Nuclear detection of Y-boxprotein-1 (YB-1) closely associates with progesterone receptor negativity and is a strong adverse survival factor in human breast cancer

Mab generation

Mouse work was in full compliance with the guidelines for animal care and was approved by the animal care committee from the government. Female BALB/c mice were immunized by intraperitoneal and intravenous injection of 100 μg recombinant YB-1 full-length protein emulsified in equal volume of Freund's adjuvant (Gibco-BRL life technologies, Karlsruhe, Germany) followed by a booster injection six weeks later containing 10 μg of antigen in Freund's adjuvant. The response was assessed by dot blot assay with recombinant YB-1 protein. Three days after the last booster injection, spleen cells were obtained and fused with X63-Ag/653 (BALB/c) mouse myeloma cells and propagated according to standard procedures. The fused cells were resuspended in DMEM medium (Gibco BRL life technologies) supplemented with 10% bovine calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM hypoxanthine, 160 μM thymidine, and 400 nM aminopterin. Aliquots of the cell suspension (100 μl) were dispensed into 96-well plates and incubated at 37°C/5% CO₂. Every week the medium was replaced with fresh selection medium supplemented with recombinant interleukin-6 (100 U/ml). After two weeks the medium was replaced by HT-medium (DMEM medium (Gibco BRL life technologies) supplemented with 10% bovine calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM hypoxanthine and 160 μM thymidine and another week later cells were cultured in standard DMEM medium (4500 mg/l D-Glucose supplemented with GlutaMax (Gibco BRL life technologies) with 10% bovine calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Hybridoma supernatants from 96-well plates were screened by dot blot assay with recombinant YB-1 protein for specific antibody synthesis. The positive-tested hybridoma cells were single-cell cloned twice by limited dilution in standard medium as described [37]. Immunoglobulins in the hybridoma cell culture supernatants were purified using protein A sepharose or protein G sepharose columns according to the immunoglobulin isotype (ÄKTA_FPLC systems, Amersham Biosciences, Freiburg, Germany). F-E2G5 was of IgG_2b isotype. Purified monoclonal antibodies were conjugated to biotin as described [38].

Plasmids, cell line and transfection

Plasmids encoding for GFP and YB-1-GFP fusion proteins (pcDNA6/V5-His-YB-1-GFP) have been described previously [20]. HEK293T cells were cultured in DME-medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Transient transfections of HEK293T cells with vectors were performed by means of calcium phosphate precipitation methodology using purified endotoxin-free plasmid DNA preparations. HEK293T cells expressing the respective proteins were harvested 48 h after transfection and cytoplasmic and nuclear fractions were prepared by cell lysis in buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM PMSF, 1 mM sodium vanadate, 0.5 mM DTT) for 10 min on ice, followed by centrifugation at 2.000 rpm for 1 minute. The supernatant corresponds to soluble cytoplasmic proteins and contains GFP and YB-1-GFP proteins.

Western Blotting

SDS-PAG electrophoresis was performed with protein extracts containing GFP or YB-1-GFP protein under reducing and non-reducing conditions. Proteins were separated on 12% SDS-PAG and transferred to nitrocellulose membranes (Schleicher-Schuell, Dassel, Germany). Membranes were blocked in TTBS (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.2% Tween-20) containing 5% non-fat dry milk for 1 h at room temperature. After three washing steps in TTBS, blots were incubated with biotin-labeled YB-1 Mab F-D2G5 diluted 1:2,000 and StreptABComplex diluted 1:50 (DAKO Cytomation, Glostrup, Denmark) was added. Following two more washes in TTBS the peroxidase reaction was visualized by ECL system (Amersham, Freiburg, Germany). Immunoprecipitation experiments were performed with the indicated antibodies that were incubated with pre-cleared pansorbin.

ELISA

Recombinant YB-1 protein was expressed in E. coli and affinity purified as previously described [39]. For ELISA 96-well polystyrene plates were incubated with 200 μl of recombinant YB-1 protein solution (3 ng/μl) in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃) overnight at 4°C. After four washing steps with phosphate-buffered saline with 0.05% Tween-20 (50 mM sodium phosphate, 3 mM potassium phosphate, 150 mM sodium chloride [pH 7.4]), blocking with 2.5% low fat milk powder in PBS was performed at 37°C for 1 h. After four additional washing steps Mabs dissolved in washing buffer at 1:50 were added to each well (200 μl) and incubated another hour at 37°C and washed four times. Bound Mabs were detected either by incubation with 100 μl HRP-conjugated polyclonal anti-mouse IgG diluted 1:1,000 in PBS/Tween (non-biotin-labelled Mabs) or by incubation with 100 μl StrepABComplex diluted 1:50 (DAKO Cytomation, Glostrup, Denmark) with biotin-labelled Mabs for 1 h at 37°C and developed with ABTS (2,2'-azino-di-3-ethylbenzthiazoline-6-sulfonic acid)/H₂O₂. One row was used as blank containing immobilised YB-1 protein but with no addition of Mabs.

