Kaiso depletion attenuates the growth and survival of triple negative breast cancer cells

Abstract

Triple negative breast cancers (TNBC) are highly aggressive and lack specific targeted therapies. Recent studies have reported high expression of the transcription factor Kaiso in triple negative tumors, and this correlates with their increased aggressiveness. However, little is known about the clinical relevance of Kaiso in the growth and survival of TNBCs. Herein, we report that Kaiso depletion attenuates TNBC cell proliferation, and delays tumor onset in mice xenografted with the aggressive MDA-231 breast tumor cells. We further demonstrate that Kaiso depletion attenuates the survival of TNBC cells and increases their propensity for apoptotic-mediated cell death. Notably, Kaiso depletion downregulates BRCA1 expression in TNBC cells expressing mutant-p53 and we found that high Kaiso and BRCA1 expression correlates with a poor overall survival in breast cancer patients. Collectively, our findings reveal a role for Kaiso in the proliferation and survival of TNBC cells, and suggest a relevant role for Kaiso in the prognosis and treatment of TNBCs.

Figure 1

Kaiso depletion attenuates the proliferation of TNBC cells. Growth curve (a), MTT assays (b) and colony formation assays (c) were conducted on Ctrl and Kaiso-depleted (sh-K1 and sh-K2, hereafter referred to as sh-K) cells, and reveal that Kaiso depletion inhibits proliferation of MDA-231 and Hs578T cells. (d) Soft agar assays were also conducted on Ctrl, sh-K1 and sh-K2 MDA-231 and Hs578T cells and revealed that Kaiso-depletion diminished anchorage-independent growth of MDA-231 but not Hs578T cells. (e) Kaiso depletion resulted in decreased c-Myc and cyclin D1 expression in MDA-231 and Hs578T cells as detected by IB analysis and densitometry analysis. The reduction in c-Myc levels in response to Kaiso depletion was more significant in MDA-231 compared to Hs578T cells. GraphPad Prism software was used to generate graphs and for all statistical calculations. Data shown are representative of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001

Figure 2

Kaiso-depleted MDA-231 cells exhibit delayed tumor onset in mouse xenografts. (a) Kaiso-depleted MDA-231 xenografts (sh-K) are delayed ~3 weeks in tumor onset and development compared to control (Ctrl) MDA-231 xenografted tumors as seen by time-course analysis of the tumor volume of Ctrl and sh-K MDA-231 xenografted cells. (b) IHC-stained images of MDA-231 xenograft tissues with Ki-67 and PCNA antibodies show a marked decrease in proliferating cells in MDA-231 Kaiso-depleted tumor tissues as indicated by the reduced expression of the proliferation markers Ki-67 and PCNA. (c and d) IHC-stained images of MDA-231 xenograft tissues with c-Myc and Cyclin D1 antibodies show that Kaiso-depletion results in reduced numbers of c-Myc and cyclin-D1 stained cells and reduced staining intensity. Representative images shown from 3 or more independent experiments

Figure 3

Kaiso depletion results in increased apoptosis of TNBC cells. (a) IHC-stained images of control (Ctrl) and Kaiso-depleted (sh-K) MDA-231 tumor tissues with cleaved (c)-Caspase 3 antibody show that Kaiso-depletion results in increased c-Caspase 3 expression in MDA-231 tumor tissues. (b) Caspase 3 assay conducted on Ctrl, sh-K1 and sh-K2 MDA-231 and Hs578T cells show that Kaiso depletion resulted in increased Caspase 3 activity in MDA-231 and Hs578T sh-K1 and sh-K2 cells compared to their control counterparts. (c) Kaiso-depleted TNBC cells (sh-K MDA-231 & Hs578T) expressing mut-p53 exhibit increased apoptosis as revealed by Annexin V-FITC staining. (d) Expression of a sh-resistant Kaiso cDNA in Kaiso-depleted MDA-231 and Hs578T cells mitigates the apoptosis induced by Kaiso depletion as assessed by Annexin V-FITC staining. Data shown are representative of three independent experiments. *P<0.05, **P<0.01, ****P<0.0001

