Histone methyltransferase Dot1L inhibits pancreatic cancer cell apoptosis by promoting NUPR1 expression

Abstract

Objective: To explore functions of the histone H3 lysine 79 (K79) methyltransferase Dot1L in the development of pancreatic cancer and evaluate the possibility of targeting Dot1L to inhibit pancreatic cancer progression.

Methods: Patient samples were used to detect differences in Dot1L expression between tumor and adjacent tissues and to determine correlations between Dot1L expression in patients with different stages of pancreatic cancer. Lentiviral-mediated knockdown of Dot1L expression and flow cytometry were used to detect apoptosis in pancreatic cancer lacking Dot1L expression; chromatin immunoprecipitation and quantitative PCR were used to detect downstream target genes of Dot1L.

Results: We show that Dot1L is highly expressed in pancreatic cancer, and that its expression is related to pancreatic cancer stage. Knocking down Dot1L significantly promoted apoptosis in pancreatic cancer cells, while overexpressing Dot1L inhibited apoptosis. Mechanistically, Dot1L regulated apoptosis in pancreatic cancer cells by promoting NUPR1 expression. The enriched H3K79 trimethylation in the transcription initiation region of NUPR1 promoted its expression. Overexpressing NUPR1 inhibited the pancreatic cancer cell apoptosis caused by Dot1L knockdown.

Conclusions: Dot1L inhibits pancreatic cancer cell apoptosis by targeting NUPR1; thus, Dot1L is a promising target for pancreatic cancer treatment.

Keywords: Dot1L; NUPR1; chromatin immunoprecipitation; histone methyltransferase; pancreatic cancer; trimethylation.

Figure 1.

Dot1L expression in pancreatic cancer cell lines and clinical specimens. (a) Dot1L mRNA levels in 30 matched tumor and paracarcinoma tissues (paired t-test). (b) The box plot of Dot1L mRNA expression as assessed by quantitative PCR at different clinical stages. (c) Quantitative PCR showing the expression of Dot1L in untransformed (HPDE) or transformed pancreatic cell lines. (d) Kaplan–Meier plot of overall survival in pancreatic cancer patients on the basis of Dot1L expression. Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 2.

Knocking down Dot1L promoted apoptosis in pancreatic cancer cells. (a) Western blot and quantitative PCR analysis of the indicated proteins in control (CTRL) and Dot1L-knockdown (Dot1L-KD) SW1990 and BxPC3 cells. (b) Cell viability assays were performed in CTRL and Dot1L-KD SW1990 and BxPC3 cells. (c) SW1990 and BxPC3 cells were depleted of Dot1L, and the percentage of cells entering apoptosis was determined by flow cytometry using APC-labeled Annexin V and 7-AAD staining. All experiments were performed at least three times. Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 3.

Overexpressing Dot1L inhibited apoptosis in pancreatic cancer cells. (a) Western blot and quantitative PCR analysis of the indicated proteins in control (CTRL) and Dot1L-overexpressing (Dot1L) SW1990 and BxPC3 cells. (b) Cell viability assays were performed in CTRL and Dot1L SW1990 cells and BxPC3 cells. (c) SW1990 and BxPC3 cells overexpressing Dot1L were sorted by flow cytometry to investigate the percentage of cells entering apoptosis using APC-labeled Annexin V and 7-AAD staining. All experiments were performed at least three times. Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 4.

Dot1L regulates apoptosis by promoting NUPR1 expression. (a) Correlation between Dot1L and NUPR1 expression in pancreatic cancer patients (n = 50); each point represents one independent sample, and the correlation coefficient (R) and P-value is presented. (b) Chromatin immunoprecipitation and quantitative PCR analysis of H3K79me3 binding to the NUPR1 promoter in SW1990 cells. (c) Western blot analysis of Dot1L, NUPR1, and pAKT in SW1990 and BxPC3 cells. (d) SW1990 cells depleted of Dot1L and overexpressing NUPR1. The percentage of cells entering apoptosis was determined by flow cytometry using APC-labeled Annexin V and 7-AAD staining. All experiments were performed at least three times. Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).