOXF1 regulates angiogenesis and drug sensitivity. Our study may provide

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insights into a new potential therapeutic strategy and targets for patients with CRC.

ACCEPTED MANUSCRIPT Materials and methods Patients and tissue specimens One hundred eighty-two paraffin-embedded CRC samples and 43

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paraffin-embedded CRC samples collected from patients who underwent first-line chemotherapy combined with bevacizumab treatment were used in this study. The response to the bevacizumab treatment was assessed according to the Response

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Evaluation Criteria in Solid Tumors (RECIST 1.1) and progression-free survival [26,

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27]. Evaluations were based on clinical and radiological examinations. The clinical samples were gathered from Nanfang Hospital, Southern Medical University between 2010 and 2013. Prior approval was obtained from the Southern Medical University Institutional Board (Guangzhou, China). Fresh biopsies of 10 CRC tissues and

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matched normal mucosa tissues were frozen and stored in liquid nitrogen until further use. Medical records for the patients who provided the samples were reviewed, and

Cell culture

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the clinical information is summarized in the Supplementary Materials and Methods.

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The human CRC cell lines SW480, HCT116, HCT15 and SW620 and human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection. SW620 cells were cultured in DMEM (Gibco), whereas SW480, HCT116 and HCT15 cells were cultured in RPMI 1640 medium (Gibco). All media were supplemented with 10% FBS (Gibco). HUVECs were cultured in DMEM containing F12K medium (Gibco) and 20% FBS (Gibco). All cells were cultured at 37°C in a 5% CO2 atmosphere.

ACCEPTED MANUSCRIPT Real-time quantitative PCR, Western blot and immunohistochemistry Real-time quantitative PCR (RT-PCR), Western blotting (WB) and immunohistochemistry (IHC) assays were performed using previously described

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methods [28]. Details are provided in the Supplementary Materials and Methods. Plasmids

The FOXF1 or VEGFA construct was generated by sub-cloning the PCR-amplified

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full-length human FOXF1 cDNA into the plent-EF1α-Flag-puro plasmid. We cloned 2

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short hairpin RNA (shRNA) oligonucleotides into pSuper-retro-puro (Oligo-Engine, Seattle, WA, USA) to generate pSuper-retro-FOXF1-shRNA (FOXF1 shRNA#1: 5’-CGAAAGGAGTTTGTCTTCT-3’; FOXF1 shRNA#2:

5’-GCATGATGAACGGCCACTT-3’) for the knockdown of FOXF1 expression. The

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shVEGFA plasmid was purchased from Vigene Biosciences. Retrovirus production and infection were performed as previously described [29]. Luciferase assays

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The luciferase assays were performed using previously described methods [28, 30,

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31]. Further details are provided in the Supplementary Materials and Methods section. Human umbilical vein endothelial cell tube formation assay

First, 200 µL of Matrigel was added to the wells of 24-well plates and incubated at

37°C for 30 min. Then, 5×104 HUVECs (expressing GFP) suspended in 200 µL of conditioned medium were plated in each well and incubated for 8 h at 37°C. Then, a picture was captured in a 100×green fluorescence field, and the tubule formation ability was measured by determining the length of the tubules.

ACCEPTED MANUSCRIPT Chicken chorioallantoic membrane assays Six-day-old fertilized chicken embryos were assayed. A 1-cm-diameter hole was opened in the upper shell of each egg (Yueqin Breeding Co., Ltd.), and the surface of the dermic sheet

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was removed to expose the chorioallantoic membrane. A 0.5-cm-diameter filter paper was added to the surface of the chorioallantoic membrane (CAM), and 70 µL of conditioned

medium was added to the paper in a dropwise manner. Then, the egg was incubated at 37°C

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for 72 h. A stationary solution (methanol: acetone=1:1) was used to fix the CAM for 15 min;

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then, the CAMs were cut, and samples were collected and imaged with a digital camera (SONY). The number of second- and third-order vessels were calculated to evaluate angiogenesis in each treatment group. ELISAs and MTT assay

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The luciferase assays were performed according to previously described methods [28, 30, 31]. Further details are provided in the Supplementary Materials and Methods section.

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Chromatin immunoprecipitation

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ChIP assays were performed using a commercial kit (ACTIVE MOTIF, ChIP-IT Express, catalogue # 53008). Briefly, cells (2×107) plated in a 10-cm culture dish were treated with 1% formaldehyde to cross-link chromatin-associated proteins to DNA. Cell lysates were subjected to ultrasound for 9-10 sets of 10-s pulses at a 40% output to shear the DNA into fragments ranging between 200 and 1,000 bp. Equal amounts of cell lysates were incubated with either 1 µg of an anti-Flag antibody (Sigma) or an anti-IgG antibody (Millipore), which served as a negative control. All chromatin

ACCEPTED MANUSCRIPT supernatants were incubated with 20 µL of magnetic Protein G beads overnight at 4°C with rotation. On the second day, the protein-DNA complexes were reversed and purified to obtain pure DNA. The human VEGFA promoter was amplified by

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RT-PCR. Bevacizumab

Bevacizumab (an anti-VEGFA monoclonal antibody purchased from Selleck,

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A2006) was added to the indicated cells at concentrations of 125 µg/mL or 250

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µg/mL and incubated for 48 h. Tumour-bearing nude mice were treated with bevacizumab at a dose of 1-2 mg/kg twice per week. Mouse model of xenograft tumours

A mouse model of CRC xenograft tumours was established as previously described

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[32]. All mice were housed and maintained under specific pathogen-free conditions and used in accordance with institutional guidelines and approved by the Use

Methods.

