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Cancer Research 68, 1504, March 1, 2008. doi: 10.1158/0008-5472.CAN-07-2362
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Expression of HER2 and Estrogen Receptor {alpha} Depends upon Nuclear Localization of Y-Box Binding Protein-1 in Human Breast Cancers

Teruhiko Fujii1,3,5, Akihiko Kawahara1,4, Yuji Basaki6, Satoshi Hattori2, Kazutaka Nakashima1,4, Kenji Nakano1, Kazuo Shirouzu3, Kimitoshi Kohno7, Takashi Yanagawa2, Hideaki Yamana1,3, Kazuto Nishio8, Mayumi Ono6, Michihiko Kuwano1 and Masayoshi Kage1,4

1 Center for Innovative Cancer Therapy of the 21st Century Center of Excellence Program for Medical Science; 2 Biostatistics Center, Kurume University; 3 Department of Surgery, Kurume University School of Medicine; 4 Department of Pathology, Kurume University Hospital, Kurume, Japan; 5 National Hospital Organization Kyushu Medical Cancer; 6 Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; 7 Department of Molecular Biology, University of Occupation and Environmental Health, Kitakyushu, Japan; and 8 Department of Genome Biology, Kinki University School of Medicine, Osakasayama, Japan

Requests for reprints: Masayoshi Kage, Department of Pathology, Kurume University Hospital, Kurume 830-0011, Japan. Phone: 81-942-31-7651; Fax: 81-942-31-7651; E-mail: masakage{at}med.kurume-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In our present study, we examined whether nuclear localization of Y-box binding protein-1 (YB-1) is associated with the expression of epidermal growth factor receptors (EGFR), hormone receptors, and other molecules affecting breast cancer prognosis. The expression of nuclear YB-1, clinicopathologic findings, and molecular markers [EGFR, HER2, estrogen receptor (ER){alpha}, ERβ, progesterone receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), phosphorylated Akt, and major vault protein/lung resistance protein] were immunohistochemically analyzed. The association of the expression of nuclear YB-1 and the molecular markers was examined in breast cancer cell lines using microarrays, quantitative real-time PCR, and Western blot analyses. Knockdown of YB-1 with siRNA significantly reduced EGFR, HER2, and ER{alpha} expression in ER{alpha}-positive, but not ER{alpha}-negative, breast cancer cell lines. Nuclear YB-1 expression was positively correlated with HER2 (P = 0.0153) and negatively correlated with ER{alpha} (P = 0.0122) and CXCR4 (P = 0.0166) in human breast cancer clinical specimens but was not correlated with EGFR expression. Nuclear YB-1 expression was an independent prognostic factor for overall (P = 0.0139) and progression-free (P = 0.0280) survival. In conclusion, nuclear YB-1 expression might be essential for the acquisition of malignant characteristics via HER2-Akt–dependent pathways in breast cancer patients. The nuclear localization of YB-1 could be an important therapeutic target against not only multidrug resistance but also tumor growth dependent on HER2 and ER{alpha}. [Cancer Res 2008;68(5):1504–12]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nuclear localization of Y-box binding protein-1 (YB-1) is required for its transcriptional control of multidrug resistance–related genes and for its action in repairing DNA damage induced by anticancer agents and radiation in cancer cells; as a result of these actions, it is responsible for the acquisition of global drug resistance to a wide range of anticancer agents (1, 2). Immunohistochemical analyses have shown that nuclear YB-1 localization is a target marker of intrinsic importance for global drug resistance in cancer (2). Bargou et al. (3) reported that nuclear localization of YB-1 was associated with P-glycoprotein expression in human primary breast cancers, and other immunohistochemical studies have shown an association between YB-1 and P-glycoprotein in osteosarcoma, synovial sarcoma, breast cancer, ovarian cancer, and prostate cancer (4–12). Fujita et al. (13) reported that the increase in P-glycoprotein expression when patients were treated with paclitaxel was accompanied by nuclear YB-1 localization in breast cancers.

