ZNF217 confers resistance to the pro-apoptotic signals of paclitaxel and aberrant expression of Aurora-A in breast cancer cells

Abstract

Background: ZNF217 is a candidate oncogene located at 20q13, a chromosomal region frequently amplified in breast cancers. The precise mechanisms involved in ZNF217 pro-survival function are currently unknown, and utmost importance is given to deciphering the role of ZNF217 in cancer therapy response.

Results: We provide evidence that stable overexpression of ZNF217 in MDA-MB-231 breast cancer cells conferred resistance to paclitaxel, stimulated cell proliferation in vitro associated with aberrant expression of several cyclins, and increased tumor growth in mouse xenograft models. Conversely, siRNA-mediated silencing of ZNF217 expression in MCF7 breast cancer cells, which possess high endogenous levels of ZNF217, led to decreased cell proliferation and increased sensitivity to paclitaxel. The paclitaxel resistance developed by ZNF217-overexpressing MDA-MB-231 cells was not mediated by the ABCB1/PgP transporter. However, ZNF217 was able to counteract the apoptotic signals mediated by paclitaxel as a consequence of alterations in the intrinsic apoptotic pathway through constitutive deregulation of the balance of Bcl-2 family proteins. Interestingly, ZNF217 expression levels were correlated with the oncogenic kinase Aurora-A expression levels, as ZNF217 overexpression led to increased expression of the Aurora-A protein, whereas ZNF217 silencing was associated with low Aurora-A expression levels. We showed that a potent Aurora-A kinase inhibitor was able to reverse paclitaxel resistance in the ZNF217-overexpressing cells.

Conclusion: Altogether, these data suggest that ZNF217 might play an important role in breast neoplastic progression and chemoresistance, and that Aurora-A might be involved in ZNF217-mediated effects.

Figure 1

ZNF217 expression in MCF7, MDA-MB-231 and pcDNA6/V5-His-ZNF217-transfected MDA-MB-231 cells. (A) ZNF217 mRNA expression was analyzed by RTQ-PCR in MCF7 and MDA-MB-231 cells (means ± s.d. of three independent experiments). (B) western-blot analysis of ZNF217 in MCF7 and MDA-MB-231 cell lines. Histograms represent quantification of protein expression levels normalized to tubulin expression (means ± s.d. of three independent experiments). ***, P < 0.001 (Student's t-test). (C) The same as (A) in ZNF217-overexpressing MDA-MB-231 cells, ZNF217-1 and ZNF217-2. (D) The same as (B) in ZNF217-1, ZNF217-2 and in control MDA-MB-231/pcDNA6 cells.

Figure 2

Constitutive expression of ZNF217 stimulates cell proliferation in vitro. (A) Cell proliferation was assessed at different time points by BrdU labeling (means ± s.d. of three independent experiments). **, P < 0.01 versus MDA-MB-231/pcDNA6 cells (Student's t-test). (B) Western-blot analysis of Cyclin D1, Cyclin E1, Cyclin E2 and Cyclin A2. Histograms represent quantification of protein expression levels normalized to tubulin expression (means ± s.d. of three independent experiments). **, P < 0.01 and ***, P < 0.001 versus MDA-MB-231/pcDNA6 cells (Student's t-test).

Figure 3

Decreased levels of ZNF217 negatively regulates cell proliferation in vitro. (A) Western-blot analysis of ZNF217 expression in non-transfected (NT) or transfected MDA-MB-231/pcDNA6 cells with either scrambled RNA, siRNA-B or siRNA-A. Histograms represent quantification of protein expression levels normalized to tubulin expression (means ± s.d. of three independent experiments). (B) the same as (A) using the ZNF217-1 cell line. (C) Cell proliferation of non-transfected (NT) or transfected MDA-MB-231/pcDNA6 cells with either scrambled RNA, siRNA-B or siRNA-A as assessed by BrdU test. (D) the same as (C) using the ZNF217-1 cell line. (E) and (F) respectively the same as (A) and (C) using MCF7 cells. *, P < 0.05, **, P < 0.01 and ***, P < 0.001 versus cells transfected with scrambled RNA (Student's t-test).

Figure 4

Constitutive expression of ZNF217 stimulates tumor growth in vivo. (A) Growth curves of control xenografts (n = 6) and ZNF217 xenografts (n = 7) in nude mice. Data are presented as means ± s.d. of tumor volumes. *, P < 0.05, **, P < 0.01 and ***, P < 0.001 (Student's t-test). (B) Representative total protein extracts from control and ZNF217 xenografts were analyzed by western-blot with anti-ZNF217 and anti-Cyclin D1 antibodies. Histogram represents quantification of protein expression levels normalized to tubulin expression (means ± s.d. of three independent experiments). ***, P < 0.001 (Student's t-test).

