Background
Esophageal cancer is one of the most aggressive malignancies in the world [
1]. Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer that occurrs in the Chinese population [
2]. ESCC has a quite poor survival rate due to local invasion and remote metastasis; however, the molecular mechanisms responsible for the malignant behaviors of ESCC cells have not been elucidated [
3].
ANXA2 (also called Annexin A2), a member of the annexin family, is a 36-kDa calcium-dependent phospholipid binding protein and is ubiquitously expressed in various eukaryotic cells. As a multifunctional protein, ANXA2 can interact with various ligands and affects diverse cellular processes, such as membrane trafficking, endocytosis, exocytosis, tissue remodeling, angiogenesis and immune regulation [
4‐
6]. Aberrantly expressed ANXA2 is observed in a wide range of malignancies, including ESCC, and plays pivotal roles in tumor formation and progression by modulating cell proliferation, apoptosis, adhesion, invasion, metastasis, and tumor neovascularization [
4,
6‐
11]. Moreover, inhibition of ANXA2 can suppress tumor cell proliferation, survival and metastasis [
4,
8,
12‐
15]. Altogether, these studies suggest that ANXA2 can be used as a prospective biomarker and therapeutic target for cancer treatment.
To date, dysregulation of ANXA2 and its implication in ESCC remain controversial [
16‐
19], and thus is worthy of further investigation. Our previous work revealed that ANXA2 is overexpressed in ESCC tissues [
20]. To clarify the functional role of ANXA2 in ESCC cells, in the present study, we investigated the effects of ANXA2 overexpression on malignant phenotypes of ESCC cells and the underlying mechanism.
Methods
Tissue specimens
Fresh ESCC tissues and adjacent non-tumorous tissues were procured from surgical resection specimens collected by the Department of Pathology at Linzhou People’s Hospital, Henan province, China. None of the patients received treatment before surgery, and all patients signed informed consent forms provided by the Cancer Hospital, CAMS & PUMC for tissue sampling and isolation and storage of DNA, RNA and protein. Primary tumor regions and morphologically normal operative margin tissues from the same patients were separated by experienced pathologists and immediately stored at − 80 °C until use. The study was approved by the Ethics Committee of the Cancer Institute (Hospital), CAMS & PUMC (NCC2015G-06).
Cell culture and treatments
The human ESCC cell lines KYSE30, KYSE70, KYSE150, KYSE180, KYSE410, KYSE450 and KYSE510 were provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan). All cell lines were authenticated through short tandem repeat DNA fingerprinting by Peking Union Medical College (Beijing, China) before the study. The ESCC cell lines were cultured as described previously [
21].
ESCC cell lines were incubated with the SRC inhibitor dasatinib (Selleck, Houston, TX, USA) under different concentrations for 24 h, with MG132 (Selleck) at 10 nM for 10 h, or with cycloheximide (Sigma, St. Louis, MO, USA) at 100 μg/mL for different lengths of time.
Small interfering RNA synthesis and plasmid construction
Small interfering RNA (siRNA) against human ANXA2, HIF1A, MYC and control non-silencing siRNA were synthesized by GenePharma (Shanghai, China). Lentivirus vector expressing ANXA2 or control scramble short hairpin RNA (shRNA) were constructed by GenePharma. All targeted sequences are provided in the Additional file
1: Table S1. All plasmid constructs generation are described in the Additional file
1.
Transfection and lentiviral transduction
Cells were transfected with siRNA or overexpression constructs using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The final concentration of siRNA used for gene silencing is 100 nM. At 48 h post-transfection, cells were collected for subsequent analyses. The lentiviruses (GenePharma) were used to transduce ESCC cells, and stable cell strains expressing ANXA2-shRNA (shANXA2) or control scramble-shRNA (sh-scramble) were selected using puromycin (2 μg/mL, Gibco) for at least 1 week.
