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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-depletion attenuates TGFβ signaling and transcriptional responses. Cells were treated with 10 ng/ml of TGFβ for 1 h before assaying for TGFβ activity. (a) TGFβ treatment of control MDA-231 and Hs578T cells results in increased p-Smad2 levels. However, Kaiso-depleted MDA-231 and Hs578T cells treated with TGFβ display reduced p-Smad2 levels. (b) Kaiso-depleted MDA-231 and Hs578T xenografts exhibit decreased TGFβ signaling as evidenced by reduced p-Smad2 protein levels. (c) TGFβ-induced expression of ANGPTL4 is attenuated in Kaiso-depleted cells treated with 10 ng/ml of TGFβ for 24 h. Interestingly, Kaiso expression is significantly increased by TGFβ treatment in MDA-231 cells. (d) Immunoblot analysis revealed a peak in Kaiso protein levels at 12 h in both MDA-231 and Hs578T cells in response to TGFβ treatment. All experiments were performed in triplicate. Representative images from all experiments are shown. *P<0.05, **P<0.005, ***P<0.0001, NS, not significant. β-Actin serves as a loading control.
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fig4: Kaiso-depletion attenuates TGFβ signaling and transcriptional responses. Cells were treated with 10 ng/ml of TGFβ for 1 h before assaying for TGFβ activity. (a) TGFβ treatment of control MDA-231 and Hs578T cells results in increased p-Smad2 levels. However, Kaiso-depleted MDA-231 and Hs578T cells treated with TGFβ display reduced p-Smad2 levels. (b) Kaiso-depleted MDA-231 and Hs578T xenografts exhibit decreased TGFβ signaling as evidenced by reduced p-Smad2 protein levels. (c) TGFβ-induced expression of ANGPTL4 is attenuated in Kaiso-depleted cells treated with 10 ng/ml of TGFβ for 24 h. Interestingly, Kaiso expression is significantly increased by TGFβ treatment in MDA-231 cells. (d) Immunoblot analysis revealed a peak in Kaiso protein levels at 12 h in both MDA-231 and Hs578T cells in response to TGFβ treatment. All experiments were performed in triplicate. Representative images from all experiments are shown. *P<0.05, **P<0.005, ***P<0.0001, NS, not significant. β-Actin serves as a loading control.

Mentions: The TGFβR1 and TGFβR2 serine/threonine kinases are essential for activation of the TGFβ signaling cascade.14, 35, 36 Hence, loss of either the expression or function of TGFβR1 or TGFβR2 perturbs TGFβ signaling.37, 38, 39, 40 As our Kaiso-depleted cells displayed decreased TGFβR1 and TGFβR2 expression, we hypothesized that suppressing Kaiso would attenuate TGFβ signaling. Indeed, Kaiso-depleted MDA-231 and Hs578T cells treated with recombinant human TGFβ1 had negligible levels of phosphorylated Smad2 (p-Smad2) that is indicative of active TGFβ signaling. This was in striking contrast to TGFβ1-treated MDA-231 and Hs578T control cells that exhibited increased p-Smad2 (Figure 4a). Consistent with our in vitro results, Kaiso-depleted MDA-231 and Hs578T mouse xenografts displayed reduced p-Smad2 expression in vivo compared with control MDA-231 and Hs578T xenografts (Figure 4b). To further validate the role of Kaiso in TGFβ-mediated signaling, we examined Kaiso-depletion effects on TGFβ-target gene expression. We chose ANGPTL4 that is involved in TGFβ-mediated breast tumor cell homing to lungs18 as both control MDA-231 and Hs578T cells displayed a proclivity for lung metastasis. Silencing Kaiso significantly reduced TGFβ-induced expression of ANGPTL4 (Figure 4c). Similarly, Kaiso depletion also attenuated TGFβ induction of ZEB1 (Supplementary Figure 2) that participates in TGFβ-mediated EMT.41 Unexpectedly, we observed increased Kaiso (ZBTB33) transcript levels in response to TGFβ treatment in both cell lines (Supplementary Figure 3). This increase in Kaiso transcripts was abrogated by Kaiso-specific shRNA in Kaiso-depleted cells (Figure 4c). Persistent TGFβ treatment (1–24 h) also resulted in increased Kaiso protein levels that peaked at ~12 h in both cell lines (Figure 4d). Together, these results hint at a positive feedback loop between Kaiso expression and TGFβ signaling.