Nuclear expression of YB-1 is often associated with poor prognosis in various human malignancies, including breast cancer (3, 6), ovarian cancer (8, 11), synovial sarcoma (5), and lung cancer (14). In a study using molecular profiling, Faury et al. (15) recently showed that overexpression of YB-1 is a novel prognostic target for pediatric glioblastoma; however, the intracellular localization of YB-1 was not determined. These clinical studies suggest the close involvement of YB-1 in the acquisition of global drug resistance (2); however, it remains unclear whether the association of YB-1 with poor prognosis is due to this effect, as YB-1 nuclear localization is also a prognostic marker irrespective of P-glycoprotein expression (8, 14, 16). This suggests that other factors affecting tumor growth, invasion, and metastasis could also be involved in the association of YB-1 with poor prognosis in malignant cancers (8, 14).

YB-1 gene induced the development of breast cancers of many histologic types in an experimental animal model (17), suggesting that YB-1 is oncogenic (18). YB-1 overexpression in human mammary epithelial cells induced epidermal growth factor (EGF)-independent growth by activating the EGF receptor (EGFR) pathway (18). Jurchott et al. (19) reported that nuclear localization of YB-1 was induced during G1-S phase transition, accompanied by increased expression of cyclin A and B. These studies suggest a close link between YB-1 expression and the growth potential of breast cancer cells, which might contribute to poor prognosis. Wu and colleagues (20) established a close correlation between YB-1 expression and the expression of EGFR and HER2 in breast cancer patients (n = 389) using tumor tissue arrays. Knock-out of YB-1 in mice caused some embryonic lethality, severe growth retardation, and exencephaly (21, 22). Moreover, fibroblasts derived from YB-1–/– knockout embryos had slower growth rates than those from wild-type embryos, and failed to undergo morphologic transformation in vitro (22, 23). Sutherland et al. (24) have also shown that breast cancer cells with defective nuclear localization of YB-1 multiply slowly in monolayers and during anchorage-independent growth. Taken together, these findings suggest that YB-1 plays a key role in the expression of not only drug resistance–related genes but also cell growth–related genes.

In the present study, we determined whether nuclear YB-1 localization influenced the expression of growth factor and hormone receptors, EGFR, HER2, estrogen receptor (ER){alpha}, and ERβ, in human breast cancers. In addition, we used molecular profiling to examine whether nuclear YB-1 localization affected the expressions of major vault protein/lung resistance protein (MVP/LRP), phosphorylated Akt (p-Akt), progesterone receptor (PgR), and chemokine (C_X_C motif) receptor 4 (CXCR4).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, protein extraction, and immunoblotting. Human breast cancer cell lines, T-47D, MCF-7, KPL-1, MDA-MB231, and SKBR-3 were obtained from the American Type Culture Collection and were grown as described elsewhere (25). Anti–YB-1 was generated as described previously (26). Anti-EGFR and anti-PTEN antibodies were obtained from Cell Signaling Technology. Anti-HER2 was purchased from Upstate, Inc. Anti-ER{alpha} was obtained from Santa Cruz Biotechnology, Inc. Anti-CXCR4 was obtained from Abcam plc. Anti– glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Trevigen, Inc. Anti-MVP/LRP was a kind gift from Professor S. Akiyama (Kagoshima University, Kagoshima, Japan). LY294002 was obtained from Sigma Co. Trastuzumab was purchased from Chugai Pharmaceutical Company. Cells were lysed in cold protein extraction reagent (M-PER; Pierce) with protease inhibitors and phosphatase inhibitors. Nuclear and cytoplasmic fractions were prepared as described previously (27). Lysates were subjected to SDS-PAGE and blotted onto Immobilon membrane (Millipore Corp.). After transfer, the membrane was incubated with the primary antibody and visualized with secondary antibody coupled to horseradish peroxidase and Supersignal West Pico Chemiluminescent Substrate (Pierce). Bands on Western blots were analyzed densitometrically using Scion Image software (version 4.0.2; Scion Corp.).

Microarray analysis. The small interfering RNA (siRNA) corresponding to nucleotide sequences of YB-1 (5'-GGU UCC CAC CUU ACU ACA U-3') was purchased from QIAGEN Inc. SiRNA duplexes were transfected using Lipofectamine and Opti-MEM medium (Invitrogen) according to the manufacturer's recommendations. Duplicate samples were prepared for microarray hybridization. Forty-eight hours after siRNA transfection, total RNA was extracted from cell cultures using ISOGEN (Nippon Gene Co. Ltd.). Two micrograms of total RNA were reverse transcribed using GeneChip 3'-Amplification Reagents One-Cycle cDNA Synthesis kit (Affymetrix, Inc.) and then labeled with Cy5 or Cy3. The labeled cRNA was applied to the oligonucleotide microarray (Human Genome U133 Plus 2.0 Array; Affymetrix). The microarray was scanned on a GeneChip Scanner3000, and the image was analyzed using a GeneChip Operating Software ver1.

