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. 2024 Jan 10;12(1):143.
doi: 10.3390/biomedicines12010143.

Suppression of Bcl3 Disrupts Viability of Breast Cancer Cells through Both p53-Dependent and p53-Independent Mechanisms via Loss of NF-κB Signalling

Daniel J Turnham  1 , Hannah Smith  1 , Richard W E Clarkson  1
Affiliations

Suppression of Bcl3 Disrupts Viability of Breast Cancer Cells through Both p53-Dependent and p53-Independent Mechanisms via Loss of NF-κB Signalling

Daniel J Turnham et al. Biomedicines. .

Abstract

The NF-κB co-factor Bcl3 is a proto-oncogene that promotes breast cancer proliferation, metastasis and therapeutic resistance, yet its role in breast cancer cell survival is unclear. Here, we sought to determine the effect of Bcl3 suppression alone on breast cancer cell viability, with a view to informing future studies that aim to target Bcl3 therapeutically. Bcl3 was suppressed by siRNA in breast cancer cell lines before changes in viability, proliferation, apoptosis and senescence were examined. Bcl3 suppression significantly reduced viability and was shown to induce apoptosis in all cell lines tested, while an additional p53-dependent senescence and senescence-associated secretory phenotype was also observed in those cells with functional p53. The role of the Bcl3/NF-κB axis in this senescence response was confirmed via siRNA of the non-canonical NF-κB subunit NFKB2/p52, which resulted in increased cellular senescence and the canonical subunit NFKB1/p50, which induced the senescence-associated secretory phenotype. An analysis of clinical data showed a correlation between reduced relapse-free survival in patients that expressed high levels of Bcl3 and carried a p53 mutation. Together, these data demonstrate a dual role for Bcl3/NF-κB in the maintenance of breast cancer cell viability and suggests that targeting Bcl3 may be more beneficial to patients with tumours that lack functional p53.

Keywords: Bcl3; NF-κB; SASP; apoptosis; breast cancer; p53; senescence.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Bcl3 suppression reduces cell confluency over time. (A) Relative Bcl3 expression 6 days post-Bcl3 siRNA transfection in 4 breast cancer cell lines was assessed using qRT-PCR. (B) Cell titre blue was used to assess cell viability following 2 and 6 days of siRNA treatment, displayed as relative viability loss compared to that of scRNA controls. (C–F) IncuCyte analysis software (version 2022B) was used to analyse differences in cell confluency in (C) MCF-7, (D) ZR751, (E) T47D and (F) BT474 cells following Bcl3 suppression with data normalised to the confluency at the time of siRNA transfection. Error bars represent ± SEM of n = 3 minimum, with statistical differences determined using a 2-tailed t test; * p < 0.05. ** p < 0.01, and *** p < 0.001.
Figure 2
Figure 2
Bcl3 loss induces p53-independent apoptosis and p53-dependent senescence in breast cancer cells. Bcl3 was suppressed in breast cancer cell lines with the addition of Annexin V reagent to determine apoptosis using IncuCyte analysis software over 6 days. (A) MCF-7, (B) ZR751, (C) T47D and (D) BT474 cells each showing a significant increase in Annexin V staining; (E) representative images of each cell line at the endpoint. Following 6 days of siRNA, cells were harvested or fixed for further analysis. (F) PUMA expression determined by qRT-PCR showing an increase in three of the cell lines following Bcl3 suppression. Immunofluorescence staining for cell cycle markers (G) ki67 and (H) pH3 showed a reduction in the percentage of positively stained MCF-7 and ZR751 cells. (I) Senescence-associated β-gal staining was increased in Bcl3 suppressed MCF-7 and ZR751 cells, (J) representative images at endpoint, scale bar = 100 µm. qRT-PCR analysis showed a significant increase in (K) p21 expression in MCF-7 and ZR751 cells, while (L) p15 expression was upregulated in each cell line following Bcl3 suppression. SASP genes (M) IL-6, (N) IL-8 and (O) CXCL10, were also upregulated in p53 wildtype MCF-7 and ZR751 cells but not in p53-mutant T47D or BT474 cells. Error bars represent ± SEM of n = 3 minimum, with statistical differences determined using a 2-tailed unpaired t test, * p < 0.05. ** p < 0.01. Expanded versions of (E,J) are available in Supplementary Figures S1 and S3, respectively.
Figure 3
Figure 3
Loss of p53 rescues senescence in Bcl3-suppressed cells. Bcl3 and p53 were suppressed either alone or together in MCF-7 and ZR751 cells for 6 days. The suppression of Bcl3 and p53 was confirmed via qRT-PCR in (A) MCF-7 and (E) ZR751 cells. (B,F) Senescence-associated β-gal staining was increased following Bcl3 suppression, with no change observed when p53 was suppressed alone or with Bcl3. The percentage of cells positively stained for the cell cycle markers (C,G) ki67 and (D,H) pH3 was reduced in MCF-7 and ZR751 cells following Bcl3 inhibition, with no change following p53 inhibition alone or with Bcl3. (I,J) qRT-PCR analysis of p15 and p21 expression showed that the significant increase in p21 expression following Bcl3 inhibition was mitigated when p53 was also suppressed in both cell lines. (K) The expression of SASP genes IL-6, IL8 and CXCL10 was not rescued in MCF-7 cells following the combined suppression of p53 and Bcl3. Cell titre blue analysis demonstrated no further loss in cell viability in (L) MCF-7 or (M) ZR751 cells following combined Bcl3 and p53 suppression Error bars represent ± SEM of n= 3 with statistical differences determined using a one-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 4
Figure 4
Loss of p52 and p50 induces senescence and SASP, respectively. NF-κB family members p50 and p52 were suppressed by siRNA for 6 days in (A) MCF-7 and (B) ZR751 cells and confirmed via qRT-PCR. (C,D) Senescence-associated β-gal staining was significantly increased following p52 suppression compared to that of scRNA controls in both cell lines in a similar response to that seen following Bcl3 knockdown. Analysis via qRT-PCR identified a significant increase in (E) p15 and (F) p21 expression in MCF-7 cells following p52 inhibition, with a similar trend in p15 expression also observed in ZR751 cells. The percentage of ki67- (G,H) and pH3- (I,J) positive cells was reduced in both cell lines treated with p52 siRNA in a similar manner to that of Bcl3 suppression, with no change observed following the suppression of p50. The expression of SASP genes (K) IL-6, (L) IL-8 and (M) CXCL10 was increased following p50 suppression and not following p52 knockdown. (N) Graphical illustration of how Bcl3 suppression in p53 wildtype breast cancer cells results in the induction of senescence and SASP through inhibiting p52 and p50 signalling, respectively. Error bars represent ± SEM of n = 3 with statistical differences determined using a one-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.01.
Figure 5
Figure 5
High Bcl3 expression is associated with reduced relapse-free survival in breast cancer patients with p53 mutations. The effect of high or low Bcl3 expression on RFS was assessed in clinical cohorts of breast cancer patients split based on the presence or absence of p53 mutations. (A) Graph showing that patients who received chemotherapy and maintained wildtype p53 had no difference in RFS; however, those with (B) p53 mutations and high levels of Bcl3 had significantly reduced RFS. A similar trend was observed in (C) p53 wildtype and (D) p53 mutant breast cancer patients who had not previously received any form of systemic treatment and in those that had received endocrine treatment (E,F). Analysis was performed on KM-plotter, which automatically calculates statistical significance using a log-rank test.
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