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-depletion attenuates TGFβ signaling and transcriptional responses. Cells were treated with 10 ng/ml of TGFβ for 1 h before assaying for TGFβ activity. (a) TGFβ treatment of control MDA-231 and Hs578T cells results in increased p-Smad2 levels. However, Kaiso-depleted MDA-231 and Hs578T cells treated with TGFβ display reduced p-Smad2 levels. (b) Kaiso-depleted MDA-231 and Hs578T xenografts exhibit decreased TGFβ signaling as evidenced by reduced p-Smad2 protein levels. (c) TGFβ-induced expression of ANGPTL4 is attenuated in Kaiso-depleted cells treated with 10 ng/ml of TGFβ for 24 h. Interestingly, Kaiso expression is significantly increased by TGFβ treatment in MDA-231 cells. (d) Immunoblot analysis revealed a peak in Kaiso protein levels at 12 h in both MDA-231 and Hs578T cells in response to TGFβ treatment. All experiments were performed in triplicate. Representative images from all experiments are shown. *P<0.05, **P<0.005, ***P<0.0001, NS, not significant. β-Actin serves as a loading control.
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fig4: Kaiso-depletion attenuates TGFβ signaling and transcriptional responses. Cells were treated with 10 ng/ml of TGFβ for 1 h before assaying for TGFβ activity. (a) TGFβ treatment of control MDA-231 and Hs578T cells results in increased p-Smad2 levels. However, Kaiso-depleted MDA-231 and Hs578T cells treated with TGFβ display reduced p-Smad2 levels. (b) Kaiso-depleted MDA-231 and Hs578T xenografts exhibit decreased TGFβ signaling as evidenced by reduced p-Smad2 protein levels. (c) TGFβ-induced expression of ANGPTL4 is attenuated in Kaiso-depleted cells treated with 10 ng/ml of TGFβ for 24 h. Interestingly, Kaiso expression is significantly increased by TGFβ treatment in MDA-231 cells. (d) Immunoblot analysis revealed a peak in Kaiso protein levels at 12 h in both MDA-231 and Hs578T cells in response to TGFβ treatment. All experiments were performed in triplicate. Representative images from all experiments are shown. *P<0.05, **P<0.005, ***P<0.0001, NS, not significant. β-Actin serves as a loading control.
Mentions: The TGFβR1 and TGFβR2 serine/threonine kinases are essential for activation of the TGFβ signaling cascade.14, 35, 36 Hence, loss of either the expression or function of TGFβR1 or TGFβR2 perturbs TGFβ signaling.37, 38, 39, 40 As our Kaiso-depleted cells displayed decreased TGFβR1 and TGFβR2 expression, we hypothesized that suppressing Kaiso would attenuate TGFβ signaling. Indeed, Kaiso-depleted MDA-231 and Hs578T cells treated with recombinant human TGFβ1 had negligible levels of phosphorylated Smad2 (p-Smad2) that is indicative of active TGFβ signaling. This was in striking contrast to TGFβ1-treated MDA-231 and Hs578T control cells that exhibited increased p-Smad2 (Figure 4a). Consistent with our in vitro results, Kaiso-depleted MDA-231 and Hs578T mouse xenografts displayed reduced p-Smad2 expression in vivo compared with control MDA-231 and Hs578T xenografts (Figure 4b). To further validate the role of Kaiso in TGFβ-mediated signaling, we examined Kaiso-depletion effects on TGFβ-target gene expression. We chose ANGPTL4 that is involved in TGFβ-mediated breast tumor cell homing to lungs18 as both control MDA-231 and Hs578T cells displayed a proclivity for lung metastasis. Silencing Kaiso significantly reduced TGFβ-induced expression of ANGPTL4 (Figure 4c). Similarly, Kaiso depletion also attenuated TGFβ induction of ZEB1 (Supplementary Figure 2) that participates in TGFβ-mediated EMT.41 Unexpectedly, we observed increased Kaiso (ZBTB33) transcript levels in response to TGFβ treatment in both cell lines (Supplementary Figure 3). This increase in Kaiso transcripts was abrogated by Kaiso-specific shRNA in Kaiso-depleted cells (Figure 4c). Persistent TGFβ treatment (1–24 h) also resulted in increased Kaiso protein levels that peaked at ~12 h in both cell lines (Figure 4d). Together, these results hint at a positive feedback loop between Kaiso expression and TGFβ signaling.

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