BRD4 inhibitor inhibits colorectal cancer growth and metastasis

Abstract

Post-translational modifications have been identified to be of great importance in cancers and lysine acetylation, which can attract the multifunctional transcription factor BRD4, has been identified as a potential therapeutic target. In this paper, we identify that BRD4 has an important role in colorectal cancer; and that its inhibition substantially wipes out tumor cells. Treatment with inhibitor MS417 potently affects cancer cells, although such effects were not always outright necrosis or apoptosis. We report that BRD4 inhibition also limits distal metastasis by regulating several key proteins in the progression of epithelial-to-mesenchymal transition (EMT). This effect of BRD4 inhibitor is demonstrated via liver metastasis in animal model as well as migration and invasion experiments in vitro. Together, our results demonstrate a new application of BRD4 inhibitor that may be of clinical use by virtue of its ability to limit metastasis while also being tumorcidal.

Figure 1

BRD4 is highly expressed in colorectal cancer (CRC). (A) mRNA expression levels of BRD2, BRD4 short isoform and BRD4 long isoform in normal colon epithelial cell lines and colon cancer cell lines; (B) Protein levels of BRD4 in normal and colon cancer human tissues (tumor and healthy control); (C) Protein levels of BRD4 according to different ages; (D) Protein levels of BRD4 according to the stage of CRC (per AJCC guidelines). BRD4S: BRD4 short isoform; BRD4L: BRD4 long isoform; BRD4C: Commercial BRD4 primer as positive control; CRC: colorectal cancer.

Figure 2

BRD4 inhibition attenuates proliferation, migration and invasion in vitro. (A) 72 h survival rate curve of HT29, HCT8, HCT116, SW480 and SW620 treated with vehicle (DMSO) or MS417 (1, 3, 6, 9, 12, 24, 30 µM), measured by MTT; (B) IC₅₀ of HT29, HCT8, HCT116, SW480 and SW620 following vehicle/MS417 for 24, 48 and 72 h; (C,D) macroscopic and microscopic images and quantification of colonies formed by HT29 or SW620 cell lines treated with MS417 (1 µM); (E,F) Cell migration and invasion of HT29 or SW620 cell lines were inhibited by MS417 (1 µM). n = 3 repeats with similar results. ** p < 0.01; *** p < 0.001. Values are depicted as Mean ± SEM.

Figure 2

BRD4 inhibition attenuates proliferation, migration and invasion in vitro. (A) 72 h survival rate curve of HT29, HCT8, HCT116, SW480 and SW620 treated with vehicle (DMSO) or MS417 (1, 3, 6, 9, 12, 24, 30 µM), measured by MTT; (B) IC₅₀ of HT29, HCT8, HCT116, SW480 and SW620 following vehicle/MS417 for 24, 48 and 72 h; (C,D) macroscopic and microscopic images and quantification of colonies formed by HT29 or SW620 cell lines treated with MS417 (1 µM); (E,F) Cell migration and invasion of HT29 or SW620 cell lines were inhibited by MS417 (1 µM). n = 3 repeats with similar results. ** p < 0.01; *** p < 0.001. Values are depicted as Mean ± SEM.

Figure 3

BRD4 inhibition impacts transcriptional programs that control cell proliferation and EMT. (A) Apoptosis of HT29 and SW620 cell lines treated with vehicle (DMSO), MS417 (1 µM) or MS417 (12 µM); (B) Protein levels of candidate apoptosis-related factors following vehicle/MS417 treatment (1 and 12 µM) for 48 h; (C) Protein levels of E-cadherin and Vimentin in HT29 or SW620 cell lines treated with vehicle or MS417 (1 and 12 µM). The expression level of each protein was normalized against GAPDH. * p < 0.01; ** p < 0.001; n.s. not significant.

Figure 3

BRD4 inhibition impacts transcriptional programs that control cell proliferation and EMT. (A) Apoptosis of HT29 and SW620 cell lines treated with vehicle (DMSO), MS417 (1 µM) or MS417 (12 µM); (B) Protein levels of candidate apoptosis-related factors following vehicle/MS417 treatment (1 and 12 µM) for 48 h; (C) Protein levels of E-cadherin and Vimentin in HT29 or SW620 cell lines treated with vehicle or MS417 (1 and 12 µM). The expression level of each protein was normalized against GAPDH. * p < 0.01; ** p < 0.001; n.s. not significant.

Figure 4

BRD4 inhibition impairs CRC tumor growth in vivo. (A) Macroscopic images of resected tumors at the conclusion of the experiment; (B) average weight of resected tumors; (C) average tumor volume of mice injected with either vehicle (DMSO) or MS417 (20 mg/kg, n = 4/treatment); (D) average mice weights with every 2-day injection of vehicle/MS417 over a time course of 3 weeks. *, p < 0.05; **, p < 0.05; ***, p < 0.001. Values are depicted as Mean ± SEM.

Figure 5

BRD4 inhibition suppresses CRC liver metastasis in vivo. (A,B) Macroscopic images of resected livers at the conclusion of the experiment in mice injected by HT29 or SW620 cell lines and then treated by vehicle/MS417 (20 mg/kg, n = 5/treatment); (C,D) Representative microscopic H&E images of livers. Metastatic focus is circled. *** p < 0.001. Values are depicted as Mean ± SEM.

Figure 5

BRD4 inhibition suppresses CRC liver metastasis in vivo. (A,B) Macroscopic images of resected livers at the conclusion of the experiment in mice injected by HT29 or SW620 cell lines and then treated by vehicle/MS417 (20 mg/kg, n = 5/treatment); (C,D) Representative microscopic H&E images of livers. Metastatic focus is circled. *** p < 0.001. Values are depicted as Mean ± SEM.