LSD1 regulates Notch and PI3K/Akt/mTOR pathways through binding the promoter regions of Notch target genes in esophageal squamous cell carcinoma

Abstract

Background: The aberrant activation of Lysine-specific demethylase 1(LSD1), Notch and PI3K/Akt/mTOR signaling pathways were frequently happened in many cancers, including esophageal squamous cell carcinoma (ESCC). However, the regulatory relationship between LSD1 and Notch as well as PI3K/Akt/mTOR pathways is still unclear. Purpose: This study aimed to explore the regulatory effects and mechanisms of LSD1 on Notch and PI3K/Akt/mTOR pathway in ESCC. Results: Firstly, we demonstrated that LSD1 and proteins in Notch and PI3K/Akt/mTOR pathway were expressed in ESCC cells. Secondly, inhibition of LSD1 by tranylcypromine (TCP) or shRNA could decrease the expressions of related proteins in Notch and PI3K/Akt/mTOR signaling pathways in ESCC cells. Finally, we found that LSD1 could bind to the promoter regions of Notch3, Hes1 and CR2, and the combinations between them were reduced by TCP in ESCC. Conclusion: Summarily, this study indicated that LSD1 might positively regulate Notch and PI3K/Akt/mTOR pathways through binding the promoter regions of related genes in Notch pathway in ESCC.

Keywords: LSD1; Notch; PI3K/Akt/mTOR; esophageal squamous cell carcinoma.

Figure 1

Expressions of LSD1 and proteins in Notch and mTOR pathway in ESCC cells. Total proteins of KYSE450, KYSE790, ECa109, EC9706 and TE-1 cells were extracted and the expressions of LSD1. Related proteins in Notch pathway such as Notch1 and Hes1 (A) as well as in mTOR pathway such as Rictor and p-Akt (Ser473) (B) were detected by Western blot.

Figure 2

TCP decreased LSD1 but increased H3K4me2 expression in ESCC cells. After KYSE450, KYSE790, ECa109, EC9706 and TE-1 cells were treated with different concentration of TCP (0, 10 and 50 µM) for 48 h, total and histone proteins were extracted for analysis the expressions of LSD1 (A) and histone H3K4me2 (B) by Western blot. *P<0.05; **P<0.01; ***P<0.001 versus untreated cells.

Figure 3

Downregulation of LSD1 by TCP or shRNA decreased the expressions of related proteins in the Notch pathway in ESCC cells. (A) ECa109 and EC9706 cells were treated with different concentrations of TCP (0, 10 and 50 µM) for 48 h, and total proteins were extracted for analysis the expressions of Notch3, Notch1, DTX1 and Hes1 by Western blot. (B) Total proteins of ECa109 and EC9706 cells with control shRNA or LSD1 shRNA were extracted, and the expressions of LSD1, Notch3, Notch1, DTX1 and Hes1 were analyzed by Western blot. *P<0.05, **P<0.01; ***P<0.001 versus control group.

Figure 4

Downregulation of LSD1 by TCP or shRNA decreased the expressions of related proteins in the PI3K/Akt/mTOR signaling pathway in ESCC cells. (A) ECa109 and EC9706 cells were treated with different doses of TCP (0, 10 and 50 µM) for 48 h, and total proteins were extracted for analyzing the expressions of PI3K, p-Akt (Ser473), Akt, p-mTOR (Ser2448), p-mTOR (Ser2481), mTOR and Rictor by Western blot. (B) Total proteins of ECa109 and EC9706 cells with control shRNA or LSD1 shRNA were extracted, and the expressions of LSD1, PI3K, p-Akt, Akt, p-mTOR (Ser2448), p-mTOR (Ser2481), mTOR and Rictor were investigated by Western blot. *P<0.05; **P<0.01; ***P<0.001 versus control group.

Figure 5

LSD1 bound to the promoter regions of Notch target genes in ECa109 cells. The chromatin from ECa109 cells treated with TCP (50 μM) for 48 h was subjected to ChIP analysis. (A) The binding of LSD1 to the promoter sequences of Notch target genes is illustrated (red dash). (B) Occupancy of LSD1 at the promoter regions of Notch target genes was shown as %input. *P<0.05; **P<0.01; ***P<0.001 versus control group. Abbreviation: TSS: transcription start site.