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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2021 Jan 1;11(1):61–78.

Radiation engenders converse migration and invasion in colorectal cancer cells through opposite modulation of ANXA2/AKT/GSK3β pathway

Han Pan 1, Yimeng Song 1, Hang Zhang 1, Yang Bai 1, Teruaki Konishi 2,3, Alisa Kobayashi 2,3, Chunlin Shao 1, Yan Pan 1
PMCID: PMC7840724  PMID: 33520360

Abstract

Radiation therapy is an effective non-surgical means to achieve local control for various solid tumors including colorectal cancer (CRC), but metastasis and recurrences after conventional radiotherapy remains a major obstacle in clinical practice, and the knowledge concerning the changes of metastatic potential after heavy ion radiation is still limited. This study investigated how radiation, including γ- and carbon ion radiation, would change the metastatic capacity of two CRC cell lines, HCT116 and DLD-1, and examined the underlying molecular mechanisms. We found that the migration and invasion was enhanced in DLD-1 cells but impaired in HCT116 cells in vitro and in vivo after radiation of γ-rays or carbons, and radiation induced epithelial mesenchymal transition (EMT) in DLD-1 cells but mesenchymal epithelial transition (MET) in HCT116 cells. The expression of snail, a key inducer of EMT, was significantly enhanced by inhibition of glycogen synthase kinase-3β (GSK3β) in both cell lines, suggesting the modulation of snail was alike in the two CRC cell lines. However, radiation inactivated GSK3β through stimulating the phosphorylation of AKT and GSK3β at Ser473 and Ser9 in DLD-1 cells respectively, but activated GSK3β by decreasing the expression of pAKTSer473 and pGSK3βSer9 or increasing the phosphorylation of GSK3β at Tyr216 in HCT116 cells. Therefore, the above inverted motility changes was due to the opposite modulation of AKT/GSK3β signaling pathway by radiation, which was further verified in other type of cancer cell lines including MCF-7, U251 and A549 cells. Moreover, it was found that annexin A2 (ANAX2) directly bound with GSK3β and acted as a negative regulator of GSK3β upon radiation. Knocking-down ANXA2 gene reversed the enhanced migration of the irradiated DLD-1 cells and strengthened radiation-impaired migration of HCT116 cells. Collectively, this study reveals that the change of cellular motility after radiation is independent of radiation type but is correlated with the inherent of cells.

Keywords: Colorectal cancer cells, radiation, migration and invasion, EMT, ANXA2/GSK3β

Introduction

Colorectal cancer (CRC) is one of the most prevalent cancer worldwide, which ranks third among all cancers in terms of incidence and second in terms of mortality [1]. CRC patients often present with overt metastases (~20%) or develop metastasis (20%-50%) during disease progression [2,3]. Local invasion and distant metastasis of tumor cells are the main cause of CRC related death. Currently, surgical resection is still the main clinical treatment for CRC [4], however only 10%-20% of patients are candidates for curative resection.

Radiotherapy is one of the most common therapeutic approaches in clinical oncology. Conventional radiotherapy, including the treatments of γ-ray, X-ray and electron beam, has proved its benefits on overall survival in numerous types of cancers. For CRC, accumulating evidence indicates that radiotherapy can provide similar benefits to surgery in CRC. For example, local control up to 92% at 2 years can be achieved with stereotactic body radiation therapy (SBRT) in the treatment of oligometastatic CRC [5]. However, as a low-LET radiation, SBRT has limitation in cell killing efficiency due to the nature of X-ray. It is now clear that X-ray or γ-ray radiation can either promote (in most cases) or inhibit the metastatic potential of malignance, as observed in various types of tumor cell lines [6], so the recurrence or distant metastases after local treatment with photon radiation remains a major therapeutic challenge. In contrast, high energy carbon ion (C-ion) radiation offers substantial potential clinical advantages over photon radiation due to its high relative biological effectiveness (RBE), low oxygen enhancement ratio (OER) and less variation in cell cycle related radiosensitivity. Now the heavy ion therapy has been expected to improve the clinical outcome of CRC patients. Hirokazu et al. reported that after C-ion radiotherapy, the 3-year actuarial overall survival rate approached to 78% for 29 CRC patients who could not receive resection, and the median survival time was 65 months [7]. An investigation of C-ion radiotherapy for pelvic recurrence of rectal cancer from 180 patients showed that the local control and survival rates at 5 years were 88% and 59%, respectively [8]. Unexpectedly, a few studies reported that C-ions could not hamper the motility of glioblastoma cells [9] or even be pro-invasive in pancreatic cancer cells [10]. These uncertainties highlight the necessity to unravel the remaining unknown mechanisms involved in the changes of metastasis potential after conventional and C-ion radiation.

