Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer

NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction in breast cancer.


Introduction
Breast cancer is the most common carcinoma in women and the second most common cause of cancer death in females [1]. Early detection in conjunction with screening programs and the advent of more efficacious and targeted adjuvant systemic therapy have contributed to the decrease in breast cancer mortality [1]. The effectiveness of pathway-specific targeted and patient-tailored therapeutics demands the need for continued advances in our understanding of the molecular biology of breast cancer progression and discovery of new prognostic markers [1].
The ductal and lobular subtype constitute the majority of all breast cancers worldwide, with the ductal subtype accounting for 40-75% of all diagnosed cases [2][3][4][5]. Nearly 80% of all diagnosed in situ and invasive breast cancers are of ductal origin [1,6]. In 2012, an estimated 229,060 new cases of breast cancer were expected to be diagnosed and approximately 39,920 deaths were expected to occur in the United States alone [7]. Breast cancer is the most common malignant disease in Western women, and distant metastasis are the main cause of death [6]. Here, we reveal a new potential diagnosis marker for breast invasive ductal carcinoma (IDC).
The Nogo isoforms-A, -B and -C are members of the reticulon family of proteins. Nogo-A and Nogo-C are highly expressed in the central nervous system (CNS), with Nogo-C also uniquely found in skeletal muscle, while Nogo-B is found in most tissues [8,9]. Nogo-A (also called RTN4-A) binds its specific receptors, such as NgR and LiNGO1, and acts as a negative regulator of axon sprouting [10][11][12][13]. Nogo-B was previously identified as a protein that is highly expressed in caveolin-1 enriched microdomains of endothelial cells (EC) [14]. The amino terminus (residues1-200) of Nogo-B (AmNogo-B) serves as a chemoattractant for EC [14]. Mice deficient in Nogo-A/B show exaggerated neointimal proliferation, abnormal remodeling [14] and a deficit in ischemia induced arteriogenesis and angiogenesis [15]. NgBR was identified as a receptor specific for AmNogo-B by an expression cloning approach [16]. High affinity binding of AmNogo-B to NgBR is sufficient for AmNogo-B mediated chemotaxis and tube formation of endothelial cells [16]. We have previously demonstrated that NogoB-NgBR ligand-receptor pair is necessary for in vivo angiogenesis in zebrafish [17]. Genetic knockdown of NogoB or NgBR by antisense morpholinos abolished intersomitic vessel (ISV) formation during developmental angiogenesis [17]. Our recent studies further demonstrated that NgBR is essential for Ras activation in breast tumor cells [unpublished data]. However, there is no information regarding the roles of Nogo-B and NgBR in any kind of cancers, including breast cancer. Here, we demonstrate the expression patterns of Nogo-B and NgBR, their relationships with different molecular subtypes of breast cancer, and their possible roles in promoting tumor cell growth in breast cancer.

Tissue Microarray Slides
Three cohorts for a total of 656 breast tumor tissues and 15 normal breast tissues on tissue microarray (TMA) slides were used in this study. The first cohort, composed of 190 breast tumors and 15 normal breast tissues with duplicate cores for each case, was purchased from Shanghai Biochip Co [18]. The second cohort composed of 210 breast tumors with a single core for each case was obtained from the breast tissue bank at the Baylor College of Medicine. The third cohort composed of 256 breast tumor tissues with pathological information was purchased from BioChain (Newark, CA). All these breast cancer cases were histopathologically re-evaluated on hematoxylin and eosin-stained slides by two pathologists (BW and JH). These breast tissue specimens are anonymous and have institutional IRB (Institutional Review Board for Baylor College of Medicine) exemption.

