Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression

Abstract

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

Trial registration: ClinicalTrials.gov NCT02404480.

Fig. 1.. BMI1 expression is inversely associated with C/EBPα expression in human adenocarcinoma cells.

(A) Left: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on experimental samples (n=3) from the human lung adenocarcinoma cell line H358 transfected with a rapamycin-inducible C/EBPα-expressing vector or a rapamycin-inducible empty vector (as control), and induced with rapamycin (for 36 hours). The mean expression is presented as a percentage of 18S RNA. The P values for each sample are indicated on the basis of a two-sided Welch’s t test. Right: Western blot analysis was carried out in H358 cells transfected with the rapamycin-inducible C/EBPα-expressing vector or the rapamycin-inducible empty vector by using anti-C/EBPα and anti-BMI1 antibodies. Loading was assessed, after complete stripping of the membrane, with an anti-actin antibody. The expected size in kilodaltons is indicated. (B) Left: Pie chart demonstrating BMI1 protein expression in patient-derived adenocarcinomas that are negative or low for C/EBPα (staining intensity, 0 or 1⁺). BMI1 was considered positive when staining was scored as 1⁺ to 3⁺. Right: Pie chart showing C/EBPα protein expression in patient-derived adenocarcinomas, subdivided as negative/low (staining intensity, 0or 1⁺) or positive (staining intensity, 2⁺ or 3⁺). (C) Representative examples of IHC data from three patients’ tissues (each enclosed in a box), which were independently and blindly scored by two pathologists. Staining intensity was scored as follows: 0 (no staining); 1⁺ (mild staining); 2⁺ (moderate staining); and 3⁺ (strong staining). Scale bar, 25 μm. (D) Overall survival curves for 490 patients from TCGA lung adenocarcinoma cohort (https://tcga-data.nci.nih.gov/tcga/). Only 490 of the 521 TCGA samples have both RNA-sequencing and survival data. Patients are stratified according to low or high C/EBPα expression, defined as log₂ expression < 9.0 or not, respectively. Survival probability is higher (P = 0.041) in BMI1^low patients, as compared to BMI1^high patients in the C/EBPα^low subgroup. The median expression of BMI1 used to define BMI1^low and BMI1^high patient subgroups was 10.2 in the C/EBPα^low group and 10.1 in the C/EBPα^high group. The method of Kaplan and Meier was used for graphical displays of overall survival. The log-rank test was used to assess differences in overall survival. A similar approach was also applied for C/EBPα^high samples. The P value and the sample size (n) for each subgroup are indicated on each plot. Among C/EBPα^low patients, 30 death events/109 patients presented with low BMI1 expression, and 39 death events/ 109patientspresentedwithhighBMI1expression. Among C/EBPα^high patients, 40 death events/136 patients presented with low BMI1 expression, and 39 death events/136 patients presented with high BMI1 expression. Asterisk indicates statistical significance.

Fig. 2.. Lung-specific C/EBPα knockout mice develop pulmonary adenocarcinoma.