Induction of epithelial mesenchymal transition (EMT) is closely related to the distant metastasis in tumor progression. Conventional radiation-mediated EMT has been widely studied in various types of tumors both in vitro and in vivo [11-13]. However, the role of C-ion radiation in EMT remains largely an unexplored area. In addition, the serine/threonine kinase, glycogen synthase kinase-3β (GSK3β) is a positive regulator of cancer cell proliferation and survival in advanced cancer. Recently, it is shown that GSK3β controls EMT process and tumor metastasis by the degradation of slug [14] and dual regulation of snail [15], both are key transcription factors for EMT induction. Inhibition of GSK3β could preferentially attenuate the survival and proliferation of multiple types of tumor cells but induced EMT [16,17] which may increase tumor invasion. Therefore, the role in regulating cell motility and EMT process after different types of radiation needs to be clarified.

Annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein and belongs to a highly conserved protein family of Annexins, and it can modulate multiple cell activities including proliferation, adhesion, migration, invasion and angiogenesis. Overexpression of ANXA2 is linked to tumorigenesis in many types of tumors including CRC [18]. Moreover, the increase of ANXA2 has been correlated with rapid recurrence and metastasis, poor response to chemotherapy and poor prognosis [19]. However, its role in radiation associated cell behavior remains unknown.

Here, we found that the migration and invasion potential was promoted in DLD-1 cells but decreased in HCT116 cells after γ- or C-ion radiation, and radiation induced EMT in DLD-1 cells, but mesenchymal epithelial transition (MET) in HCT116 cells. Further investigation revealed that the above converse motility changes in the two CRC cells was attributed to the opposite modulation of ANXA2/AKT/GSK3β signaling pathway after radiation, which was further confirmed in other cancer type cell lines. Moreover, we identified that ANAX2 bound directly with GSK3β and acted as a negative regulator of the activation of GSK3β. Knocking-down ANXA2 gene reversed the enhanced migration in the irradiated DLD-1 cells and in the meantime strengthened radiation-impaired migration in HCT116 cells.

Methods and materials

Cell culture

Human CRC cell lines DLD-1, HCT116, lung cancer cell line A549 and breast cancer cell line MCF-7 were purchased from Shanghai Cell Bank. Radioresistant glioblastoma cell line U251R were previously established from its parental cell line U251 in our laboratory by exposing to 2 Gy X-ray/day (0.883 Gy/min) for 30 fractions (5 fractions/weekly in general) with a total dose of 60 Gy [20]. All cells were maintained in RMPI-1640 and DMEM (Hyclone Co., Beijing, China) medium, respectively, supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% O2 at 37°C.

Cell irradiation

Carbon ion irradiation was conducted at Heavy Ion Medical Accelerator of National Institute of Radiological Sciences in Japan [21]. The 290-MeV/u mono energetic carbon ion beam with a LET of 70 keV/mm was used at an average dose rate of 0.5-1 Gy/min. Cells were also irradiated with γ-rays at a dose rate of 0.73 Gy/min using a 137Cs γ-irradiator (Gammacell-40, Nordion Inc., Toronto, Canada) at room temperature.

Cell proliferation assay

Cell proliferation was measured using a Cell Counting Kit-8 (CCK8) assay (Dojindo Laboratories, Kumamoto, Japan) as described previously [22]. Briefly, after different treatment, 2000 cells were seeded and grew for 6 days before the cell viability was determined. CCK-8 assay of cell proliferation was repeated four times with eight replicates for each cell sample.

Drug treatment

Lithium chloride (LiCl, Sinopharm Chemical Reagent Co., Ltd., China) was dissolved in ddH2O and sterilized through a 0.22-µm filter to have a stock solution. To inhibit the activity of GSK3β, cells were pretreated with 40 mM LiCl for 5 h before exposure to other treatments.

Migration and invasion assays in vitro

Cell migration and invasion were determined with wound healing and transwell assay. For the wound healing assay, MCF-7 cells were grown till full confluence, then exposed to 3 Gy of γ-radiation and scratched using a 200 µL pipette tip. The cells was subsequently maintained in serum-free medium and photographed after 0 h and 48 h of wound formation. The cell coverage area compared to the initial scratch wound was quantified using ImageJ software and defined as migration rate.

The migration and invasion assays for DLD-1, HCT116, U251R and A549 cells were performed using a 24-well transwell chamber (Corning, NY, USA) with an 8-μm-pore PET membrane as described before [23]. The invasion and migration of cells was photographed at 40× magnification in order to cover ~90% of the bottom surface of the transwell. The extents of invasion and migration were determined by counting cells at 200× magnification in at least 8 different fields of each well and were expressed as the average number of cells per field.

