Synthesis and Characterization of Novel BMI1 Inhibitors Targeting Cellular Self-Renewal in Hepatocellular Carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) represents one of the most lethal cancers worldwide due to therapy resistance and disease recurrence. Tumor relapse following treatment could be driven by the persistence of liver cancer stem-like cells (CSCs). The protein BMI1 is a member of the polycomb epigenetic factors governing cellular self-renewal, proliferation, and stemness maintenance. BMI1 expression also correlates with poor patient survival in various cancer types.

Objective: We aimed to elucidate the extent to which BMI1 can be used as a potential therapeutic target for CSC eradication in HCC.

Methods: We have recently participated in characterizing the first known pharmacological small molecule inhibitor of BMI1. Here, we synthesized a panel of novel BMI1 inhibitors and examined their ability to alter cellular growth and eliminate cancer progenitor/stem-like cells in HCC with different p53 backgrounds.

Results: Among various molecules examined, RU-A1 particularly downregulated BMI1 expression, impaired cell viability, reduced cell migration, and sensitized HCC cells to 5-fluorouracil (5-FU) in vitro. Notably, long-term analysis of HCC survival showed that, unlike chemotherapy, RU-A1 effectively reduced CSC content, even as monotherapy. BMI1 inhibition with RU-A1 diminished the number of stem-like cells in vitro more efficiently than the model compound C-209, as demonstrated by clonogenic assays and impairment of CSC marker expression. Furthermore, xenograft assays in zebrafish showed that RU-A1 abrogated tumor growth in vivo.

Conclusions: This study demonstrates the ability to identify agents with the propensity for targeting CSCs in HCC that could be explored as novel treatments in the clinical setting.

Fig. 1

BMI1 expression in HCC. a) Left panel: Immunohistochemistry staining for BMI1 in HCC and normal liver tissues. Representative images from normal liver tissue (negative), and HCC tissues with weak (BMI1 1+ or +/−) or strong (BMI1 2+ or 3+) BMI1 expression. Right panel: Quantification of BMI1 expression in cohorts of normal liver tissues and HCC. Staining percentages and statistical analysis are described in Supplementary Table 1. b) Oncomine analysis of BMI1 expression in HCC vs normal liver tissues. Four datasets (identified as A-D) are included. Dataset description and patient characteristics are described in Supplementary Table 2

Fig. 2

Downregulation of BMI1 expression in HCC cells by the BMI1 inhibitor RU-A1. a) Side-by-side electrostatic potential plots for RU-A1 and RU-A5. The plots indicate that RU-A5 is more electron-rich than RU-A1. The electrostatic potential was computed from a B3LYP/6-31G* single point calculation using Spartan software. b) The Glide (Grid-based ligand docking with energetics) algorithm [21] approximates the position, orientation, and conformation of C-209 and the RU compounds to the BMI1 RNA binding site on precomputed van der Waals and electric grids to generate binding free energy. c) Cell viability evaluated in HepG2, PLC/PRF/5, and Huh1 cell lines treated with the BMI1 inhibitors RU-A1 and C-209 (0.01–10 μM) for 72 h. d) Left panel: Protein lysates derived from HepG2, PLC/PRF/5, and Huh1 cells treated with either RU-A1 or C-209 at their respective IC₅₀ (Table 1) and probed for BMI1 and β-actin (loading control). Right panel: Quantification of BMI1 expression levels relative to the displayed western blot. All experiments were done three times

Fig. 3

Specificity and cellular effects of the RU-A1 inhibitor. a) Left panel: WB analysis showing BMI1 expression modulation in Sh-Luc and Sh-BMI1 infected, untransduced, and BMI1-transduced (FLAG-BMI1 and Sh – +FLAG-BMI1) HepG2 cells. Right Panel: cell viability performed on the indicated cells after 48 h of exposure to 10 μM RU-A1. Data show the results of three independent experiments with three replicates for each condition. b) Annexin V/PI staining performed on HepG2 and PLC/PRF/5 cells treated with RU-A1 and C-209. Camptothecin (Campt) was used as an apoptosis-inducing control. c) Representative image and quantification of cell cycle distribution assessed in HepG2 and PLC/PRF/5. In experiments displayed in b and c, HepG2 cells were treated with RU-A1 and C-209 both at 10 μM, while PLC/PRF/5 with 300 nM RU-A1 and 5 μM C-209 for 72 h. Altogether experiments were performed at least three times