(A) Left: Representative histological lung sections of C/EBPα^Lung-Δ mice and control littermates, stained with hematoxylin and eosin (H&E). “Control” indicates littermates that do not have at least one of the transgenic alleles (CCSP-rtTA, Cre, or C/EBPα^loxP/loxP). Right: Representative IHC data showing C/EBPα protein expression (brown and indicated by arrows). The dashed line separates the C/EBPα^Lung-Δ tumor area (to the left of the dashed line) fromthe normal-appearing tissue, which surrounds the tumor. Scale bar, 50 μm. (B) qRT-PCR analysis was performed in duplicate using RNA from lineage-depleted (CD45.1⁻,CD45.2⁻,andCD31⁻) pulmonary cells from five doxycycline-untreated (Doxy −) (n = 5) and five doxycycline-treated (Doxy +) (n=5) C/EBPα^loxP/loxP CCSP-rtTA⁺ Cre⁺ mice, as well as fluorescence-activated cell sorting–purified lineage-depleted pulmonary cells from five tumors (n = 5). Adjacent tissue represents the “normal-appearing” area in the vicinity of the adenocarcinoma, which does not display a transformed phenotype. The mean expression is presented as a percentage of 18S RNA amount. Two-sided Welch’s t test showed a significant statistical difference among uninduced tissue versus tumor (P = 0.0001) and induced normal-appearing tissue versus tumor (P = 0.00004). (C) Representative Southern blot analysis of lung genomic DNA from doxycycline-untreated (Doxy −) and doxycycline-treated (Doxy +) C/EBPα^loxP/loxP CCSP-rtTA⁺ Cre⁺ mice. In doxycycline-treated mice, both tumor adjacent tissue and adenocarcinoma tissue were aluated, as shown, indicating nearly complete excision in tumor cells. No unexcised allele could be detected in tumors, as compared to uninduced pulmonary tissue, in which excision is undetectable. The sizes of the targeted (10.9-kb) and excised (4.7-kb) alleles are shown. Lu, lung; Adj. tiss., adjacent nontumor tissue; Tum., tumor tissue. (D) qRT-PCR was performed in duplicate using RNA extracted from three lineage-depleted (CD45.1⁻, CD45.2⁻, and CD31⁻) tumors (n = 3) and wild-type (WT) pulmonary cells (n = 3), as well as the C/EBPα-null cell line (n = 3). The mean expression is presented as a percentage of 18S RNA. P values for each sample are indicated on the basis of a two-tailed Welch’s t test. (E) IHC analysis performed on C/EBPα^Lung-Δ adenocarcinomas and the C/EBPα-null pulmonary tumor cell line confirmed a reciprocal relationship between C/EBPα and BMI1 protein expression. Representative C/EBPα (dark brown) and BMI1 (brown) IHC in lung sections from control, C/EBPα^Lung-Δ, and the C/EBPα-null tumor cell line derived from C/EBPα^Lung-Δ tumors are shown. Filled arrowheads indicate alveolar epithelial cells, and triangles indicate bronchioepithelial cells. Tumor tissue is to the left of the dashed line in the C/EBPα^Lung-Δ sections (middle panels). Scale bar, 40 μm.

Fig. 3.. C/EBPα negatively affects BMI1 expression.

(A) qRT-PCR of C/EBPα (black circles) and Bmi1 (white circles) was performed in C/EBPα-null cell lines transduced with either a retroviral construct to overexpress murine C/EBPα (MSCV-C/EBPα-IRES-GFP) or a control vector (MSCV-IRES-GFP). qRT-PCR was performed on GFP⁺ cells 3 days after the infection. Mean expression is presented as a percentage of 18S RNA. Assays were performed in duplicate on two independent biological replicates (n = 2). Data were compared by Welch’s t test, and the P value for Bmi1 expression is indicated. (B) Western blot analysis carried out in C/EBPα-null cell lines transduced with either a retroviral construct to overexpress murine C/EBPα (MSCV-C/EBPα-IRES-GFP) or a control vector (MSCV-IRES-GFP). Total protein lysates were immunoblotted with anti-C/EBPα and anti-BMI1 antibodies. Loading was assessed with an anti-actin antibody. The expected size in kilodaltons is indicated.

Fig. 4.. BMI1 is required for lung tumor formation in C/EBPα^Lung-Δ mice.

(A) Detection of tumorigenic foci in lungs of mice with reduced Bmi1 expression. Pups were treated with doxycycline, and the presence of tumors was scored between 7.5 and 9.5 months after treatment termination, when mice were 9 to 11 months old. The difference in tumorigenesis was estimated to be statistically significant by the two-sided Fisher’s exact test (P = 0.0006). (B) Representative pictures of tumors growing subcutaneously in NSG mice after injection of cells infected with control shRNA lentivirus (Luc-sh), Bmi1-sh1, and Bmi1-sh2. Three mice were injected on each flank with each lentiviral construct (n = 6 measurements, two per mouse). The mean weight and P values, analyzed by a two-sided Welch’s test, are indicated. There was no difference between Bmi1-sh1 and Bmi1-sh2 (P = 0.681). (C) Tumor size was measured with calipers at 10, 20, and 30 days after injection of cells infected with control shRNA lentivirus (Luc-sh), Bmi1-sh1, or Bmi1-sh2 into immunocompromised mice (three mice per lentiviral construct; mice were injected on both flanks, n = 6). The figure indicates the tumor burden volume versus time since injection (in days). Data normalized to tumor size at day 10 are shown for each time point. At day 30, each of the Bmi1-sh constructs was significantly different from controls, using a two-sided Welch’s t test. In the control animals, tumor burden was 4.95 times that of day 10, compared to 1.69 times that of day 10 for Bmi1-sh1 (P = 0.0036), and 1.77 times that of day 10 for Bmi1-sh2 (P = 0.033). There was no difference between Bmi1-sh1 and Bmi1-sh2 (P = 0.453). (D) Tumor size was measured with calipers at 10, 20, and 30 days after injection of GFP⁺-sorted cells infected with control retrovirus (MSCV) or C/EBPα-expressing retrovirus (MSCV-C/EBPα) into immunocompromised mice (n = 3 mice per retroviral construct). The figure indicates tumor burden versus time since injection (in days). Data normalized to tumor size at day 10 are shown for each time point. The P value is indicated in the figure, as calculated by the Welch’s two-sided t test.