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Committee for Animal Care. Details are provided in the Supplementary Materials and

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Statistical analysis

All statistical analyses were performed using SPSS 20.0 for Windows. The statistical tests conducted included Fisher’s exact test, the log-rank test, χ2 test, ANOVA and Student’s t-test. Bivariate correlations between study variables were determined by calculating Spearman’s rank correlation coefficients. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Data are presented as means ± SD. A p-value of p<0.05 was considered statistically significant.

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ACCEPTED MANUSCRIPT Results High FOXF1 expression is associated with an advanced progression, an increased microvessel density and poorer survival of patients with CRC Western blotting and RT-PCR were used to examine FOXF1 expression in 10

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paired primary tumours (T) and adjacent normal intestinal mucosa (N), and the results showed significantly higher levels of the FOXF1 protein and mRNA in tumour tissues

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than in normal tissues (Figure 1A and Supplementary Figure S1A).

Next, the expression pattern of the FOXF1 protein was examined in 182

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paraffin-embedded CRC tissues by immunohistochemistry. FOXF1 expression was upregulated in 55.5% (101/182) of CRC tissues compared to the expression in their matched adjacent normal tissues (Figure 1B and Supplementary Table 1). The analysis of the relationship between FOXF1 expression and the clinicopathological parameters

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showed that higher FOXF1 expression was associated with more aggressive tumour phenotypes, such as poorly differentiated tumours (p=0.015), advanced Dukes stage

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(p<0.001), T classification (p=0.004), lymph node involvement (p<0.001) and distant metastasis (p=0.044, Supplementary Table S2). The microvessel density (MVD) was

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evaluated by CD31 staining [33]. The MVD in tumour tissues with high FOXF1 expression was significantly increased compared with tumour tissues with low FOXF1 expression (Figure 1C and D). The correlation between levels of the FOXF1 mRNA and the microvessel biomarker CD31 was analysed in The Cancer Genome Atlas (TCGA) database and the public GEO database, and the results revealed a positive correlation between FOXF1 mRNA expression and CD31 expression (Figure 1E r=0.49, p<0.001; Supplementary Figure S1C, GSE39582, r=0.41, p<0.001;

ACCEPTED MANUSCRIPT GSE41258, r=0.47, p<0.001; GSE41258, r=0.53, p<0.001). The results from Kaplan-Meier survival analyses revealed shorter survival times for patients with high FOXF1 expression levels than for patients with low FOXF1 expression (p<0.001,

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Figure 1F); similar results were obtained from the published CRC dataset (GSE17538, Supplementary Figure S1B). Based on these results, FOXF1 expression was increased in CRC tumour tissues, and that its high expression pattern was significantly

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correlated with advanced progression, a higher microvessel density and poorer

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survival of patients with CRC.

High FOXF1 expression is associated with angiogenesis and drug resistance to bevacizumab in CRC tissues

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A gene set enrichment analysis (GSEA) and GO analysis were used to examine the FOXF1-regulated gene signatures. According to the results of the GSEA, higher FOXF1 expression was positively correlated with the enrichment of

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angiogenesis-related gene signatures (GSE17538, GSE41258 and GSE13294) (Figure

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2A). The results of GO analysis of a public mouse embryonic lung FOXF1 ChIP-seq database (GSE77951, n=3) revealed that genes that are potentially transcriptionally regulated by FOXF1 were mainly enriched in the GO terms “transcription factor activity”, “RNA polymerase II distal enhancer sequence-specific binding” and “positive regulation of angiogenesis” (Figure 2B). A protein network analysis of genes that are potential transcriptional targets of FOXF1 (GSE77951) showed VEGFA in the core location of the network (Figure 2C). VEGFA is an essential regulator of both

ACCEPTED MANUSCRIPT physiological and pathological angiogenesis, including angiogenesis occurring during tumourigenesis. VEGFA is a particular pharmacological target, and many anti-angiogenic drugs target VEGFA, including bevacizumab. Bevacizumab

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specifically binds to VEGFA and blocks its biological activity; thus, it is a conventional anti-angiogenic agent used to treat CRC [34]. We collected data from 43 patients with CRC who received first-line chemotherapeutic regimens in combination

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with bevacizumab treatment and divided them into two groups (bevacizumab-resistant

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or -sensitive). A greater proportion of patients in the bevacizumab-resistant group expressed FOXF1 at high levels than patients in the bevacizumab-sensitive group (Figure 2D). Thus, FOXF1 might promote angiogenesis by binding to VEGFA DNA, and the proangiogenic effects of FOXF1 may accelerate the resistance of CRC to

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bevacizumab.

FOXF1 transcriptionally activates VEGFA and leads to increased VEGFA

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secretion from CRC cells

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We established stable FOXF1-overexpressing or knockdown CRC cell lines (Supplementary Figure S2A), and the overexpression or knockdown of FOXF1 increased or decreased VEGFA expression, respectively, at both the mRNA and protein levels (Figure 3A and B). As a transcription factor, FOXF1 binds to a target gene with the specific DNA sequence RTAAAYA to promote gene expression [4]. Furthermore, we analysed the promoter sequence of the VEGFA gene and detected the sequence of the FOXF1 binding site (Figure 3C). We hypothesized that FOXF1 might

ACCEPTED MANUSCRIPT promote angiogenesis by directly binding to the VEGFA promoter to induce its expression. Therefore, we performed chromatin immunoprecipitation (ChIP) assays of FLAG-FOXF1 in SW480 cells, followed by QPCR of the VEGFA promoter and

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upstream regions, and the results revealed that the FOXF1 protein bound to the VEGFA promoter at the candidate site P2 (Figure 3D). Moreover, the results of the dual luciferase reporter assay also revealed that FOXF1 activated the wild-type

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VEGFA promoter but not the mutant promoter (Figure 3E).

