Ubiquitination of Oncogenic Mutant p53 via Attenuation of Ribosome Biogenesis Machinery Effectively Inhibits Pancreatic Tumor Growth

Abstract

Dysregulated ribosome biogenesis and p53 mutations are known to play oncogenic roles in various cancers, including pancreatic cancer. In this study, we demonstrated the therapeutic potential of BMH-21, a pharmacologic inhibitor of RNA polymerase I, against pancreatic cancer by uncovering a novel molecular mechanism involving RPA194-mediated ubiquitination of mutant p53 without affecting the ubiquitination of wild-type p53. Our key findings are that (i) BMH-21 selectively induces apoptosis and cell growth inhibition of pancreatic cancer cells with no effect on normal human pancreatic ductal epithelial cells; (ii) BMH-21 degrades RPA194; (iii) BMH-21 inhibits recruitment of both RPA194 and RPA135 on rDNA to suppress pre-rRNA synthesis; (iv) RPA194 physically interacts with p53 and BMH-21-induced degradation of RPA194 selectively exposes truncated and mutated p53 for ubiquitination with no effect on ubiquitination of wild-type p53 in pancreatic cancer cells; and (v) BMH-21 treatment significantly reduces the growth of orthotopic xenograft pancreatic tumors in athymic nude mice with no observed toxicity. Altogether, these findings suggest that BMH-21 is a promising, nontoxic therapeutic agent for patients with pancreatic cancer with aberrant ribosome biogenesis and mutant p53, offering a potential new avenue for targeted treatment.

Figure 1.

BMH-21 preferentially inhibits cell viability and induces apoptosis in pancreatic cancer cells. A, Effect of BMH-21 on cell viability of HPDEC and pancreatic cancer cells (Capan-2, AsPC-1, BxPC-3, PANC-1, HPAF-II, and MIA PaCa-2). Cells were treated with indicated concentrations of BMH-21 for 24 hours, and cell viability was analyzed via MTT assay. B–E, Effect of BMH-21 on apoptosis induction in HPDEC and pancreatic cancer cells. B, Effect of BMH-21 on PARP in HPDEC and pancreatic cancer cells as assessed using WB. Briefly, cells were treated with indicated concentrations of BMH-21 for 12 hours. Total PARP and cleaved PARP (cl-PARP) were detected by WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin antibody. C and D, Flow cytometric analysis to detect apoptotic cells in control and BMH-21 (2 μmol/L)–treated HPDEC and MIA PaCa-2 cells using Alexa Fluor 488–labeled Annexin V. Representative flow cytometric images showing percent Alexa Fluor 488–tagged Annexin V–positive cells. E, The bar graph represents the percentage of live and apoptotic HPDEC and MIA PaCa-2 cells after BMH-21 treatment. The data represent the mean ± SEM of three samples. The P values were expressed as follows: **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. PI, propidium iodide.

Figure 2.

Expression pattern of RPA194 and targeting RiBi in pancreatic cancer. A,In silico analysis of RPA194 expression in human normal and pancreatic tumor tissues. The box plot shows significant (P = 0.01) overexpression of RPA194 in pancreatic tumor tissues compared with normal pancreatic tissues. B, Differential expression of RPA194 in various pancreatic cancer cell lines by WB analysis. C, Effect of BMH-21 on the RPA194 protein level in normal (HPDEC) and pancreatic cancer cells (Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells). Briefly, cells were treated with indicated concentrations of BMH-21 for 3, 6, and 12 hours, and the RPA194 protein level was determined by WB analysis. D, Effect of BMH-21 (2 μmol/L) on the localization of RPA194 (red) and UBTF (green) in Capan-2, MIA PaCa-2, and HPAF-II cells as determined by immunofluorescence. Scale bar, 5 μm. The quantification of immunofluorescence images is provided in Supplementary Fig. S3. E, Effect of BMH-21 on pre-rRNA synthesis in HPDEC, Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Cells were treated with BMH-21 (2 μmol/L) for 6 hours. qPCR was performed using primers for 5′ ETS expression at 851 regions. The bar graph indicates fold expression of 5′ ETS in control and BMH-21–treated cells. F–I, Effect of BMH-21 on the recruitment of RNA Pol I catalytic subunits (RPA194 and RPA135) on rDNA by ChIP assay. The cells were grown in 150-mm dishes to 90% confluency and treated with 2 μmol/L BMH-21 for 24 hours. IP was performed using RPA194 or RPA135 antibodies, and purified chromatin was amplified by qPCR. F and G, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in Capan-2 cells. H and I, Effect of BMH-21 on the recruitment of RPA194 and RPA135 on rDNA in MIA PaCa-2 cells. Values in bar graphs represent the mean ± SEM of three replicates. ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 3.

BMH-21 disperses the nucleolar proteins in pancreatic cancer cells. A and B, Effect of BMH-21 on the localization of nucleolar proteins FBL and NCL in pancreatic cancer cells. The cells were grown on poly-L-lysine–coated coverslips and treated with BMH-21 (2 μmol/L) for 12 hours and subjected to confocal microscopy. Representative confocal images show the diffusion of FBL and NCL in Capan-2, MIA PaCa-2, and HPAF-II cells as indicated by white arrows. Scale bar, 5 μm. C and D, The quantification of immunofluorescence images of FBL and NCL in the nuclei of Capan-2, MIA PaCa-2, and HPAF-II cells was done using ImageJ software. ns, P ≥ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. E and F, Dose- and time-dependent effects of BMH-21 on the expression of FBL and NCL in Capan-2 and MIA PaCa-2 cells as analyzed by WB analysis.