Fig. 5.. BMI1 pharmacological inhibition reduces tumorigenicity of C/EBPα-null tumor cells.

(A) Western blot assay with an anti-BMI1 antibody was carried out in the C/EBPα-null tumor cell line treated for 48 hours with the BMI1 inhibitor (0.7 and 1.5 μM) or 0.5% DMSO vehicle as a control. Loading was assessed, after complete stripping of the membrane, with an anti-actin antibody. The expected size in kilodaltons is indicated. (B) GSEA shows enrichment of BMI1-activated targets (P = 0.002) in vehicle (DMSO)–treated cells, as compared to PTC-209–treated cells (left panel). GSEA also shows enrichment in BMI1-repressed targets in PTC-209–treated cells, as compared to vehicle (DMSO)–treated cells (right panel, P < 0.001). Normalized enrichment score (NES) is indicated in each panel. Fold change cutoff value of 2 and false discovery rate–adjusted P value cutoff of 0.05 were used for heat presentation. (C) Cell cycle analysis of the C/EBPα-null tumor cell line after treatment for 48 hours with 0.7 and 1.5 μM BMI1 inhibitor and vehicle as control. Cell cycle status was determined by pyronin Y/Hoechst 33342 staining (Hoechst 33342 stains DNA and pyronin Y stains RNA). Percentages of cells in G₀ (double-negative population), G₁ (pyronin Y⁺ population), and S/G₂-M phases (double-positive population) are indicated. Specifically, the average percentage of the population in cell cycle arrest is 61 ± 4.3% (cells treated with 0.7 μM PTC-209, P = 0.05), 76.4 ± 7.6% (cells treated with 1.5 μM PTC-209, P = 0.01), and 46 ± 7% (DMSO-treated cells). Assays were performed as triplicate and analyzed by running three two-sample one-sided t tests, corrected by a Bonferroni adjustment. (D) Tumor size was measured with calipers at the beginning of the treatment and every 15 days from initiation of treatment until termination. Mice were treated daily for 1 month with PTC-209 (50 mg/kg) or vehicle control. The left panel indicates tumor burden versus time of treatment (in days). Growth curves of six vehicle-treated mice are shown on the left, and those of six PTC-209–treated mice are on the right. Data (n = 6 per group) are normalized to the tumor burden detected at the beginning of treatment and are shown for each time point. The normalized tumor volume at day 30 is indicated in the histogram (right panel). The difference in tumor growth is statistically significant (P = 0.00001), as calculated by the two-sided Welch’s t test. (E) The graph indicates the percentages of cells in G₀ (white), G₁ (striped), and S/G₂-M phases (black). Cell cycle analysis was performed by pyronin Y/Hoechst 33342 staining of subcutaneously transplanted tumors treated with the PTC-209 compound (n = 4) or the vehicle only (n = 4) as control. Welch’s two-sided t test was used to calculate differences in G₀ (P = 0.002) between PTC-209– and vehicle-treated cells. (F) Western blot analysis of the indicated human adenocarcinoma cell lines treated for 48 hours with the BMI1 inhibitor (1.5 μM, +) or 0.5% DMSO vehicle as control (−). Protein lysates were immunoblotted with an anti-BMI1 antibody, and loading was assessed with an anti-actin antibody. The expected size in kilodaltons is indicated. Treatment with PTC-209 at 1.5 μM reduces the expression of BMI1 to 4.2% (H322), 1.87% (A549), 6.33% (H23), 9.08% (H1975), 6.1% (H358), and 5.4% (PC9), as compared to the expression with 0.5% DMSO. (G) NSG mice were subcutaneously injected with different human adenocarcinoma cell lines, as indicated on each plot. Mice were treated daily for 2 weeks with PTC-209 (50 mg/kg) or vehicle control. Tumor burden was measured with calipers at the beginning of the pharmacological treatment and after 7 and 15 days from initiation of treatment. The figure indicates tumor volume versus time of treatment (in days). Each graph shows growth curves of three vehicle-treated mice on the left and curves from three PTC-209–treated mice on the right. Data (n = 3 per group) are normalized to the tumor volume measured at the beginning of treatment and are shown per time point. The difference in tumor size at day 15 was statistically significant (P values indicated in the figure), as calculated by Welch’s two-sided t test, in every xenograft model.