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ZNF217, a candidate breast cancer oncogene amplified at 20q13, regulates expression of the ErbB3 receptor tyrosine kinase in breast cancer cells.

Krig SR, Miller JK, Frietze S, Beckett LA, Neve RM, Farnham PJ, Yaswen PI, Sweeney CA - Oncogene (2010)

Bottom Line: In particular, the ErbB3 gene is not significantly amplified, raising the question as to how ErbB3 overexpression is achieved.Although ZNF217 has previously been linked with transcriptional repression because of its close association with C-terminal-binding protein (CtBP)1/2 repressor complexes, our results show that ZNF217 also activates gene expression.In addition, we identify ErbB3 as one of the mechanisms by which ZNF217 augments PI-3K/Akt signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Medicine, Divison of Basic Sciences, University of California at Davis Cancer Center, Sacramento, CA 95817, USA. skrig@ucdavis.edu

ABSTRACT
Understanding the mechanisms underlying ErbB3 overexpression in breast cancer will facilitate the rational design of therapies to disrupt ErbB2-ErbB3 oncogenic function. Although ErbB3 overexpression is frequently observed in breast cancer, the factors mediating its aberrant expression are poorly understood. In particular, the ErbB3 gene is not significantly amplified, raising the question as to how ErbB3 overexpression is achieved. In this study we showed that the ZNF217 transcription factor, amplified at 20q13 in ∼20% of breast tumors, regulates ErbB3 expression. Analysis of a panel of human breast cancer cell lines (n = 50) and primary human breast tumors (n = 15) showed a strong positive correlation between ZNF217 and ErbB3 expression. Ectopic expression of ZNF217 in human mammary epithelial cells induced ErbB3 expression, whereas ZNF217 silencing in breast cancer cells resulted in decreased ErbB3 expression. Although ZNF217 has previously been linked with transcriptional repression because of its close association with C-terminal-binding protein (CtBP)1/2 repressor complexes, our results show that ZNF217 also activates gene expression. We showed that ZNF217 recruitment to the ErbB3 promoter is CtBP1/2-independent and that ZNF217 and CtBP1/2 have opposite roles in regulating ErbB3 expression. In addition, we identify ErbB3 as one of the mechanisms by which ZNF217 augments PI-3K/Akt signaling.

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ZNF217 depletion suppresses ErbB3-mediated downstream signaling(A) ZR75-1 cells were transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum starved overnight and then treated with two different concentrations of Neuregulin1βfor twenty minutes. Lysates were prepared and blotted for ZNF217, p-AKT and actin (loading control). A representative western blot is shown. (B) Densitometric analysis of phospho Akt signal from three independent experiments. p < 0.01 for all comparisons between scramble and siZNF217. (C) Cell viability was determined in ZR75-1 cells transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum-starved overnight followed by treatment with Neuregulin1β for an additional 48h. Proliferation of cells at 96h post-transfection was measured by MTT assay.
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Figure 7: ZNF217 depletion suppresses ErbB3-mediated downstream signaling(A) ZR75-1 cells were transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum starved overnight and then treated with two different concentrations of Neuregulin1βfor twenty minutes. Lysates were prepared and blotted for ZNF217, p-AKT and actin (loading control). A representative western blot is shown. (B) Densitometric analysis of phospho Akt signal from three independent experiments. p < 0.01 for all comparisons between scramble and siZNF217. (C) Cell viability was determined in ZR75-1 cells transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum-starved overnight followed by treatment with Neuregulin1β for an additional 48h. Proliferation of cells at 96h post-transfection was measured by MTT assay.

Mentions: To examine the functional impact of ZNF217 modulation of ErbB3, ErbB3-mediated activation of the PI3K/Akt signaling cascade was examined in ZR75-1 breast cancer cells. ErbB3 is unique amongst the ErbB family in its propensity to potently activate the PI3K/Akt signaling cascade. To determine whether ZNF217 status impacts downstream signaling through ErbB3, ZR75-1 cells were treated with either scramble control or siZNF217 and then stimulated with two different concentrations of the ErbB3 activating ligand, Neuregulin-1β. Lysates were probed for ZNF217, phospho-Akt (S473) and actin control and a representative immunoblot is shown in Figure 7A with quantification of several experiments in Figure 7B. Depletion of ZNF217 decreased basal Akt phosphorylation and limited ligand-stimulated Akt phosphorylation such that at both Neuregulin 1β concentrations, Akt phosphorylation was significantly diminished. This attenuation of Neuregulin 1β-stimulated Akt phosphorylation is likely a direct consequence of the ErbB3 depletion observed following ZNF217 knockdown, although other means by which ZNF217 may influence ErbB3 signaling through the PI3K/Akt pathway cannot presently be ruled out. To examine whether this attenuation of signaling has consequences for tumor cell growth, an MTT assay was performed. ZR75-1 cells were treated with either scramble control or ZNF217 siRNA and then stimulated with a sub-saturating concentration of Neuregulin-1β (necessary to appreciate the differences between ZNF217-replete and -depleted cells). Although ZR75-1 cells have a high basal rate of proliferation, they demonstrated a modest but reproducible response to a low concentration of Neuregulin-1β(p = 0.005). Conversely, in ZNF217-depleted cells, the proliferative response to Neuregulin-1β was abolished and cells instead proliferated less in the presence of Neuregulin-1β that in its absence (p = 0.009). Similar results were observed in MCF7 cells (data not shown). These findings have important implications for future therapeutic approaches which aim at targeting ErbB3. Depletion of ZNF217 may be beneficial in this regard.


