GASC1 Promotes Stemness of Esophageal Squamous Cell Carcinoma via NOTCH1 Promoter Demethylation

Ruinuo Jia, Li Yang, Xiang Yuan, Jinyu Kong, Yiwen Liu, Weijiao Yin, Shegan Gao, Yi Zhang, Ruinuo Jia, Li Yang, Xiang Yuan, Jinyu Kong, Yiwen Liu, Weijiao Yin, Shegan Gao, Yi Zhang

Abstract

The highest incidence of esophageal squamous cell carcinoma (ESCC) occurs in China. Cancer stem cells play key roles for tumor progression. Gene amplified in squamous cell carcinoma 1 (GASC1) is essential to maintain self-renewal and differentiation potential of embryonic stem cells. This study aimed to reveal the effect and mechanism of GASC1 on ESCC stemness. The biological function of GASC1 in ESCC was evaluated both in vitro and in vivo. ChIP assay was performed to determine the molecular mechanism of GASC1 in epigenetic regulation of NOTCH1. We found that GASC1 expression was increased in poor differentiated ESCC cells and tissues. ESCC patients with a high level of GASC1 presented a significantly worse survival rate. GASC1 expression in purified ALDH+ ESCC cells was significantly higher than that in ALDH- cells. The stemness of ESCC was dramatically decreased after GASC1 blockade. Furthermore, blockade of GASC1 decreased NOTCH1 expression via increase of NOTCH1 promoter H3K9me2 and H3K9me3. Moreover, the impaired stemness after blockade of GASC1 could be reversed after transfection of NOTCH1 overexpression lentiviral vector. GASC1 promoted stemness in ESCC cells via NOTCH1 promoter demethylation. Therefore, GASC1/NOTCH1 signaling might be a potential therapeutic target for the treatment of ESCC patients.