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VEGFA functions as paracrine effector and binds to the corresponding VEGFR on the surface of endothelial cells as a ligand, thus activating its downstream signalling pathway (e.g., ERK signalling pathway, PI3K/AKT signalling pathway and P38/MAPK signalling pathway). Therefore, we determined the VEGFA concentration

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in the supernatants of the indicated cells, and paracrine VEGFA secretion was increased in FOXF1-overexpressing CRC cells compared with control cells (Figure 3F). In contrast, the concentration of VEGFA in the supernatant was significantly

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reduced in FOXF1 knockdown cells (Figure 3G). After human umbilical vein

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endothelial cells (HUVECs) were incubated with the indicated cell supernatant for 48 h, the activity of the corresponding signalling pathway in endothelial cells was detected by WB. The expression of the key regulatory molecules P-ERK, P-AKT and P-P38 was significantly increased in HUVECs that were incubated with supernatants collected from FOXF1-overexpressing CRC cells compared with control cells. In contrast, the levels of crucial signalling molecules were decreased in HUVECs incubated with FOXF1 knockdown cell supernatants (Figure 3H).

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FOXF1 overexpression induces bevacizumab resistance in CRC cells through VEGFA

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Next, we detected the effects of FOXF1 upregulation on bevacizumab resistance in CRC cells through the transcriptional activation of VEGFA. FOXF1-overexpressing, knockdown or control CRC cells were treated with the indicated dose of bevacizumab

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for 48 h. The level of the VEGFA protein in vector-transfected SW480 or HCT116

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cells was significantly reduced by the administration of 250 µg/mL bevacizumab (Figure 4A, top panel); the expression level was evaluated using Quantity One Software (Supplementary Figure S3A, left panel). The same dose had less of an effect on VEGFA levels in FOXF1-transfected SW480 or HCT116 cells unless VEGFA

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expression was simultaneously silenced (Figure 4A, top panel, and Supplementary Figure S3A, left panel). HUVECs were incubated with the indicated conditioned medium, and the expression of downstream intermediates in the VEGFR signalling

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pathway was detected. The results showed significant inactivation of VEGFR

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signalling in vector-transfected SW480 or HCT116 cells after treatment with the indicated supernatants, as evidenced by the lower levels of P-ERK, P-AKT and P-P38, while the same concentration had less of an effect on VEGFA levels in FOXF1-transfected SW480 or HCT116 cells unless VEGFA expression was simultaneously decreased (Figure 4B, top panel, and Supplementary Figure S3B). Furthermore, the concentration of VEGFA in the indicated CRC cell supernatants exhibited a similar trend (Figure 4C, top panel).

ACCEPTED MANUSCRIPT In contrast, the treatment of HCT15 or SW620 cells transfected with shFOXF1 with 125 µg/mL bevacizumab exerted a greater effect on VEGFA expression than the same treatment in control cells (Figure 4A, bottom panel, and Supplementary Figure

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S3A, right panel), similar to the levels of P-ERK, P-AKT and P-P38 in HUVECs incubated with the indicated supernatants (Figure 4B, bottom panel and

Supplementary Figure S3C). The concentration of VEGFA in the supernatant

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collected from FOXF1 knockdown cells was substantially decreased compared with

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the concentration in the supernatant from control cells after treatment with the same dose of bevacizumab (Figure 4C, bottom panel). Meanwhile, ectopic VEGFA expression in the indicated cells reduced the effect. The same effect was observed on the proliferation of HUVECs cultured with the conditioned medium collected from

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the indicated tumour cells (Figure 4D and E). Based on these results, FOXF1 overexpression increased bevacizumab resistance, while FOXF1 knockdown exerted

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transcription.

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the opposite effect on bevacizumab resistance in CRC cells by inducing VEGFA

Downregulation of FOXF1 inhibits angiogenesis and bevacizumab resistance in CRC cells through VEGFA Next, we detected the effects of FOXF1 downregulation on angiogenesis and

bevacizumab resistance in CRC cells through the transcriptional activation of VEGFA. Tubule formation and chicken CAM assays were performed by treating the cells/CAMs with the indicated cell supernatants. Ectopic expression of FOXF1

ACCEPTED MANUSCRIPT significantly increased the formation of tubules by HUVECs and promoted angiogenesis in CAM (Figure 5A and C, top panels). The same dose of bevacizumab exerted a significantly smaller effect on the FOXF1 overexpression group than on the

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control group, unless VEGFA expression was simultaneously blocked in the FOXF1 overexpression group (Figure 5A and C, top panels). The same phenomenon was

observed after increasing the dose of bevacizumab. Low-dose bevacizumab was more

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effective in the control group, and the same effect was only achieved when the dose of

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bevacizumab was increased in the FOXF1 overexpression group (Supplementary Figure S4A and B). In contrast, FOXF1 knockdown decreased the tubule length and inhibited angiogenesis in CAMs (Figure 5A and C, bottom panels). The same concentration of bevacizumab exerted greater effects on tubule formation and

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angiogenesis in the FOXF1 knockdown group than in the control group. Meanwhile, the overexpression of exogenous VEGFA partially reversed the effects (Figure 5A and C, bottom panels). Thus, FOXF1 regulates VEGFA transcription to further promote

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angiogenesis and bevacizumab resistance in CRC cells.