NgBR and Nogo-B Antibody Generation
The peptide (AHHRMRWRADGRSLEK, residues from 81-96 of NgBR) was used to immunize rabbits (Epitomics, Burlingame, CA). The antiserum was purified using the same peptide-conjugated SulfoLink Coupling Gel (Pierce, Rockford, IL). The peptide recognizing epitope 14 to 30 of human Nogo-B was used to immunize rabbits (IMG-5346A, Imgenex, San Diego, CA). In addition, NgBR rabbit monoclonal antibody (Clone ID: EPR8668) also was generated by Epitomics as a collaboration project and was used for Western blot analysis.
Cell growth assay. MCF-7 and T47D cells were subcultured to each well of 12 wells plate. After overnight culture, cells were transfected with non-silencing siRNA (NS, negative controls) or siRNA specifically targeting NgBR (siNgBR). The next day after transfection, cells were treated with 10 nM b-estradiol (Sigma). After 24 hours or/and 48 hours treatment, the total viable cell number was determined using Bio-Rad TC10 TM automated cell counter Bio-Rad. Western blot analysis. Total cell lysates were prepared by adding 200 ml of cell lysate buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100, and 1 mg/ml leupeptin. Total cell extract (50 mg) was separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). Total levels of survivin, ER and NgBR were determined by using specific antibodies, survivin rabbit polyclonal antibody from Novus, ER rabbit monoclonal antibody from Dako and NgBR rabbit monoclonal antibody from Epitomics.
Real-time PCR. Survivin and NgBR transcripts in breast cancer were determined by real-time PCR. Normalized breast cancer cDNA arrays (BCRT101, BCRT102 and BCRT104), survivin primer and survivin standard were utilized (Origene). Total RNA was also isolated from T47D, MDA-MB-468 and MCF-7 cell lines using RNeasy mini plus kit (Qiagen). One mg RNA was used for reverse transcription (RT) using iScript cDNA synthesis kit (BioRad). The forward and reverse primers for NgBR are 59-tgccagttagtagcccagaagcaa-39 and 59-tgatgtgccagggaagaaagccta-39, respectively. The forward and reverse primers for survivin are 59-caaggagctggaaggctg-39 and 59-ttcttggctctttctctgtcc-39, respectively. Beta-actin was used as a normalized control. The forward and reverse primers for Beta-actin are 59-ttctacaatgagctgcgtgtggct-39 and 59-tagcacagcctggatagcaacgta-39 respectively. Real-time PCR analysis was performed with Bio-Rad MyiQ detection system.

Statistical Analysis
Histological data was analyzed using statistical software SPSS 16.0 for Windows. The relationship was tested using Pearson Chisquare tests. A p-value,0.05 defined statistical significance. Quantitative scoring of NgBR and survivin immunostaining, real-time PCR and cell growth data are presented as mean 6 the standard error of the mean (SEM) and the statistical significance of differences was evaluated with the ANOVA analysis. Significance was defined as p,0.05. Correlation of NgBR and Survivin were analyzed using Pearson's correlation coefficient analysis.

Specificity of Nogo-B and NgBR IHC Staining
To confirm the specificity of NgBR and Nogo-B IHC, we performed IHC staining in human IDC tissue sections and used primary antibodies preabsorbed with their corresponding epitope peptide-conjugated beads as negative controls. As shown in Figure  S1, expression of both NgBR and Nogo-B proteins were observed only in the cytoplasm or in membrane/cytoplasm of cancer cells, and smooth muscle cells or endothelial cells of blood vessels. The expression of NgBR in smooth muscle cells was much stronger than endothelial cells. On the contrary, the expression of Nogo-B in endothelial cells was stronger than in smooth muscle cells. As negative controls, the same primary NgBR and Nogo-B antibodies were preabsorbed using their corresponding epitope peptideconjugated beads. There are no specific staining signals in cancer cells or smooth muscle cells as well as endothelial cells of blood vessels. IHC staining using preabsorbed NgBR and Nogo-B antibodies confirmed the specificity of NgBR and Nogo-B IHC staining.