Figure 4

Kaiso depletion increases expression of pro-apoptotic proteins in TNBC cells lacking wt p53. (a) Kaiso co-precipitates with mutant p53 in MDA-231 and Hs578T cell lysates. Parental MDA-231 and Hs578T cells were subjected to immunoprecipitation with anti-Kaiso, anti-p53 and anti-IgG antibodies, and immunoblotted with the indicated antibodies. Kaiso-depleted MDA-231 and Hs578T cells express increased amounts of Bax and PUMA protein compared to control cells (b), that is decreased upon expression of a sh-resistant Kaiso cDNA in the MDA-231 and Hs578T sh-K cells (c). Graphical representation of the quantitated protein values is shown. (d) Kaiso-depleted MDA-231 and Hs578T cells (that express mut-p53) exhibit a statistically significant increase in PUMA mRNA levels as measured by qRT-PCR. Although Bax mRNA levels were also slightly increased, it was not significant. (e) ChIP-PCR analysis of MDA-231 and Hs578T chromatin revealed that Kaiso and mut-p53 associate endogenously with the PUMA promoter in TNBC cells. (f) Transcript profiles of patients from the GEO (GSE20685, GSE21653, GSE16446, GSE19615 and GSE9195) BCa data sets were pooled and segregated into Kaiso high/PUMA low, and Kaiso low/PUMA high groups. Kaplan–Meier survival curves revealed a correlation between high Kaiso and low PUMA expression with poor overall survival in all BCa cases. However, it was not statistically significant. Data representative of three independent experiments. **P<0.01 and NS, not significant. For Kaplan–Meier survival curves, log-rank test was performed to determine statistical significance. P<0.05 is considered statistically significant

Figure 5

Kaiso depletion enhances the sensitivity of TNBC cells to Cisplatin. (a) Kaiso-depletion sensitizes TNBC cells to Cisplatin treatment, as demonstrated by the increased cleaved-PARP expression observed in Kaiso-depleted MDA-231, Hs578T and MDA-157 cells treated with Cisplatin (Cis) for 48 h compared to similarly treated control cells. UT, untreated cells. (b) Graphical representation of the fold change in protein expression is shown. All experiments were conducted independently at least three times. Representative images shown. *P<0.05

Figure 6

Kaiso depletion attenuates BRCA1 expression in sporadic TNBC cells. (a) BRCA1 mRNA expression was significantly reduced in Kaiso-depleted MDA-231 and Hs578T cells compared to controls as measured by qRT-PCR. (b) WB analysis with a BRCA1-specific antibody shows decreased BRCA1 protein expression in Kaiso-depleted MDA-231 and Hs578T cells, which is partially rescued by expression of a sh-resistant Kaiso cDNA in the MDA-231 and Hs578T sh-K cells (c). (d) Schematic illustration of the minimal BRCA1 promoter region showing the location of a core KBS (cKBS) that was amplified by ChIP-PCR. Kaiso was enriched at the BRCA1 promoter region indicated in MDA-231 and Hs578T cells. Data representative of three independent experiments. *P<0.05, **P<0.01

Figure 7

High Kaiso and BRCA1 expression correlates with poor prognosis in BCa patients. Transcript profiles of patients from the TCGA and GEO (GSE20685, GSE21653, GSE16446, GSE19615 and GSE9195) BCa data sets were pooled and segregated into Kaiso/BRCA1 high or Kaiso/BRCA1 low, and BRCA1 high or BRCA1 low groups. Kaplan–Meier survival curves revealed that high Kaiso and BRCA1 expression significantly correlates with poor overall survival in TNBC patients specifically (a) or all BCa cases (b) whereas increased BRCA1 expression did not correlate with poor overall survival in BCa patients (c). Log-rank test was performed to determine statistical significance. P<0.05 is considered statistically significant

Figure 8

Schematic diagram of proposed model for Kaiso's role in TNBC. (a) Kaiso interacts with both wt p53 and mutant p53 in BCa cells and this differential interaction may modulate Kaiso's function in apoptosis. (b) In TNBC cells lacking wt p53 (but expressing mutant p53), Kaiso might directly or indirectly inhibit the activation of the pro-apoptotic genes Bax and PUMA, which leads to tumor survival. However, Kaiso's inhibitory effect on Bax protein expression may be attenuated by Kaiso interaction with other proteins like p120^ctn. Kaiso may also activate c-Myc, Cyclin D1 and BRCA1 expression independently or in collaboration with other cofactors in TNBC cells, which would also promote TNBC cell growth and survival