Figures

Figure 1
Figure 1
The expression level of GASC1 in ESCC cells. (a) Relative expression of GASC1 in established cell lines was analyzed by qPCR. ESCC cell lines: KYSE30, KYSE70, KYSE140, and KYSE150; human immortalized esophageal epithelial cell line: SHEE. (b) The protein level of GASC1 expression in ESCC cell lines and SHEE cell line was analyzed by western blotting. (c) GASC1 protein level in primary ESCC cells (ECs) from tumor tissues of patients with ESCC was analyzed by western blotting. Data are represented as means ± SD. ∗ = P < 0.05, ns = nonsignificant.
Figure 2
Figure 2
The expression level of GASC1 in ESCC tissues. (a) Relative expression of GASC1 in tumor and peritumor tissues from ESCC patients was analyzed by qPCR. (b) Relative expression of GASC1 in different grade tissues (G1, G2+G3) from ESCC patients was analyzed by qPCR. (c) GASC1 protein level in tumor and peritumor tissues from ESCC patients was analyzed by western blotting. Four representative patients are shown. (d) Western blotting results of GASC1 expression in tumor and peritumor tissues from ESCC patients are presented as a histogram. (e) Western blotting results of GASC1 expression in different grade tissues from ESCC patients are presented as a histogram. Data are represented as means ± SD. ∗ = P < 0.05, ns = nonsignificant.
Figure 3
Figure 3
The correlation between GASC1 level and clinical parameters in ESCC patients. GASC1 expression in all ESCC tissues was measured by immunohistochemistry. (a) The expression of GASC1 in peritumor and different grade tumor tissues from ESCC patients was detected. One representative micrograph is shown. Scale bar represents 30 μm. (b) The expression of GASC1 in tumor and peritumor tissues from ESCC patients is presented as a histogram. (c) The expression of GASC1 in different grade tissues (G1, G2+G3) from ESCC patients is presented as a histogram. (d) GASC1 expression in ESCC tissues with positive and negative lymph node metastasis is shown as a histogram. (e) GASC1 expression in different tumor tissues based upon T score (T1+T2, T3+T4) is shown as a histogram. (f) GASC1 expression in ESCC tissues with different clinical parameters analyzed by immunohistochemistry is shown as a histogram with staining score. (g) Kaplan-Meier survival curves for ESCC patients with lower and higher GASC1 expression (immunohistochemistry analysis). Data are represented as means ± SD. ∗ = P < 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001, and ns = nonsignificant.
Figure 4
Figure 4
GASC1 is involved in CSC-like properties of ESCC cells. (a) Relative expression of GASC1 in purified ALDH-/+ cells from primary ECs. (b) Sphere forming ability of KYSE150 cells with GASC1 knockdown (shGASC1-5 and shGASC1-7) and usage of CA (5, 10, and 20 μM) was analyzed. One representative micrograph is shown. The results are presented as histograms. (c) The percentages of ALDH+ cells before and after knockdown of GASC1 (shGASC1-5 and shGASC1-7) and treatment with CA (2, 5, 10, and 20 μM) in KYSE150 cells were analyzed by flow cytometry. (d) ALDH+ KYSE150 cells before and after GASC1 knockdown and treatment with CA (10 μM) subjected to double immunofluorescence for GASC1 (green), ALDH (red), and DAPI (blue). One representative micrograph is shown. (e) Tumor volumes were measured after ALDH+ KYSE150 cell implantation with GASC1 knockdown (shGASC1-5 and shGASC1-7) and CA usage (5 and 10 μM). (f) The numbers of metastatic lesions in lung were measured in groups before and after GASC1 knockdown and CA usage (10 μM). Data are represented as means ± SD. Scale bar represents 30 μm. ∗ = P < 0.05.
Figure 5
Figure 5
NOTCH1 is decreased after GASC1 knockdown in KYSE150 cells. (a) Heatmap showing the expression of transpiration-related genes in shGASC1 and scramble shRNA KYSE150 cells. (b) Relative expression of NOTCH1, POU5F1, SOX2, MYC, and ALDH1A1 in shGASC1 and scramble shRNA KYSE150 cells was analyzed by qPCR. (c) shGASC1 and scramble shRNA KYSE150 cells subjected to double immunofluorescence for GASC1 (green), NOTCH1 (red), and DAPI (blue). One representative micrograph is shown. Scale bar represents 30 μm. Data are represented as means ± SD. ∗ = P < 0.05.
Figure 6
Figure 6
Blockade of GASC1 induces NOTCH1 promoter methylation. (a-c) ChIP analysis of H3K9 methylation levels at NOTCH1 promoter region after GASC1 knockdown (shGASC1) in ALDH+ KYSE150 cells was quantified by qPCR. Relative promoter occupancies (% input) are shown with error bars based on standard errors calculated from at least three replicates. The input signal is set as 100% (not depicted in graphs) for each set of assays. Specific antibodies that individually recognize either the di- (H3K9me2) or trimethylated form of H3K9 (H3K9me3) were used. GST antibody was used as a control. (d-e) ChIP analysis of H3K9me2 and H3K9me3 levels at the NOTCH1 promoter after CA treatment (2, 5, 10, and 20μM) in ALDH+ KYSE150 cells was quantified by qPCR. ALDH+ KYSE150 cells before and after GASC1 knockdown and treatment with CA (10 μM) subjected to double immunofluorescence for GASC1 (green), H3K9me2 (g) / H3K9me3 (h) (red), and DAPI (blue). One representative micrograph is shown. Scale bar represents 30 μm. Data are represented as means ± SD. ∗ = P < 0.05; ns = nonsignificant.
Figure 7
Figure 7
NOTCH1 is required for GASC1-induced CSC-like properties in ESCC. Sphere forming ability of shNOTCH1 and shRNA scramble KYSE150 cells was analyzed. (a) One representative micrograph is shown. Scale bar represents 20 μm. (b) Sphere forming efficiency is shown as a histogram. (c) shGASC1 KYSE150 cells before and after NOTCH1 vector infection were capable of self-renewal in vitro, as shown by similar esosphere-initiating capacity in serial passages. (d) The percentage of ALDH+ cells in shGASC1 KYAE150 cells before and after infection of NOTCH1 vector was analyzed by flow cytometry. Sphere forming ability (e) and ALDH+ cell frequency (f) of KYSE150 cells (with or without NOTCH1 overexpression) before and after treatment with CA (10 μM) were analyzed. (g) Tumor volumes were measured after implantation of purified ALDH+ KYSE150 cells (with or without NOTCH1 overexpression) treated with CA (10 mg/kg). Data are represented as means ± SD. ∗ = P < 0.05.

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