Downregulation of BCL10 attenuates the proliferation of PDAC cells
To investigate the functions of BCL10 in PDAC cells, we first validated the localization of BCL10. The three PDAC cell lines were infected with BCL10 shRNA lentivirus, and then, assessed by western blot analysis. The data showed efficient knockdown of BCL10 after transfection with BCL10 shRNA (Fig. 1a). The immunofluorescence image from confocal microscopy showed that BCL10 was localized in nucleus and cytoplasm of all three PDAC cell lines (PANC-1, AsPC-1, and BxPC-3) (Fig. 1b). After transfection with BCL10 shRNA (shBCL10), the fluorescence intensity of BCL10 in cytoplasm and nucleus was markedly reduced in BXPC-3 cells, and BCL10 expression was mainly restricted to cytoplasm in PANC-1 and AsPC-1 cells (Fig. 1c).
We further examined whether the growth of PDAC cells was inhibited after BCL10 knockdown. As shown in Fig. 1c, transfection with shBCL10 significantly reduced cell proliferation in three PDAC cell lines. These results suggested that nuclear translocation of BCL10 may be a key event in the promotion of proliferation of these PDAC cells.
Downregulation of BCL10 causes cell cycle arrest and enhances the cytotoxicities of chemotherapeutic agents in PDAC cells
To investigate whether BCL10-mediated suppression of proliferation of pancreatic cancer cells is via the cell cycle arrest, we assessed the effects of shBCL10 transfection on cell cycle regulation in three PDAC cell lines. As shown in Fig. 2a, in K-RAS mutant PDAC cells, PANC-1 and AsPC-1, there was a significant increase in the number of cells in the G2/M phase and significant decrease in the number of cells in the G1 phase. In the K-RAS wild-type PDAC cell line, BxPC-3, we observed that there was a significant increase in the number of cells in the G1 phase and significant decrease in the number of cells in the G2/M phase (Fig. 2a). These findings indicated that knockdown of BCL10 differentially inhibited cell cycle progression in PDAC cell lines; G1 phase arrest was noted in wild-type K-RAS cell lines, whereas G2/M phase arrest was noted in mutant K-RAS cell lines.
We further evaluated whether PDAC cells are sensitive to conventional chemotherapy after silencing of BCL10 using shBCL10. The PDAC cell lines were treated with various doses of gemcitabine and oxaliplatin for 72 h, respectively. The cytotoxicity assay showed that BCL10-silenced cells exhibited significantly lower cell survival rate compared with scrambled cells after treatment with the same drug doses of gemcitabine (Fig. 2b) and oxaliplatin (Fig. 2c) in the three PDAC cell lines.
Downregulation of BCL10 significantly decreases the expression of cell cycle-regulatory and NF-κB-related proteins, and inhibits NF-κB activation
It has previously been demonstrated that cyclins D1 and D3 are involved in the G1 phase, while cyclin E participates in the transition to the G1/S phase [25]. In contrast to G1/S phase, Cdc2 and cyclin B1 are involved in the G2/M phase [26]. We then assessed whether cyclins D1, D3, and E, and Cdc2 in PANC-1 (mutant K-RAS) cells were affected by inhibition of BCL10; we found that the expression of Cdc2 was markedly reduced after shBCL10 transfection, while the expressions of cyclins D1, D3, and E were not affected (Fig. 3a). In contrast to PANC-1, we found that in BxPC-3 (wild type K-RAS) cells, shBCL10 downregulated cyclin D1 expression but did not affect the expression of cyclin D3, cyclin E, and Cdc2 (Fig. 3a). These results indicated that BCL10 inhibition can result in differential cell cycle arrest in the two different pancreatic cancer cell lines: mutant K-RAS cell lines (G2/M arrest) and wild-type K-RAS cell lines (G1 arrest).
