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ETV1 inhibition depressed M2 polarization of tumor-associated macrophage and cell process in gastrointestinal stromal tumor via down-regulating PDE3A
Associated Data
Abstract
M2-type polarization of tumor associated-macrophage (TAM) is involved in the malignancy of gastrointestinal stromal tumor (GIST) progression. ETS variant 1 (ETV1) has been previously validated to regulate GIST pathogenesis. Our study intended to explore the role and mechanism of ETV1 in mediating the M2-polarization of TAM in GIST progression. First, we analyzed the correlation between ETV1 expression and M2-polarization in GIST tissues. IL-4 was used to treat THP-1-derived TAM cells and IL-4-stimulated TAM were co-cultured with GIST-T1 cells to mimic the GIST microenvironment. A loss-of-function assay was performed to explore the role of ETV1. Results showed that ETV1 elevation was positively correlated with M2-polarization. IL-4-induced TAM promoted ETV1 expression, silencing ETV1 inhibited proliferation, invasion and KIT activation in IL-4-treated GIST cells, while cell apoptosis was enhanced. Besides, co-culture of ETV1-silenced GIST cells significantly depressed M2-polarization in TAM, presented as decreased levels of CD206, Agr-1 and cytokines, as well as the proportion of CD206-positive TAM. PDE3A was positively correlated with ETV1 and M2-polarization. Overexpressing PDE3A reversed the inhibitory effects of ETV1 silencing. Generally, ETV1 inhibition depressed M2-polarization of TAM in GIST and its promotion on pathological aggravation via down-regulating PDE3A. This evidence may provide a new target for GIST regulation.
Introduction
Gastrointestinal stromal tumor (GIST) is the most common gastrointestinal stromal tissue-derived tumor it originates from gastric pacemaker cells and precursor cells in the gastric muscularis propria and muscularis mucosae.(1,2) Evidence has indicated that GIST is characterized by non-directional differentiation and potential malignant tendency leading to a prognosis mainly depending on early detection and timely treatment.(3) It is widely accepted that the functional acquired mutation of KIT is closely related to the pathological progression of GIST.(4) GIST can be alleviated using imatinib, a small-molecule inhibitor targeted to KIT, with beneficial effects on GIST.(5) However, secondary mutations in KIT resulted in secondary drug resistance. Therefore, it is of significant clinical significance to further explore the pathogenesis of GIST and seek for a new targeted therapy.
ETS variant 1 (ETV1) belongs to the ETS family, which is one of the largest families of signal-dependent transcriptional regulators.(6) More and more studies have shown that the overactivation of ETV1 is involved in the malignant initiation, development and progression of various tumors, including colorectal cancer, pancreatic cancer and breast cancer.(7–9) Recently, the specific expression of ETV1 has been discovered in ICCs cells,(10) and strong ETV1 expression has been demonstrated to give rise to the abnormal functions of tumor cells during GIST by cooperating with KIT activation.(11)
Phosphodiesterase (PDE) 3A encodes a cyclic nucleotide phosphodiesterase that is associated with multiple cellular processes, such as proliferation, inflammation, metabolism and differentiation, by regulating the intracellular concentration, localization and signal transduction of cyclic AMP (cAMP).(12,13) Recent studies have indicated that PDE3A is expressed in a variety of cells and tissues, and ectopic regulation of PDE3A contributes to pathological formation and development.(14) Abnormally high expression of PDE3A has been found in GIST and treatment with PDE3 inhibitor weakens the vitality of GIST cells and plays a role in cell death.(15,16)
Tumor associated macrophages (TAM) is a major component of the inflammatory cells those infiltrate in the tumor microenvironment, derived from the white blood cells from bone marrow and evolved from peripheral blood monocytes.(17) Studies have shown that there are two polarization types of TAM in different environmental conditions, i.e., M1 and M2.(18) Among them, M2 polarization inhibits the anti-tumor immune response and releases various factors, to directly or indirectly promote tumor angiogenesis, tumor growth and tumor metastasis.(19) Moreover, M2-type TAM has been validated as the high-density component in TAM which mainly infiltrates within or around tumor tissues.(20) Poor prognosis of GIST patients is also associated with M2-type TAM infiltration.(21)
Our study aimed to explore the potential impact and mechanism of ETV1 exerted on GIST progression. The expression profiles and regulation between ETV1 and PDE3A were validated by a loss-of-function assay. Besides, a co-culture system of TAM and GIST cells were constructed to illustrate the role of ETV1/PDE3A in mediating TAM M2 polarization and cell processes during GIST development.
