The estrogen and c-Myc target gene HSPC111 is over-expressed in breast cancer and associated with poor patient outcome

Abstract

Introduction: Estrogens play a pivotal role in the initiation and progression of breast cancer. The genes that mediate these processes are not fully defined, but potentially include the known mammary oncogene MYC. Characterization of estrogen-target genes may help to elucidate further the mechanisms of estrogen-induced mitogenesis and endocrine resistance.

Methods: We used a transcript profiling approach to identify targets of estrogen and c-Myc in breast cancer cells. One previously uncharacterized gene, namely HBV pre-S2 trans-regulated protein 3 (HSPC111), was acutely upregulated after estrogen treatment or inducible expression of c-Myc, and was selected for further functional analysis using over-expression and knock-down strategies. HSPC111 expression was also analyzed in relation to MYC expression and outcome in primary breast carcinomas and published gene expression datasets.

Results: Pretreatment of cells with c-Myc small interfering RNA abrogated estrogen induction of HSPC111, identifying HSPC111 as a potential c-Myc target gene. This was confirmed by the demonstration of two functional E-box motifs upstream of the transcription start site. HSPC111 mRNA and protein were over-expressed in breast cancer cell lines and primary breast carcinomas, and this was positively correlated with MYC mRNA levels. HSPC111 is present in a large, RNA-dependent nucleolar complex, suggesting a possible role in ribosomal biosynthesis. Neither over-expression or small interfering RNA knock-down of HSPC111 affected cell proliferation rates or sensitivity to estrogen/antiestrogen treatment. However, high expression of HSPC111 mRNA was associated with adverse patient outcome in published gene expression datasets.

Conclusion: These data identify HSPC111 as an estrogen and c-Myc target gene that is over-expressed in breast cancer and is associated with an adverse patient outcome.

Figure 1

Identification of HSPC111 as an estrogen-regulated target of c-Myc. Cells were pretreated with ICI 182780 for 48 hours. Parental MCF-7 cells were then treated with either 17β-estradiol (diamonds) or vehicle (squares), and MCF-7/MycWT (triangles) and empty vector (squares) cells were treated with zinc. (a) HSPC111 mRNA expression in two probe sets from HG-U133 Plus V2.0 microarray platforms, 6 hours after treatment with estradiol (E₂) or vehicle, or after expression of c-Myc (MycWT) or zinc treatment of empty vector cells (empty). (b) RNA was isolated at various time points as indicated and analyzed in triplicate by reverse transcription PCR with HSPC111-specific primers. Expression of HSPC111 is presented normalized to RPLP0. (c) immunoblot analysis of endogenous HSPC111 expression in whole cell lysates at time points up to 24 hours. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Representative blots and densitometric analyses from three independent experiments are shown.

Figure 2

Estrogen regulation of HSPC111 expression is dependent upon direct transcriptional activation by Myc. (a) Left panel: MCF-7 cells were arrested with ICI 182780 for 48 hours and then treated with cycloheximide (black bars) or control (white bars) before addition of estrogen or vehicle for 3 and 6 hours; levels of HSPC111 mRNA were determined by quantitative real-time PCR. Middle panel: MCF-7 cells were transfected with an HSPC111-luciferase reporter construct in the presence of increasing amounts of the c-Myc expression construct pCDNA3.1-cMyc. Right panel: MCF-7/MycWT (black bars) and empty vector controls (white bars) were transfected with the HSPC111-luciferase reporter construct and stimulated with increasing concentrations of zinc. Values are expressed as means ± standatrd deviation of triplicate samples from three independent experiments. (b) Left panel: MCF-7 cells were transfected with Myc-specific small interfering (si)RNA (siMyc17), RISC-free (RF), or nontargeting (NT) siRNA controls, or mock transfected with no siRNA. Transfected cells were arrested with ICI 182780 for 48 hours. Levels of MYC and HSPC111 mRNA were determined by quantitative real-time PCR after 24 hours of treatment with estradiol (black bars) or vehicle control (white bars). (c) Schematic showing the structure of the HSPC111 proximal promoter with the location of putative Myc-binding sites (E-boxes). Electrophoretic mobility shift assays demonstrate specific binding of c-Myc to E-boxes within the HSPC111 promoter. Radiolabeled oligonucleotides, as indicated above each gel, were incubated with nuclear extract from MCF-7 cells. Lane NC indicates no competitor oligonucleotides were added. The nonlabeled competitor oligonucleotides are indicated below each lane. Lane NS indictates nonspecific competitor oligonucleotide. (d) Chromatin was obtained from MCF-7/MycWT cells after 6 hours of treatment with zinc, and immunoprecipitated with c-Myc-specific or nonspecific (NS) antibodies as indicated. Left panel: Chromatin immunoprecipitation (ChIP) assay demonstrating the binding of c-Myc to the endogenous HSPC111 promoter. Lane I contains input chromatin that was not immunoprecipitated. Specific regions were then amplified by PCR using primers specific for site 1 or site 3, as indicated. Right panel: ChIP assay demonstrating the recruitment of c-Myc to the endogenous HSPC111 promoter in response to treatment with estradiol (E₂) at 3 and 6 hours. Chromatin was immunoprecipitated with either a c-Myc-specific (C33; black bars) or a nonspecific (white bars) antibody and analyzed by quantitative real-time PCR using primers specific for site 1.