Mouse model for in vivo metastasis assay

Male BALB/c nu/nu mice (Slac laboratory animal center, Chinese Academy of Science, Shanghai, China) in 4-5 weeks old were used for tumor metastasis assay. Control or irradiated (3 Gy) CRC cells (3×106 cells in 100 μl of 0.9% NaCl solution) were injected into mice through tail vein. After 8 weeks, all the mice were anesthetized with ketamine and sacrificed by cervical dislocation and the lungs from each mouse were dissected and stained with Bouin’s solution. The number of macroscopically visible pulmonary metastatic nodules was then counted. All in vivo experiments were repeated twice and carried out in accordance with the guidelines issued by the Institutional Animal Care and Use Committee.

Western blot assay

Whole cell protein extracts were analyzed by western-blot assay as described before [24]. The primary antibodies were anti-E-cadherin, anti-N-cadherin, anti-snail, anti-vimentin, anti-phosphorylated GSK3β (p-Ser9 or p-Tyr216), anti-ANXA2 (1:1000, Cell Signaling Technology), and anti-phosphorylated AKT (p-Ser473) (1:1000; Abcam, Cambridge, MA). The proteins were detected by the enhanced chemiluminescence system (Millipore, Merck, German) and their band images were recorded by the Bio-Rad ChemiDoc XRS system and analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA). For the detection of proteins with similar or the same molecular weight, after the first chemiluminescence, the original antibody was stripped and the membrane was incubated with other antibodies.

Immunoprecipitation (IP) and label-free LC-MS/MS analysis

Cell samples were lysed in an IP buffer (Beyotime Biotech., Haimen, China) containing protease inhibitor cocktails (Sigma, USA). The homogenates were incubated on ice for 45 min. Subsequently, the samples were centrifuged at 10000 g for 15 min at 4°C. The total cell lysates were incubated with GSK3β primary antibodies or IgG on protein G magnetic beads (Cell Signaling, USA) overnight. Then the immunoprecipitates were collected for label free LC-MS/MS measurement performed by Genechem Co., Ltd. (Shanghai, China) and western blot assay.

Immunofluorescence staining

Immunofluorescence assay of ANXA2 was carried out as described before [25]. Briefly, after the indicated treatments, cells were fixed in situ with 4% paraformaldehyde for 15 min and permeabilized with the enhanced immunostaining permeabilization buffer (Beyotime Biotech.) for 5 min at room temperature. Then the non-specific sites were blocked at room temperature for 1 h and the cells were incubated with the primary anti-ANXA2 antibody at a dilution of 1:100 at 4°C overnight. Subsequently, cells were incubated with the secondary antibodies anti-IgG rabbit Alexa Alexa Fluor®555 (Thermo Fisher Scientific, USA) at 1:600 in the dark for 1 h and then the cell nuclei were counterstained with DAPI (5 µg/ml) for 5 min. The samples were examined and recorded using a fluorescence microscope (Olympus, Japan).

RNA interference

Specific knockdown of ANAX2 was achieved using two siRNA oligonucleotides with the following sequences, ANXA2 #1: 5-CGG GAT GCT TTG AAC ATT GAA-3, and ANXA2 #2: 5-TGAGGGTGACGTTAGCATTAC-3. Cells were transfected with 40 nM siRNA using RNAiMAX (Thermo, USA) 24 h before radiation according to the manufacturer’s introduction. The efficiency of siRNA knockdown was checked using western blot assay 48 h after transfection.

Statistical analysis

All cellular experiments were repeated independently at least three times with three to four replicates. Data are presented as means ± SE. Comparison is performed by two-sample student’s t-test. P<0.05 was considered as statistically significant.

Results

Effects of γ-ray or C-ion radiation on the migration and invasion of DLD-1 and HCT116 cells

Figure 1A and 1B illustrated that the viabilities of DLD-1 and HCT116 cells were significantly reduced by γ-ray or carbon ion radiation in a dose-dependent manner, and DLD-1 cells were more radioresistant than HCT116 cells. Then we detected the migration and invasiveness of the two cell lines after 3 or 6 Gy γ-radiation. Interestingly, Figure 1C showed that γ-radiation significantly promoted the migration and invasiveness of DLD-1 cells, in contrast, it inhibited the migratory and invasive capacity of HCT116 cells. Then DLD-1 and HCT116 cells were exposed to C-ions of 0.5 or 1 Gy dose, which rendered similar cell viability as exposed to the above γ-radiation. We supposed that the increased migration and invasiveness of DLD-1 cells could be eliminated or reversed by heavy ion radiation, however, as shown in Figure 1D, C-ion radiation still enhanced the migratory and invasive potential of DLD-1 cells, although the increased number of migration cells was lower than γ-radiation, and in the meantime it significantly decreased the migration and invasiveness of HCT116 cells.