Immunohistochemistry

Immunohistochemistry was performed as described recently [40] using an established breast tissue microarray [41]. The breast tissue samples of this tissue microarray were obtained from patients treated by primary surgery for breast cancer at the Department of Gynecology, University Hospital Regensburg, Germany, with institutional review board approval. All patients gave informed consent to the study for retention and analysis of their tissue for research purposes. Briefly, tissue sections were deparaffinized and rehydrated, and the endogenous peroxidase activity was quenched by treatment with 3% H₂O₂. Antigen retrieval was performed by pretreatment in citrate buffer [pH 6.0] in a microwave oven (three times for 8 minutes at 600 W). Slides were incubated with a 1:25 dilution of monoclonal biotin-labeled YB-1 antibody in 2% milk powder dissolved in PBS in a humidified chamber overnight at 4°C. After another wash Vectastain avidin-biotin complex (Vector Laboratories, California, USA) was added for 10 minutes. Immunostaining was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma, Deisenhofen, Germany) and hydrogen peroxide for 5 minutes. Finally, sections were counterstained with Mayer's hematoxylin. The primary antibody was omitted for negative controls. Tissue microarray slides were analyzed by a pathologist without knowledge of clinicopathological parameters of the tumors. Nuclear YB-1 staining was simply scored as positive (1) or negative (0), depending on visual inspection of tumor nuclei under high magnification (400×). The decision to score the staining pattern according to a dichotomous criterium, nuclear versus non-nuclear staining seemed justified as the pattern was evident throughout all specimens. There was no tissue with restricted focal nuclear staining. Cytoplasmic YB-1 staining was scored according to the immunoreactivity score (IRS) system developed by Remmele and Stegner [42], subsequently tumors were grouped into low expressers (IRS 0-3) and high expressers (IRS 4-12). For comparison of the F-E2G5 tissue staining pattern with peptide-derived affinity-purified YB-1 antibodies, the following antibodies were utilized: YB-1#1 has been generated by immunization and purification with a peptide corresponding to an N-terminal YB-1 epitope and was purchased from antibodies-online; YB-1#2 has been described by Janz et al. [1])

Statistical analyses of tissue microarray data

Statistical analyses were performed with SPSS version 17.0 (SPSS, Chicago, IL). Differences were considered statistically significant when p < 0.05. Contingency table analyses and two-sided Fisher's exact tests were used to study the statistical association between clinicopathologic and immunohistochemical variables. Recurrence-free and disease-specific survival curves comparing patients with or without any of the factors were calculated using the Kaplan-Meier method, with significance evaluated by two-sided log-rank statistics. Disease-specific survival and recurrence-free survival were measured from time of surgery. For the analysis of recurrence, patients were censored at the time of their last tumor-free clinical follow-up appointment. For disease-specific survival analysis, patients were censored at the time of their last tumor-free clinical follow-up appointment or at their date of death not related to the tumor. A stepwise multivariable Cox regression model was adjusted, testing the independent prognostic relevance of nuclear YB-1 immunoreactivity. The limit for reverse selection procedures was p = 0.01. The proportionality assumption for all variables was assessed with log-negative-log survival distribution functions.

Multiple logistic regression model

Regression analyses were conducted with R 2.8 (r-project.org). The binary response variable y is regressed on the explanatory variables, x₁, x₂, ..., x_q. The model for n observations is defined by ln(p/(1-p)) = Xβ + ε, where ε contains the residual error terms and p = Pr(y = 1). The parameters are estimated by maximum likelihood estimation. The regression coefficients β1, β₂, ..., βq give the change in the response variable corresponding to a unit change in the appropriate explanatory variable, conditional on the other variables remaining constant. The intercept corresponds to the expected value of the response variable y when all the explanatory variables are zero. Significance tests of whether the coefficients take the value zero can be derived on the assumption that for a given set of values of the explanatory variables, y has a normal distribution with constant variance. The data was centered and scaled previous to applying logistic regression. Centering is performed by subtracting the column means (omitting missing values) of x from their corresponding columns, and scaling is done by dividing the (centered) columns of x by their root-mean-square. Explanatory variables were considered statistically significant when P < 0.05.