FOXF1 overexpression promotes angiogenesis and tumourigenesis and facilitates bevacizumab resistance in CRC cells by regulating VEGFA transcription Next, we used the SCID mouse tumourigenesis assay to investigate the functional

roles of FOXF1 in tumourigenesis and angiogenesis in vivo. The indicated cells were subcutaneously injected into each mouse, and tumours were allowed to develop. FOXF1 overexpression promoted tumour growth (Figure 6A and B) and increased the

ACCEPTED MANUSCRIPT MVD (as indicated by CD31-positive cells, Figure 6C and D) compared with the control, and the opposite results were obtained after silencing FOXF1 expression. The bevacizumab treatment (2 mg/kg for indicated group) exerted a greater effect on the

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tumour volume and in vivo angiogenesis in the control group than in the FOXF1 overexpression group, while the simultaneous knockdown of VEGFA partially

abrogated this effect. Moreover, the FOXF1 knockdown group showed the same

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effects on tumourigenesis and angiogenesis as treatment with a lower dose of

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bevacizumab (2 mg/kg for the control group and 1 mg/kg for the knockdown group). In addition, overexpression of VEGFA partially reversed this effect. Taken together, FOXF1 promoted tumourigenesis and angiogenesis in vivo by regulating VEGFA

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expression and increasing the bevacizumab resistance in vivo.

Clinical relevance of the expression of FOXF1 and VEGFA in human CRC tissues

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As mentioned above, the upregulation of FOXF1 promoted angiogenesis and

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induced bevacizumab resistance in CRC cells by transcriptionally activating VEGFA. RT-PCR, Western blot and IHC analyses were used to verify this result. The analyses of protein (n=10 and 2 normal tissues as control) and mRNA expression (n=30) revealed a positive correlation between FOXF1 and VEGFA levels in human CRC tissues (Figure 7A, r=0.849, p<0.001; Figure 7B, r=0.813, p<0.001, Supplementary Figure S5A and B). Moreover, the analysis of a CRC tissue chip including 2 normal tissues and 80 CRC tissues exhibited a similar correlation between FOXF1 and

ACCEPTED MANUSCRIPT VEGFA levels (Figure 7C and D, r=559, p<0.001). We also evaluated the levels of the FOXF1 and VEGFA mRNAs in breast cancer and renal cell carcinoma samples from patients who also used bevacizumab in combination with chemotherapy as a first-line

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treatment in TCGA dataset. A positive correlation was observed between FOXF1 and VEGFA levels in breast cancer (r=0.13, p<0.001) and renal cell carcinoma (r=0.56, p<0.001; Supplementary Figure S5C). Taken together, these data support the

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hypothesis that the upregulation of FOXF1 directly transcriptionally activates VEGFA,

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subsequently promoting angiogenesis and inducing bevacizumab resistance in CRC

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cells (Figure 7E).

ACCEPTED MANUSCRIPT Discussion FOXF1 is a member of the forkhead box family of transcription factors and has been reported to participate in multiple human cancers, such as prostate cancer [11]

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and breast cancer [13]. According to the results from the present study, the upregulation of FOXF1 was correlated with advanced aggressive characteristics of CRC, such as poor differentiation and advanced Dukes and TNM stages. High

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FOXF1 expression was significantly correlated with a poorer prognosis for patients

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with CRC, indicating that FOXF1 might be a candidate tumour oncogene in CRC. Tumour is currently considered a developmental biology problem. Many similarities exist between tumourigenesis and the biological behaviours observed during embryonic development [35]. Signalling pathways involved in embryonic

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development (including Wnt, Notch, and VEGF) are abnormally activated during the development and progression of various malignancies, including CRC. Since FOXF1 plays an essential role in regulating angiogenesis during embryonic development [36],

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we hypothesized that FOXF1 may participate in angiogenesis to influence the

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progression of CRC.

The sustained growth and metastasis of CRC depends on sufficient angiogenesis

and blood supply. The generation of the vasculature depends on the balance of various pro-angiogenic and anti-angiogenic regulators. Both tumour cells and stromal cells secrete multiple proangiogenic factors to stimulate the germination of new vessels, which in turn leads to the persistent proliferation of tumour cells and a greater probability of invasion and metastasis [37]. Various growth factor pathways

ACCEPTED MANUSCRIPT participate in modulating the growth and maintenance of angiogenesis. VEGFA is one of most important growth factors that regulates angiogenesis [38]. VEGFA directly binds to VEGFR1 or 2 on the surface of endothelial cell (EC) membranes and

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activates the downstream signalling pathways to promote EC proliferation and survival through autocrine or paracrine pathways [20]. VEGFA overexpression is

associated with the development of multiple tumours and malignancies, including

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colorectal cancer [39], breast cancer [40], prostate cancer [41], lung cancer [42] and

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melanoma [43]. As shown in the present study, high FOXF1 expression showed a strong positive correlation with the microvessel density, and the upregulation of FOXF1 promoted angiogenesis in CRC samples in vivo and in vitro. Moreover, FOXF1 promoted angiogenesis by inducing VEGFA transcription. We provided

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several lines of evidence to prove our hypothesis. First, VEGFA was upregulated by the ectopic expression of FOXF1 and downregulated by FOXF1 knockdown. Second, the exogenous overexpression of FOXF1 in CRC cells increased the secretion of

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VEGFA and continued to activate the VEGFR signalling pathway in endothelial cells.