Figure 3

HSPC111 resides in high molecular weight protein complexes in the nucleolus. (a) Detection of endogenous and tagged HSPC111 by indirect immunofluorescence. Upper panels: Immunostaining of MCF-7/HSPC-NV5 cells with purified antibodies against endogenous protein (HSPC111; green) and the V5 tag (V5; red). Middle panels: Parental MCF-7 cells were stained with anti-HSPC111 (green) and anti-nucleophosmin (NPM; red) antibodies. Lower panels: Parental MCF-7 cells stained with anti-HSPC111 (green) and anti-fibrillarin (red) antibodies. DNA was counterstained with DAPI (4,6-diamidino-2-phenylindole; blue). Images are representative of at least two independent experiments. Bar = 10 μm. (b) Nuclear extracts of MCF-7 cells treated with or without RNase A were fractionated on sucrose density gradients. The trace from continuous monitoring of absorbance at 254 nm is shown. Fractions were precipitated and immunoblotted for HSPC111 and fibrillarin.

Figure 4

Effects of modulation of HSPC111 expression on cell proliferation. (a) Immunoblot analysis of whole cell lysates from MCF-7 clones stably expressing HSPC111 (HSPC#1 and HSPC#4) or LacZ controls at various passages after transfection. Blots were analyzed for expression of tagged HSPC111 protein using V5 antibody or actin as a loading control. (b) Left panel: Growth curves of HSPC111 over-expressing clones and LacZ controls. Right panel: Stable transfectants were treated with tamoxifen (TAM), ICI 182780 (ICI), or vehicle for 48 hours, and then S phase was determined by flow cytometric analysis of propidium iodide-stained cells. (c) Upper panel: Endogenous HSPC111 mRNA and protein expression in MCF-7 cells 24 and 48 hours after transfection with HSPC111-specific small interfering (si)RNA (siHSPC2 and siHSPC4) determined by quantitative real-time PCR and immunoblot analysis with HSPC111 antibody, respectively. NS indicates mock transfection with no siRNA, RF indicates RISC-free control siRNA, and NT indicates nontargeting control siRNA. Lower panel: S phase was determined 48 hours after transfection by flow cytometric analysis of propidium iodide-stained cells.

Figure 5

Correlation between HSPC111 and c-Myc expression in human breast cancer cell lines and tumor samples. (a) Expression of HSPC111 and Myc mRNA and protein was determined by quantitative real-time PCR and immunoblot, respectively, in a panel of 16 breast cancer cell lines with either estrogen receptor (ER)-negative or ER-positive status. (b) Correlation between HSPC111 and MYC mRNA expression in primary breast cancers, and distribution of HSPC111 mRNA expression levels amongst ER-negative and ER-positive cancers.

Figure 6

High HSPC111 expression is associated with poor survival in breast cancer cohorts. Kaplan-Meier survival curves of the relationship between HSPC111 and MYC mRNA expression and survival in two independent publicly available breast cancer cohorts: (a) Uppsala cohort and (b) the Nederlands Kanker Instituut (NKI) cohort.