Background
Colorectal cancer (CRC) is a highly prevalent malignancy in the worldwide [
1,
2]. The development of CRC occurs as a result of activation of multiple signaling pathways, which stimulate proliferation, invasion and metastasis as well as angiogenesis [
3‐
5]. While important efforts in the prevention and early detection of CRC are ongoing, a majority of patients with metastatic colorectal cancer (mCRC) will face poor prognosis in part due to tumor angiogenesis, which is required for tumor growth and metastasis [
6‐
9]. Therefore, the use of anti-angiogenic agent for mCRC will promise to further improve our treatment of this prevalent disease.
Angiogenesis plays a crucial role in the initiation and progression of malignant tumors, and thus is considered as an initial cancer hallmark [
10,
11]. Although many studies have reported that various signaling molecules and growth factors are participated in angiogenesis, vascular endothelial growth factor (VEGF) family members are the most important pro-angiogenic factors that have been validated to date [
12]. In tumors, the cancer cells are suffered from hypoxia, and vascular destabilization allows VEGF to activate dormant endothelial cells to obtain oxygen and energy [
13,
14]. Then, the activation of VEGF pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature [
15]. In recent years, VEGF has been extensively studied in relation to CRC and corresponding hematogenous metastasis [
16‐
18].
Grb2-associated binder 2 (Gab2), a key member of the Gab family proteins, mainly mediates phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways [
19]. It is well documented that Gab2 is involved in human tumorigenesis, especially in leukemia, breast and ovarian cancers [
20‐
22]. Interestingly, overexpression of Gab2 induces endothelial cell migration in response to VEGF, whereas its depletion using siRNAs results in its reduction, suggesting that Gab2 may play an important role in tumor angiogenesis [
23]. Subsequently, this assumption has gradually been explored and confirmed. For example, Gab2 induces tumor angiogenesis in NRAS-driven melanoma through the RAS/ERK signaling to upregulate hypoxia inducible factor-1a (HIF-1a) [
24]. In addition, Duckworth C, et al. also found that amplification of Gab2 promotes ovarian tumor growth and angiogenesis by upregulating inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ)-dependent chemokine expression [
25].
We previously found that Gab2 induces epithelial-to-mesenchymal-transition (EMT) and CRC metastasis by mitogen-activated protein kinase (MEK)/ERK/matrix metalloproteinase (MMP) signaling pathway [
26]. However, the relative molecular mechanisms by which Gab2 overexpression contributes to tumorigenesis and metastasis of CRC remain not well defined. In this study, we examined the roles of Gab2 in human CRC growth and angiogenesis, as well as its underlying mechanism. Our results showed that elevated Gab2 induced colorectal carcinoma growth and vascularization through upregulation of VEGF expression mediated by ERK/c-Myc signaling pathway. The current findings show for the first time that Gab2 plays a vital role in regulating CRC angiogenesis.
Methods
Cell culture and tissue collection
The human CRC cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SW480 and SW620 cells were cultured in Leibovitz’s L-15 medium (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 100 U/ml penicillin and 100μg/ml streptomycin. All the cells were cultured at 37 °C in a humidified air atmosphere containing 5% carbon dioxide. 30 consecutive specimens were collected form CRC patients undergoing surgical resection at Department of Gastrointestinal Surgery, Affiliated Hospital of Zunyi Medical College, between May 2015 and September 2015. None of these patients had received chemo-, radio- or immunotherapy prior to surgery. Informed consent was obtained from all patients before surgery, and our study were approved by the Ethics Committee of Affiliated Hospital of Zunyi Medical College according to the 1975 Declaration.
Immunohistochemistry and quantification of vascular density
For the immunohistochemistry, we performed as previously described [
26,
27]. The Gab2 (OriGene Technologies, USA) and VEGF (Abcam, UK) primary antibodies were used at a 1:150 dilution in the immunohistochemistry analysis. The immunostaining intensity and average percentage of positive cells were evaluated as previous reported [
26]. Immunostaining reactions were evaluated by staining intensity (0, no staining; 1, light yellow; 2, buffy; and 3, brown) and the percentage of stained cells (0, ≤5%; 1, 6–25%; 2, 26–50%; 3, > 51%). Then, the staining intensity and the percentage of positive cells were multiplied to generate the immunoreactivity score for each case. Tumor vascular density was determined as previously described [
28,
29]. The Ki67 (OriGene Technologies, USA), CD34 (Invitrogen, USA) and VEGFR2 (Invitrogen, USA) primary antibodies were used at a 1:75 dilution in the study of tumor growth and vascular density.