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Most importantly, FOXF1 directly bound to a specific region in the VEGFA promoter and induced VEGFA transcription. Thus, we speculated that FOXF1 upregulation plays an essential role in the VEGFA-dependent pro-angiogenesis effect on CRC. VEGFA plays an essential role in angiogenesis, and its expression is upregulated

in CRC tumour cells. Blockade of VEGFA signalling inhibits tumour angiogenesis and thus inhibits tumour growth and metastasis [44]. Bevacizumab is a monoclonal immunoglobulin G antibody that specifically binds to and neutralizes human VEGFA

ACCEPTED MANUSCRIPT to prevent the activation of VEGFR signalling [45]. Although bevacizumab is a molecular-targeted therapy that can be used to treat metastatic colorectal cancer [46], bevacizumab resistance limits its therapeutic efficacy in these patients. The

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mechanism of bevacizumab resistance may be related to the bypass of the angiogenesis pathway [47], and it has been shown to involve EGF [48], FGF1 [49],

FGF2, and interleukin-8 [50]. In addition, researchers have proposed some possible

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mechanisms for drug resistance, such as the activation of cancer stem cells, changes

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in blood flow, the acquisition of blood vessels in the normal tissue around the tumour, hypoxia, the progression and invasion of the tumour, and the inhibition of immune surveillance [51]. However, a key, universal mechanism has not been elucidated, and biological markers that predict the resistance to bevacizumab have not yet been

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discovered [52]. Here, we discovered that patients presenting bevacizumab resistance expressed higher levels of FOXF1. The exogenous overexpression of FOXF1 promoted bevacizumab resistance by regulating angiogenesis and tumourigenesis in

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vivo and in vitro. Silencing VEGFA partially reversed the effects, suggesting that

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FOXF1 increased bevacizumab resistance by targeting VEGFA during angiogenesis in CRC.

In summary, FOXF1 promotes angiogenesis during CRC progression and then

induces tumour resistance to bevacizumab by inducing VEGFA transcription. These data highlight the FOXF1-VEGFA axis as a potential therapeutic target in CRC and provide a new biological marker for selecting suitable patients for anti-angiogenic therapy.

ACCEPTED MANUSCRIPT Acknowledgements This work was supported by grants from the National Key R&D Program of China (2017YFC1309002), the National Basic Research Program of China (973 program,

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2015CB554002), the National Natural Science Foundation of China (81472313, 81172055, 81472710, 81773196, 81702915, 81773101 and 81672886), the

Postdoctoral Science Foundation of China (2016M592511 and 2018M633080), the

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Guangdong Provincial Natural Science Foundation of China (2016A030310395,

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2016A030310394, 2016A030310392, 2017A030310117, 2017A030313463, and 2017A030313583), the Science and Technology Innovation Foundation of Guangdong Higher Education (CXZD1016), and Guangdong Medical Research Fund (A2017302 and A2016218).

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Competing interests

The authors have no competing financial interests to disclose. Conflict of Interest

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The authors of this manuscript have no conflict of interest.

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References [1] R.L. Siegel, K.D. Miller, S.A. Fedewa, D.J. Ahnen, R.G.S. Meester, A. Barzi, et al., Colorectal cancer statistics, 2017, CA: a cancer journal for clinicians, 67 (2017) 177-193. [2] R. Ronca, M. Benkheil, S. Mitola, S. Struyf, S. Liekens, Tumor angiogenesis revisited: Regulators and clinical implications, Medicinal research reviews, 37 (2017) 1231-1274. [3] I. Dimova, G. Popivanov, V. Djonov, Angiogenesis in cancer - general pathways and their therapeutic

RI PT

implications, Journal of B.U.ON. : official journal of the Balkan Union of Oncology, 19 (2014) 15-21. [4] S. Pierrou, M. Hellqvist, L. Samuelsson, S. Enerback, P. Carlsson, Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending, The EMBO journal, 13 (1994) 5002-5012.

[5] X. Ren, V. Ustiyan, A. Pradhan, Y. Cai, J.A. Havrilak, C.S. Bolte, et al., FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells,

SC

Circulation research, 115 (2014) 709-720.

[6] N. Nagano, K. Yoshikawa, S. Hosono, S. Takahashi, T. Nakayama, Alveolar capillary dysplasia with misalignment of the pulmonary veins due to novel insertion mutation of FOXF1, Pediatrics

M AN U

international : official journal of the Japan Pediatric Society, 58 (2016) 1371-1372.

[7] H.M. Luk, T. Tang, K.W. Choy, M.F. Tong, O.K. Wong, F.M. Lo, Maternal somatic mosaicism of FOXF1 mutation causes recurrent alveolar capillary dysplasia with misalignment of pulmonary veins in siblings, American journal of medical genetics. Part A, 170 (2016) 1942-1944. [8] Y. Cai, C. Bolte, T. Le, C. Goda, Y. Xu, T.V. Kalin, et al., FOXF1 maintains endothelial barrier function and prevents edema after lung injury, Science signaling, 9 (2016) ra40.

[9] C. Bolte, H.M. Flood, X. Ren, S. Jagannathan, A. Barski, T.V. Kalin, et al., FOXF1 transcription factor

TE D

promotes lung regeneration after partial pneumonectomy, Scientific reports, 7 (2017) 10690. [10] L. Ran, Y. Chen, J. Sher, E.W.P. Wong, D. Murphy, J.Q. Zhang, et al., FOXF1 Defines the Core-Regulatory Circuitry in Gastrointestinal Stromal Tumor, Cancer discovery, 8 (2018) 234-251. [11] L. Fulford, D. Milewski, V. Ustiyan, N. Ravishankar, Y. Cai, T. Le, et al., The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5, Science

EP

signaling, 9 (2016) ra48.

