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Kaiso depletion attenuates transforming growth factor-β signaling and metastatic activity of triple-negative breast cancer cells.

Bassey-Archibong BI, Kwiecien JM, Milosavljevic SB, Hallett RM, Rayner LG, Erb MJ, Crawford-Brown CJ, Stephenson KB, Bédard PA, Hassell JA, Daniel JM - Oncogenesis (2016)

Bottom Line: Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT.Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses.Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, Ontario, Canada.

ABSTRACT
Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-β (TGFβ) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs.

No MeSH data available.


Related in: MedlinePlus

Kaiso associates with the endogenous TGFβR1 and TGFβR2 promoter in breast cancer cell lines. (a) Schematic illustration of the TGFβR2 promoter highlighting multiple KBS. Four double-stranded oligonucleotides were designed to contain core KBS from different regions of the promoter and utilized in electrophoretic mobility shift assay (EMSA) to determine Kaiso binding. (b) EMSA shows that Kaiso binds the proximal TGFβR2 promoter in a KBS-dependent manner (lanes 6, 10 and 14). This interaction was abolished (lanes 8, 12 and 16) upon introduction of a point mutation in the core KBS sequence of these probes or competition with the cold unlabeled wild-type probe. (c) ChIP of MDA-231 and Hs578T chromatin revealed that Kaiso binds the TGFβR2 promoter endogenously. (d) ChIP experiments of MDA-231 and Hs578T chromatin shows that Kaiso also interacts with the TGFβR1 promoter endogenously even after 5′-aza-cytidine treatment. Representative images are shown. All experiments were conducted in triplicate. H3, Histone 3 positive control; Input, 10% input. MUT, mutated; NTC, no template control; WT, wild type.
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fig5: Kaiso associates with the endogenous TGFβR1 and TGFβR2 promoter in breast cancer cell lines. (a) Schematic illustration of the TGFβR2 promoter highlighting multiple KBS. Four double-stranded oligonucleotides were designed to contain core KBS from different regions of the promoter and utilized in electrophoretic mobility shift assay (EMSA) to determine Kaiso binding. (b) EMSA shows that Kaiso binds the proximal TGFβR2 promoter in a KBS-dependent manner (lanes 6, 10 and 14). This interaction was abolished (lanes 8, 12 and 16) upon introduction of a point mutation in the core KBS sequence of these probes or competition with the cold unlabeled wild-type probe. (c) ChIP of MDA-231 and Hs578T chromatin revealed that Kaiso binds the TGFβR2 promoter endogenously. (d) ChIP experiments of MDA-231 and Hs578T chromatin shows that Kaiso also interacts with the TGFβR1 promoter endogenously even after 5′-aza-cytidine treatment. Representative images are shown. All experiments were conducted in triplicate. H3, Histone 3 positive control; Input, 10% input. MUT, mutated; NTC, no template control; WT, wild type.

Mentions: As Kaiso depletion attenuated TGFβR1 and TGFβR2 expression, we next assessed whether Kaiso promotes TGFβ signaling through regulation of TGFβR1 and TGFβR2. We performed electrophoretic mobility shift assay analyses using purified GST-Kaiso-ΔPOZ fusion proteins as previously described,42, 43 and oligonucleotides derived from the TGFβR1 (KBS 1–4) and TGFβR2 (KBS1–4) promoters that each contains several KBS and/or CpGs (Tables 1 and 2). Kaiso bound the core KBS in proximal TGFβR2 oligonucleotides (TβR2-KBS-2, 3, 4) but not the distal TβR2-KBS1 probe (Figures 5a and b). However, Kaiso binding was abolished upon introduction of a point mutation in the core KBS in these probes (Figure 5b; Supplementary Figure 4). Surprisingly, despite the strong correlation between Kaiso and TGFβR1 expression in the TCGA BCa dataset, no binding was observed between Kaiso and any of the TβR1-KBS-1–4 probes even after methylation of the CpG sequences found in the TGFβR1 (KBS2-4) probes (data not shown).


Kaiso depletion attenuates transforming growth factor-β signaling and metastatic activity of triple-negative breast cancer cells.