Quantitative real-time PCR. RNA was reverse transcribed from random hexamers using avian myeloblastosis virus reverse transcriptase (Promega). Real-time quantitative PCR was performed using the Real-time PCR system 7300 (Applied Biosystems). In brief, the PCR amplification reaction mixtures (20 µL) contained cDNA, primer pairs, the dual-labeled fluorogenic probe, and Taq Man Universal PCR Master Mix (Applied Biosystems). The thermal cycle conditions included maintaining the reactions at 50°C for 2 min and at 95°C for 10 min, and then alternating for 40 cycles between 95°C for 15 s and 60°C for 1 min. The primer pairs and probe were obtained from Applied Biosystems. The relative gene expression for each sample was determined using the formula 2 (–{delta} Ct) = 2 (Ct (GAPDH)–Ct (target)), which reflected the target gene expression normalized to GAPDH levels.

Immunohistochemistry. Anti-EGFR, anti-HER2, anti-ER{alpha}, and anti-PgR were obtained from Ventana Medical Systems. Anti-CXCR4 was purchased from Prosci, Inc. Anti-MVP/LRP was obtained from Chemicon. Tissue sections were taken from 73 breast cancer patients who underwent radical surgery in the Department of Surgery, Kurume University Hospital, Japan, between 1993 and 1999. The 4-µm tissue sections were deparaffinized, and the slides were heated in a Cell Conditioning Solution buffer for 60 min at 100°C. The sections were stained using the BenchMark XT (IHC Automated System) and ChemMate ENVISION method (Dako Corporation). BenchMark XT was used for staining anti–YB-1, anti-ER{alpha}, anti-EGFR, anti-HER2, and anti-PgR. The ChemMate ENVISION method was used for immunochemical staining of anti-ERβ, anti–p-Akt, anti-CXCR4, and anti-MVP/LRP. The samples were viewed using an Olympus BX51 fluorescence microscope (Olympus). The extent of staining of YB-1, ER{alpha}, ERβ, and PgR proteins was classified based on the percentage of cells with strongly stained nuclei: ≥10% indicated that a gland was positive for YB-1, and ≤9% indicated that it was negative. EGFR and HER2 expressions were classified into four categories: score 0, no staining at all or membrane staining in <10% of tumor cells; score 1+, faint/barely perceptible partial membrane staining in >10% of tumor cells; score 2+, weak to moderate staining of the entire membrane in >10% of tumor cells; and score 3+, strong staining of the entire membrane in >10% of tumor cells. The extent of immunohistologic staining for EGFR was defined as follows: scores of 2+ or 3+ were regarded as positive, and scores of 0 or 1+ were regarded as negative. The extent of immunohistologic staining for HER2 was defined as follows: scores of 3+ were regarded as positive, and scores of 0 or 1+ or 2+ were regarded as negative. Immunohistochemical staining of p-Akt and CXCR4 was defined based on the percentage of cells with strong cytoplasmic staining as follows: ≥10% indicated that a gland was positive, whereas ≤9% indicated that it was negative. MVP/LRP staining was defined as follows: ≥50% of cells with a strongly stained cytoplasm indicated that a gland was positive, whereas ≤49% indicated that it was negative. All immunohistochemical studies were evaluated by two experienced observers who were blind to the conditions of the patients.

Statistical analysis. The associations between YB-1 and clinicopathologic findings (age, tumor size, menopausal status, histologic grade, and lymph node metastasis) and molecular markers were tested by Fisher's exact test, the {chi}2 test, or the Wilcoxon rank-sum test, depending on the type of data. A P value of <0.05 was regarded as significant unless otherwise indicated. The relationships between YB-1 expression and overall survival/progression-free survival, as well as other clinicopathologic findings and molecular markers, were examined by the Kaplan-Meier method and the log-rank test. Hazard ratios (HR) were estimated by Cox regressions.