Figure 5

ZNF217 expression induces resistance to paclitaxel. Cell viability of (A) MDA-MB-231/pcDNA6, ZNF217-1 and ZNF217-2 cells and (B) non-transfected (NT) or transfected MCF7 cells with either scrambled RNA or siRNA-B was assessed by cytotoxicity assay (means ± s.d. from three independent experiments). **, P < 0.01, ***, P < 0.001 (Student's t-test).

Figure 6

ABCB1/PgP transporter does not mediate ZNF217-induced resistance to paclitaxel. (A) ABCB1 protein levels were analyzed by flow cytometry in control K562-R7, MDA-MB-231/pcDNA6, ZNF217-1 and ZNF217-2 cells. Representative FACS histograms of the three cell lines after incubation with or without ABCB1 antibody were superimposed according to increased PE fluorescence. (B) Intracellular DNR efflux was assessed by flow cytometry. Maximal DNR accumulation (100%) is represented by the DNR fluorescence median after 30 min accumulation. After DNR removal, cells were incubated 1 h in the absence (white columns) or in the presence of CSA (black columns). Results are means ± s.d. from three independent experiments.

Figure 7

ZNF217 overexpression alters paclitaxel-induced apoptosis. (A) MDA-MB-231/pcDNA6, ZNF217-1 and ZNF217-2 cells were untreated (-) or treated with 10 or 100 nM paclitaxel, stained with Annexin-V-Fluos and propidium iodide and analyzed by flow cytometry (means ± s.d. from three independent experiments). **, P < 0.01 and ***, P < 0.001 versus the corresponding MDA-MB-231/pcDNA6 cells (Student's t-test). (B) MDA-MB-231/pcDNA6, ZNF217-1 and ZNF217-2 cells were treated with 100 nM paclitaxel. Caspase 3 activity was assessed (means ± s.d. from three independent experiments). ***, P < 0.001 versus MDA-MB-231/pcDNA6 cells (Student's t-test). (C) Western-blot analysis of PARP cleavage in response to 100 nM paclitaxel. Histogram represents quantification of the cleaved PARP in 100 nM paclitaxel-treated cells and tubulin expression was used for normalization (means ± s.d. of three independent experiments). ***, P < 0.001 versus paclitaxel-treated MDA-MB-231/pcDNA6 cells (Student's t-test). (D) Non-transfected (NT) or transfected MCF7 cells with either scrambled RNA or siRNA-B were treated with 100 nM paclitaxel. Caspase 3 activity was assessed (means ± s.d. from three independent experiments). *, P < 0.05 versus MCF7 cells transfected with scrambled RNA (Student's t-test).

Figure 8

ZNF217 overexpression is associated with altered expression of Bcl-2 family proteins. (A) Western-blot analysis of Bcl-2, Bcl-x_L, Bad, Bak, Bax at basal level or (B) after 3 days of paclitaxel treatment. Histograms represent quantification of protein expression levels normalized to tubulin expression (means ± s.d. of three independent experiments). *, P < 0.05, **, P < 0.01 and ***, P < 0.001 versus the corresponding MDA-MB-231/pcDNA6 cells (Student's t-test).

Figure 9

ZNF217 modulates Aurora-A protein expression and ZNF217-mediated paclitaxel resistance is reversed by an Aurora-A inhibitor. Western-blot analysis of Aurora-A expression in (A) MDA-MB-231/pcDNA6, ZNF217-1 and ZNF217-2 cells, (B) in representative control and ZNF217 xenograft cells, (C) in ZNF217-1 or MCF7 cells transfected or not (NT) with either scrambled RNA or siRNA-B. (A), (B) and (C) Histograms represent quantification of protein expression levels normalized to tubulin expression (means ± s.d. of three independent experiments). *, P < 0.05 and ***, P < 0.001 (Student's t-test). (D) The viability of MDA-MB-231/pcDNA6 and ZNF217-1 cells treated with 5 μM Aurora-A kinase inhibitor III, 2.5 nM paclitaxel or a combination of both was assessed by cytotoxicity assay (means ± s.d. from three independent experiments). ***, P < 0.001 (Student's t-test). (E) MCF7 cells transfected with either scrambled RNA or siRNA-B were treated with 1 μM Aurora-A kinase inhibitor III, 2.5 nM paclitaxel or a combination of both. Cell viability was assessed by cytotoxicity assay (means ± s.d. from three independent experiments). **, P < 0.01 (Student's t-test).