Fig. 4

BMI1 inhibition reduces the self-renewal capacity and CSCs content in vitro. a) Left panel: Representative image showing clonogenic capability of untreated, RU-A1-, and C-209-treated HepG2 and PLC/PRF/5 cells. Right panel: Colony quantification for each cell line. Three independent experiments with six replicates for each condition were performed. Data are displayed as mean percentage ± S.D. b) Upper panel: limiting dilution analysis (LDA) plot used to assess the frequency of self-renewing TICs in HCC. Frequencies were calculated using LDA with 99% confidence interval. Correlation coefficient lower, estimate and upper values are displayed. Lower panel: graphic representation of limiting dilution assay in RU-A1- and C-209-treated or untreated HCC cells. c) qPCR showing stem cell marker expression variation in HCC cells following treatments with BMI1 inhibitors. Data are plotted as results of two independent experiments. *p < 0.05, ***p < 0.001 and ****p < 0.0001. In all experiments, HepG2 cells were treated with both RU-A1 and C-209 at 10 μM, while PLC/PRF/5 cells were exposed to 300 nM RU-A1 and 5 μM C-209, respectively

Fig. 5

BMI1 inhibition negatively impacts CSC features. a) Cell motility of HCC cells in vitro. Following short-term exposure (24 h) to RU-A1, both HepG2 and PLC/PRF/5 cells were washed and replated in fresh media to assess cell migration at 24 h. b) Viability of HepG2 and PLC/PRF/5 cells treated with 5-FU (10 μg/ml and 50 μg/ml) for 48 h and subsequently treated with RU-A1 and C-209 for an additional 72 h. c) Survival of therapy-resistant HCC cells. HepG2 and PLC/PRF/5 cells, treated as described above, were washed and replated in fresh media for an additional 3 days. Experiments in b and c were done at least 3 times in triplicates. d) Cell clonogenicity of therapy-resistant HCC cells. HepG2 and PLC/PRF/5 cells treated as described above, were washed and plated in soft agar. After 2–3 weeks, media were removed, colonies were stained with crystal violet and counted. e) Q-PCR analysis of stem cell markers in untreated and treated HCC cells. Results are indicated as mean ± S.D. of two independent experiments with 6 replicates for each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Establishment of human tumor xenograft in zebrafish. a) Representative images of HepG2 EGFP+ cells injected in the perivitelline space of 2dpf embryos and their fate after 2 days. At least 50 embryos were utilized for either control untreated or each treatment with DMSO or RU-A1. Xenograft take rates were 70–80% and were confirmed by fluorescence before initiation of treatment. Untreated cells (upper) and RU-A1 treated (lower). Red circles outline the engrafted tumor areas. b) Graphical representation showing area of fluorescence of untreated cells and RU-A1-treated cells when measured at 4dpf. To confirm that fluorescent area measurements correlate with cell counts, matching cell numbers were injected and cell counts representing tumor formation in these zebrafish xenografts between untreated and RU-A1 treated cells on day 0 were not different. c) Kaplan–Meier survival analysis of casper zebrafish embryos after injecting untreated and RU-A1 treated HepG2 cells. The survival rate for embryos with untreated cells (n = 33) was significantly lower due to continued tumor cell growth when compared to that of embryos with RU-A1 treated cells (n = 43) (p < 0.001). Survival data are representative for six independent experiments performed each with >50 embryos/treatment. *p < 0.05, **p < 0.01 and ***p < 0.001. All experiments were done at least 3 times