[12] G. Nilsson, M. Kannius-Janson, Forkhead Box F1 promotes breast cancer cell migration by upregulating lysyl oxidase and suppressing Smad2/3 signaling, BMC cancer, 16 (2016) 142.

AC C

[13] J. Nilsson, K. Helou, A. Kovacs, P.O. Bendahl, G. Bjursell, M. Ferno, et al., Nuclear Janus-activated kinase 2/nuclear factor 1-C2 suppresses tumorigenesis and epithelial-to-mesenchymal transition by repressing Forkhead box F1, Cancer research, 70 (2010) 2020-2029. [14] H.J. Wei, J.A. Nickoloff, W.H. Chen, H.Y. Liu, W.C. Lo, Y.T. Chang, et al., FOXF1 mediates mesenchymal stem cell fusion-induced reprogramming of lung cancer cells, Oncotarget, 5 (2014) 9514-9529.

[15] R.A. Saito, P. Micke, J. Paulsson, M. Augsten, C. Pena, P. Jonsson, et al., Forkhead box F1 regulates tumor-promoting properties of cancer-associated fibroblasts in lung cancer, Cancer research, 70 (2010) 2644-2654. [16] Z.G. Zhao, D.Q. Wang, D.F. Hu, Y.S. Li, S.H. Liu, Decreased FOXF1 promotes hepatocellular carcinoma tumorigenesis, invasion, and stemness and is associated with poor clinical outcome, OncoTargets and therapy, 9 (2016) 1743-1752. [17] A. Mihalache, I. Rogoveanu, Angiogenesis factors involved in the pathogenesis of colorectal

ACCEPTED MANUSCRIPT cancer, Current health sciences journal, 40 (2014) 5-11. [18] P. Carmeliet, R.K. Jain, Molecular mechanisms and clinical applications of angiogenesis, Nature, 473 (2011) 298-307. [19] S.M. Weis, D.A. Cheresh, Tumor angiogenesis: molecular pathways and therapeutic targets, Nature medicine, 17 (2011) 1359-1370. [20] L. Claesson-Welsh, M. Welsh, VEGFA and tumour angiogenesis, Journal of internal medicine, 273 (2013) 114-127.

RI PT

[21] M. Presta, P. Chiodelli, A. Giacomini, M. Rusnati, R. Ronca, Fibroblast growth factors (FGFs) in

cancer: FGF traps as a new therapeutic approach, Pharmacology & therapeutics, 179 (2017) 171-187. [22] D. Bruce, P.H. Tan, Vascular endothelial growth factor receptors and the therapeutic targeting of angiogenesis in cancer: where do we go from here?, Cell communication & adhesion, 18 (2011) 85-103.

[23] V.V. Orlova, Z. Liu, M.J. Goumans, P. ten Dijke, Controlling angiogenesis by two unique TGF-beta

SC

type I receptor signaling pathways, Histology and histopathology, 26 (2011) 1219-1230. [24] D.J. Kerr, Targeting angiogenesis in cancer: clinical development of bevacizumab, Nature clinical practice. Oncology, 1 (2004) 39-43.

M AN U

[25] G. Ruan, L. Ye, G. Liu, J. An, J. Sehouli, P. Sun, The role of bevacizumab in targeted vascular endothelial growth factor therapy for epithelial ovarian cancer: an updated systematic review and meta-analysis, OncoTargets and therapy, 11 (2018) 521-528.

[26] E.A. Eisenhauer, P. Therasse, J. Bogaerts, L.H. Schwartz, D. Sargent, R. Ford, et al., New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1), European journal of cancer, 45 (2009) 228-247.

[27] S. Stintzing, D.P. Modest, L. Rossius, M.M. Lerch, L.F. von Weikersthal, T. Decker, et al., FOLFIRI

TE D

plus cetuximab versus FOLFIRI plus bevacizumab for metastatic colorectal cancer (FIRE-3): a post-hoc analysis of tumour dynamics in the final RAS wild-type subgroup of this randomised open-label phase 3 trial, The Lancet. Oncology, 17 (2016) 1426-1434.

[28] W.T. Liao, Y.P. Ye, N.J. Zhang, T.T. Li, S.Y. Wang, Y.M. Cui, et al., MicroRNA-30b functions as a tumour suppressor in human colorectal cancer by targeting KRAS, PIK3CD and BCL2, The Journal of pathology,

EP

232 (2014) 415-427.

[29] S.Y. Wang, K. Gao, D.L. Deng, J.J. Cai, Z.Y. Xiao, L.Q. He, et al., TLE4 promotes colorectal cancer progression through activation of JNK/c-Jun signaling pathway, Oncotarget, 7 (2016) 2878-2888.

AC C

[30] H.L. Jiao, Y.P. Ye, R.W. Yang, H.Y. Sun, S.Y. Wang, Y.X. Wang, et al., Down-regulation of SAFB sustains the NF-kappaB pathway by targeting TAK1 during the progression of colorectal cancer, Clinical cancer research : an official journal of the American Association for Cancer Research, (2017). [31] W.T. Liao, D. Jiang, J. Yuan, Y.M. Cui, X.W. Shi, C.M. Chen, et al., HOXB7 as a prognostic factor and mediator of colorectal cancer progression, Clinical cancer research : an official journal of the American Association for Cancer Research, 17 (2011) 3569-3578. [32] W. Tseng, X. Leong, E. Engleman, Orthotopic mouse model of colorectal cancer, Journal of visualized experiments : JoVE, (2007) 484. [33] C. Huang, Z. Li, N. Li, Y. Li, A. Chang, T. Zhao, et al., Interleukin 35 Expression Correlates With Microvessel Density in Pancreatic Ductal Adenocarcinoma, Recruits Monocytes, and Promotes Growth and Angiogenesis of Xenograft Tumors in Mice, Gastroenterology, 154 (2018) 675-688. [34] P. Saharinen, L. Eklund, K. Pulkki, P. Bono, K. Alitalo, VEGF and angiopoietin signaling in tumor angiogenesis and metastasis, Trends in molecular medicine, 17 (2011) 347-362.