Dicer shRNA and cell transductions
Lentiviral constructs containing Gab2 gene (LV-Gab2) and a negative control (LV-NC) were designed and provided by Cyagen Biosciences Inc. (Guangzhou, China). On the basis of the Gab2 sequence, three short hairpin RNAs were designed using the siRNA Target Finder (InvivoGene, San Diego, CA, USA). The effective Gab2-shRNA and negative control-shRNA sequence is 5′-GCACCAATTCTGAAGACAA-3′ and 5′-TTCTCCGAACGTGTCACGT-3′, respectively. Lentiviral vectors encoding short haipin RNAs were generated using GV248 vector (Genechem lnc. Shanghai, China). 70–80% confluent cells were transfected with lentivirus at multiplicity of infection (MOI) of 80 with enhanced infection solution (ENI.S) and 6μg/ml polybrene according to the manufacturer’s instructions.
Western blot analysis
Western blot analysis was performed as previously described [
27]. The following commercial antibodies were used in this study: Gab2 (OriGene Technologies, USA), VEGF and c-Myc (Abcam, UK), phospho-ERK1/2 and total ERK1/2 (Invitrogen, USA), β-actin (Immunology Consultants Laboratory, USA).
Total RNA isolation and qRT-PCR assays
Total RNA was isolated using Trizol (Invitrogen, USA) according to the manufacturer’s instructions. The obtained RNA was first reversely transcribed into cDNA by using RT reagent Kit (TakaRa, Japan). Quantitative reverse transcription-PCR analysis was performed as previously described [
26,
27]. GAPDH was used as an internal control. The sequences of primers in this section are the followings: (1) Gab2: 5′-GTGGGGGATCTGAATGTTTTTATG-3′ (forward) and 5′-GCCCCAGGGTAGAATGAAACG-3′ (reverse); (2) VEGF: 5′-CTTGCCTTGCTGCTCTACCT-3′ (forward) and 5′-CTGCATGGTGATGTTGGACT-3′ (reverse); (3) c-Myc: 5′-ACAGCAAACCTCCTCACAG-3′ (forward) and 5′-CGCAACAAGTCCTCTTCAG-3′ (reverse); (4) GAPDH: 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse).
Cell counting kit‑8 assay
The Cell Counting Kit-8 (CCK-8) assay kit (Dojindo, Kumamoto, Japan) was performed as reported [
28]. Transfected cells were plated in 96-well plates at a density of 4 × 10
3/well with triplicate. At indicated time points, 10 μl CCK-8 solution was added to the cells for 2.5 h at 37 °C, and the absorbance of the cells was measured at 450 nm using an ELISA reader (BioTek, Winooski, VT, USA) according to the manufacturer’s instructions. The experiments were repeated 3 times for 5 days.
To determine clonogenic ability, cells were trypsinized and placed in each well of a 6-well plate at a density of 4 × 102 cells per well. Cells were allowed to grow for 2 weeks to form visible cell colonies, which were then fixed with methanol for 15 min and stained with 0.1% crystal violet for 20 min.
Experiments in mice
Female BALB/C nude mice (5–6 weeks old) were purchased from CAVENS (Changzhou, China) and used for xenograft studies. 3 × 10
6 of control and experiemental cells (SW480-NC, SW480-Gab2, SW620-si-Ctrl and SW620-Gab2si) suspended in phosphate-buffered saline (PBS) were injected subcutaneously into the right armpit of mice (six mice each group). In targeting MEK experiment, 3 × 10
6 SW480-Gab2 cells were injected into nude mice subcutaneously. Mice were treated or not with U0126 every 5 days via tail vein for 5 weeks (25 mg/kg of U0126, six mice each group). Tumor volume was determined by external measurement according to the formula d
2 × D/2 [
30]. Mice were sacrificed after 35 days, the xenograft tumors were harvested and examined histologically. All animal experiments were approved by the Ethics Committee for Animal Experimentation of Zunyi Medical College.
ELISA assay
Supernatants collected from CRC cells xenografts were assayed by the VEGF ELISA Kit (Invitrogen) according to the manufacturer’s instructions. Three independent experiments were performed with triplicate wells.
Statistical analysis
All values were reported as mean ± SEM. Student’s t-test and one-way analysis of variance analysis were used to determine the significance of two groups and multiple groups, respectively. Correlation parameters were submitted to Pearson and non-parametric Spearman correlations. All statistical tests were two-sided and p-values < 0.05 were considered to be statistically significant.