As YB-1 and the expression of receptors of the EGFR family and hormone receptors, as well as the clinicopathologic findings, were all correlated, we summarized them by means of their principal components and investigated the relationship between these components and overall survival/progression-free survival by Cox regression. The relationship between the principal components was found to be related to overall survival/progression-free survival, and the clinicopathologic findings and molecular markers were investigated by studying their correlations. In addition, to obtain a direct representation of the relationship between molecular markers, we used a graphical modeling technique incorporating logistic regressions; a path was drawn between two markers only if these markers were conditionally associated with a significance level of 0.1, given the other markers. The data on overall survival and progression-free survival in this analysis were updated on February 27, 2007.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The knock-down of YB-1 alters the expression of EGFR, HER2, ER{alpha}, CXCR4, and MVP/LRP genes. We initially compared the expressions of YB-1 siRNA–treated and control MCF-7 breast cancer cells using a high-density oligonucleotide microarray. Of the 54,675 RNA transcripts and variants in the microarray, we identified differentially expressed genes containing 43 genes that were up-regulated >2-fold and 203 genes that were down-regulated 0.5-fold or less (Supplementary Table S1). It has been reported that the activity of PI3K/Akt was required for translocation of YB-1 into the nucleus (24, 27). We therefore investigated the effect of LY294002, a selective inhibitor of PI3K, in both T-47D and MDA-MB231 cells. LY294002 inhibited the nuclear expression of YB-1 in both cell lines (Supplementary Fig. S1A), consistent with previous reports (24, 27). We also examined whether PTEN status was correlated with nuclear YB-1 expression in breast cancer cells. Of the five human breast cancer cell lines used, cellular levels of the PTEN were not significantly correlated with nuclear expression levels of YB-1 protein (Supplementary Fig. S1B and C).

The differentially expressed genes included MVP/LRP and CXCR4, consistent with our previous study on human ovarian cancer cells (27). We next tested, by quantitative real-time PCR (qRT-PCR), whether the expression of CXCR4 and MVP/LRP was affected by knock-down of YB-1 in various human breast cancer cell lines. We also examined the expressions of growth factor receptors and hormone receptors, such as EGFR, HER2, ER{alpha}, and ERβ, which are thought to be important target genes in breast cancer. The five cell lines used were as follows: T-47D, MCF-7, and KPL-1, which are ER{alpha}-positive; and MDA-MB231 and SKBR-3, which are ER{alpha}-negative. Transfection of YB-1 siRNA decreased the expression of YB-1 mRNA by ≥70% in all five cell lines (Fig. 1A ). Both EGFR and HER2 mRNA levels were found to be decreased in YB-1 siRNA–treated T-47D and KPL-1 cells but not in MDA-MB231 and SKBR-3 cells (Fig. 1B). EGFR and HER2 mRNAs were not detected in MCF-7 cells. It has been reported that the 5' regulatory region of the ER{alpha} gene contains several Y-box–like sequences. Cellular mRNA levels of ER{alpha} were reduced by YB-1 siRNA in T-47D, KPL-1, and MCF-7 cells by 74%, 75%, and 40%, respectively, (Fig. 1B). CXCR4 and MVP/LRP mRNA levels were also decreased in YB-1 siRNA–treated T-47D and KPL-1 cells but not in MDA-MB231 and SKBR-3 cells (Fig. 1B).


Figure 1
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  		Figure 1. Effect of YB knock-down on expression of EGFR, HER2, ER{alpha}, CXCR4, and MVP/LRP in ER{alpha}-positive and ER{alpha}-negative breast cancer cells. A, YB-1 knock-down by treatment of YB-1 siRNA for 48 h. Relative mRNA expression was measured by qRT-PCR. Columns, mean of three independent experiments; bars, SD. B, levels of EGFR, HER2, ER{alpha}, ERβ, PgR, CXCR4, and MVP/LRP mRNA expression in YB-1 siRNA–treated cells. Changes of mRNA expression were expressed as the log of relative expression. ND, not detected. C, T-47D and MDA-MB231 cells were incubated with YB-1 siRNA for 72 h, and lysates were prepared. D, levels of YB-1, EGFR, HER2, ER{alpha}, CXCR4, and MVP/LRP expression were measured by densitometry.