Bassey-Archibong BI, Kwiecien JM, Milosavljevic SB, Hallett RM, Rayner LG, Erb MJ, Crawford-Brown CJ, Stephenson KB, Bédard PA, Hassell JA, Daniel JM - Oncogenesis (2016)

Kaiso associates with the endogenous TGFβR1 and TGFβR2 promoter in breast cancer cell lines. (a) Schematic illustration of the TGFβR2 promoter highlighting multiple KBS. Four double-stranded oligonucleotides were designed to contain core KBS from different regions of the promoter and utilized in electrophoretic mobility shift assay (EMSA) to determine Kaiso binding. (b) EMSA shows that Kaiso binds the proximal TGFβR2 promoter in a KBS-dependent manner (lanes 6, 10 and 14). This interaction was abolished (lanes 8, 12 and 16) upon introduction of a point mutation in the core KBS sequence of these probes or competition with the cold unlabeled wild-type probe. (c) ChIP of MDA-231 and Hs578T chromatin revealed that Kaiso binds the TGFβR2 promoter endogenously. (d) ChIP experiments of MDA-231 and Hs578T chromatin shows that Kaiso also interacts with the TGFβR1 promoter endogenously even after 5′-aza-cytidine treatment. Representative images are shown. All experiments were conducted in triplicate. H3, Histone 3 positive control; Input, 10% input. MUT, mutated; NTC, no template control; WT, wild type.
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fig5: Kaiso associates with the endogenous TGFβR1 and TGFβR2 promoter in breast cancer cell lines. (a) Schematic illustration of the TGFβR2 promoter highlighting multiple KBS. Four double-stranded oligonucleotides were designed to contain core KBS from different regions of the promoter and utilized in electrophoretic mobility shift assay (EMSA) to determine Kaiso binding. (b) EMSA shows that Kaiso binds the proximal TGFβR2 promoter in a KBS-dependent manner (lanes 6, 10 and 14). This interaction was abolished (lanes 8, 12 and 16) upon introduction of a point mutation in the core KBS sequence of these probes or competition with the cold unlabeled wild-type probe. (c) ChIP of MDA-231 and Hs578T chromatin revealed that Kaiso binds the TGFβR2 promoter endogenously. (d) ChIP experiments of MDA-231 and Hs578T chromatin shows that Kaiso also interacts with the TGFβR1 promoter endogenously even after 5′-aza-cytidine treatment. Representative images are shown. All experiments were conducted in triplicate. H3, Histone 3 positive control; Input, 10% input. MUT, mutated; NTC, no template control; WT, wild type.
Mentions: As Kaiso depletion attenuated TGFβR1 and TGFβR2 expression, we next assessed whether Kaiso promotes TGFβ signaling through regulation of TGFβR1 and TGFβR2. We performed electrophoretic mobility shift assay analyses using purified GST-Kaiso-ΔPOZ fusion proteins as previously described,42, 43 and oligonucleotides derived from the TGFβR1 (KBS 1–4) and TGFβR2 (KBS1–4) promoters that each contains several KBS and/or CpGs (Tables 1 and 2). Kaiso bound the core KBS in proximal TGFβR2 oligonucleotides (TβR2-KBS-2, 3, 4) but not the distal TβR2-KBS1 probe (Figures 5a and b). However, Kaiso binding was abolished upon introduction of a point mutation in the core KBS in these probes (Figure 5b; Supplementary Figure 4). Surprisingly, despite the strong correlation between Kaiso and TGFβR1 expression in the TCGA BCa dataset, no binding was observed between Kaiso and any of the TβR1-KBS-1–4 probes even after methylation of the CpG sequences found in the TGFβR1 (KBS2-4) probes (data not shown).

Bottom Line: Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT.Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses.Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, Hamilton, Ontario, Canada.

ABSTRACT
Triple-negative breast cancers (TNBCs) represent a subset of breast tumors that are highly aggressive and metastatic, and are responsible for a disproportionate number of breast cancer-related deaths. Several studies have postulated a role for the epithelial-to-mesenchymal transition (EMT) program in the increased aggressiveness and metastatic propensity of TNBCs. Although EMT is essential for early vertebrate development and wound healing, it is frequently co-opted by cancer cells during tumorigenesis. One prominent signaling pathway involved in EMT is the transforming growth factor-β (TGFβ) pathway. In this study, we report that the novel POZ-ZF transcription factor Kaiso is highly expressed in TNBCs and correlates with a shorter metastasis-free survival. Notably, Kaiso expression is induced by the TGFβ pathway and silencing Kaiso expression in the highly invasive breast cancer cell lines, MDA-MB-231 (hereafter MDA-231) and Hs578T, attenuated the expression of several EMT-associated proteins (Vimentin, Slug and ZEB1), abrogated TGFβ signaling and TGFβ-dependent EMT. Moreover, Kaiso depletion attenuated the metastasis of TNBC cells (MDA-231 and Hs578T) in a mouse model. Although high Kaiso and high TGFβR1 expression is associated with poor overall survival in breast cancer patients, overexpression of a kinase-active TGFβR1 in the Kaiso-depleted cells was insufficient to restore the metastatic potential of these cells, suggesting that Kaiso is a key downstream component of TGFβ-mediated pro-metastatic responses. Collectively, these findings suggest a critical role for Kaiso in TGFβ signaling and the metastasis of TNBCs.

No MeSH data available.


Related in: MedlinePlus