ACCEPTED MANUSCRIPT [35] R.S. Peterson, L. Lim, H. Ye, H. Zhou, D.G. Overdier, R.H. Costa, The winged helix transcriptional activator HFH-8 is expressed in the mesoderm of the primitive streak stage of mouse embryos and its cellular derivatives, Mechanisms of development, 69 (1997) 53-69. [36] . [37] J. Folkman, Role of angiogenesis in tumor growth and metastasis, Seminars in oncology, 29 (2002) 15-18. [38] J. Holash, P.C. Maisonpierre, D. Compton, P. Boland, C.R. Alexander, D. Zagzag, et al., Vessel

RI PT

cooption, regression, and growth in tumors mediated by angiopoietins and VEGF, Science, 284 (1999) 1994-1998.

[39] M.L. Slattery, A. Lundgreen, R.K. Wolff, VEGFA, FLT1, KDR and colorectal cancer: assessment of

disease risk, tumor molecular phenotype, and survival, Molecular carcinogenesis, 53 Suppl 1 (2014) E140-150.

[40] L.V. Santos, M.R. Cruz, L. Lopes Gde, J.P. Lima, VEGF-A levels in bevacizumab-treated breast cancer

SC

patients: a systematic review and meta-analysis, Breast cancer research and treatment, 151 (2015) 481-489.

[41] F. Botelho, F. Pina, N. Lunet, VEGF and prostatic cancer: a systematic review, European journal of

M AN U

cancer prevention : the official journal of the European Cancer Prevention Organisation, 19 (2010) 385-392.

[42] D. Frezzetti, M. Gallo, M.R. Maiello, A. D'Alessio, C. Esposito, N. Chicchinelli, et al., VEGF as a potential target in lung cancer, Expert opinion on therapeutic targets, 21 (2017) 959-966. [43] C. Cui, B. Tang, J. Guo, Chemotherapy, biochemotherapy and anti-VEGF therapy in metastatic mucosal melanoma, Chinese clinical oncology, 3 (2014) 36.

[44] K.J. Kim, B. Li, J. Winer, M. Armanini, N. Gillett, H.S. Phillips, et al., Inhibition of vascular 841-844.

TE D

endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo, Nature, 362 (1993) [45] N. Ferrara, K.J. Hillan, H.P. Gerber, W. Novotny, Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer, Nature reviews. Drug discovery, 3 (2004) 391-400. [46] A. Gerger, A. El-Khoueiry, W. Zhang, D. Yang, H. Singh, P. Bohanes, et al., Pharmacogenetic

EP

angiogenesis profiling for first-line Bevacizumab plus oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer, Clinical cancer research : an official journal of the American Association for Cancer Research, 17 (2011) 5783-5792.

AC C

[47] S. Kopetz, P.M. Hoff, J.S. Morris, R.A. Wolff, C. Eng, K.Y. Glover, et al., Phase II trial of infusional fluorouracil, irinotecan, and bevacizumab for metastatic colorectal cancer: efficacy and circulating angiogenic biomarkers associated with therapeutic resistance, Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 28 (2010) 453-459. [48] T. Cascone, M.H. Herynk, L. Xu, Z. Du, H. Kadara, M.B. Nilsson, et al., Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor-resistant human lung adenocarcinoma, The Journal of clinical investigation, 121 (2011) 1313-1328. [49] O. Casanovas, D.J. Hicklin, G. Bergers, D. Hanahan, Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors, Cancer cell, 8 (2005) 299-309. [50] D. Huang, Y. Ding, M. Zhou, B.I. Rini, D. Petillo, C.N. Qian, et al., Interleukin-8 mediates resistance to antiangiogenic agent sunitinib in renal cell carcinoma, Cancer research, 70 (2010) 1063-1071. [51] N.S. Vasudev, A.R. Reynolds, Anti-angiogenic therapy for cancer: current progress, unresolved questions and future directions, Angiogenesis, 17 (2014) 471-494.

ACCEPTED MANUSCRIPT [52] H.I. Hurwitz, N.C. Tebbutt, F. Kabbinavar, B.J. Giantonio, Z.Z. Guan, L. Mitchell, et al., Efficacy and safety of bevacizumab in metastatic colorectal cancer: pooled analysis from seven randomized

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EP

TE D

M AN U

SC

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controlled trials, The oncologist, 18 (2013) 1004-1012.

ACCEPTED MANUSCRIPT Figure legends Figure 1. FOXF1 expression is upregulated and correlates with the microvessel density in CRC tissues. A, Expression of the FOXF1 protein in 10 paired primary CRC tissues (T) and

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adjacent noncancerous tissues (N) from the same patient. FOXF1 expression was normalized to α-tubulin. B, Representative image of IHC staining showing the

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upregulation of FOXF1 expression in CRC tissues. C and D, CRC tissues with high

FOXF1 expression displayed an increased microvessel density compared with CRC

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tissues expressing low levels of FOXF1, p<0.001. E, Correlation between the levels of FOXF1 and CD31 mRNA in samples from TCGA (r=0.49, p<0.001, n=633). F, Patients with high FOXF1 expression exhibited a poor overall survival, as determined

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by the Kaplan-Meier analysis (p<0.05, log-rank test).