Materials and Methods
Tissue sample collection and ethic approval
Before the initiation of the research, we obtained approval from the Ethic Committee of Shaanxi Provincial People’s Hospital. All procedures were performed in line with the guideline of the Declaration of Helsinki. In addition, GIST patients (32 cases), who were diagnosed and received surgery at Shaanxi Provincial People’s Hospital, all provided written informed consents for tissue studies. None of them suffered any kind of chemo- or radiation-therapy, as well as imatinib treatment, before sample collection. The stored GIST and adjacent non-tumor tissues were immediately cryopreserved at −80°C after excision.
Cell culture and treatment
GIST-T1, GIST-882 (GIST cell lines), human normal gastric epithelial cells (GES-1) and human mononuclear cell line (THP-1) were cultured in a humified incubator (5% CO2, 7°C). Dulbecco’s modified Eagle’s medium (DMEM) was used to grow cells with the supplementations of 1% penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA). Besides, THP-1 cells were cultured in RPMI-1640 medium containing 10% FBS. Cells involved in the present study were all purchased from the Shanghai Institutes for Biological Sciences (Shanghai, China).
To construct a co-culture system of TAM and GIST cells, a previous study was referenced.(22) THP-1 cells in the logarithmic phase were seeded into a Transwell chamber whose basement membrane contained a 0.4 μm microporous polyester film (24 mm). Each chamber was inoculated with 1 × 106 cells and then maintained in 6-well plates. RPMI-1640 medium was added into both outer chamber (2.5 ml) and inner chamber (1.5 ml). Subsequently, cells were transformed into undifferentiated macrophages (uTAM) following a 12 h-treatment with 200 nmol/L PMA (phorbol myristrate acetate). To induce M2-polarization, IL-4 was utilized to treat uTAM at different concentrations and durations. After removing the inducing regents, the chamber was transported into 6-well plates where GIST cells were cultured. Forty-eight hours later, the system was analyzed.
Genomeditech Co. (Shanghai, China) was the provider of design and synthesis services of small interfering RNAs (siRNAs), as well as the overexpression plasmid. According to the manufacturers’ protocol, siRNA (50 nM) and PDE3A overexpression vector (0 μg, 0.2 μg, and 0.5 μg) were transfected into GIST cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA). Additionally, C646 (Selleck Chemicals, Houston, TX), an inhibitor of CBP/p300 which also deregulated ETV1,(23) was used to treat cells at a concentration of 25 μM for 2 h after cell transfection.
RT-qPCR
To extract total RNA and transform it into cDNA from tumor cells and homogenates, TRIzol reagent (Life Technologies, Carlsbad, CA) and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) were used in line with the manufacturers’ instruction. A system containing 2 μl cDNA, 0.4 μl primer (×2), 0.4 μl ROX Dye (Takara) and 10 μl SYBR Taq (Takara) was used to perform the qPCR procedure on a StepOnePlusTM System (Applied Biosystems, Waltham, MA). Finally, GAPDH was set as the internal control and the 2−ΔΔCt method was used to quantify the relative expressions of target genes.
Western blot assay
RIPA buffer (Thermo Scientific, Shanghai, China) along with protease and PMSF was prepared for protein lysis. To separate (equal amount) and immunoblot target proteins, SDS-PAGE (12%) and primary antibodies were used after which the incubation of HRP-conjugated secondary antibody was conducted. At last, ECL reagent (Thermo Scientific) and ImageJ software (Bethesda, MD) were utilized for band visualization and quantification, respectively. Primary antibodies included in this study were purchased from Cell Signaling Technology (Danvers, MA) and listed as follows: anti-ETV1, anti-KIT, anti-p-KIT, anti-CD206, anti-Agr-1, and anti-PDE3A.
