MLN4924 inhibits cell proliferation by targeting the activated neddylation pathway in endometrial carcinoma

Abstract

Objective: To explore the neddylation pathway, found to be highly activated in various cancers, as a potential therapeutic target in endometrial carcinoma, one of the three most frequent malignant tumours in the female reproductive system.

Methods: Data from The Cancer Genome Atlas were analysed using online servers. Expression levels of key neddylation genes were validated by reverse-transcription polymerase chain reaction and western blots of tumour and adjacent tissues. Underlying mechanisms and the effects on cell activities of the neddylation pathway-specific inhibitor, MLN4924, were investigated in endometrial cancer cell lines.

Results: Key neddylation enzymes, ubiquitin conjugating enzyme E2 M (UBC12), ubiquitin conjugating enzyme E2 F (UBE2F), ring-box 1 (RBX1) and ring finger protein 7 (RBX2), were significantly overexpressed in endometrial carcinoma tissues versus normal tissues, but only UBE2F and RBX2 positively correlated with patient survival. MLN4924 significantly suppressed proliferation and colony formation in EC cells by inducing DNA re-replication, cell cycle arrest and apoptosis. Mechanism study revealed that MLN4924 induced the accumulation of cullin-RING ligase substrates in vitro.

Conclusions: The neddylation pathway was identified to play an important role in endometrial cancer. The neddylation specific inhibitor, MLN4924, may be a potential therapeutic drug for endometrial carcinoma.

Keywords: Endometrial cancer; MLN4924; apoptosis; cell cycle arrest; cullin-RING ligase; neddylation.

Figure 1.

Expression and somatic variations of E1, E2 and E3 enzymes of neddylation in samples from The Cancer Genome Atlas (TCGA): (a) NEDD8-activating enzyme E1 regulatory subunit (NAE1) and NEDD8-activating enzyme E1 catalytic subunit (UBA3) expression; (b) ubiquitin conjugating enzyme E2 M (UBC12) and ubiquitin conjugating enzyme E2 F (UBE2F) expression; (c) ring-box 1 (RBX1) and ring finger protein 7 (RBX2) expression; (d) NEDD8 and COP9 signalosome subunit 5 (CSN5) expression; and (e) somatic variations in neddylation genes. BLCA, bladder urothelial carcinoma; CHOL, cholangiocarcinoma; ESCA, oesophageal carcinoma; HNSC, head and neck squamous cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; BRCA, breast invasive carcinoma; KICH, kidney chromophobe; COAD, colon adenocarcinoma; KIRC, kidney renal clear cell carcinoma; and THCA, thyroid carcinoma. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

Vertical scatter plots showing expression of E1, E2 and E3 neddylation enzymes in endometrial cancer (EC) tissues and adjacent normal tissues: (a) reverse transcription (RT)-polymerase chain reaction (PCR) results showing NEDD8-activating enzyme E1 regulatory subunit (NAE1) and NEDD8-activating enzyme E1 catalytic subunit (UBA3) expression; (b) RT-PCR results showing ubiquitin conjugating enzyme E2 M (UBC12) and ubiquitin conjugating enzyme E2 F (UBE2F) expression; (c) RT-PCR results showing ring-box 1 (RBX1) and ring finger protein 7 (RBX2) expression; and (d) western blot analysis showing representative immunoblot panels and relative UBE2F and RBX2 protein levels. T, tumour tissue; N, normal adjacent tissue; GAPDH, loading control. Data presented as mean ± SD.

Figure 3.

Kaplan–Meier curves showing the correlation between neddylation enzyme expression and survival of patients with endometrial cancer (EC) from The Cancer Genome Atlas dataset: (a) E1 enzymes NEDD8-activating enzyme E1 regulatory subunit (NAE1) and NEDD8-activating enzyme E1 catalytic subunit (UBA3); (b) E2 enzymes ubiquitin conjugating enzyme E2 M (UBC12) and ubiquitin conjugating enzyme E2 F (UBE2F); and (c) E3 enzymes ring-box 1 (RBX1) and ring finger protein 7 (RBX2). Kaplan–Meier curves were analysed using the UALCAN online server.

Figure 4.

MLN4924 suppressed proliferation and colony formation of endometrial cancer cells in vitro: (a) cell counting kit-8 assay showing reduced proliferation of HHUA and AN3CA cells treated with 0.3 µM MLN4924. Data presented as mean ± SD of three replicates; (b) representative images of crystal violet-stained cells, and colony forming data showing reduced colony forming ability, after 12 days in culture, of HHUA and AN3CA cells treated with 0.3 µM MLN4924 for 48 h. Data presented as mean ± SD of three independent experiments; (c) Representative flow cytometry images and data showing MLN4924-induced cell cycle arrest and apoptosis of HHUA and AN3CA cells treated with 0.3 µM MLN4924 or DMSO for 48 h. The percentages of cells in G₂/M and subG₁ phases were determined. Data presented as mean ± SD of three replicates. MLN, MLN4924; OD, optical density; PI, propidium iodide.

Figure 5.

Representative western blot panels showing relative protein levels in HHUA and AN3CA cells in response to treatment with 0–1.0 µM MLN4924 for 48 h. MLN4924 was shown to inhibit cullin 5 neddylation and induce cullin-RING ligase substrate accumulation. CUL5, cullin 5; CDT1, DNA replication factor Cdt1; ORC1, origin recognition complex subunit 1; P21, cyclin dependent kinase inhibitor 1A; P27, cyclin dependent kinase inhibitor 1B; IκBα, nuclear factor κB inhibitor alpha; p-IκBα, phosphorylated-IκBα; P-H2AX, phosphorylated Histone H2A.X; c-Caspase3, cleaved-Caspase-3; and GADPH loading control.