PRMT1 inhibition induces differentiation of colon cancer cells

Abstract

Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.

Figure 1

ALP activity in normal epithelial cell is much higher than in colon cancer cells; sodium butyrate (SB) increases ALP activity in colon cancer cells. (a) ALP activity in colon cancer cells (HT-29 and HCT-116) and normal colon epithelial cells (CCD-841). Graph represents mean ± SEM from three independent experiments. Statistical significances of ALP activity in cancer cells versus normal cells are indicated. (b) Effect of SB in the indicated concentrations on ALP activity and colon cancer cell (HT-29) growth, 5 days after treatment. Graphs represent mean ± SEM. from three independent experiments. Statistical significances of ALP activity and cell viability between non-treated and SB treated cells are indicated.

Figure 2

MS023 and entinostat significantly increase ALP activity and delay growth of colon cancer cells in 2D and 3D cultures. (a) Effect of PRMT type 1 inhibitor (MS023) and HDAC inhibitor (entinostat) on normal (CCD-841) and colon cancer (HT-29) cell growth and ALP activity in 2D culture, 5 days after treatment. The data is normalized relatively to DMSO treated cells. Graphs represent mean ± SEM from four independent experiments. Statistical significances of ALP activity in cancer cells versus normal cells are indicated. (b) Effect of PRMT type 1 inhibitor (MS023) and HDAC inhibitor (entinostat) on growth kinetics of HT-29 GFP expressing colon cancer spheroids (1–6 days after treatment). Graphs represent total mean spheroid area ± SEM. Statistical significances of spheroid cell growth in DMSO versus compounds treated spheroids are indicated. Graphs in (a) and (b) were made using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com). (c) Representative pictures of the spheroids, 6 days after treatment. Green—HT-29-GFP spheroids, red—propidium iodide staining (dead cells), × 10 magnification, scale bar 200 µm.

Figure 3

PRMT1 inhibition/knock down mediates differentiation phenotype in colon cancer cells. (a) Effect of PRMT type 1 inhibitor (MS023) and its inactive analog (MS094) on colon cancer cell (HT-29) growth and ALP activity, 5 days after treatment. The data is normalized relatively to DMSO treated cells. Graphs represent mean ± SEM from three independent experiments. Graph was made using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com). Statistical significances of ALP activity and cell viability between MS023 and MS094 treated cells are indicated. Chemical structures of the compounds are shown on the right side of the graph. (b) Effect of PRMT1 knock down on HT-29 growth and ALP activity, 5 days after treatment. Groups: non treated cells, DharmaFECT – cells treated with transfection reagent alone, siCon, cell transfected with relevant control siRNA, siPRMT1—cells transfected with siPRMT1. Graphs represent mean ± SEM from three independent experiments. Statistical significance of ALP activity and cell viability between siPRMT1 and others groups is indicated. (c) Western blot analysis of PRMT1 expression in the indicated groups of HT-29 cells, 5 days after transfection. Full-length blots are presented in Supplementary Fig. S2.

Figure 4

MS023 stabilizes PRMT1. (a) Cellular thermal shift assay of MS023 in HT-29 cells. Graph represents relative PRMT1 band intensities of the indicated samples ± SEM from three independent experiments. Representative PRMT1 Western blots of each sample are shown. Statistical significances between MS023 and DMSO are indicated. The analysis was performed simultaneously in the MS023 and DMSO samples with same time of the exposure. Full-length blots are presented in Supplementary Fig. S3. (b) Isothermal (at 63 °C) dose response fingerprints in HT-29 cells. Graph represents relative PRMT1 band intensities of the indicated samples ± SEM from three independent experiments. Graphs in a and b were made using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com). Representative PRMT1 and actin Western blots of each sample are shown. Full-length blots are presented in Supplementary Fig. S4.

Figure 5

MS023 treatment drives colon cancer cell differentiation: genomic analysis. (a) Overlap between differentially expressed genes, 5 days after entinostat (1.5 µM) or MS023 (5 µM) treatments (n = 4/group). (b) Heat map of the differential expressed genes. The values are counts on normalized log2 scale. (c) Log2 fold changes in expression of genes, which are positively involved in the differentiation of normal intestinal cells. Comparison of samples derived from HT-29 cells treated with MS023 (5 µM, n = 4) and DMSO (n = 4), 5 days after treatment, mean ± SEM. Bold grey color columns represent differentially expressed genes. Statistical significances (pV) of all differentially expressed genes between MS023 and DMSO treated samples are < 0.001.

Figure 6

MS023 treatment drives colon cancer cell differentiation: proteomic analysis. Graph represents changes in pathways associated with cancer progression (grey columns) and cell differentiation (black columns). Comparison of MS023 treated (5 µM, n = 8) and DMSO treated (n = 8) samples of HT-29 cells, 5 days after treatment. Statistical significances of the indicated pathways changes upon MS023 treatment are indicated.

Figure 7

MS023 and entinostat treatment increase expression of junction adhesion molecules (JAMs) E-cadherin and ZO-1 and their genes in HT-29 colon cancer cells. (a) Representative pictures of HT-29 cells treated with indicated compounds and stained for E-cadherin (red) and ZO-1 (green), × 40magnification, scale bar 100 µM. (b) Graph represents mean ± SEM of normalized fluorescence derived from pictures presented in “a”. Statistical significances of JAMs induction upon MS023 and entinostat treatments are indicated. (c) Effect of MS023 and entinostat treatments on changes in the expression of E-cadherin and ZO-1 genes in HT-29 cells. Graph represents log2 change of normalized fluorescence ± SEM in the cells treated with indicated compounds in comparison to DMSO treated cells. Statistical significances of gene induction of JAMs upon MS023 and entinostat treatments are indicated.

Figure 8

MS023 significantly inhibits HT-29 xenograft growth and delays cancer cell proliferation in-vivo. (a) Kinetics of HT-29 xenograft growth in nude mice (n = 5/group). Data represented as tumor volume means ± SEM. Grey bright diamonds—MS023, 30 mg/kg; grey bold tringles – MS023, 15 mg/kg; black squares—saline control. Statistical significances of tumor volumes between saline treated and MS023 treated groups are indicated. (b) Representative images of Ki67—DAB stained HT-29 xenograft taken from saline (n = 3) and MS023 treated mice (n = 5), scale bar 50 µm. (c) Graph represents present of Ki67 positive cells ± SEM. Statistical significance of Ki67 changes upon MS023 treatment is indicated.