Figure 2. Upregulation of FOXF1 is positively correlated with angiogenesis and

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bevacizumab resistance in CRC samples. A, Results of the GSEA analysis of the published CRC gene expression database

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(GSE17538, n=177; GSE13294, n=155 and GSE41258, n=390) showing that the upregulation of FOXF1 was positively correlated with the enrichment of angiogenesis-related genes (HALLMARK_ANGIOGENESIS). B, GO analysis of genes to which FOXF1 potentially bound to the promoter in the published mouse embryonic lung FOXF1 ChIP-seq database (GSE77951, n=3). C, Protein network analysis of possible FOXF1 transcriptional target genes (GSE77951). D, The proportion of patients with CRC presenting high FOXF1 who were sensitive or

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Figure 3. FOXF1 transcriptionally activates VEGFA and leads to increased

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secretion of VEGFA from CRC cells. A and B, VEGFA levels were detected by Western blotting or RT-PCR and normalized to GAPDH or α-tubulin in the indicated cells, **p<0.01. C, Diagram of the VEGFA

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promoter, with the yellow rectangle showing the FOXF1 binding sites and red

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highlighting the mutated FOXF1 binding motif in the P2 promoter. D, ChIP was performed with an anti-Flag antibody in SW480-Flag-FOXF1 cells to analyse FOXF1 binding to the VEGFA promoter. RT-PCR experiments were performed using primers against the indicated area in the VEGFA promoter, and the indicated region showed

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significant enrichment compared with the GAPDH control. E, Analysis of the luciferase activation of the wild-type (WT) VEGFA promoter-driven luciferase reporter in the indicated cells (SW480 and HCT116, left panel) or WT and mutant

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(MUT) VEGFA promoter-driven luciferase reporters in FOXF1-overexpressing cells

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(right panel). Error bars represent the means of 3 independent experiments, * p<0.05 and ** p<0.01. F and G, ELISAs were used to determine the VEGFA concentrations in the supernatants of the indicated cells, * p<0.05 and ** p<0.01. H, HUVECs were incubated with the indicated supernatants and the activation of VEGFR downstream signalling pathway was determined by measuring the levels of P-ERK, P-AKT and P-P38. α-tubulin was used as a loading control.

ACCEPTED MANUSCRIPT Figure 4. Ectopic overexpression of FOXF1 increases bevacizumab resistance in CRC cells through VEGFA. A, VEGFA expression in the indicated cells treated with or without bevacizumab or

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transfected with VEGFA or shVEGFA. B, VEGFA expression in HUVECs cultured with the indicated conditioned medium; α-tubulin was used as a loading control. C, VEGFA concentrations in the indicated supernatants were explored by ELISA,

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*p<0.05 and **p<0.01. D and E, The MTT assay was used to explore the proliferation

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of HUVECs incubated with the conditioned medium collected from the indicated CRC cells, p<0.01.

Figure 5. FOXF1 downregulation inhibits angiogenesis and bevacizumab

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resistance in CRC cells through VEGFA.

A and B, A representative image of the formation of HUVEC tubules following an incubation with supernatants collected from the indicated cells (A); the length of the

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tubes was calculated (B), *p<0.05 and **p<0.01. C and D, The CAM assay was used

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to examine blood vessel formation after stimulation with the supernatants from the indicated cells.

Figure 6. Upregulation of FOXF1 promotes angiogenesis and tumourigenesis and enhances bevacizumab resistance in CRC cells through the transcriptional regulation of VEGFA. A, Xenograft models were established by subcutaneously injecting mice with the

ACCEPTED MANUSCRIPT indicated cells (n=4). B, Tumour volumes were calculated to measure the tumourigenesis ability, **p<0.01. C, Representative images of H&E (left panel) and IHC staining (right panel) of tumour sections. A CD31 antibody was used to evaluate

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the microvessel density. D, Analysis of the microvessel density in the indicated tumour group, **p<0.01.

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Figure 7. FOXF1 expression is positively correlated with VEGFA levels in CRC

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tissues.

A, The levels of the FOXF1 and VEGFA proteins in 10 fresh human CRC tissues were positively correlated (2 normal tissues as control), as determined by an analysis of the grey level using Quantity One software (r=0.849, p<0.001). B, RT-PCR was

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used to examine mRNA expression in 30 fresh human CRC samples, and the results showed a significant positive correlation between FOXF1 and VEGFA (r=0.813, p<0.001). C and D, IHC staining showed a correlation between levels of the FOXF1

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and VEGFA proteins in 80 CRC and 2 normal tissues. The results of Spearman’s

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correlation analysis are shown on the right (r=0.559, p<0.001). E, Model: The upregulation of FOXF1 induced VEGFA transcription, thus promoting angiogenesis and inducing bevacizumab resistance in CRC.

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ACCEPTED MANUSCRIPT Highlights FOXF1 could promote angiogenesis and tumorigenesis in CRC. FOXF1 transcriptional activates VEGFA by directly binding to its promoter and

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then induces angiogenesis in CRC FOXF1 could promote bevacizumab resistance by targeting VEGFA during angiogenesis in CRC.

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FOXF1-VEGFA axis could be a potential therapeutic target in selecting

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bevacizumab treatment for anti-angiogenic therapy in CRC.