Expression of Nogo-B and NgBR in Normal Breast Tissue
As shown in Figure 1 (panel A-C), expression of NgBR, Nogo-B and survivin proteins were detected in most of the epithelial and myoepithelial cells in the normal breast tissue. The staining intensity in myoepithelial cells was stronger than in epithelial cells. In addition, the staining intensity was variable with gland-to-gland, cell-to-cell, and regional heterogeneity within a case. There is no obvious difference of expression locations between NgBR and Nogo-B in breast tissue. In addition, it is consistent with our previous discovery that expression of Nogo-B and NgBR are also detected in interstitial blood vessels of breast tissues. Based on the scoring system previously described in the methods section, we analyzed the relationship of Nogo-B and NgBR expression with survivin expression as well as other well known breast cancer molecular subtype markers, such as ER, PR, Her2 and CK5/6. The statistical analysis results ( Table 2) showed that higher expression of NgBR is frequently detected in ER-positive, and HER2-negative IDC. The expression pattern of survivin is also consistent with higher NgBR expression, namely in ERpositive, and HER-2 negative IDC. Based on molecular subtypes of IDC, NgBR is highly expressed in non-triple negative breast cancer, particularly in luminal A subtype (ER-positive and/or PRpositive, HER2-negative) of breast cancer. Although there is a strong correlation between Nogo-B and NgBR, the presence of Nogo-B as determined by IHC staining is higher than NgBR in IDC. The breakdown of the distribution of Nogo-B in IDC tumors was as follows: 8.2% negative staining, 23.5% weak and 68.2% strong. As shown in Table 2, expression of Nogo-B only has correlation with survivin, but does not have significant correlation with ER, PR, HER2 and any molecular subtypes. We further analyzed the association of NgBR and survivin expression with the progression of breast cancer. As shown in Table 3, the score of NgBR and survivin IHC staining increased in the later stages of breast cancer and the correlation of their expression in different stages of breast invasive ductal adenocarcinoma is statistically significant.

Expression of Nogo-B and NgBR in Invasive Ductal Carcinoma
To further confirm the correlation of NgBR and survivin expression in breast cancer, we used a real-time PCR approach to determine the copy number of NgBR and survivin transcripts in normal and different stages of breast cancers. Three human breast tumor qPCR panels (BCRT101, BCRT102, BCRT104) were used (Origene). The panels contained a total of 136 normalized cDNAs prepared from pathologist-verified human breast tumor specimens, including 16 normal breast tissue samples, and 120 ductal adenocarcinoma tissue samples. Accompanying pathology reports were used to categorize the 120 ductal adenocarcinoma specimens into four different disease stages from I to IV. Real-time PCR results ( Figure 2A) show that NgBR expression is significantly higher in Stage II (53 samples) and Stage III-IV (44 samples) ductal adenocarcinoma specimens when compared with both normal breast samples (16 samples) and Stage I ductal adenocarcinoma samples (23 samples). Consistent with NgBR expression pattern, survivin expression ( Figure 2B) is significantly higher in Stage II (53 samples) and Stage III-IV (44 samples) ductal adenocarcinoma specimens when compared with normal breast samples (16 samples) and Stage I ductal adenocarcinoma samples (23 samples). We also found that expression of NgBR and survivin has statistically significant correlations in the Stage II (correlation = 0.448, p,0.05) and Stage III-IV (correlation = 0.386, p,0.05) of ductal adenocarcinoma samples, but there are no statistically significant correlations in normal and Stage I groups (Table 4). Combined with IHC staining results, our data clearly demonstrated that NgBR expression is strongly associated with survivin expression in later stages of ductal carcinomas.  To confirm the specificity of NgBR siRNA, we used a second siRNA (siNgBR2) targeting the coding region of NgBR to confirm both siNgBR1 and siNgBR2 can efficiently knock down NgBR and specifically abolished the estradiol-induced expression of survivin in MCF-7 breast tumor cells (Fig. S2). In addition, we used realtime PCR approach to examine the change of survivin gene expression. As shown in the Figure S3A, estradiol treatment increases the survivin gene expression in ER-alpha positive MCF-7 and T47D breast tumor cells but not in ER-alpha negative MDA-MB-468 cells. As shown in the Figure S3B, estradiol increases the survivin gene expression in MCF-7 cells (24 hour: 1.49060.084 fold increase) and NgBR knockdown reduces estradiol-induced survivin gene expression (24 hour: 1.02160.096 fold increase). As shown in Fig. 3A and 4A, NgBR knockdown did not reduce estrogen receptor expression. However, estradiol treatment caused the decrease of ER-alpha levels because of ER-alpha recycling [20] , [21]. In addition, we also examined the effects of NgBR knockdown on estradiol-stimulated cell growth. As shown in Figure 4C, estradiol treatment increases the growth of MCF-7 cells by 11.8% at 24 hours and 29.1% at 48 hours (n = 3, p,0.05), respectively, and NgBR knockdown abolishes the estradiolstimulatory effects (    As shown in Figure 4C and Figure S4A, NgBR knockdown does not reduce the basal growth of both MCF-7 and T47D breast tumor cells, respectively. These results demonstrate that NgBR is essential for estrogen-depended survivin induction and ER positive breast tumor cell growth.