Figure 1.

Figure 1

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Radiation induced converse migration and invasion capability in DLD-1 and HCT116 cells in vivo and vitro. (A, B) Relative cell viability of HCT116 and DLD-1 cells after high or low-LET radiation, (A) γ-radiation, (B) C-ion radiation. (C, D) Effects of γ-radiation (C) or C-ion radiation (D) on the migration and invasiveness of DLD-1 and HCT116 cells. Migration and invasion were assayed using transwell coated with or without matrigel, and the photographs are the representative images of the migratory and invaded cells of DLD-1 and HCT116 cells. Data represent the mean ± SE for at least three independent experiments. (E) Lung metastasis from i.v. injected DLD-1 or HCT116 cells with or without 3 Gy of γ-radiation before injection. Data represent the mean ± SE generated from 2 independent experiments with 7-10 mice/group. (F) Images of H&E staining of lungs from i.v. injected DLD-1 or HCT116 cells with or without 3 Gy of γ-radiation before injection. Arrows show the typical lung metastases. *P<0.05 and **P<0.01 compared with non-irradiated group.

The i.v. model was then applied to test the metastatic ability of the irradiated CRC cells. As shown in Figure 1E and 1F, two months after i.v. injection, 100% mice (10/10) injected with 3 Gy γ-ray irradiated DLD-1 cells developed metastatic lung lesions and the average number of nodules per lung was 16 ± 2, whereas 6 out 7 mice injected with non-irradiated DLD-1 cells developed lung metastasis and the average number of nodules per lung was 6 ± 2. For HCT116 cells, 6 out 9 mice injected with 3 Gy γ-ray irradiated cells developed lung metastasis and the average number of metastatic nodules per lung was only 4 ± 1, whereas all mice injected with non-irradiated cells had lung metastasis and the average number of metastatic nodules per lung increased to 13 ± 2. Accordingly, radiation increased the metastatic ability of DLD-1 cells but decreased the metastasis of HCT116 cells in vivo.

Effects of γ-ray or C-ion radiation on EMT process of DLD-1 and HCT116 cells

EMT is known to play critical and intricate roles in tumor invasion and metastasis of various types of cancers, thus we checked the expression of EMT-related proteins in these two CRC cells. As shown in Figure 2A, a typical EMT phenotype including decreased-expression of E-cadherin and up-regulation of N-cadherin, vimentin and snail was identified in DLD-cells at 24-72 h after 3 Gy and 6 Gy of γ-radiation. In contrast, a typical MET phenotype, which is shown as increased expression of E-cadherin and down-regulated N-cadherin, vimentin and snail was observed in the irradiated HCT116 cells (Figure 2B). Similarly, the expression of E-cadherin was down-regulated and the expressions of N-cadherin and snail were elevated in the C-ion irradiated DLD-1 cells, but all of these proteins in C-ion irradiated HCT116 cells had opposite expression patterns in comparison with DLD-1 cells (Figure 2C, 2D). Taken together, the converse migration and invasion behaviors of the irradiated DLD-1 and HCT116 cells may result from their EMT or MET alteration after radiation.

Figure 2.

Figure 2

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Carbon ion or γ-radiation triggers EMT in DLD-1 but MET in HCT116 cells. (A, B) Expressions of EMT-related proteins 24-72 h in DLD-1 (A) and HCT116 (B) cells after γ-radiation. (C, D) Expression of EMT-related proteins in DLD-1 (C) and HCT116 (D) cells at 24-72 h after C-ion radiation. The original uncropped gels of Western blot assay were shown in Supplementary Figure 1. The column plots show the relative amounts of E-cadherin, N-cadherin, Vimentin and snail that were normalized to tubulin first and then the ratio of each normalized value to the control value was calculated. Data were generated from at least three independent experiments. *P<0.05 and **P<0.01 compared with non-irradiated control.

Inhibition of GSK3β stimulated the migration and invasion of the irradiated CRC cells by regulating snail expression

Snail is a key transcription factor of EMT and plays critical role in CRC progression. Since GSK3β can negatively regulate the stabilization of snail [15], we wonder whether GSK3β impacts EMT process in the irradiated CRC cells. Figure 3A and 3B showed that LiCl, a potent inhibitor of GSK3β, strongly increased the expressions of snail and N-cadherin in HCT116 and DLD-1 cells, indicating that GSK3β inversely regulated snail protein level in both cell lines. Meanwhile, Figure 3C showed that inhibition of GSK3β significantly increased the migratory and invasive potential in DLD-1 cells with or without radiation, but it effectively weakened the radiation suppressed migration and invasion in HCT116 cells, compared to those without LiCl treatment.