MTT cell proliferation assay
After cultured cells (2 × 104 cells/ml) were planted into 96-well plates, MTT solution (5 mg/ml) was supplemented into the medium, which was cultured overnight, for 4 h-incubation at 37°C. Subsequently, the treated cells were treated with 150 μl DMSO to dissolve the formazan crystals. Proliferation was expressed as the optical density at 490 nm which was determined on a microplate reader (Thermo Scientific).
Transwell invasion assay
A Transwell chamber (BD Biosciences, San Jose, CA), in which 2 × 104 cells were seeded, was pre-treated in the upper chamber with Matrigel (50 μl), while 600 μl 10% FBS was supplemented in the medium. The incubation lasted for 24 h. Then, fixation (30 min) and staining (crystal violet) were performed. The analysis of invaded cells was conducted at three randomly selected fields under a microscope.
Cell apoptosis
Based on the DNA fragmentation process of cells in apoptosis can be represented by oligomer generation, we evaluated the apoptosis rate using a Cell Death Detection ELISA kit (Merck, Darmstadt, Germany). All procedures of the operation proceeded according to its manufacturers’ protocol.
Flow cytometry
To analyze the proportion of CD206 positive TAMs, Flow cytometry was performed according to a previous study.(24) Briefly, the isolated cells were stained by being incubated with a monoclonal antibody, anti-CD206 (BD Biosciences), which was conjugated with FITC/Annexin (30 min, 4°C, dark). Then, after acquisition on a Fortessa flow cytometer (BD Biosciences), data were analyzed on a TreeStar FlowJo platform (Becton Dickinson & Company, Franklin Lakes, NJ).
Enzyme-linked immunosorbent assay (ELISA)
The supernatants of conditioned cell culture systems were isolated after all treatments were performed. Then, ELISA was used to evaluate the contents of pro-inflammatory cytokines and soluble CD206 (sCD206). According to the instruction of manufacturers, the final OD data of absorbance was measured at 470 nm.
Statistical analysis
The data were obtained from at least three repeats and the analysis was conducted using SPSS statistics 19.0 (IBM, Chicago, IL). Two-tailed Student’s t test and one-way ANOVA analysis were utilized for the significance detection of differences between various sets. The final data were expressed as mean ± SD and significance was represented as p<0.05.
Results
ETV1 was significantly up-regulated and positively correlated with the M2-type of TAM in GIST
We first evaluated the expression of ETV1 in paired tissues from 32 GIST patients. As Fig. 1A showed, ETV1 was dramatically unregulated in tumor tissues compared with adjacent non-tumor tissues. Likewise, the expression of ETV1 in two GIST cell lines was also significantly elevated as GIST-T1 cell presented the highest expression of ETV1 (Fig. 1B and C). Subsequently, we explored the association between abnormal ETV1 expression and M2 polarization of TAM cells in the GIST microenvironment. The expression of CD206, a biomarker related to M2-type polarization of macrophages, were significantly upregulated in GIST tissues compared with normal tissues (Fig. 1D). We also found that CD206 expression was positively correlated with ETV1 expression (Fig. 1E).
Expression of ETV1 in GIST tissues and cells and its association with CD206. (A, B, D) The expressions of ETV1 and CD206 in adjacent non-tumor and GIST tissues, as well as GES-1, GIST-T1, and GIST-882 cells, were detected by RT-qPCR method. (C) Western blotting presented the expression of ETV1 protein. (E) Pearson’s correlation analysis was performed for ETV1 and CD206 expression association. The data was presented as the mean ± SEM. **p<0.01 and ***p<0.005 (vs non-tumor) in (A) and (D); **p<0.01 (vs GES-1), #p<0.05 (vs GIST-T1).
Silencing ETV1 reduced TAM M2 polarization-induced GIST malignancy
Next, we used IL-4 to induce M2 polarization of TAM cells. As shown in Fig. 2A and B, IL-4 stimulation dramatically promoted the expression of CD206 in TAM in a dose- and time-dependent manner. In the co-culture system of TAM and GIST cells, ETV1 (both mRNA and protein) was upregulated in GIST-T1 cells after IL-4 treatment, as the treated concentration increasing and time prolonging (Fig. 2C–F). Subsequently, we performed a loss-of-function assay to illustrate the role of ETV1 in TAM M2 polarization-induced GIST progression. After transfection, si-ETV1-3 was the most effective at depressing ETV1 expression; meanwhile, C646 also directly declined both mRNA and protein expressions of ETV1 (Fig. 2G and H). Then, the results indicated that TAM significantly increased the proliferation, invasion and KIT activation of GIST cells while the apoptosis rate was decreased; ETV1 knockdown, induced by si-ETV1-3 or C646, suppressed the effects of IL-4-induced TAM on GIST-T1 cells (Fig. 2I–L). Taken together, these results showed that ETV1 silencing inhibited TAM M2 polarization-induced GIST progression.
