Depletion of H3K79 methyltransferase Dot1L promotes cell invasion and cancer stem-like cell property in ovarian cancer

Abstract

DOT1-like protein (Dot1L) is the sole methyltransferase for methylation of lysine 79 in histone H3. Dot1L-dependent H3K79 methylation is involved in many biological processes, including telomeric silencing, cell cycle regulation, transcriptional activation and DNA repair. Genome-wide sequencing studies have revealed recurrent deletion and mutations of Dot1L gene in many types of human malignancies including ovarian cancer, however the role of Dot1L in ovarian cancer are largely unknown. To demonstrate the role of Dot1L in ovarian cancer, the expression of Dot1L was knocked out in ovarian cancer cells using CRISPR/Cas9 technology in the present study. Dot1L loss showed minimal effect on cell growth, but significantly promoted cell invasion and induced cancer stem-like cell property in ovarian cancer cells. Mechanistically, loss of Dot1L downregulated the expression of tight junction makers E-Cadherin and TJP1 and upregulated the expression of ALDH1A1 through Wnt signaling activation. Our data indicate potential tumor suppressor function of Dot1L in ovarian cancer, which is correlated with observed deletion of Dot1L gene in ovarian cancer patients, further study is granted to elucidate the function of Dot1L in tumorigenesis and progression in ovarian cancer.

Keywords: DOT1-like protein; Wnt signaling; cancer stem cell; cell invasion; ovarian cancer.

Figure 1

Dot1L knockout in ovarian cancer cells. (A) Dot1L mutations in multiple type of cancers. Dot1L mutations and CNV alterations were analyzed in multiple cancers from TCGA database. Sample number ≥ 50, mutation frequency > 2% were shown here. (B) Dot1L expression was knockout in ovarian cancer cells OVCAR3 and OVCAR4 with two different sgRNAs using the CRISPR methodology. Cells were selected in puromycin for 3 days, the expression of Dot1L and it mediated H3K79 Methylation was examined in Dot1L knockout cells by western blotting, total histone H3 and β-actin are used as loading controls. (C) Colony formation assay of OVCAR3 and OVCAR4 cells with Dot1L knockout by CRISPR. 1000 of indicated cells were plated into 24-well plate, and 7 days later cells were fixed and stained with 0.1% crystal violet. (D) Quantification of (C). Integrated intensity was quantified by Image J software. Mean of three independent experiments with SD were shown.

Figure 2

Dot1L knockout promotes cell migration of ovarian cancer cells. (A) OVCAR3 and OVCAR4 cells were infected with the Cas9 and sgRNA-expressing or control (Cas9 only) lentivirus, after selected in puromycin, the cells were subjected to trans-well migration assays. Representative images of cell migration were shown. (B and C) Same as (A) but quantified for crystal violet staining of OVCAR3 cells (B) and OVCAR4 cells (C). A total of 200 cells were examined for each of the indicated groups, error bars represent mean of three independent experiments with SD. **P < 0.01.

Figure 3

Dot1L knockout and restoration in ovarian cancer cells. Dot1L was knockout in Dot1L-wildtype OVCAR3 and OVCAR4 cells with two different sgRNAs using CRISPR technology, the mRNA and protein level of E-Cadherin and TJP1 were examined by qPCR and western blotting. CDH1 and TJP1 are genes encoding E-Cadherin and TJP1 protein respectively. A and B. Protein and mRNA level of E-Cadherin and TJP1 in OVCAR3 cells with Dot1L knockout. C and D. Protein and mRNA level of E-Cadherin and TJP1 in OVCAR4 cells with Dot1L knockout. E and F. Dot1L expression was restored in Dot1L-deficient CAOV4 ovarian cancer cells, the expression of E-cadherin and TJP1 were determined by qPCR. E. The express ion of Dot1L and H3K79 Methylation was examined in Dot1L-overexpressed CAOV4 cells by western blotting, total histone H3 and β-actin are used as loading controls. F. The mRNA level of CDH1 and TJP1 in Dot1L-overexpressed CAOV4 cells.

Figure 4

Dot1L knockout promotes cancer stem cell like properties in ovarian cancer cells. (A) ALDH activity was measured by FACS using ALDEFLUOR^TM Kit in control or Dot1L knockout OVCAR3 and OVCAR4 cells. DEAB was used as negative control for ALDH activity, the percentages of positive cells are indicated. (B) Quantification of (A). Error bars represent mean of three independent experiments with SD. **P < 0.01. (C) The expression level of ALDH1A1 was detected by western blotting in control or Dot1L knockout OVCAR3 and OVCAR4 cells. β-actin was used as loading control here.

Figure 5

Dot1L knockout promote Wnt signaling pathway. A. The activity of Wnt/β-catenin signaling pathway was assessed by dual-luciferase reporter assays in control and Dot1L knockout cells (n=3). B. The expression of total and activated β-catenin (non-phosphorylation status) was detected by western blotting in control or Dot1L knockout OVCAR3 and OVCAR4 cells.