Figure 3.

Figure 3

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Inhibition of GSK3β stimulates the migration and invasion of DLD-1 and HCT116 cells after γ-radiation. (A, B) LiCl promoted the expressions of N-cadherin and snail in DLD-1 cells (A) and HCT116 (B) cells. The original uncropped gels of Western blot assay were shown in Supplementary Figure 2. The column plot shows the relative amounts of N-cadherin and snail that were normalized to tubulin first and then the ratio of each normalized value to the control value was calculated. (C) LiCl pretreatment promoted radiation-enhanced migration and invasion in DLD-1 cells and reversed radiation-decreased motility of HCT116 cells after 3 Gy of γ-radiation. The photographs are the representative images of the migration and invasion of DLD-1 and HCT116 cells. Data were generated from three independent experiments. *P<0.05 and **P<0.01 compared with the indicated control.

Since the above results indicated that the regulation of snail by GSK3β was alike in two CRC cell lines, we speculate that the cause of the inverted cellular behavior in the irradiated DLD-1 and HCT116 cells may lie in the up-stream modulation of GSK3β activity.

Radiation conversely modulated the activity of GSK3β through AKT pathways in two CRC cell lines

To test the above hypothesis, we determined the activity of GSK3β in two CRC cell lines after radiation. It is known that GSK3β can be phosphorylated by AKT at Ser9, which renders GSK3β an inactive form, while Tyr216 phosphorylation inactivates GSK3β. Figure 4A showed that, after γ-radiation, the expression of p-AKTSer473 was increased in accompany with the up-regulation of p-GSK3βSer9 but no detectable change of p-GSK3βTyr216 in DLD-1 cells could be observed. While in HCT116 cells, the expression of p-AKTSer473 was significantly decreased in accompany with a drastic increase of p-GSK3βTyr216 expression but the level of p-GSK3βSer9 did not change significantly in comparison with their non-irradiated control (Figure 4B). We further detected the changes of the phosphorylation of these proteins after C-ion radiation and found that the expression of p-AKTSer473 and p-GSK3βSer9 were increased in DLD-1 cells but obviously decreased in HCT116 cells, which had similar trend with that after γ-radiation (Figure 4C, 4D).

Figure 4.

Figure 4

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Carbon or γ-radiation conversely modulates the activity of GSK3β in DLD-1 and HCT116 through AKT pathway. (A, B) Expressions of p-GSK3β (Ser9 and Tyr216) and p-AKT (Ser473) in DLD-1 cells (A) and HCT116 cell (B) at 24-72 h after 3 Gy of γ-radiation. (C, D) Expressions of p-GSK3β (Ser9 and Tyr216) and p-AKT (Ser473) in DLD-1 cells (C) and HCT116 cells (D) at 24-72 h after 0.5 Gy of C-ion radiation. The original uncropped gels of Western blot assay were shown in Supplementary Figure 3. The column plots show the relative amounts of phosphorylated GSK3β (Ser9 and Tyr216) and AKT (Ser473) that were normalized to tubulin first and then the ratio of each normalized value to the control value was calculated. Data were generated from three independent experiments. *P<0.05 and **P<0.01 compared with un-irradiated control.

Effect of radiation on the migration of U251, A549 and MCF-7 cells

To broaden our findings, we tested radiation-induced migration in other three cell lines, including radioresistant glioblastoma cell line U251R, lung adenocarcinoma cell line A549 and breast cancer cell line MCF-7. As shown in Figure 5A, the number of the migratory cells from U251R was much higher than its parental counterpart. Similarly, radiation obviously increased the migration of A549 cells (Figure 5B). In contrast, the migratory capacity was significantly impaired in the irradiated MCF-7 cells in comparison with its unirradiated control (Figure 5C). In addition, the expressions of p-AKTSer473 and p-GSK3βSer9 were increased in U251R cells and the irradiated A549 cells, while the expressions of p-GSK3βTyr216 had no alteration. However, in MCF-7 cells, radiation significantly increased the expression of p-GSK3βTyr216 but decreased the phosphorylation of p-AKT and p-GSK3β at Ser473 and Ser9, respectively. These results were consistent with that in DLD-1 and HCT116 cells respectively, i.e., these data validated our findings concerning CRC migration in other tumor type cells.

Figure 5.