ETV1 mediated GIST aggravation induced by M2-polarization of TAM. TAM differentiated from THP-1 cells were stimulated with IL-4. GIST-T1 cells were transfected with si-ETV1-3 at the concentrations of 50 nM for 48 h or treated with C646 (10 μM/6 h). GIST-T1 and TAM cells were co-cultured for 48 h. (A–C, E, G) RT-qPCR analysis showed the expression levels of CD206 in TAM and ETV1 in GIST-T1. *p<0.05 and **p<0.01 (vs control); #p<0.05 and ##p<0.01 [vs 5 ng/ml in (A) and (C), vs 24 h in (B) and (E), vs NC siRNA in (G), respectively]; †p<0.05 and ††p<0.01 [vs 10 ng/ml in (A) and (C), vs 48 h in (B) and (E), vs si-ETV1-2 in (G)]; §§p<0.01 [vs 72 h in (E)]. (D, F, H, L) The protein expressions of ETV1 and KIT activation were measured using Western blotting. (I) MTT assay evaluated cell proliferation. (J) Cell invasion was detected by Transwell assay. (K) ELISA Cell Death kit was used to measure cell apoptosis rate. *p<0.05 and **p<0.01 (vs GES-1); #p<0.05 and ##p<0.01 (vs untreated GIST-T1); ††p<0.01 (vs IL-4-TAM treated group). Data was presented as the mean ± SEM (n≥3).
Inhibiting ETV1 depressed M2 polarization of TAM in GIST
To further illustrate whether the abnormal expression of ETV1 in GIST was involved in the regulation of TAM M2 polarization in the GIST microenvironment, we co-cultured uTAM with GIST cells in which ETV1 was silenced. The results showed that co-culture of GIST-T1 cells significantly elevated the protein expressions of CD206 and Agr-1 (proteins related to M2 polarization in TAM), as well as the proportion of CD206 positive cells and the content of sCD206 compared with the GES-1 co-culture system which was dramatically inhibited by ETV1 silencing (Fig. 3A–C). In addition, the mRNA expressions and contents of M2-type TAM cytokines, i.e., TGF-β and IL-6, in the ETV1 silenced GIST-T1 group were dramatically lower than untreated group and much higher than in the GES-1 group (Fig. 3D and E).
ETV1 silence affected M2-polarization of TAM in GIST microenvironment. TAM differentiated from THP-1 cells were co-cultured with GES-1 or GIST-T1 cells transfected with si-ETV1-3 (50 nM/48 h) or treated with C646 (25 μM/2 h) for 48 h. (A) Western blotting showed CD206 and Agr-1 protein expressions (B) Flow cytometry was performed to evaluate the proportion of CD206+ cells. (C, E) Contents of sCD206, TGF-β, and IL-6 were measured using ELISA kits. (D) The mRNA expressions of TGF-β and IL-6 were detected by RT-qPCR. Data was presented as the mean ± SEM (n≥3). *p<0.05, **p<0.01 and ***p<0.005 (vs GES-1); #p<0.05, ##p<0.01 and ###p<0.005 (vs untreated GIST-T1).
Upregulation of PDE3A in GIST was positively related to ETV1 and TAM M2-polarization
Subsequently, we evaluated the expression of PDE3A in GIST to illustrate the regulation mechanism of ETV1 during pathogenesis. As shown in Fig. 4A–C, the mRNA expression levels of PDE3A in GIST tissues and cells were dramatically upregulated compared with non-tumor tissues and cells. Besides, PDE3A expression was prominently positive-correlated with both ETV1 and CD206 at mRNA level (Fig. 4D and E). Therefore, we inferred that PDE3A was associated with ETV1 and the M2-polarization of TAM in GIST.
