Negative transcriptional control of ERBB2 gene by MBP-1 and HDAC1: diagnostic implications in breast cancer

Backgound: The human ERBB2 gene is frequently amplified in breast tumors, and its high expression is associated with poor prognosis. We previously reported a significant inverse correlation between Myc promoter-binding protein-1 (MBP-1) and ERBB2 expression in primary breast invasive ductal carcinoma (IDC). MBP-1 is a transcriptional repressor of the c-MYC gene that acts by binding to the P2 promoter; only one other direct target of MBP-1, the COX2 gene, has been identified so far. Methods: To gain new insights into the functional relationship linking MBP-1 and ERBB2 in breast cancer, we have investigated the effects of MBP-1 expression on endogenous ERBB2 transcript and protein levels, as well as on transcription promoter activity, by transient-transfection of SKBr3 cells. Reporter gene and chromatin immunoprecipitation assays were used to dissect the ERBB2 promoter and identify functional MBP-1 target sequences. We also investigated the relative expression of MBP-1 and HDAC1 in IDC and normal breast tissues by immunoblot analysis and immunohistochemistry. Results: Transfection experiments and chromatin immunoprecipitation assays in SKBr3 cells indicated that MBP-1 negatively regulates the ERBB2 gene by binding to a genomic region between nucleotide -514 and - 262 of the proximal promoter; consistent with this, a concomitant recruitment of HDAC1 and loss of acetylated histone H4 was observed. In addition, we found high expression of MBP-1 and HDAC1 in normal tissues and a statistically significant inverse correlation with ErbB2 expression in the paired tumor samples. Conclusions: Altogether, our in vitro and in vivo data indicate that the ERBB2 gene is a novel MBP-1 target, and immunohistochemistry analysis of primary tumors suggests that the concomitant high expression of MBP-1 and HDAC1 may be considered a diagnostic marker of cancer progression for breast IDC

Myc Promoter-Binding Protein-1 (MBP-1) Is a Novel Potential Prognostic Marker in Invasive Ductal Breast Carcinoma

Background Alpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. \u3b1-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of \u3b1-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features have not been undertaken. Methodology and Findings We analyzed \u3b1-enolase and MBP-1 expression in normal breast epithelium and primary invasive ductal breast carcinoma (IDC) from 177 patients by Western blot and immunohistochemical analyses, using highly specific anti-\u3b1-enolase monoclonal antibodies. A significant increase in the expression of cytoplasmic \u3b1-enolase was observed in 98% of the tumors analysed, compared to normal tissues. Nuclear MBP-1 was found in almost all the normal tissues while its expression was retained in only 35% of the tumors. Statistically significant associations were observed among the nuclear expression of MBP-1 and ErbB2 status, Ki-67 expression, node status and tumor grade. Furthermore MBP-1 expression was associated with good survival of patients with IDC. Conclusions MBP-1 functions in repressing c-myc gene expression and the results presented indicate that the loss of nuclear MBP-1 expression in a large number of IDC may be a critical step in the development and progression of breast cancer and a predictor of adverse outcome. Nuclear MBP-1 appears to be a novel and valuable histochemical marker with potential prognostic value in breast cancer

Survival analysis of IDC patients according to MBP-1 expression.

<p>Kaplan-Meier curves for disease-free survival (DFS) in 77 IDC patients relative to nuclear MBP-1 positive (+^ve) or negative (−^ve) expression. The absence or low expression (MBP-1^−ve) of nuclear MBP-1 was significantly associated with recurrence (<i>P</i> = 0.0036 by log-rank test).</p

IDC immunohistochemical staining for α-enolase and nuclear MBP-1 revealed heterogeneity in breast cancer samples.

<p><b>A</b>. Representative immunohistochemical staining of 3 out of the 177 IDC samples analyzed with mAbs ENO-19/8. Tumor sections (T) were compared with the corresponding normal breast tissue (N). All normal breast tissues showed nuclear MBP-1 expression and low expression of cytoplasmic α-enolase (sample 1–3). In sample 1 and 2, cytoplasmic staining of the invasive breast carcinoma (T) was stronger than in the matched normal breast tissues. In tumor samples 2 and 3, the loss of MBP-1 nuclear expression occurred. Sections of sample 4 were immunoassayed with antibodies blocked with a recombinant α-enolase polypeptide (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012961#s2" target="_blank">materials and methods</a>). Magnification: 250×. <b>B</b>. MBP-1 nuclear staining correlated with ErbB2 and Ki67 expression. Immunohistochemical staining for α-enolase, ErbB2 and Ki67 in MBP-1-positive (S1) and MBP-1-negative (S2) tumors. Magnification: 500×.</p

Spearman correlation analysis between MBP-1 and clinicopathological features.