 
Western blot analysis showed that siRNA to YB-1 decreased protein levels of EGFR, HER2, and ER{alpha} in T-47D cells; however, we did not observe decreased expression of EGFR and HER2 as a result of YB-1 knock-down in MDA-MB231 cells (Fig. 1C and D). These observations show that YB-1 only reduces the expression of EGFR and HER2 when ER{alpha} is present. Moreover, the expression of ER{alpha} was also affected by YB-1 knock-down. We next examined the causal relationship between HER2, ER{alpha}, and nuclear YB-1 in breast cancer cells in culture. Treatment with HER2-targeting trastuzumab blocked the nuclear localization of YB-1 in both T-47D and MDA-MB231, but the inhibitory effect was less in MDA-MB231 (Supplementary Fig. S2A). By contrast, nuclear YB-1 expression was not affected by ER{alpha} knock-down in T-47D cells in culture (Supplementary Fig. S2B). HER2 might directly modulate the cellular localization of YB-1 in breast cancer cells; however, ER{alpha} might not directly affect nuclear YB-1 localization.

Immunostaining of EGFR, HER2, ER{alpha}, ERβ, CXCR4, p-Akt, and MVP/LRP in human breast cancers. To examine which genes are specifically associated with nuclear YB-1 localization in human breast cancers, we selected eight molecular markers: EGFR, HER2, ER{alpha}, ERβ, PgR, CXCR4, p-Akt, and MVP/LRP. Representative immunohistochemical staining patterns in the presence and absence of nuclear YB-1 are shown in Fig. 2 . Expression of nuclear YB-1 was detected in 30 of 73 patients (40%; nuclear YB-1 positive). Clinical and pathologic characteristics at diagnosis of the 73 patients in this study are summarized in Supplementary Table S2. There was no significant correlation between the expression of nuclear YB-1 and age (P = 0.2562), histologic grade (P = 0.1910), menopausal status (P = 0.1508), tumor size (P = 0.1478), or lymph node metastasis (P = 0.0620).


Figure 2
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  		Figure 2. Histologic findings and expression of YB-1, EGFR, HER2, ER{alpha}, ERβ, PgR, CXCR4, p-Akt, and MVP/LRP in human breast cancer. YB-1 expression was recognized in two patterns: nuclear positive or negative. Cancer cells showed strong expression of EGFR and HER2 in the membrane. Strong expression of ER{alpha}, ERβ, and PgR was found in the nucleus. Moderate-to-strong expressions of CXCR4, p-Akt, and MVP/LRP were found in the cytoplasm.

 
Figure 2 also shows representative examples of immunohistochemical staining for EGFR, HER2, ER{alpha}, ERβ, PgR, CXCR4, p-Akt, and MVP/LRP. There were significant correlations between the expression of nuclear YB-1 and HER2 (P = 0.0153), ER{alpha} (P = 0.0122), and CXCR4 (P = 0.0166; Table 1 ). By contrast, there was no significant correlation between nuclear YB-1 expression and the expression of EGFR (P = 1.0000), PgR (P = 0.0944), ERβ (P = 0.0576), p-Akt (P = 0.0521), or MVP/LRP (P = 0.0577).


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  		Table 1. Correlation between nuclear YB-1 expression and expression of eight target genes

 
Effects of nuclear YB-1 on survival and other molecular markers. The estimated product-limit survival functions of nuclear YB-1 are shown in Fig. 3A (overall survival) and Fig. 3B (progression-free survival), as well as the results of log-rank tests. Survival curves for patients with nuclear YB-1 were significantly different from those without nuclear expression (P = 0.0139 for overall survival; P = 0.0280 for progression-free survival). The results of log-rank tests for other factors are given in Table 2 , showing that the tests for lymph node metastasis were significant (P = 0.0001 for overall survival; P < 0.0001 for progression-free survival).


Figure 3
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  		Figure 3. Kaplan-Meier overall survival (A) and progression-free survival (B) according to nuclear YB-1 expression in 73 patients with breast cancer. Nuclear expression of YB-1 has a significant predictive value for survival.