PDE3A was correlated with ETV1 and M2-polarization of TAM. (A, B) The expression of PDE3A in GIST tissues (p<0.005) and cells (p<0.01 vs GES-1 and GIST-T1) were measured using RT-qPCR. (C) Western blotting showed the protein expression of PDE3A in cells. (D, E) The correlations between PED3A expression and ETV1 and CD206 were analyzed. All data were presented as the mean ± SEM (n≥3).
Silencing ETV1 attenuated the promotion of M2 polarization on GIST cell progress by inhibiting PDE3A
Finally, we performed a rescue assay to validate the participation of PDE3A in ETV1-regulated M2-polarization of TAM and the accelerated malignancy GIST caused by it. First, we found that ETV1 knockdown dramatically inhibited the expression of PDE3A at both mRNA and protein levels; also, the results of the overexpression assay showed that the transfection of pcPDE3A overexpression vector promoted PDE3A expression in a dose-dependent manner (Fig. 5A and B). Besides, overexpressing PDE3A reversed the inhibitory effects of ETV1 silencing on proliferation, invasion and KIT activation of GIST cells, as well as the promoted effect on cell apoptosis; importantly, these reversal effects could exert on GIST cells both under or not under IL-4 treatment (Fig. 5C–F). Next, overexpression of PDE3A was also found to promote M2-type polarization of TAM, which was depressed by ETV1 silence, presenting as the promoted expressions of CD206 and Agr-1, increased CD206-positive cell counts and elevated contents of TGF-β and IL-6 (Fig. 5G–I).
ETV1/PDE3A regulated M2-polarization of TAM-induced GIST aggravation. PDE3A overexpression vector was transfected into GIST-T1 with or without si-ETV1-3. (A, B) RT-qPCR and Western blotting showed the expression of PDE3A in GIST cells; **p<0.01 (vs NC siRNA); #p<0.05 and ##p<0.01 (vs si-ETV1-3 and NC pcDNA); ††p<0.01 (vs si-ETV1-3 and 0.2 μg pcPDE3A). TAM differentiated from THP-1 cells were stimulated with IL-4. GIST-T1 cells were transfected with si-ETV1-3. GIST-T1 and TAM cells were co-cultured for 48 h. (C–F) Cell proliferation, invasion, apoptosis and KIT activation were measured by MTT, Transwell, Elisa and Western blotting. *p<0.05 and **p<0.01 (vs control); ##p<0.01 (vs si-ETV1-3); ††p<0.01 (vs IL-4); *p<0.05 and ††p<0.01 (vs IL-4 and si-ETV1-3). TAM differentiated from THP-1 cells were co-cultured with GES-1 or GIST-T1 cells transfected with si-ETV1-3 with or without pcPDE3A. (G–I) Protein expressions of CD206/Agr-1, CD206+ cell proportion and cytokine contents were evaluated by Western blotting, flow cytometry and ELISA. **p<0.01 (vs control); ##p<0.01 (vs si-ETV1-3); ††p<0.01 (vs IL-4); *p<0.05 and ††p<0.01 (vs IL-4 and si-ETV1-3). The final data was shown as the mean ± SEM (n≥3).
Discussion
GIST has been recognized as a specific kind of cancer that frequently induces aberrant mutations in KIT or platelet-derived growth factor receptor α.(25) Evidence has already indicated that depressing KIT mutation could be an effective approach in GIST therapy.(5) However, there is a high rate of re-progression due to the drug resistance induced by secondary mutations.(12) Therefore, it is urgent to identify effective agents and investigate their regulatory mechanism in GIST development.