Discussion
Although Nogo-B and NgBR have been shown to play important roles in regulating endothelial cell migration and blood vessel formation [14,16,17], the roles of Nogo-B and NgBR in cancer cells and cancer progression are still unclear. Nogo-B (also known as ASY) was previously identified as one of the apoptosisinducing genes in human cancer [22]. Ectopic expression of the Nogo-B/ASY gene led to extensive apoptosis, particularly in cancer cells [22]. It was further demonstrated that Nogo-B/ASY overexpression contributes to endoplasmic reticulum stress and induces apoptosis through Ca 2+ depletion in endoplasmic reticulum [23]. However, at the same time, stable transfectants overexpressing high levels of Nogo-B/ASY are resistant to endoplasmic reticulum stress associated stimuli, which implies that Nogo-B/ASY overexpression activates a protective response to endoplasmic reticulum stress [23]. In addition, the osteosarcoma SaOS-2 cell lines and the CHO cell lines have been shown to express high levels of endogenous Nogo-B. Overexpression of Nogo-B in both SaOS-2 and CHO cell lines do not differ significantly from the respective parental wild-type or control cell lines both in respect to cell proliferation and to spontaneous apoptosis or cell death induced by staurosporine and tunicamycin [24]. These conflicting studies have caused the uncertainty about the precise role of Nogo-B in modulating the apoptosis of cancer cells.
Our preliminary results show that overexpression of the aminoterminal domain of Nogo-B (AmNogo-B) does not cause any significant effects on tumor cell growth and cell survival (data not shown). As shown in Figure 4C and Figure S4A, knockdown of NgBR also does not affect the growth and survival of MCF-7 and T47D cells, typical estrogen receptor alpha positive breast tumor cells, under baseline growth conditions. However, NgBR knockdown reduces estradiol-induced MCF-7 and T47D cell growth ( Fig. 4C and Fig. S4A), respectively. Further comparison of ERalpha and NgBR expression in both MCF-7 and T47D cells as well as their response to estradiol stimulation show that T47D has lower ER-alpha expression and higher NgBR expression than MCF-7 cells (Fig. S4B), but T47D has more remarked response to estradiol-induced expression of survivin (Fig. S3A) as well as cell growth as compared to MCF-7 cells (Fig. S4A vs. Fig. 4C). It indicates that higher expression of NgBR may enhance the ERalpha-mediated signaling. Our recent studies demonstrated that NgBR acts as a scaffold protein required for Ras plasma membrane translocation and Ras signaling in tumor cells [unpublished data]. Our findings suggest that NgBR may recapitulate the oncogene function of Ras and coordinate with ER to promote estrogen response. This detailed molecular mechanism needs further investigation.
Given these findings, we sought to demonstrate the significances of Nogo-B and NgBR in specific types of breast cancer. ER, PR, HER2 are three well-characterized tumor markers that are typically expressed and are strongly associated with prognosis in breast cancer. To distinguish the heterogeneity of this disease, breast cancer has been categorized as four distinct subtypes based on gene expression profiling, including luminal A (ER-positive  and/or PR-positive, HER2-negative, low Ki67 index), luminal B (ER-positive and/or PR-positive, HER2-positive or HER2 negative, higher Ki67 index), HER2 enriched (HER2-positive and ER-negative/PR-negative) and triple-negative/basal like (ERnegative, PR-negative and HER2-negative) [1,[25][26][27] , [28]. Luminal A and luminal B are the most common subtype, usually representing low-to intermediate-grade tumors characterized by the expression of genes that are commonly expressed by normal ductal epithelial cells [1]. The luminal A subtype is welldifferentiated and associated with lobular histology and more frequent co-expression of both ER and PR than the luminal B subtype. Most cases of luminal B presented as grade II or III carcinoma showing HER2 overexpression and a higher Ki67 index [28]. The HER2 enriched subtype usually represents highgrade tumors with strong HER2 expression. The triple-negative/ basal