Western blot analysis
Total protein was isolated using RIPA buffer (Applygen, Beijing, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Nuclear and cytoplasmic protein was isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Immunoblotting was performed with primary antibodies against ANXA2 (1:200), p-ANXA2 (Tyr23, 1:200), VEGF (1:200) (Santa Cruz Biotechnology, Dallas, Texas, USA), MYC (1:1000), p-SRC (Tyr418, 1:1000) (Abcam, Cambridge, UK), Ubiquitin (1:500), Histone H3 (1:1000) (CST, Danvers, MA, USA), HIF1A (1:500), SRC (1:500), HA-tag (1:3000), His-tag (1:3000) (Proteintech, Wuhan, China). GAPDH (1:500) (Proteintech) was used as a loading control. Secondary antibodies (Goat anti-Mouse IgG and Goat anti-Rabbit IgG, 1:5000) were purchased from Applygen. The signals were visualized with a super enhanced chemiluminescence (ECL) detection reagent (Applygen). Quantitative analysis of immunoblotting was performed using ImageJ (Ver. 1.52a, NIH image, Bethesda, MD, USA).
Wound-healing assay
Cells were seeded into 6-well plates. When the cells reached confluence, scrape wounds were made in each well. The cells were photographed at the indicated time points.
Cell matrigel migration and invasion assays
Migration and invasion assays were performed in Transwell plates as described previously [
21]. For the migration assay, 5 × 10
5 KYSE30 cells or 1 × 10
6 KYSE150 cells were were seeded and incubated for 16 h (KYSE30) or 24 h (for KYSE150) at 37 °C. For the invasion assay, the cells were incubated for 36 h (KYSE30) or 48 h (for KYSE150) hours at 37 °C. The cells were stained with 0.5% crystal violet (Sigma) and imaged with a microscope (Leica, Wetzlar, Germany). The percentage of stained cell area were measured by ImageJ. The data are presented as the mean ± SEM from three separate experiments.
RNA isolation and real-time RT-PCR
Total RNA was isolated using an RNApure Tissue & Cell Kit (Cwbiotech, Beijing, China). Isolated RNA was used as a template for reverse transcription reactions using a HiFiScript cDNA Synthesis Kit (Cwbiotech). Quantitative real-time RT-PCR analysis was performed using SYBR® Fast qPCR Mix (TaKaRa, Shiga, Japan) and a CFX96 Real-Time System (Bio-Rad). The relative mRNA expression of the target genes was normalized to an endogenous reference (ACTB). The primer sequences are provided in Additional file
1: Table S5.
Chromatin immunoprecipitation
A chromatin immunoprecipitation (ChIP) assay was performed with a ChIP-IT® Express Magnetic Chromatin Immunoprecipitation Kit (Active & Motif, Carlsbad, CA, USA) followed the manufacturer’s instructions. Chromatin samples were incubated with anti-MYC antibody (Abcam). Rabbit IgG (Applygen) was used as the negative control. A non-immunoprecipitated sample was used as the input control. Precipitated DNA was amplified by PCR using primers provided in the Additional file
1: Table S6.
Luciferase reporter assay
Luciferase reporter assays were performed using a Dual-Luciferase Reporter Assay System (Promega) as described previously [
21]. The data are presented as the mean ± SEM from three separate experiments.
Co-immunoprecipitation
Total protein was isolated from cells using a non-denaturing lysis buffer (Applygene) with protease inhibitors. The protein lysate was incubated with anti-MYC antibody (Abcam), Rabbit IgG (Applygen) or anti-ANXA2 (Santa Cruz), Mouse IgG (Applygen) at 4 °C overnight. Then, Protein G agarose beads (Applygene) were added and incubated at 4 °C for 4 h. The immunoprecipitates were collected by centrifugation and washed with PBS. The mixture was subjected to Western blot analysis.