Characterization of monoclonal YB-1-specific antibody F-E2G5

In a first approach the F-E2G5 antibody was examined for its substrate specificity by western blot analysis using cell extracts from HEK293 cells expressing GFP, YB-1-GFP or YB-1(21-147)-GFP proteins. GFP and YB-1-GFP fusion protein expression was ascertained by detection with monoclonal GFP antibody (Figure 1A, lanes 1-3). Immunoblotting with biotinylated F-E2G5 Mab was performed following addition of non-reducing (lanes 4-6) or reducing buffer (lanes 7-8) to protein samples. Mab F-E2G5 detects full-length YB-1-GFP fusion protein and also the truncated protein encompassing amino acids 21-147. However, detection succeeds only with non-reducing buffer and not under reducing buffer conditions with inclusion of mercaptoethanol (Figure 1A, compare lanes 5 and 7). Endogenous YB-1 protein with a relative molecular weight of 50 kDa was not detected by immunoblotting using F-E2G5 antibody, neither in denaturing nor in non-denaturing gels (data not shown). These results indicate that (i) the epitope(s) recognized by F-E2G5 resides within the YB-1 protein domains aa21-147 and (ii) detection is highly susceptible to the chosen conditions of western blotting.

One may conclude that the GFP-tag stabilizes the YB-1 protein conformation and permits successful detection by means of Mab F-E2G5, as the endogenous YB-1 protein from cell lysates (~50 kDa) was not detected by western blotting (only minute amounts were immunopositive following prolonged exposure). To ensure that Mab F-E2G5 successfully binds YB-1 protein under non-reducing and non-denaturing conditions, we thereafter established a direct ELISA with recombinant, affinity-purified YB-1 protein expressed in E. coli. Biotinylated as well as non-biotinylated Mabs F-E2G5 were tested. Under both conditions recombinant YB-1 protein was detected (Figure 1B), even better with biotinylated Mab F-E2G5. Controls included omission of antibody (Con1) or antigen (Con2) and addition of irrelevant IgG2/irrelevant biotinylated IgG2 antibody (not shown), all yielding negative results. Thus, Mab F-E2G5 binds epitope(s) that reside within YB-1 protein domains aa21 to 147. In addition, a sandwich ELISA was established with a series of GFP-tagged fusion proteins that include the following domains of YB-1 protein: aa21-147, aa146-317, aa146-225, aa146-172, aa260-317, aa146-262, aa21-262. Biotinylated Mab F-E2G5 was able to detect all fusion proteins that encompassed aa146-172. ELISA results were scored positive with an optical density of >0.4, whereas background values were below 0.05. These results support the notion that two distinct conformational epitopes are recognized by the Mab, one within the N-terminal (aa21-147) and one within the centrally localized domains (aa146-172).

To further evaluate the propensity of Mab F-E2G5 to associate with YB-1-GFP or endogenous YB-1 proteins Mab F-E2G5 was evaluated in immunoprecipitation studies (Figures 2A and 2B). Mab F-E2G5 successfully pulled down YB-1-GFP, that was subsequently detected by monoclonal anti-GFP-tag antibody (Figure 2A). Furthermore Mab F-E2G5 immunoprecipitated endogenous YB-1 protein from HEK293 whole cell extracts, with immunoprecipitated protein being detected by polyclonal peptide-derived anti-YB-1 antibody directed against the protein C-terminus (denoted YB-1(C-term), Figure 2B, lane 2). Notably, when endogenous YB-1 protein was immunoprecipitated by polyclonal anti-YB-1(C-term) immunoblotting was unsuccessful under reducing conditions (Figure 2B, lane 3).

Next immunoprecipitated proteins were loaded on gels in non-reducing buffer (omission of mercaptoethanol and EDTA). Under these conditions immunoprecipitated endogenous YB-1 pulled down by monoclonal F-E2G5 antibody was no longer detected with polyclonal anti-YB-1(C-term) antibody. Successful immunoprecipiation by anti-YB-1(C-term) antibody was however possible (Figure 2B, lane 8). Heavy and light chains from immunoglobulins, that are contaminants from the immunoprecipitation procedure, were detected in samples loaded under non-reducing buffer conditions (indicated by "*" in Figure 2B).