 

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  		Table 2. Univariate analysis of patient characteristics and target gene expression regarding overall survival and progression-free survival

 
The first eight principal components were used in the subsequent analysis, as their cumulative coefficients of variance were ~80%. Denoting the i-th principal component by PRINi, the results of Cox regression analysis were as follows. For overall survival, PRIN1 and PRIN7 were statistically significant [HR = 1.52 and P = 0.0090 (for PRIN1); HR = 2.06 and P = 0.0499 (for PRIN7); Fig. 4A ]; and for progression-free survival, PRIN1, PRIN6, PRIN7, and PRIN8 were significant [HR = 1.59 and P = 0.0009 (for PRIN1); HR = 1.86 and P = 0.0103 (for PRIN6); HR = 2.30 and P = 0.0078 (for PRIN7); HR = 1.68 and P = 0.0508 (for PRIN8); data not shown]. PRIN1 was positively correlated with YB-1 [correlation coefficient (r) = 0.593], HER2 (r = 0.397), histologic grade (r = 0.557), tumor size (r = 0.577), and lymph node metastasis (r = 0.522). PRIN1 was negatively correlated with ER{alpha} (r = –0.684), PgR (r = –0.453), CXCR4 (r = –0.460), menopausal status (r = –0.618), and age (r = –0.607). This might indicate that some effect shared by YB-1, HER2, ER{alpha}, PgR, and CXCR4 leads to poor survival. PRIN7 was positively correlated with tumor size and negatively correlated with EGFR and p-Akt. Note that PRIN7 was not correlated with YB-1; this points to the existence of different mechanisms that influence survival apart from those involving PRIN1.


Figure 4
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  		Figure 4. Statistical modeling of nuclear YB-1 localization–based network in human breast cancer. A, relationships among principal components, which were found significantly related to overall survival (PRIN1 and PRIN7) and clinicopathologic findings/molecular markers. Principal components and clinicopathologic findings or molecular markers are linked by a line if and only if the absolute value of correlation coefficient among them is >0.3. Each line is labeled by the correlation coefficient. B, relationship of molecular markers by graphical modeling incorporating with logistic regressions (+, positive correlation; –, negative correlation).

 
Stepwise variable selection was used to select the following molecular markers for graphical modeling: YB-1, HER2, ER{alpha}, ERβ, and CXCR4. Figure 4B shows the results of graphical modeling of these markers when two markers were positively correlated; a plus symbol is shown on the path, otherwise a minus symbol is shown. The relationships are indicated between markers; for example, YB-1 is related to CXCR4, ERβ, and HER2 but not directly to ER{alpha}. Note that HER2, YB-1, CXCR4, and ER{alpha} are correlated with PRIN1, emphasizing their important effects on survival.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we assessed whether the expression of EGFR and ErbB2/HER2 was affected by YB-1 in breast cancers, as this might influence prognosis. We developed two independent approaches to identify which genes are under the control of YB-1 in human breast cancer cells. One approach involved microarray analysis, qRT-PCR, and immunoblotting to determine whether the expression of EGFRs, ER{alpha}, and other YB-1–related proteins is controlled by YB-1 in culture. The other approach consisted of immunohistochemical analysis of those protein molecules closely associated with nuclear localization of YB-1 in patients with breast cancer.

The expression of EGFR and HER2 was down-regulated by YB-1 knock-down in ER{alpha}-positive, but not ER{alpha}-negative, breast cancer cell lines, suggesting that YB-1 siRNA–induced suppression depends upon the presence of ER{alpha}. By contrast, immunohistochemical analysis showed that nuclear YB-1 expression was significantly associated with the expression of HER2 but not of EGFR. Janz et al. (6) have also reported a close association of YB-1 nuclear localization with the expression of HER2 in primary breast cancers. Moreover, Wu et al. (20) found that the introduction of a p-Akt–insensitive mutation into YB-1 markedly decreased the expressions of both EGFR and HER2, suggesting a close linkage between YB-1 and EGFR/HER2 expression in breast cancer cells in culture. YB-1 overexpression in human breast epithelial cells did not affect HER2 but caused up-regulation of EGFR, with concomitant EGF-independent phosphorylation of EGFR (18). The effect of YB-1 on EGFR and/or HER2 might depend in part on the particular cell line examined.