Discussion
The mortality from CRC has decreased slightly over the past decade, but approximately 20% patients will develop into metastatic disease [
6,
35]. When colorectal carcinoma lesions have spread into blood or other organs, patients will have very limited options for target agents and conventional chemotherapy. According to the European and US guidelines, several targeting-therapies have been recommended for the treatment of mCRC [
36]. However, the most patients have not a good response to these treatments and then lead to relapse with chemo-resistance. Therefore, traditional chemotherapy need to be improved, and novel drug targets for personalized precision medicine may be the most effective strategy for each patient based on genetic characterization of the cancer [
37]. We previously performed studies have identified that Gab2 is amplified in approximately one-half of CRC tissues, and can serve as a novel oncogene for CRC metastasis [
26,
27]. In the present study, we provided additional evidence showing the involvement of Gab2 in regulation of tumor angiogenesis. We found that overexpression of Gab2 in CRC cells induced tumor growth and angiogenesis through upregulating the levels of VEGF mediated by ERK/c-Myc pathway.
It has been reported that Gab2 expression is required for human tumorigenesis and tumor growth by increasing cell proliferation and independent growth [
19]. Interestingly, a previous study has underscored the non-redundant and essential roles of Gab2 in VEGF-mediated signaling, and suggested major contributions of the protein during in vivo angiogenesis [
23]. VEGF not simply promotes tumor angiogenesis by stimulating endothelial cell proliferation and migration, altering blood vessel permeability, but controls the functional and morphological form of these vessels, which also contribute to CRC growth and progression [
35,
38]. In this study, we demonstrated that Gab2 expression was positively correlated with the levels of VEGF in CRC tissues. In addition, elevated Gab2 promoted cell proliferation and clone formation in CRC, whereas silencing of Gab2 had the opposite effects. Notably, overexpression of Gab2 in CRC cells induced tumor growth and angiogenesis in mouse xenografts through enhancing VEGF expression. Consistent with our findings, Gab2 was reported to be an inducer of tumor angiogenesis essential for melanoma and ovarian cancer [
24,
25]. Our results provided new evidence supporting the involvement of Gab2 in driving tumor angiogenesis.
Our prior study showed that Gab2 facilitates EMT and metastasis in CRC, and these functions are mainly dependent on the activation of MEK/ERK signaling [
26]. In order to further clarify the underlying mechanism by which Gab2 promotes tumor growth and angiogenesis by the ERK pathway, we first looked at the expression of c-Myc, a versatile pro-oncogene in CRC [
34]. Similarly to ERK1/2 phosphorylation, increased expression of c-Myc was been found in Gab2-upregulated CRC cells, whereas decreased levels in Gab2-downregulated ones. In addition, Gab2-overexpressing SW480-Gab2 cells treated with the inhibitor of MEK were resulted in the suppression of Gab2-enhanced cell proliferation in vitro, and the levels of VEGF and c-Myc. Although mechanistic target of MEK has an imperfect role in reducing Gab2-induced tumor growth and angiogenesis, in all, it works. The above findings implied that Gab2-induced tumor growth and angiogenesis in CRC may beyond the control of MEK/ERK signaling.
It has been widely recognized that endothelial cell growth factor receptor (EGFR) has a potent effect on tumor associated angiogenesis, and combined treatment with EGFR and VEGF signaling inhibitors has at least additive antitumor activity [
35]. Meanwhile, Gab2 is a scaffolding protein acting downstream of both EGFR and VEGF, which mediates several intracellular pathways, such as AKT, ERK and signal transducer and activator of transcription-3 (STAT3) signaling [
19,
23]. Of note, a recent study suggested that VEGF-mediated CRC cell survival is dependent on AKT and ERK1/2 signaling via an intracellular mechanism, not paracrine or autocrine model [
39]. According to these findings, we speculate that the plausible reasons for imperfect effect of MEK inhibitor in suppressing of Gab2-induced VEGF expression and tumor angiogenesis are listed as following: 1) Although Gab2 expression does not marked effect the phosphorylation of AKT in some CRC cells, the activation of AKT and other intracellular signaling may also play important roles in Gab2-induced tumor angiogenesis; 2) Mechanistic target of MEK may be only impact Gab2-induced VEGF expression via the intracellular mechanism, but not the paracrine or autocrine way in tumor microenvironment; 3) EGFR and other growth factors are also involved in Gab2-enhanced VEGF levels and tumor angiogenesis through an independent model of Gab2 status.
Acknowledgements
We thank the Department of Immunology, Zunyi Medical College, and Immunology Innovation Base of Postgraduate Education in Guizhou Province for providing the experimental platform. In addition, we also thank the Department of Gastrointestinal Surgery, the Affiliated Hospital of Zunyi Medical College for providing CRC sample and clinical data.