<p>Spearman correlation analysis between MBP-1 and clinicopathological features.</p

Monoclonal antibodies ENO-276/3 and ENO-19/8 specifically recognize α-enolase or both α-enolase and MBP-1.

<p><b>A</b>. Top, schematic representation of Flag-tagged constructs used, black boxes indicate the position of the Flag epitope, and numbers indicate the first and last amino acid in each construct. Protein regions containing the epitopes recognized by ENO-276/3 and ENO-19/8 mAbs are indicated by striped and dotted bars respectively, numbers refer to corresponding amino acids in the α-enolase sequence (Swiss-Prot: P06733). Bottom, Western blot analysis of 293T cells transiently transfected with vectors expressing Flag-tagged α-enolase (Flag-Eno), or MBP-1 (Flag-MBP-1) proteins, compared to mock transfected cells (mock). Cell lysates (20 µg) were firstly probed with polyclonal anti-Flag antibodies and, after stripping, the same filter was reacted with anti-α-enolase ENO-276/3, and finally with ENO-19/8, as indicated. Molecular weight (kDa) are indicated on the right. Relative positions of endogenous (end.) and ectopically expressed (exo.) α-enolase and MBP-1 from transfected pFlag-ENO are indicated on the left. <b>B</b>. Representative immunohistochemical staining on serial sections of a normal mammary tissue (a and b) and an IDC tumors sample (c and d) from the commercial TMA. The anti-α-enolase mAbs ENO-19/8 specifically decorated both nuclei and cytoplasm, while ENO-276/3 gave an almost exclusive cytoplasmic staining. As a control, breast cancer sections (e and f) were immunoassayed with mAbs which had been previously coincubated and thereby blocked with antigenic peptides, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012961#s2" target="_blank">materials and methods</a>. Magnification: 500× (a–d), and 200× (e and f). <b>C</b>. Representative Western blot and immunohistochemical analyses of normal and tumor tissues from IDC samples. Left, Western blot detection of α-enolase (α-eno) and MBP-1 proteins in total lysates (30 µg) from a breast tumor sample (T) and normal-matched tissue (N) using mAbs ENO-276/3 and ENO-19/8. Anti-β-actin antibodies were used as a control for loading. Right, immunohistochemical staining with mAbs ENO-276/3 and ENO-19/8 of serial sections of the tumor (b and d) and the corresponding normal breast tissue (a and c) from the same sample analyzed by Western blot. Magnification: 400×.</p

Correlation between MBP-1 expression and clinicopathological characteristics of breast cancer patients.

<p><b>NOTE</b>: Statistical analyses were done by the Fisher's exact test. A <i>P</i> value of <0.05 was considered significant.</p><p>Abbreviations: ER, estrogen receptor; PG, progesterone receptor;</p><p>NS, nonsignificant.</p

Relative expression of α-enolase, MBP-1 and Myc in primary breast tumors and adjacent normal tissue.

<p><b>A</b>. Representative Western blot analysis of α-enolase (α-eno), MBP-1, Myc and β-actin proteins in total lysates (30 µg) from breast tumors (T) and normal-matched tissues (N) was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012961#s2" target="_blank">materials and methods</a>. <b>B</b>. Graphic representation of the averaged tumor/normal (T/N) ratios of α-enolase (white bars), MBP-1 (striped bars) and Myc (black bars) protein expression quantified by the densitometric analysis of Western blot results. Expression levels were normalized to β-actin. Columns are the mean of three parallel experiments; bar, ±SD. <b>C</b>. α-Enolase protein levels in normal and breast cancer tissues. The Box plot represents the α-enolase/β-actin ratio determined in 24 breast tumors and 21 normal-matched tissues. Significantly higher levels of α-enolase were present in breast cancers than normal tissues (t-test value, <i>p</i><0.001). <b>D</b>. Myc mRNA expression levels in normal and breast cancer tissues. Transcripts were analyzed by real-time PCR and normalized with respect to TBP mRNA. Box plot of c-myc mRNA levels in MBP-1-positive (+^ve) and negative (−^ve) breast tumors. Myc mRNA levels are significantly associated with MBP-1 status (t-test value <i>p</i><0.001). In C and D bars above and below the boxes represent the maximum and minimum expression. Each box delineates the first to third quartiles of expression, and the central bar represents the median.</p