Hypermethylated RASAL1's promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

Abstract

Background: Patients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.

Methods: The expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.

Results: The expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.

Conclusion: Taken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

Keywords: Chemoresistance; Choriocarcinoma; RASAL1; TET2.

Fig. 1

Decreased RASAL1 was detected in choriocarcinoma tissues and cells. (A) IHC staining of RASAL1 in FTV (n = 18), HM (n = 18), IM (n = 11), CC (n = 11). The scale bar is 25µm. (B) Quantification analysis, (C) and percentage analysis of RASAL1 staining. (D) Expression of RASAL1 in HTR-8 and CC cells by qRT-PCR and western blotting. β-actin was used as a loading control. (E) IHC staining of RASAL1 in chemoresistant and chemosensitive CC. The scale bar is 25µm. (F) Chemotherapeutic remission rate of patients with high or low expression of RASAL1 (n = 7 versus n = 4). (G) Individual results for methylation status at each CpG site in HTR-8 and CC cells. Each circle represents a single CpG site. Filled and open circles indicate methylated and unmethylated cytosines. CpG, cytosine-phosphate-guanine. 5mC, cytosine-5’ methylation. (H) The expression of RASAL1 in CC cells incubated with 5-Aza for 48 h. ***P˂0.001 versus DMSO

Fig. 2

Downregulated RASAL1 promoted choriocarcinoma cells proliferation. (A) Western blotting and qRT-PCR of RASAL1 in JEG-3 sublines. (B) Cell viability assay. (C) Flow cytometry apoptosis analysis. (D) Tunnel assay. (E) Cell proliferation experiments. (F) Cell cycle analysis. (G) Western blotting of protein markers of EMT, proliferation, and stemness. (H) Representative images of cell morphology. (I) Colony-formation assays. (J) Representative images of sphere. **P < 0.01, ***P < 0.001

Fig. 3

Effects of RASAL1 in choriocarcinoma’s tumorigenicity and proliferation in vivo. (A) Representative images of subcutaneous xenografts. (B) Tumor growth curve and quantification analysis. Xenograft weight (mg) and volume (cm³) were measured. (C) Western blot analysis of RASAL1, β-catenin, Bcl2 and Oct4. (D) IHC staining of xenograft tissues. The scale bar is 40 μm. ***P < 0.001

Fig. 4

RASAL1 sensitizes choriocarcinoma cells to MTX. (A) Cell proliferative ability. (B) Representative images of colony-formation assays in JAR cell and JEG-3 sublines incubated with MTX. **P < 0.01, ***P < 0.001

Fig. 5

RASAL1 modulated tumorigenicity and proliferative ability via β-catenin in choriocarcinoma cells. (A) IF staining with antibodies against β-catenin (green) and DAPI (nuclei, blue) in JAR cell incubated with 5-Aza and JEG-3 sublines. The scale bar represents 25 μm. (B-C) Western blotting assay in JAR treated with both 5-Aza and BML-284, (D) and in JEG-3 sublines treated with XAV939. (E) Expression of protein markers of EMT, proliferation, and stemness in JAR treated with both 5-Aza and BML-284, (F) and in JEG-3 sublines treated with XAV939

Fig. 6

It’s TET2 not DNMTs modulating tumorigenicity via RASAL1 by targeting β-catenin in CC cells. (A) Changes of DNMTs, (B) and TETs in HTR8 and CC cells. (C) Western blotting of TET2 in cells. (D) IHC staining of TET2 in FTV (n = 18), HM (n = 18), IM (n = 11), CC (n = 11). The scale bar is 25 μm. (E) Quantification analysis, (F) and percentage analysis of TET2 staining. (G) Prognostic data for CC patients with high or low expression of TET2. (H) Expression of TET2 and the methylation frequency of RASAL1 promoter in JEG-3 and JAR sublines. (I) Expression of RASAL1 and protein markers of EMT, proliferation, and stemness, (J) and the expression of TET2, β-catenin in JAR and JEG-3 sublines. (K) Co-immunoprecipitation assay of RASAL1 and β-catenin in JAR cells. (L) Global 5-hmC levels in JEG-3 and JAR sublines. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

The proposing schematic of mechanism. Downregulated TET2 in CC cells prevents the demethylation of RASAL1 gene promoter, leads to the increase of methylated RASAL1 and results in the decrease of RASAL1. And then the rising active β-catenin promotes the proliferation, EMT, stemness, and represses apoptosis, and eventually stimulates the tumorigenesis and chemoresistance of CC cells. Black arrow-head represents promotion