From these results it is concluded that monoclonal F-E2G5 antibody has the propensity to immunoprecipitate YB-1-GFP as well as endogenous YB-1 protein, however, detection by means of immunoblotting with the utilized polyclonal anti-YB-1 antibody is susceptible to the chosen buffer conditions.

Mab F-E2G5 is suitable for YB-1 immunodetection in formalin-fixed paraffin-embedded breast cancer tissue

From our previous studies it was clear that the fixation procedure markedly affects detection of YB-1 in rat tissue [43]. In the following we performed immunohistochemistry of sequential tissue sections from patients diagnosed with invasive breast cancer. Biotinylated Mab F-E2G5 and two polyclonal peptide-derived YB-1 antibodies generated against distinct epitopes within the protein N-terminus were utilized. Notably, one of the polyclonal antibodies used (YB-1#2) has been previously applied by Janz et al. [1], the other is commercially available (YB-1#1; see Materials). Sequential sections were stained for Ki67 to identify cycling cells and exclude the possibility that biotinylated Mab F-E2G5 merely detects such cells. As can be seen in Figure 3 there was strong cytoplasmatic staining of cancerous cells with both polyclonal antibodies (#1 and #2). The overall pattern of immunopositive cells was similar with the Mab, however, a considerable fraction of cells exhibited strong nuclear staining. Ki67 positivity was observed in about 10 to 25% of all tumor cells, which was considerably lower than YB-1 positive cells detected by Mab F-E2G5. This finding excludes the possibility that the Mab only detects cycling cells.

From these results we conclude that the established Mab F-E2G5 is suitable for immunohistochemistry with formalin-fixed paraffin-embedded tissue, as routinely performed for histological analyses. The overall staining pattern with Mab and polyclonal antibodies is similar. However, Mab F-E2G5 preferentially detects nuclear YB-1.

Nuclear YB-1 staining pattern in breast cancer tissue

Immunohistochemical analysis was used to investigate YB-1 protein expression in normal breast tissue and breast cancers. We analyzed a tissue microarray with 224 breast tissue samples, i.e. 179 invasive ductal carcinomas, eight ductal carcinoma in situ (DCIS) and 37 normal breast tissue samples. To our knowledge, the latter group is larger than all other control groups analyzed by immunohistochemistry for YB-1 expression so far. Nuclear YB-1 immunohistochemical staining was only detectable in invasive ductal carcinomas, but not in DCIS and normal breast tissue. Figure 4 shows representative images of YB-1 expression in normal and malignant breast tissues. YB-1 staining was not detectable in normal breast tissue (Figures 4A and 4B). In DCIS (Figures 4C and 4D) cytoplasmic YB-1 expression was present in 75% (6/8) of cases, while nuclear YB-1 expression was not present. Invasive ductal carcinomas exhibited nuclear staining in 24% (43/179) of cases (Figures 4E and 4F).

YB-1 expression, clinico-pathological parameters and patient survival

However, nuclear YB-1 expression detected by antibody F-E2G5 was associated with shorter overall survival (OS; p = 0.0046) and exhibited a strong trend towards association with shorter recurrence-free survival (RFS; p = 0.09). These were compared by Kaplan Meier analysis between invasive breast tumours with nuclear YB-1 expression versus all other invasive breast tumours exhibiting no nuclear expression (Figure 5). Patients with nuclear YB-1 expression in the tumor had an estimated mean OS of 90 months (95% confidence interval (CI): 72-109 months) compared to 117 months (95% CI: 108-126 months) in patients with absent nuclear YB-1 immunoreactivity. Nuclear YB-1 detection also correlated with tumor stage (p = 0.004), higher (G2/G3) histological grade (p = 0.011) and negativity of progesterone receptor status (p = 0.002) (Table 1). These strong correlations are demonstrated in Figure 6. In a stratified univariate analysis, the prognostic value of nuclear YB-1 detection became even more pronounced in the clinically important subgroup of stage pT1/T2 tumors, representing ~80% of all diagnosed carcinomas, and breast tumors with negative progesterone receptor status (Figures 6 and 7). While pT1 tumours exhibit no prominent nuclear YB-1 staining (Figure 7A), distinct nuclear YB-1 staining was detectable in pT4 tumours (B). In a similar comparison nuclear YB-1 protein was not detectable in most progesterone receptor positive breast tumours (Figure 7C), while there was prominent nuclear YB-1 staining in most tumors with negative progesterone receptor status (D).