ZNF217, a candidate breast cancer oncogene amplified at 20q13, regulates expression of the ErbB3 receptor tyrosine kinase in breast cancer cells.

Krig SR, Miller JK, Frietze S, Beckett LA, Neve RM, Farnham PJ, Yaswen PI, Sweeney CA - Oncogene (2010)

ZNF217 depletion suppresses ErbB3-mediated downstream signaling(A) ZR75-1 cells were transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum starved overnight and then treated with two different concentrations of Neuregulin1βfor twenty minutes. Lysates were prepared and blotted for ZNF217, p-AKT and actin (loading control). A representative western blot is shown. (B) Densitometric analysis of phospho Akt signal from three independent experiments. p < 0.01 for all comparisons between scramble and siZNF217. (C) Cell viability was determined in ZR75-1 cells transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum-starved overnight followed by treatment with Neuregulin1β for an additional 48h. Proliferation of cells at 96h post-transfection was measured by MTT assay.
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Figure 7: ZNF217 depletion suppresses ErbB3-mediated downstream signaling(A) ZR75-1 cells were transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum starved overnight and then treated with two different concentrations of Neuregulin1βfor twenty minutes. Lysates were prepared and blotted for ZNF217, p-AKT and actin (loading control). A representative western blot is shown. (B) Densitometric analysis of phospho Akt signal from three independent experiments. p < 0.01 for all comparisons between scramble and siZNF217. (C) Cell viability was determined in ZR75-1 cells transiently transfected with either scramble control or ZNF217-specific siRNA. 48 hours after transfection, cells were serum-starved overnight followed by treatment with Neuregulin1β for an additional 48h. Proliferation of cells at 96h post-transfection was measured by MTT assay.
Mentions: To examine the functional impact of ZNF217 modulation of ErbB3, ErbB3-mediated activation of the PI3K/Akt signaling cascade was examined in ZR75-1 breast cancer cells. ErbB3 is unique amongst the ErbB family in its propensity to potently activate the PI3K/Akt signaling cascade. To determine whether ZNF217 status impacts downstream signaling through ErbB3, ZR75-1 cells were treated with either scramble control or siZNF217 and then stimulated with two different concentrations of the ErbB3 activating ligand, Neuregulin-1β. Lysates were probed for ZNF217, phospho-Akt (S473) and actin control and a representative immunoblot is shown in Figure 7A with quantification of several experiments in Figure 7B. Depletion of ZNF217 decreased basal Akt phosphorylation and limited ligand-stimulated Akt phosphorylation such that at both Neuregulin 1β concentrations, Akt phosphorylation was significantly diminished. This attenuation of Neuregulin 1β-stimulated Akt phosphorylation is likely a direct consequence of the ErbB3 depletion observed following ZNF217 knockdown, although other means by which ZNF217 may influence ErbB3 signaling through the PI3K/Akt pathway cannot presently be ruled out. To examine whether this attenuation of signaling has consequences for tumor cell growth, an MTT assay was performed. ZR75-1 cells were treated with either scramble control or ZNF217 siRNA and then stimulated with a sub-saturating concentration of Neuregulin-1β (necessary to appreciate the differences between ZNF217-replete and -depleted cells). Although ZR75-1 cells have a high basal rate of proliferation, they demonstrated a modest but reproducible response to a low concentration of Neuregulin-1β(p = 0.005). Conversely, in ZNF217-depleted cells, the proliferative response to Neuregulin-1β was abolished and cells instead proliferated less in the presence of Neuregulin-1β that in its absence (p = 0.009). Similar results were observed in MCF7 cells (data not shown). These findings have important implications for future therapeutic approaches which aim at targeting ErbB3. Depletion of ZNF217 may be beneficial in this regard.

Bottom Line: In particular, the ErbB3 gene is not significantly amplified, raising the question as to how ErbB3 overexpression is achieved.Although ZNF217 has previously been linked with transcriptional repression because of its close association with C-terminal-binding protein (CtBP)1/2 repressor complexes, our results show that ZNF217 also activates gene expression.In addition, we identify ErbB3 as one of the mechanisms by which ZNF217 augments PI-3K/Akt signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Medicine, Divison of Basic Sciences, University of California at Davis Cancer Center, Sacramento, CA 95817, USA. skrig@ucdavis.edu

ABSTRACT
Understanding the mechanisms underlying ErbB3 overexpression in breast cancer will facilitate the rational design of therapies to disrupt ErbB2-ErbB3 oncogenic function. Although ErbB3 overexpression is frequently observed in breast cancer, the factors mediating its aberrant expression are poorly understood. In particular, the ErbB3 gene is not significantly amplified, raising the question as to how ErbB3 overexpression is achieved. In this study we showed that the ZNF217 transcription factor, amplified at 20q13 in ∼20% of breast tumors, regulates ErbB3 expression. Analysis of a panel of human breast cancer cell lines (n = 50) and primary human breast tumors (n = 15) showed a strong positive correlation between ZNF217 and ErbB3 expression. Ectopic expression of ZNF217 in human mammary epithelial cells induced ErbB3 expression, whereas ZNF217 silencing in breast cancer cells resulted in decreased ErbB3 expression. Although ZNF217 has previously been linked with transcriptional repression because of its close association with C-terminal-binding protein (CtBP)1/2 repressor complexes, our results show that ZNF217 also activates gene expression. We showed that ZNF217 recruitment to the ErbB3 promoter is CtBP1/2-independent and that ZNF217 and CtBP1/2 have opposite roles in regulating ErbB3 expression. In addition, we identify ErbB3 as one of the mechanisms by which ZNF217 augments PI-3K/Akt signaling.

Show MeSH
Related in: MedlinePlus