Oda et al. (11) found a highly significant association of p-Akt with nuclear YB-1 expression in human ovarian cancers, and both p-Akt and nuclear YB-1 expression were independent prognostic biomarkers; however, we observed no statistically significant association of p-Akt expression with nuclear YB-1 expression in our immunohistochemical analysis (Table 1). Cross-talk between growth factor receptors, such as EGFR, insulin-like growth factor (IGF) receptor, and estrogen signaling cascades occurs at the level of ER{alpha} (28, 29); this leads to activation of PI3K/Akt and ultimately to activation of ER{alpha} (30, 31). Thus, activation of ER{alpha} as well as YB-1 and its translocation to the nucleus seem to be coordinately controlled in breast cancer cells by the PI3K/Akt pathway in response to growth factors such as EGF/transforming growth factor {alpha} and IGF. PI3K/Akt activation could therefore be primarily dependent on the active state of ER{alpha}, which seems to play a major role in the nuclear translocation of activated YB-1 in ER{alpha}-positive breast cancer cells. In relation to a possible association of hormone receptors with nuclear YB-1 localization, we found that the expression of ER{alpha} and ERβ was down-regulated by YB-1 knock-down. Wu and colleagues (20) have reported an inverse relationship between ER{alpha} and YB-1 in breast cancer samples. In the present study, ER{alpha} expression was inversely correlated with nuclear YB-1 localization, whereas ERβ expression was positively correlated with nuclear YB-1 localization. Like ER{alpha}, ERβ expression is closely associated with the PI3K/Akt signaling cascade (32). ERβ has emerged as an important determinant in breast cancer (33) and is a useful biomarker for breast cancer independent of ER{alpha} expression (34). The close linkage of nuclear YB-1 localization with ERβ expression points to the presence of a novel signaling pathway that could be a target for anticancer therapy in breast cancer.

We examined two targets of YB-1, MVP/LRP and CXCR4, which were identified by our expression profiling analysis. MVP/LRP expression, which is involved in drug resistance, is promoted by 5-fluorouracil and other anticancer agents in response to transcriptional activation by YB-1, suggesting a direct link between YB-1– and MVP/LRP-mediated drug resistance (35–37). MVP/LRP expression was not affected by YB-1 knock-down in ovarian cancer cells in culture, although nuclear YB-1 expression and MVP/LRP expression are closely associated in patients with ovarian cancer (11, 27). CXCR4 is also known to play a critical role in the growth and metastasis of human breast cancers (38, 39). CXCR4 expression was down-regulated in YB-1 siRNA–treated ovarian cancer cells, and nuclear YB-1 expression was closely associated with CXCR4 expression in clinical samples of human ovarian cancers (11, 27). A significant positive association of nuclear YB-1 location with CXCR4 expression in breast cancer was also shown in the present study.

Nuclear localization of YB-1, in part mediated by Akt activation, thus modulates the expressions of EGFR, HER2, ER{alpha}, ERβ, and CXCR4 in breast cancer cells. YB-1–driven cell signaling of growth, survival, and hormone responses might be mainly mediated by transcriptional activation of the above-mentioned genes (1, 2); however, from our biostatistical analysis, YB-1 nuclear expression was positively associated with the expression of HER2, and negatively associated with the expressions of CXCR4 and ERβ (Fig. 4B). Moreover, ER{alpha} expression was positively correlated with CXCR4 expression and negatively correlated with HER2 expression. Although there remain inconsistencies between the data for cultured breast cancer cells and actual breast cancers with regard to the relationship between YB-1 nuclear location and the expression of other biomarkers, our biostatistical linkage map should provide important information for the development of strategies for molecular diagnosis and therapy.

In conclusion, nuclear YB-1 expression might be a prognostic marker in breast cancer. Furthermore, YB-1 plays a key role in the network annotation of genes such as HER2, CXCR4, ER{alpha}, and ERβ (Fig. 4). In addition to YB-1–mediated acquisition of multidrug resistance, the close association of nuclear YB-1 localization with HER2 expression should be considered part of the underlying mechanism. The determination of the nuclear versus cytoplasmic localization of YB-1 might provide a useful molecular indicator for personalized therapeutics of anticancer drugs targeting HER2 and/or ER{alpha}.


   Acknowledgments
 
Grant support: Centers of Excellence program for Medical Science, Kurume University, Japan; and a Grant-in-Aid for Scientific Research on Priority Areas, Cancer, from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M. Ono); and by the Third Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare, Japan (M. Kuwano). This study was also supported, in part, by the Formation of Innovation Center for Fusion of Advanced Technologies, Kyushu University, Japan (M. Ono and M. Kuwano).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

T. Fujii, A. Kawahara, Y. Basaki, and S. Hattori contributed equally to this work.

Received 6/25/07. Revised 12/12/07. Accepted 12/27/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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