Loss of the human polycomb group protein BMI1 promotes cancer-specific cell death

Abstract

The polycomb group protein BMI1 has been shown to support normal stem cell proliferation via its putative stem cell factor function, but it is not known if BMI1 may also act as a cancer stem cell factor to promote cancer development. To determine the role of human BMI1 in cancer growth and survival, we performed a loss-of-function analysis of BMI1 by RNA interference (RNAi) in both normal and malignant human cells. Our results indicate that BMI1 is crucial for the short-term survival of cancer cells but not of normal cells. We also demonstrated that loss of BMI1 was more effective in suppressing cancer cell growth than retinoid-treatment, and surviving cancer cells showed significantly reduced tumorigenicity. The cancer-specific growth retardation was mediated by an increased level of apoptosis and a delayed cell cycle progression due to the loss of BMI1. By comparison, BMI1 deficiency caused only a moderate inhibition of the cell cycle progression in normal lung cells. In both normal and cancer cells, the loss of BMI1 led to an upregulation of INK4A-ARF, but with no significant effect on the level of telomerase gene expression, suggesting that other BMI1-cooperative factors in addition to INK4A-ARF activation may be involved in the BMI1-dependent cancer-specific growth retardation. Thus, human BMI1 is critical for the short-term survival of cancer cells, and inhibition of BMI1 has minimal effect on the survival of normal cells. These findings provide a foundation for developing a cancer-specific therapy targeting BMI1.

Figures

Figure 1

Morphological changes and gene expression analyses of BMI1 and GAPDH in RNA interference (RNAi)-treated and scrambled (scr)-treated control cells. (A) Representative morphological changes of SH-SY5Y neuroblastoma (NB) cells (a), HCN-2 cortical neurons (b), NCCIT embryonic carcinoma cells (c), and H1 ES cells (d) 3 days after RNAi treatment (magnification: × 200). All cells were grown following providers' guidelines. For all experiments, the cells were seeded at 5 × 104 cells/ml in fresh medium. To generate dsRNA targeting BMI1 mRNA, a DNA fragment was PCR amplified from human leukemic cell cDNA using primer sequences specific for the BMI1 gene (see below) but with an addition of T7 primer sequences at the 5′ end of both forward and reverse primers. The amplified DNA fragment was used to generate dsRNA targeting BMI1 mRNA using the MEGAscript RNAi kit (Ambion) following the manufacturer's instructions. Purified dsRNA was transfected into cultured cells (8 μg dsRNA/106 cells) using the X-tremeGENE siRNA Transfection Reagent (Roche Applied Science). For RNAi specificity control, an equal amount of mixed pool of scrambled (scr) siRNA molecules (commercially available from Ambion) was used for transfection in place of the BMI1 dsRNA. Detection of alkaline phosphatase activity was performed using a semi-quantitative Alkaline Phosphatase Histochemical Staining system (Sigma Diagnostics) according to the manufacturer's instructions. (B) Representative gene expression analysis of BMI1 and GAPDH in RNAi-treated and scr-treated control EC cells, normal ES cells, NB cells, and normal neurons. Extraction of total cellular RNA and cDNA synthesis were performed as described previously (Liu et al., 2004). cDNA was amplified with primers specific for either BMI1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR conditions were: 94°C for 4 min, 28–30 cycles of 94°C for 45 s, 56°C for 30 s, 72°C for 40 s, and a final extension step at 72°C for 5 min. The primer sequences for BMI1 are 5′-GTCCAAGTTCACAAGACCAGACC-3′ and 5′-ACAGTCATTGCTGCTGGGCATCG-3′. GAPDH primers were the same as used previously (Liu et al., 2004). (C) Representative Western blotting analysis of BMI1 and GAPDH in RNAi-treated and scr-treated control EC cells, NB cells, and normal neurons. Western blotting was performed using primary antibody against GAPDH (Santa Cruz Biotechnology) and monoclonal antibody against BMI1 (Abcam). Bound antibody was detected with the Immun-Star™ HRP chemiluminescent system following the manufacturer's instructions (Bio-Rad).

Figure 2

Effects of RNA interference (RNAi) of BMI1 on cell proliferation and tumorigenicity. (a) Histogram of the number of live cells remaining in the culture of control, RNAi-treated, and ATRA-treated EC cells, respectively, during the first 3 days of treatment. The cells used for growth curve studies were grown for a total of three days, and cells were counted on days 0, 1, 2 and 3 following transfection using trypan blue (0.25%) for monitoring viability. (b) Histogram of the number of live cells remaining in the culture of control and RNAi-treated normal WI-38 cells, respectively; (c) Histogram of the number of colonies formed by control and RNAi-treated neuroblastoma (NB) cells on days 5 and 10, respectively. For soft agar colony formation assays, two sets of human NB cells were prepared for normal control cells, cells treated with scrambled siRNA, and cells treated with dsRNA, respectively. Cells (1 × 104) were suspended in 1.5 ml growth medium (10% FBS-DMEM) containing 0.4% Bacto agar. The cell suspension was overlaid onto a hard agar base composed of growth medium containing 0.7% Bacto agar in six-well plates. Plates were maintained in a humidified incubator at 37°C for 5 days and 10 days, respectively. Plates were subsequently stained with 0.005% Crystal Violet (Sigma) solution for 4 h at room temperature and colonies were counted under a microscope at × 200 magnification on the fifth day and 10th day, respectively. Vertical bars represent standard deviation (s.d.) from at least three sets of experiments. HPF: high-power field.

Figure 3

Effects of RNA interference (RNAi) of BMI1 on cell cycle progression and the expression of INK4A-ARF and hTERT. (a) Cell cycle position and apoptosis of BMI1 RNAi-treated and scrambled (scr)-treated control EC cells (upper panel) and WI-38 lung cells (lower panel) were detected by using a FITC BrdU Flow system following the manufacturer's instructions (BD Pharmingen). For labeling, BrdU was added directly to the cell culture at a final concentration of 100 μm 24 h after transfection, and was incubated for another 48 h. Cells were then harvested, fixed, permeabilized, treated with DNase I and stained with FITC-conjugated anti-BrdU antibodies and 7-AAD (to display DNA content) (BD Pharmingen). A Becton Dickinson FACS apparatus was used to acquire and analyse a minimum of 10 000 events using the Cellquest program. The graphs shown are representative of similar results obtained from three independent experiments; (b) Real-time PCR was performed to quantify the relative level of gene expression for hTERT and p16 using the ABI real-time PCR apparatus as reported previously (Liu et al., 2004). As BMI1 may affect the expression of p16 and p14 differentially in a tissue-specific manner (Bruggeman et al., 2005), we have chosen primer sequences that allowed us to simultaneously assess the expression of both p16 and p14 encoded by the INK4A-ARF locus in the same real-time PCR analysis. The expression levels of both hTERT and INK4A-ARF in control cells were normalized to a relative value of 1, and the level of gene expression in the corresponding RNAi-treated cells was adjusted accordingly to reflect the fold-changes by comparison with the controls. Vertical bars represent s.d. from at least three sets of experiments.