Immunofluorescence microscopy
The cells that grew on the slides were fixed, permeabilized, blocked and incubated with MYC (1:100) (Abcam), p-ANXA2 (Tyr23, 1:100) (Santa Cruz) or His-tag (1:100) (Proteintech) at 4 °C overnight. The bound primary antibodies were detected using goat anti-mouse IgG-FITC or goat anti-rabbit IgG H&L (1:100) (Abcam) at 37 °C for 1 h. The fluorescence was detected via confocal microscopy (General Electric Company, Fairfield, CT, USA).
Animal experiments
All the animal experiments were approved by the Animal Center of the Institute of National Cancer Center/Cancer Hospital, CAMS & PUMC (NCC2015A013).
For the tumor metastasis assay, six-week-old male NOD/SCID mice (Hfkbio, Beijing, China) were injected with 1 × 106 KYSE30 cells stably expressing sh-scramble or shANXA2 via the tail vein (n = 10 per group). The mice were sacrificed after 6 weeks. Metastastic nodules in lung tissues were fixed in Bouin’s solution (Applygen), and the number of metastases was determined. The tumor samples were embedded in paraffin, cut into 5-μm sections, and stained with hematoxylin and eosin (H&E).
To inhibit tumor growth, four-week-old female BALB/c mice (Hfkbio, Beijing, China) were given a subcutaneous (s.c.) injection of 1 × 106 KYSE150 cells. After 1 week, mice were equally divided into six groups according to the mean tumor volume (n = 8 per group). The mice were injected intraperitoneally with PBS (vehicle control), or dasatinib (30 mg/kg, three times per week, Selleck) in PBS containing 5% PEG300 and 5% Tween 20, bevacizumab (20 mg/kg, three times per week, Genentech, South San Francisco, CA, USA) in PBS, or intravenously injected with ANXA2 siRNA (2OD/8 mice, twice a week) incubated with Entranster in vivo reagent (Engreen Biosystem, Beijing, China) according to the manufacturer’s instructions. For combination treatment, dasatinib and bevacizumab were administered at 20 mg/kg and 15 mg/kg, respectively. Tumor size and body weight were measured every 4 days, and tumor volume was calculated using the following formula: Volume = length × width2 × 0.52. Three weeks after drug administration, the mice were sacrificed, and the weight of the xenografts were measured.
Statistical analysis
All statistical analyses were performed using SPSS 22.0 software (SPSS Inc. Chicago, IL, USA). The experimental results were statistically evaluated using Student’s t-test for comparisons between two groups or ANOVA for comparisons between more than two groups. The Pearson’s Chi-squared test was used to assess the correlation between each two genes’ mRNA expression level in ESCC tissues. P < 0.05 was considered statistically significant.
Discussion
ESCC is a malignant cancer with poor prognosis and effective therapies for unresectable advanced ESCC are lacking at present. A comprehensive understanding of the molecular basis of the invasion and metastasis behaviors of ESCC cells will provide both useful prognostic indicators and effective therapeutic targets for the disease. Herein, we demonstrate that highly expressed p-ANXA2 promotes the invasion and metastasis of ESCC cells through upregulation of MYC-HIF1A-VEGF cascade. Moreover, this study revealed that phosphorylation of ANXA2 at Tyr23 by SRC increases MYC protein abundance through inhibiting its proteasomal degradation in ESCC cells. Particularly, our data suggest that targeting p-ANXA2-associated signaling may be a promising strategy in the treatment of ESCC.
Extensive studies have shown that ANXA2 is abnormally expressed in a variety of malignancies, and it exerts tumor promoter or suppressor functions depending on the cancer type [
4,
7]. To date, the functional significance of aberrant ANXA2 expression in ESCC is still ambiguous. An earlier study showed that overexpression of ANXA2 repressed the migration and invasion capability of the ESCC cell line Eca109 [
16]. In contrast, our present study provides evidence that a high ANXA2 level promotes cell migration and invasion in the ESCC cell lines KYSE30, KYSE150 and KYSE180 in vitro and metastasis in vivo, suggesting that upregulation of ANXA2 plays critical roles in the progression of ESCC by conferring more aggressive phenotypes on tumor cells. In line with our findings, overexpression of ANXA2 has been demonstrated to facilitate cancer cell migraton, invasion and metastasis in the majority of cancer types [
6,
8,
10,
35,
36]. Particularly, previous studies have reported that a high level of ANXA2 is associated with poor survival in ESCC patients [
18,
37], further supporting our findings in this study.