Figure 4.

BMH-21 induces WT p53 protein expression by several folds but not the mutant p53 expression in pancreatic cancer cells. A, Effect of BMH-21 on the protein level of WT and mutant p53 in Capan-2, AsPC-1, MIA PaCa-2, and HPAF-II cells. Briefly, cells were treated with 2 μmol/L BMH-21 for 24 hours, and 40–100 μg cell lysate was subjected to WB analysis. Equal loading of protein was determined by stripping and probing the blots with β-actin or cyclophilin B antibodies. B, The bar graph represents the quantification of blots represented in A. The blots were quantified using ImageJ software and normalized with respective loading controls. C and D, The effect of BMH-21 on the localization of WT and mutant p53 levels in Capan-2 and MIA PaCa-2 cells was evaluated by immunofluorescence. Scale bar, 5 μm. E, The bar graphs represent the quantification of immunofluorescence of p53 shown in C and D. The quantification was done using ImageJ software. ns, P ≥ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 5.

RPA194 interacts with both WT and mutant p53, and BMH-21–induced RPA194 degradation exposes mutant p53 for its ubiquitination. A and B, RPA194 interacts with WT p53 in Capan-2 cells, truncated p53 (Δp53) in AsPC-1, and mutant p53 R248W in MIA PaCa-2 cells. Cells were treated with BMH-21 (2 μmol/L) or BMH-21 + MG132 (10 μmol/L) for 24 hours. A, RPA194 immunoprecipitated samples were subjected to WB analysis using p53 (HRP-conjugated) antibody (top). IgG antibody was used as the isotype control for IP. Eighty micrograms of protein for Capan-2 and 40 μg of protein for AsPC-1 and MIA PaCa-2 cells were used as inputs. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). B, Effect of BMH-21 on ubiquitination of RPA194 and RPA194 binding partners in pancreatic cancer cells (bottom). RPA194 was immunoprecipitated using the RPA194 antibody, and WB analysis was performed using the anti-ubiquitin antibody. The results indicate that RPA194 or its binding partners are ubiquitinated by BMH-21. The RPA194 antibody was used for WB to indicate the position of RPA194 in the same immunoprecipitated samples (bottom). C, Effect of BMH-21 on the ubiquitination level of RPA194 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. Briefly, untreated, BMH-21, and BMH-21 + MG132–treated samples were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB analysis using the RPA194 antibody. D, Effect of BMH-21 on the ubiquitination level of p53 in Capan-2 and MIA PaCa-2 cells was analyzed by TUBEs. The cells were treated as mentioned in C, and protein lysates were incubated overnight with HCMB with rotation at 4°C. The unbound and bound fractions were collected and processed for WB using HRP-conjugated p53 antibody. Hundred micrograms of protein for Capan-2 and 40 μg protein for MIA PaCa-2 cells were used as inputs.

Figure 6.

Effect of BMH-21 on pancreatic tumor growth in the orthotopic xenograft mouse models. A and B, Schematic diagram depicting the experimental plan of the mouse xenograft study to investigate the therapeutic efficacy of BMH-21 against pancreatic cancer. Luciferase-labeled MIA PaCa-2 and AsPC-1 cells (1 × 10⁶) were implanted in the pancreas of athymic nude mice (n = 8/group). The vehicle and BMH-21 (25 mg/kg body weight) treatments started at 4–6 days after implantation of pancreatic cancer cells. Bioluminescence imaging of all the mice was performed at indicated days. C, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the MIA PaCa-2 luciferase xenograft at various time points. D and E, The average radiance (photons/second/cm²/sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the MIA PaCa-2 luciferase xenograft model. F, Representative bioluminescence images of vehicle-treated and BMH-21–treated live mice from the AsPC-1 luciferase xenograft at various time points. G and H, The average radiance (photons/second/cm²/sr) of the pancreatic region of each mouse from vehicle-treated and BMH-21–treated groups was recorded and plotted over time in separate graphs for the AsPC-1 luciferase xenograft model. I, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the MIA PaCa-2 luciferase xenograft mouse model. J, The bar graphs indicate the weight of excised pancreatic tumors from the vehicle-treated and BMH-21–treated groups in the AsPC-1 luciferase xenograft mouse model. K, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated groups in MIA PaCa-2 luciferase xenograft mice. L, The bar graphs indicate tumor volume from vehicle-treated and BMH-21–treated AsPC-1 luciferase xenograft mice. Tumor volume was calculated using the ellipsoid formula [0.5 × (W² × L)], where W and L represent the shortest and longest tumor diameters, respectively. M, Representative IHC staining images showing the expression of RPA194, Ki-67, PCNA, and mutant p53 in excised MIA PaCa-2 luciferase cell–derived xenograft tumors of vehicle-treated and BMH-21–treated mice. Magnification: 20×; scale bar, 50 μm. N, The bar graphs represent the quantification of IHC staining of RPA194, PCNA, Ki-67, and mutant p53 in pancreatic tumors excised from mice orthotopically xenografted with MIA PaCa-2 luciferase cells represented in M. The quantification of IHC staining was done using ImageJ software. Values in bar graphs represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P = 0.0003; ****, P ≤ 0.0001.

Figure 7.

Schematic representation shows the proposed molecular mechanisms of BMH-21–induced ubiquitination of mutant p53 via RPA194. We proposed that the interaction of RPA194 with mutant p53 masks the ubiquitination sites of mutant p53 and BMH-21–mediated degradation of RPA194 unmasks these ubiquitination sites on mutant p53, making mutant p53 susceptible to ubiquitination (Ub) and degradation.