Figure 5

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Effect of radiation on the migration of U251, A549 and MCF-7 cells. (A) The migration of U251 cells and its radioresistant subline U215R, and the expressions of p-AKT (Ser473), p-GSK3β (S9 and Y216) in U251 and U251R cells. (B, C) The effect of radiation on the migratory capacity of A549 (B) and MCF-7 (C) cells, and the expressions of p-AKT, p-GSK3β (S9 and Y216) in A549 and MCF-7 cells at 72 h after 3 Gy of γ-radiation. Cellular migration were detected using transwell (U251, U251R, and A549) or wound healing (MCF-7) assay. The original uncropped gels of Western blot assay were shown in Supplementary Figure 4. The photographs are the representative images of the migration of U251, U251R, A549 and MCF-7 cells. The column plots show the number of migratory cells per field or the relative migration rate, and the relative amounts of phosphorylated GSK3β (Ser9 and Tyr216) and AKT (Ser473) that were normalized to tubulin first and then the ratio of each normalized value to the control value was calculated. Data were generated from three independent experiments. *P<0.05 and **P<0.01 compared with un-irradiated control.

Identification of ANXA2 that can bind with GSK3β in CRC cells

Next, endogenous GSK3β was immunoprecipitated in HCT116 and DLD-1 cells at 24 h after γ-radiation, and the obtained protein complexes were subject to LC-MS/MS analysis (Figure 6A). A total of 155 proteins were identified (Supplementary Table 1), among which ANXA2 is involved. GEPIA database analysis shows that ANXA2 is highly expressed in CRC tissue in comparison with normal mucosae (Figure 6B). Hence we speculated that ANXA2 might be a direct modulator of GSK3β. To verify this assumption, the cellular protein extracts were detected by co-IP assay using GSK3β antibody followed by western blot assay. Figure 6C showed that ANXA2 was detected in GSK3β precipitates, indicating that ANXA2 binds with GSK3β directly. This protein interaction was further confirmed by a reciprocal experiment where the protein lysate was immunoprecipitated by anti-ANXA2 monoclonal antibody and the binding protein was recognized by anti-GSK3β antibody (Figure 6D).

Figure 6.

Figure 6

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ANXA2 interacts directly with GSK3β. A. Experimental procedure to identify the proteins directly binding to GSK3β. B. Comparison of the expression of ANXA2 between tumor and normal tissue. C. Endogenous ANXA2 were detected by co-IP assay with anti-GSK3β antibody in DLD-1 and HCT116 cells. D. Endogenous GSK3β were detected by co-IP assay with anti-ANXA2 antibody in DLD-1 and HCT116 cells. E. Expression of ANXA2 in DLD-1 and HCT116 cells at 24-72 h after 3 Gy of γ-ray or 0.5 Gy of C-ion radiation. F. Fluorescent staining for ANXA2 in DLD-1 and HCT116 cells at 72 h after 0 or 3 Gy of γ-radiation. The original uncropped gels of Western blot assay were shown in Supplementary Figure 5.

Then we wonder how ANXA2 responses toward radiation in HCT116 and DLD-1 cells. Figure 6E showed that, when cells were exposed to γ-rays or C-ions, the expression of ANXA2 in DLD-1 cells increased significantly over time after radiation, while it slightly decreased in the irradiated HCT116 cells. Immunofluorescence staining also demonstrated the opposite changes of ANXA2 expression in these two CRC cell lines after radiation (Figure 6F).

ANXA2 negatively regulate the activity of GSK3β

The crosstalk between ANXA2 and GSK3β was then determined. As shown in Figure 7A, LiCl treatment did not alter the expression level of ANXA2 in HCT116 and DLD-1 cells whether they were irradiated or not, but the radiation-enhanced expressions of p-AKTSer473 and p-GSK3βSer9 were more prominent in LiCl-treated DLD-1 cells. While in HCT116 cells, the radiation-decreased expression of p-AKTSer473 was totally reversed by LiCl treatment. However, the original changes of p-GSK3βTyr216 in these two CRC cells after radiation were not altered. To further confirm the relationship between ANXA2 and AKT/GSK3β, the expression of ANXA2 was effectively knocked down in DLD-1 and HCT116 cells by ANXA2 siRNAs (Figure 7B). Figure 7C showed that, in comparison with the negative control (si-control), silencing ANXA2 significantly decreased the expressions of p-AKTSer473 and GSK3βSer9 at 24-72 h after radiation, but it did not change the expression level of p-GSK3βTyr216 in DLD-1 cells. As for HCT116 cells, ANXA2 knockdown significantly reduced the expression of p-AKTSer473 and GSK3βSer9 but weakened the up-regulation of p-GSK3βTyr216 by radiation. Moreover, silencing ANXA2 drastically decreased the migratory capacity of both non-irradiated cell lines, and totally reversed radiation-enhanced migration in DLD-1 cells while significantly strengthened radiation impaired migration in HCT116 cells (Figure 7D).