As shown in Fig. 3b, shBCL10 transfection inhibited the expression of nuclear BCL3 and p-IκBα, both of which are recognized as important regulators of NF-κB in both PANC-1 and BxPC-3 cells. In addition, shBCL10 transfection downregulated nuclear expression of NF-κB (p65) and c-Myc in both PANC-1 and BxPC-3 cells. As indicated by the results from the NF-κB-Luc promoter activity assay, the transcription of NF-κB-dependent genes, led by the nuclear translocation and DNA binding of NF-κB, was downregulated in both PANC-1 and BxPC-3 cells after transfection with shBCL10 (Fig. 3c).
To confirm the role of BCL10-mediated activation of NF-κB in cell cycle regulation, we treated PANC-1 and BxPC-3 cells with an NF-κB inhibitor, BAY117082 (2 µM), for 24 h; cell cycle distribution analysis, as shown in Fig. 3d, revealed that the inhibition of NF-κB did cause cell cycle arrest at G2/M in PANC-1 cells and at G1 arrest in BxPC-3 cells. As shown in Fig. 3e, we showed that expression of cyclin B1 (PANC-1), cyclin D1 (BxPC-3), and nuclear BCL10, BCL3, c-Myc, and p-p65 (both BxPC-3 and PANC-1) was downregulated after BAY117082 treatment. We concluded that BCL10, at least in part, regulates the growth of PDAC cells via NF-κB-dependent signaling.
Inhibition of BCL10 expression reduces tumor growth of PDAC in a xenograft model
To assess the anti-tumor effect of BCL10 knockdown in PDAC xenograft model, PANC-1 cells treated with or without shBCL10 transfection were inoculated into the flanks of mice. Tumor volume was recorded from appearance of initial tumor burden (Fig. 4a and 4b). When compared with scrambled group of PANC-1 xenograft tumors, we observed that the tumor growth was significantly inhibited after transfection with shBCL10 (Fig. 4a). The shBCL10-transfected group exhibited 78% reduction of tumor volume compared with scrambled group at 5 weeks (Fig. 4b). There were no significant differences in the body weight between scrambled PANC-1 group and shBCL10-transfected PANC-1 group (Fig. 4c). The nuclear expression levels of BCL10, BCL3, and NF-κB (p65) in tumor cells were also downregulated in shBCL10-transfected PANC-1 group when compared with scrambled PANC-1 group (Fig. 4d).
Nuclear expression of BCL10 in clinical sample is closely associated with the poor overall survival of patients with recurrent, advanced, and metastatic PDAC
The patients’ clinical characteristics are summarized in Table 1. The median age for all the patients was 59 years old (range: 27–82) and all patients had histologically confirmed PDAC. Approximately one third of the patients (49 of 136 cases, 36%) had a history of smoking tobacco. Of 136 patients, 80 patients (58.8%) were initially diagnosed with stage IV pancreatic cancer, and the most common metastatic sites were liver, whereas 19 patients had locally advanced pancreatic cancer. Of 136 patients, 37 patients had local recurrence (n = 15) and/or distant metastasis (n = 22) after undergoing surgery with curative intent. After a median follow-up of 24.8 months (95% confidence interval [CI]: 13.2–36.4 months), the median OS after commencing chemotherapy for locally advanced and metastatic PDAC or developing recurrence and/or distant metastasis of the entire group was 7.54 months (95% CI: 6.11–8.98 months).