In this study, we validated the association between ETV1 upregulation and GIST pathogenesis; also, it was proved that ETV1 knockdown blocked the progression of GIST, as it depressed cell proliferation and invasion, and promoted apoptosis. Previously, the crucial role of ETV1 in the pathogenesis regulation of multiple cancer diseases had been discovered. The amplification of ETV1 caused by its translocation was found in prostate tumor tissues and contributed to the initiation and progression of prostatic intraepithelial neoplasia in mice.(26) Heeg and colleagues(7) demonstrated that overexpressing ETV1 triggered cellular epithelial-mesenchymal transition and stromal expansion, leading to tumor invasion and metastasis, in pancreatic cancer. Moreover, a study that was consistent with our results illustrated the upregulation of ETV1 in GIST and fluctuating the stability of ETV1 effectively inhibited both in vitro cell viability and in vivo tumor growth.(27)
Immune infiltration, the assemble of macrophages and T cells occurs in tumor microenvironment, has been identified as an important feature of various cancers and mediates tumor growth and aggravation.(28) Besides, an increased number of anti-inflammatory M2-type TAM, which suggested as an immune suppressive mechanism, is disadvantageous to cancer therapy.(29) The study of Martínez et al.(30) indicated that enhanced polarization of M2-like TAM was facilitated to tumor growth and metastasis by depressing the adaptive immunity. M2-type of TAM was also proved to harbor more than two folds in the advanced GIST lesions compared with the primary ones.(31) In addition, by inhibiting the IL-4-induced TAM M2-polarization, lnc-NIFK-AS1 hindered cellular proliferation, migration and invasion in endometrial cancer.(32) Takemoto and colleagues(33) validated the crucial role of lysyl oxidase derived from M2-like macrophage in promoting BC progression. Consistent with the previous study, our study demonstrated that ETV1 silence dramatically depressed the malignant progression of GIST tumor induced by M2-polarized-TAM. Interestingly, the M2-like polarization of TAM was also depressed by knocking-down ETV1 in GIST-co-cultured system. These may indicate the important role of ETV1 exerted on the interaction between TAM and GIST cells. Similarly, epigallocatechin gallate suppressed the infiltration and polarization of M2-type TAM under breast cancer microenvironment by decreasing exosome secretion derived from breast cancer cells.(34) Hsieh and colleagues(35) also indicated that Snail over-amplification in tumor cells increased the content of miR-21-containing exosome which was engulfed by TAM and led to its M2-polarization. Therefore, we suspected that ETV1 could regulate M2-type polarization of TAM under tumor microenvironment by mediating the extracellular secretion of GIST cells. However, the specific mechanism should be explored more detailly in further research.
Additionally, our research also investigated the potential mechanism of ETV1 regulating GIST progression, in which PDE3A was a positive-related gene with and was mediated by ETV1 leading to M2-polarization of TAM and aggravation of GIST. Previously, the important role of PDE3A in cancer development had been emerging. The hyper-activation of PDE3A was proved to be correlated with poor diagnosis and adverse overall survival of lung squamous cell carcinoma patients.(36) In a recent research, PDE3A upregulation was identified in breast cancer and drove cell stemness and metastasis of tumor.(37) An et al.(38) indicated that the inhibitor used to depress PED3A, anagrelide, showed effective effects on cancer treatment during which cell death-associated signaling pathways and cytokines were activated. Supporting our findings, Pierre and colleagues demonstrated that PE3A was involved in Cajal interstitial cell development and exerted as a contributor of GIST.(39)
All in all, the present study validated the upregulation of ETV1 in GIST development and depressing ETV1 dramatically inhibited the malignant progression of GIST cells induced by M2-polarizated TAM. Moreover, we also demonstrated the association between ETV1 and M2-polarization of infiltrated TAM in GIST microenvironment, emphasizing the crucial role of ETV1 in the interaction between TAM and GIST cells, as well as GIST pathogenesis. This evidence may provide a new target of GIST regulation.
Author Contributions
XG and PW conceived of the project; XG, YL, BW, YL, XW, and GL collected and assembled the data; XG conducted the data analysis, interpretation and manuscript writing. All of the authors read and approved the manuscript.
Acknowledgments
This work was supported by grants from the Chinese Nationl Foundation of Natural Sciences (No. 81201924) and the Natural Science Foundation of Shanxi Province (No. 2020JQ-944).
Abbreviations
| ELISA | enzyme-linked immunosorbent assay |
| ETV1 | ETS variant 1 |
| FBS | fetal bovine serum |
| GIST | gastrointestinal stromal tumor |
| PDE3A | phosphodiesterase 3A |
| sCD206 | soluble CD206 |
| siRNAs | small interfering RNAs |
| TAM | tumor associated macrophages |
| uTAM | undifferentiated macrophages |
Conflict of Interest
No potential conflicts of interest were disclosed.
Availability of Data and Materials
The data used to support the findings of this study are available from the corresponding author upon request.