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

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.

Figure 1. ErbB3 and ZNF217 mRNA expression in 50 breast cancer cell lines

Expression data for 50 breast cancer cell lines were determined using Affymetrix U133A arrays. A single probe set represents ErbB3 expression (202454_s_at) and ZNF217 expression (203739_at) in this dataset (Chin et al., 2006). The breast cancer cell lines are listed with the corresponding relative log2 ratio for ZNF217 and ErbB3 in Supplementary Table 1. The correlation coefficient (determined by Pearson's method of correlation analysis) between ErbB3 and ZNF217 was 0.69 (R²=0.47), indicating a significant linear association between the expression of these two genes.

Figure 2. ErbB3 and ZNF217 protein expression in human and murine specimens

(A) An immunoblot of 15 human primary breast tumor lysates probed with antibodies to ZNF217, ErbB3 and actin (loading control). (B) The intensities of the ZNF217 and ErbB3 signals relative to actin were quantified by densitometry and plotted. (C) Immunoblot of lysates from wild-type FvB mouse mammary tissue versus matched normal and tumor tissues from NDL 2-5 mice probed with antibodies to ErbB3, ZNF217 and actin.

Figure 3. Exogenous ZNF217 gene expression enhances endogenous ErbB3 expression in HMECs

(A) Protein expression levels of ZNF217 and ErbB3 in HMEC 184 before (passage 9) and after infection (passage 10) with ZNF217-encoding retroviruses, and after immortalization (passage 28). (B) Immunoblot of ZNF217 and ErbB3 protein expression in HMECs infected with control or ZNF217- encoding adenoviruses (C) Relative levels of ErbB3 mRNA in HMECs infected with control or ZNF217-encoding adenoviruses, determined at indicated time-points by Taqman real-time PCR analyses. Actin was used as an internal control. Note that error bars are present but very small.

Figure 4. Silencing ZNF217 reduces ErbB3 transcript and protein expression

(A) Scrambled control or ZNF217 siRNAs were transiently transfected into MCF7 and ZR75-1 cells. Lysates were harvested at 72h post-transfection and subjected to immunoblotting with ZNF217, ErbB3 and actin antibodies. Densitometric analysis of ZNF217 and ErbB3 relative to actin from three independent experiments is shown for both cell lines. (B) RNA samples from MCF7 cells treated in triplicate for 72 h with scrambled control or ZNF217-specific siRNAs were subjected to Taqman real-time PCR to determine relative ZNF217 and ErbB3 mRNA levels. Actin mRNA levels were used as an internal standard. Note that error bars are present but very small. (C) MCF7 cells were co-transfected with control or ZNF217 expression construct and an ErbB3-promoter driven luciferase reporter construct. Lysates were harvested 24h post-transfection and assayed for firefly and renilla luciferase activity using Promega Dual Luciferase Kit. Relative luciferase activity is shown. (D) ZR75-1 cells were co-transfected with scrambled control or ZNF217 siRNA and an ErbB3-promoter driven luciferase reporter construct. Cells were lysed at 24h post-transfection and assayed for firefly luciferase and beta-galactosidase activity (as an internal control). Relative luciferase activity is shown for a representative experiment performed in triplicate. p values for all comparisons in (A) through (D) are < 0.01.

Figure 5. ZNF217 and CtBP2 occupy the ErbB3 proximal promoter in MCF7 breast cancer cells

(A) Cartoon illustrating the locations of the sequences amplified in the ChIP studies shown in B and C. (B) Far right: PCR-amplified DNA enriched by immunoprecipitation with antibodies to ZNF217 or CtBP2, or with IgG (negative control) and 1:50 dilutions of un-enriched lysate (Input). Left: Quantitative PCR on ZNF217 ChIP (far left) and CtBP2 ChIP (middle) at the erbB3 proximal promoter versus 3kb upstream. (C) PCR-amplified DNA enriched by immunoprecipitation with antibodies to ZNF217, CtBP1, CTBP2, coREST, PolII, indicated histone modification marks, IgG (negative control), or 1:50 dilution of input are shown for comparison at the proximal erbB3 promoter. Results for ChIP-qPCR with primers amplifying the proximal region of the ErbB3 promoter are shown below gel panels. (D). ChIP-qPCR was performed on DNA enriched by immunoprecipitation with antibodies to ZNF217, CtBP2, AcH3K9, or TrimeK4 from MCF7 cells treated with siRNA to ZNF217 or scrambled control. Relative enrichment between siZNF217 and scramble control is compared (also graphed as fold change in Supplementary Figure 1). For (B) through (D), all qPCR was performed in triplicate and graphed as averaged relative DNA over 1:50 input for each ChIP sample (relative enrichment). A representative experiment is shown.

Figure 6. CtBP2 represses ErbB3 expression

(A) Protein lysates from wildtype and CtBP1/2 null MEFs were immunoblotted for ErbB3, CtBP1, CtBP2, and actin. (B) Mouse primers for the comparable ErbB3 proximal promoter sequence were PCR-amplified on DNA from wildtype or CtBP1/2 null MEFs enriched by immunoprecipitation with antibodies to ZNF217, CtBP2, indicated histone modification marks or IgG (negative control). 1:50 dilutions of input also shown. ChIP-qPCR was performed in triplicate and is shown as relative enrichment of immunoprecipitated DNA for mek27 and ZNF217 (left lower graph) and AcH3K9 and trimeK4 (right lower graph). (C) CtBP1/2 null MEFs were transiently transfected with a CtBP2 expression construct or empty control vector and the levels of ErbB3, CtBP2, and actin were analyzed by immunoblotting. Densitometric analysis of ErbB3 from three independent experiments is shown in the right panel. p < 0.05. (D) CtBP1/2 null MEFs were transiently transfected with a CtBP2 expression construct and an ErbB3-promoter driven luciferase reporter construct. Lysates were collected at 24h and assayed for firefly luciferase and beta-galactosidase activity. Relative luciferase activity is shown for a representative experiment performed in triplicate. p < 0.01.

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.