Overexpression of MEF2D contributes to oncogenic malignancy and chemotherapeutic resistance in ovarian carcinoma

Abstract

The transcription factor MEF2 promotes survival in various cell types and a number of studies indicate that abnormal regulation of MEF2 is linked to oncogenicity in several carcinomas. We have found that MEF2D, a member of the MEF2 family, is upregulated in Ovarian Cancer (OC). Immunohistochemistry analysis of tumor sections of 402 OC patients revealed that MEF2D is significantly elevated at the protein level. We have also found that the expression level of MEF2D is associated with cisplatin-resistance and poor prognosis by a retrospective analysis. Furthermore, Downregulation of MEF2D by siRNA reduces proliferation and invasiveness of OC cells SKOV3 and OVCAR3, induces apoptosis in vitro, and abolishes OVCAR3 tumorigenicity in xenograft model. Mechanistic study via ChIP analysis identified two of MEF2D-targeted genes, HPSE and IKBKE, which are associated with tumor invasion and chemotherapy-resistance, in accord with MEF2D expression in OC. Remarkably, knock-down of MEF2D invariably lead to the downregulation of IKBKE and reversed cisplatin (DDP)-resistance in cisplatin-resistant cells SKOV3-DDP. Our results suggest that MEF2D promotes malignant biological behaviors and cisplatin-resistance in OC and establish MEF2D as a new therapeutic target in OC treatment.

Keywords: HPSE; IKBKE; MEF2D; Ovarian cancer (OC); cisplatin-resistant.

Figure 1

Analyses of MEF2D expression in OC cell lines and patient samples. A. Data on MEF2D DNA copy number and mRNA expression in ovarian cancer and normal ovarian tissue from several study groups deposited in the Oncomine database (www.oncomine.org). B. The protein expression of MEF2D in normal human ovarian epithelial cells (IOSE) and human OC cells (OVCAR3, SKOV3) was determined by western blot assays. C. The protein expression of MEF2D in normal ovarian tissues (N1-N4) and human OC tissues (T1-T12) was determined by western blot assays. Error bars represent the s.d. of triplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001. D. IHC analysis of MEF2D protein expression in normal ovarian tissues, and OC tissues Original magnifications: × 200 and × 400. E. IHC analysis of IKBKE and HPSE protein expression in normal ovarian tissues, and OC tissues Original magnifications: × 200 and × 400.

Figure 2

The relationship between MEF2D overexpression and cisplatin resistance & prognosis of OC patients. A. The representative microscopic photographs of negative and positive MEF2D expression in OC tissues by immunohistochemistry. B. Percentages of positive and negative MEF2D expression in different groups are shown in the charts. It showed that high level of MEF2D was correlated with chemotherapy resistance (P < 0.001), clinical stage (P = 0.046), pathological grade (P = 0.033) and histologic type (P = 0.028). C. 3-year Kaplan-Meier analysis of overall survival of patients during 2010-2015 with OC according to the expression level of MEF2D protein. D. 5-year Kaplan-Meier analysis of overall survival of patients during 2010-2013 with OC according to the expression level of MEF2D protein.

Figure 3

Effect of MEF2D knockdown on proliferation and apoptosis of OC cells in vitro. A. CCK-8 assays were performed to determine the effects of MEF2D knockdown on the proliferation of SKOV3 and OVCAR3 cells. Cell viability was determined at 0, 12, 24, 48 and 72 h. B. Flowcytometry assays were performed to determine the effects of MEF2D knockdown on the apoptosis of SKOV3 and OVCAR3 cells. C. Effects of MEF2D knockdown on proliferation-associated protein cyclin-D1 and c-myc and apoptosis-related protein caspase3 and cleaved caspase3 were analyzed by western blotting in SKOV3 and OVCAR3 cells. Error bars represent the s.d. of triplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Effect of MEF2D knockdown on invasion and migration of OC cells in vitro. A. The migration abilities of SKOV3 and OVCAR3 were measured through testing the wound closure after MEF2D knockdown using wound healing assays. B. Transwell assays were used to detect the migration and invasion abilities after MEF2D knockdown in SKOV3 and OVCAR3 cells. Original magnifications, × 200 and × 400. C. Effects of MEF2D knockdown on migration associated protein MMP9 were analyzed by western blotting in SKOV3 and OVCAR3 cells. Error bars represent the s.d. of triplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

ChIP analysis of MEF2D targeted genes in OC cells. A. Silencing MEF2D expression significantly reduced the level of HPSE and IKBKE transcript and upregulated DUSP6 transcript examined by qRT-PCR. Error bars represent the s.d. of triplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001. B. Reducing MEF2D level also significantly downregulated the expression of HPSE and IKBKE protein level determined by Western blot. Error bars represent the s.d. of triplicate measurements. *P < 0.05; **P < 0.01; ***P < 0.001. C. Analysis of the HPSE and IKBKE gene promoter reveals the presence of a putative MEF2D binding site (5’-ACTAAAAATAGA-3’) shared by the two genes and putative states of MEF2D binding on HPSE and IKBKE promoter were predicted through prediction websites www.jasper.com). D. In ChIP assays the anti-MEF2D antibody actually immunoprecipitated MEF2D protein. PCR product of HPSE and IKBKE were both observed in the presence of MEF2D antibody and anti-RNA Polymerase II but not in negative control IgG.

Figure 6

Effect of MEF2D knockdown on IKBKE expression and cisplatin resistance in OC cells. A. The sensitivity of SKOV3 parental and DDP-resistant cells (SKOV3/DDP) were evaluated by use of CCK-8 kits. B. siMEF2D can significantly increase the sensitivity of SKOV3/DDP cells to DDP treatment (P < 0.05). C. SKOV3/DDP cells were transfected with siMEF2D001 or NC and the effect of MEF2D knockdown was identified by western blots. D. Knocked down MEF2D also reduced IKBKE expression in SKOV3/DDP cells.

Figure 7

Effect of MEF2D knockdown on tumorigenecity of ovarian carcinoma cells in vivo. A, B. OVCAR3 cells were subcutaneously inoculated in SCID mice, which were randomly grouped to NC or siMEF2D001 (n = 6 for each group) and then injected with NC or siMEF2D001 every 3 days. 28 days later tumors were removed for analysis. Each tumor formed was volumed and weighted. The weight and volume of established tumors was measured and is shown in a scatter plot. C. Immunohistochemical analysis of MEF2D expression was performed on OVCAR3 tumor xenografts. The representative images are shown (with original magnification, of × 200 and × 400).

Figure 8

Signaling and transcription network of MEF2D and its deregulation in oncogenesis. A. In normal cells the activity of MEF2D is regulated by a number of co-factors, including co-repressors Cabin1/Cain, class IIa HDACs and co-activators CBP/p300, leading to the proper regulation of downstream genes. B. In diseased cells such as OC cells, the activity of MEF2D is deregulated leading to the abnormal expression of its downstream genes.