Figure 7.

Figure 7

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ANXA2 negatively regulates the activity of GSK3β upon radiation. A. Expressions of p-AKT (Ser473), p-GSK3β (S9 and Y216) and ANXA2 in DLD-1 and HCT116 cells at 24-72 h after 3 Gy of γ-radiation with or without LiCl pretreatment. B. The efficiency of ANXA2 knockdown was examined by western blot assay, both siRNAs against ANXA2 significantly decreased its expression in HCT116 and DLD-1 cells. C. Expressions of ANXA2, p-AKT (Ser473), p-GSK3β (S9 and Y216) in ANXA2 knockdown DLD-1 and HCT116 cells at 24-72 h after 3 Gy of γ-radiation. The original uncropped gels of Western blot assay were shown in Supplementary Figure 6. The column plots show the relative amounts of the indicated proteins that were normalized to tubulin first and then the ratio of each normalized value to the control value was calculated. Data were generated from three independent experiments. *P<0.05 and **P<0.01 compared with the respective control. D. Representative images showing the migration of DLD-1 and HCT116 cells pretreated with siANXA2 or si-control followed by 3 Gy of γ-radiation, and the number of migratory cells per field were counted. Data were generated from three independent experiments. *P<0.05 and **P<0.01 compared with non-irradiated si-control.

Discussion

It is well accepted that conventional radiation can paradoxically improve or impair the metastasis of various types of tumor cells [26]. In contrast, C-ion radiation is known to diminish the invasive potential of cancer cells in vitro and in vivo and has advantage over photon radiation [27,28]. We found that γ-radiation can suppress the migration and invasion of HCT116 cells but strengthens the metastasis capacity of DLD-1 cells both in vivo and in vitro. Surprisingly, the increased motility was still observed in DLD-1 cells after C-ion radiation, although this beam decreased the migration and invasion in HCT116 cells. Similar to our findings, it was reported that the migration of HCT116 and MCF-7 cells significantly decreased after X-ray or C-ion radiation [29,30]. Other literatures showed that C-ion radiation enhanced the invasive potential in A549 cells [31] and increased the metastatic rate in rats bearing prostate adenocarcinoma [32], which is consistent with our results in DLD-1 cells. Based on the above data, we speculate that whether the motility of tumor cells is enhanced or decreased after radiation may be independent of radiation type but is correlated with the inherent of cells. Therefore, the comparison of molecular changes between HCT116 and DLD-1 cells might yield critical information on the signaling pathways upon radiation and may allow for developing new clinical strategies to improve the outcome of radiotherapy.

EMT is a reversible biological process and closely involved in the initial cell-migratory phenotype in various neoplasia. Radiation can induce EMT and enhance metastasis in several kind of cancers but decrease the metastasis in other kinds of cancers [26,33]. Consistent with the motility changes of the above two CRC cell lines, we found that C-ion or γ-ray radiation induced EMT in DLD-1 cells but induced MET in HCT116 cells (Figure 2). Of particular note, snail is a key factor that regulates EMT and an early marker of this program [34]. During EMT, snail represses E-cadherin transcription by directly binding to its promoter and activates the expressions of mesenchymal phenotype related proteins such as N-cadherin and vimentin [35]. Data mining from TCGA database showed that snail has the highest frequency of overexpression in colorectal cancer compared with other EMT-inducers including ZEB1, slug and ZEB2 [36], suggesting that snail may also play vital role in radiation-modulated EMT in our experiment. In the present study, we found that snail expression was elevated in DLD-1 cells but declined in HCT116 cells after C-ion or γ-radiation, indicating the inversed regulation of snail by radiation in these two CRC cell lines may be responsible for the above different changes of cell motilities.

GSK3β can phosphorylate snail and regulate its function in mediating EMT [15]. We found that the regulatory mechanism of snail by GSK3β was alike in the two CRC cell lines. When GSK3β was inhibited, the expression of snail was up-regulated and the migration and invasion were stimulated in both cell lines after radiation. In consistent with our findings, inactivation of GSK3β by LiCl treatment notably increased EMT of MCF-7 cells [37], abolished the suppression of cell invasion mediated by HOXA4 and increased the migration and invasion of lung cancer cells [38]. Hence, the likely possibility that radiation induced opposite expression patterns of snail in DLD-1 and HCT116 cells is due to the converse regulation of GSK3β in these CRC cells.