To date, the elaborate molecular mechanisms by which ANXA2 mediates invasive and metastatic phenotypes has not been elucidated. In the current study, HIF1A-VEGF was identified as a key singling pathway downstream of ANXA2, which accounts for the high migratory and invasion potential of ESCC cells. Our earlier and current study demonstrate that both VEGF and HIF1A overexpression potentiates the migratory and invasive ability of ESCC cells [
21], which coincides with observations in other malignancies [
38‐
43]. Furthermore, our results reveal that ANXA2 activates the HIF1A-VEGF axis by increasing the MYC protein abundance. Consistently, MYC is associated with the invasion and metastasis potential of multiple types of cancer cells, and it can be upregulated by ANXA2 [
44‐
46]. Altogether, our data suggest that the ANXA2-MYC-HIF1A-VEGF axis is a crucial signaling pathway in regulation of the prometastatic phenotypes of ESCC cells.
As a multifunctional molecule, the ANXA2 protein has been observed in multiple cellular compartments, including the extracellular space, plasma membrane, cytoplasm and nucleus. The subcellular distribution and function of ANXA2 are largely affected by post-translational modification and are dependent on cell type [
4,
6,
28]. Of note, we observed that Tyr23-phosphorylated ANXA2 accumulated in the nucleus, where it promoted cell motility and invasion. Likewise, previous studies indicate that Tyr23 phosphorylation of ANXA2 accelerates cancer cell migration, invasion and metastasis [
9,
36,
47,
48]. Furthermore, our results revealed that a high p-ANXA2 (Tyr23) level prevents MYC protein from ubiquitin-dependent proteasomal degradation, thus uncovering a novel regulatory effect of p-ANXA2 (Tyr23) on MYC protein stability. More importantly, the p-ANXA2 (Tyr23) and MYC levels were consistently altered in the majority of the nuclear extract of primary ESCC tissues, which further supports the functional link between p-ANXA2 (Tyr23) and MYC. Notably, we observed that p-ANXA2 (Tyr23) was mainly localized on the nuclear membrane of ESCC cells. It is well established that ANXA2 can interact with S100A10 (P11) to form heterotetramer (containing an S100 protein dimer and two ANXA2 molecules) [
28], which may facilitate the association of ANXA2 with nuclear membrane. We speculate that nuclear membrane-bound ANXA2/S100A10 heterotetramer can intereact with MYC and protect it from ubiquitination and proteasomal degradation, hence we will investigate the interaction of ANXA2 and S100A10 in the future studies. Taken together, this study provides novel insight into a molecular mechanism underlying the invasion and metastasis capacities of ESCC cells.
At present, identification of effective chemotherapeutic and targeted drugs is an urgent need for ESCC therapy. Our current study not only illustrates that the SRC-ANXA2-MYC-HIF1A-VEGF signaling pathway contributes to the progression of ESCC but also provides novel molecular targets and therapeutic strategies for the treatment of ESCC. Considering that abnormally expressed p-ANXA2 (Tyr23) confers metastatic potential to ESCC cells, specific blocking of ANXA2 phosphorlation of Tyr23 using dasatinib or another inhibitor might be a feasible therapeutic strategy for patients with unresectable advanced ESCC. Intriguingly, our data further showed that combination therapy with bevacizumab and dasatinib elicited a robust anti-tumor effect on ESCC xenograft tumors, suggesting that it is a promising regimen for ESCC therapy. Notably, both bevacizumab and dasatinib have entered clinical trials for multiple types of malignancies and have been approved for therapy of certain malignant tumors [
32,
33,
49], and hence, our findings could easily be translated into the clinic.