Table 1
Clinicopathologic features between nuclear BCL10-negative and nuclear BCL10-positive groups of patients with PDAC
| | Nuclear BCL10 expression | |
| Total (N) | Negative | Positive | p-value |
Number | 136 | 78 (57.4%) | 58 (42.6%) | |
Age | | | | 0.837† |
Median | 59.0 | 58.0 | 61.0 | |
Range | 27–82 | 35–82 | 27–76 | |
Sex | | | | 0.102‡ |
Men | 83 (61.0%) | 43 (55.1%) | 40 (69.0%) | |
Women | 53 (39.0%) | 35 (44.9%) | 18 (31.0%) | |
Smoking | | | | 0.262‡ |
No | 87 (64.0%) | 53 (67.9%) | 34 (58.6%) | |
Yes | 49 (36/0%) | 25 (32.1%) | 24 (41.4%) | |
Alcohol | | | | 0.937‡ |
No | 98 (72.1%) | 56 (71.8%) | 42 (72.4%) | |
Yes | 38 (27.9%) | 22 (28.2%) | 16 (27.6%) | |
Stage | | | | 0.743§ |
Recurrent | 37 (27.2%) | 23 (29.5%) | 14 (24.1%) | |
Stage IIIB | 19 (14.0%) | 9 (11.5%) | 10 (17.2%) | |
Stage IV | 80 (58.8%) | 46 (59.0%) | 34 (58.6%) | |
Metastases | | | | 0.667§ |
Peritoneal | 12 (8.8%) | 7 (9.0%) | 5 (9.6%) | |
Liver | 83 (61.0%) | 47 (60.2%) | 36 (62.1%) | |
Other sites* | 23 (16.9%) | 12 (15.4%) | 11 (19.0%) | |
Regional LNs | 18 (13.2%) | 12 (15.4%) | 6 (10.3%) | |
Chemotherapy | | | | 0.306‡ |
Gem-alone | 44 (32.4%) | 28 (35.9%) | 16 (27.6%) | |
Gem-based | 92 (67.6%) | 50 (61.1%) | 42 (72.4%) | |
Response | | | | 0.423§ |
CR + PR | 11 (8.1%) | 6 (7.7%) | 5 (8.6%) | |
SD + PD | 85 (62.5%) | 51 (65.4%) | 34 (58.6%) | |
NA | 40 (29.4%) | 21 (26.9%) | 19 (32.8%) | |
NF-κB expression** | | | | < 0.001‡ |
Negative | 58 (61.1%) | 49 (84.5%) | 9 (24.3%) | |
Positive | 37 (38.9%) | 9 (15.5%) | 28 (75.7%) | |
The nuclear expression of BCL10 was detected in tumor cells of 58 patients (42.6%) (Fig. 5a, b, c, and d). The difference in the demography, including age, sex, smoking history, and alcoholism were not significant between patients with nuclear BCL10 expression in tumors and those without nuclear BCL10 expression in tumors (Table 1). Of 136 patients, 96 patients (70.6%) with available response to gemcitabine-based chemotherapy; however, patients without nuclear BCL10 expression did not exhibit better response (complete and partial remission) to gemcitabine-based chemotherapy than those with nuclear BCL10 expression (5/39 [12.8%] versus 6/57 [10.5%], p = 0.753). Patients with nuclear BCL10 expression had significantly worse prognoses than those without nuclear BCL10 expression (median OS after starting chemotherapy: 6.90 months [95% CI: 5.77–8.03] versus 9.53 months [95% CI: 5.89–13.18], p = 0.019). The 1-year OS rates for patients with nuclear BCL10 expression and for patients without nuclear BCL10 expression were 23.6% (95% CI: 12.4–34.8%) and 43.7% (95% CI: 32.1–55.3%), respectively (Fig. 5d).
Among 95 patients with available tissues to assess the expression of NF-κB (p65), we found that the nuclear BCL10 expression was significantly associated with nuclear NF-κB expression (p < 0.001, Table 1). Similarly, patients with nuclear NF-κB expression (n = 37) had a shorter OS than those without nuclear NF-κB expression (n = 58) (median survival: 6.50 months [95% CI: 3.22–9.78] versus 9.90 months [95% CI: 3.96–15.84], p = 0.025). The 1-year OS rates for patients with nuclear NF-κB expression and for patients without nuclear NF-κB expression were 31.9% (95% CI: 16.2–47.6%) and 46.5% (95% CI: 32.6–53.6%), respectively.