It is demonstrated that the activity of GSK3β is inhibited by the phosphorylation of Ser9 [39] but triggered by the phosphorylation at Try216 site [40]. Our results showed that γ-ray or C-ion radiation inactivated GSK3β in DLD-1 cells by up-regulating the phosphorylation of AKT, a mediator of GSK3β. Similarly, radiation inactivated GSK3β by phosphorylation of Ser9 and induced EMT in RLE-6TN cells [41]. In contrast, the same type and dose of radiation down-regulated p-AKT at Ser473 and then activated GSK3β in HCT116. More importantly, we verified these findings in other tumor type cells (Figure 5A-C). Therefore, the radiation-enhanced or decreased cellular motility is reasoned from the activation or inhibition of AKT/GSK3β signal pathway after radiation, which then changed the expression of snail and induced EMT or MET process.

Our another important finding is the identification of protein ANXA2 that negatively mediates GSK3β by directly binding to GSK3β protein. To the best of our knowledge, there is few literature reporting the relationship between these two proteins, except one paper reported that ANXA2 could bind to GSK3β and disrupt the formation of GSK3β/β-catenin complex in hepatocarcinoma cells [42]. In addition, we found that the expression of ANXA2 was increased in DLD-1 but slightly decreased in HCT116 cells, which exhibited similar trend with the change of AKT/GSK3β pathway in the two CRC cells after radiation. The regulation between AKT and ANXA2 remains controversial. ANXA2 depletion could enhance the expression of p-AKT in H2O2 treated HT1080 and MDA-MB231 cells [43], however other reports showed that ANXA2 activated AKT pathway [44] and ANXA2 knockdown inhibited p-AKT in the MHCC-97L cells [45]. In accordance, our results also demonstrated that silencing of ANXA2 impaired radiation-induced phosphorylation of AKT and increased the activation of GSK3β in DLD-1 cells. Notably, inhibition of GSK3β by LiCl had a feedback on the activation of p-AKT but had no effect on the expression of ANXA2, indicating ANXA2 was up-stream of GSK3β and negatively regulated its activity upon radiation. Jurcak et al. reported a role for tumoral ANXA2 in the paracrine signaling mechanism of tumor cell migration toward nerves [46] and an increased expression of ANXA2 was detected in aggressive MDA-MB231 cells compared to non-invasive MCF-7 cells [47]. Consistently, silencing of ANXA2 in our experiment drastically decreased the migration of unirradiated CRC cells, reversed the radiation-enhanced migration in DLD-1 cells but strengthened the radiation-decreased migration in HCT116 cells. Taken together, the above results indicate that the up- or down-regulation of ANXA2 may serve as a biomarker for evaluating the outcome of radiotherapy regarding metastasis and hence is a potential therapeutic target in radiotherapy.

In collusion, this study demonstrated that DLD-1 and HCT116 cells had converse responses to γ-ray or C-ion radiation in cellular migration and invasion both in vivo and vitro, which suggests the independence of radiation type on the change of cell motility. We also demonstrated that the opposite cellular motilities were resulted from the different EMT or MET process after radiation, which was modulated by AKT/GSK3β signaling pathway. Interestingly, we identified a new regulator of GSK3β, ANXA2, which can directly bind to GSK3β and negatively regulate its activation upon radiation (Figure 8). These findings provide a new insight into the mechanism of radiation-altered cell migration and invasion and suggest that ANXA2 is applicable as a potential target of radiotherapy.

Figure 8.

Figure 8

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Schematic model for radiation induced opposite invasion and migration potential in HCT116 and DLD-1 cells. Radiation increases the phosphorylation of AKT at Ser473 and the expression of ANXA2 overtime in DLD-1 cells, and inactivates GSK3β by up-regulating its phosphorylation at Ser9. The inactive form of GSK3β can’t initiate the ubiquitylation of snail, resulting in the accumulation of snail, which promotes EMT process and stimulates the cellular migration and invasion. In HCT116 cells, radiation decreases the phosphorylation of AKT at Ser473 and does not change the expression level of ANXA2, but increases the activation of GSK3β by up-regulating its phosphorylation at Tyr216. The active form of GSK3β promotes the ubiquitylation of snail, results in the increase of E-cadherin expression and finally impairs the cellular migration and invasion.

Acknowledgements

This study was mainly supported by the National Natural Science Foundation (Nos. 11775052, 31770910), Shanghai Science and Technology Commission (No. 19411950902) and the Research Project with Heavy Ions at NIRS-HIMAC of Japan (Proposal Nos. 17J121).

Disclosure of conflict of interest

None.

Supporting Information

ajcr0011-0061-f9.pdf (2.1MB, pdf)

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