like subtype usually represents high-grade tumors displaying necrosis, prominent lymphocytic infiltration and a pushing border, carrying a poor prognosis [1,29,30]. Our results suggested that high expression of NgBR is positively associated with ER-positive and HER2 negative breast cancers. Our results further indicate that high expression of NgBR in ER positive breast cancer may promote tumor cell growth and division by increasing the expression of survivin via an estrogen-dependent manner. These data strongly suggest that there is a close relationship among ER alpha, NgBR, survivin and their associated signaling pathways in breast cancer. Further experiments are needed to confirm this hypothesis.
Our results clearly demonstrated that both NgBR and survivin are highly expressed in ER positive IDC ( Fig. 1 and Table 2). NgBR knockdown reduced the estradiol-induced expression of survivin in ER positive breast tumor cells but not in ER-negative breast tumor cells (Fig. 3 and Fig. 4). Survivin was first identified as a baculovirus anti-apoptotic protein and is a member of the inhibitor of apoptosis proteins (IAP) family, which specifically inhibits caspases 3, 7 and 9 [31][32][33] and is involved in acquiring resistance to apoptosis. It has been shown that survivin inhibits apoptosis, regulates cell division and enhances angiogenesis [33]. Survivin is rarely expressed in terminally differentiated adult tissues, however high expression of survivin is found in most cancers [33][34][35]. High expression of survivin has been found to be related to poor survival in breast cancer patients [36,37] and progression of breast cancer [38]. Survivin is also associated with resistance to chemotherapy and hormone therapy, and predicts a poor clinical outcome in breast cancer [37,39]. Recent metaanalysis of survivin expression in breast cancer patients also demonstrated a significant association between positive survivin expression and a poor overall survival consequence in breast cancer patients [40]. Decreased survivin expression was found to increase sensitivity to chemotherapy drugs [41,42] and ionizing radiation [43]. It has been shown that estrogen upregulates the expression of survivin in ER positive MCF-7 breast cancer cells [44]. This finding might suggest that there is a positive association between ER and survivin expression in breast cancer. In the context of our pathological findings in 656 specimens of breast cancer patients and in vitro results, high expression of NgBR in ER-positive breast cancer may contribute to the survivin induction caused by estrogen stimulation. Our findings further indicate that the signaling to control tumor growth may be partially mediated through the ER-NgBR-survivin pathway in ER-positive breast cancer. This pathway may serve as a potential target for directed therapy. These results suggest that NgBR may play an important role in ER-positive breast cancer growth via increasing survivin expression.
In summary, our study is the first to investigate the expression and localization of Nogo-B protein and NgBR receptor in human breast cancer. The findings from this study demonstrate: (a) NgBR is highly expressed in ER positive and Her2 negative IDC breast cancer, whereas Nogo-B is ubiquitously expressed in IDC; (b) expression of NgBR is correlated with survivin expression in IDC as well as in later stages of breast cancer; (c) NgBR is essential for estradiol-induced survivin expression in ER positive breast tumor cells; (d) and finally, NgBR is also required for estradiol-stimulated ER positive breast tumor cell growth. Although we need further investigation to reveal the molecular mechanism by which NgBR promotes survivin expression in ER-positive breast cancer cells and the potential roles of NgBR in ER-positive breast cancer progression, current findings suggest high expression of NgBR may be a novel diagnosis marker or a potential therapeutic target for ER-positive breast cancer. Figure S1 Immunohistochemical (IHC) staining of NgBR and Nogo-B in invasive ductal carcinoma (IDC). Staining was developed using NovaRed as described in methods. Images were taken using an Olympus microscope with x20 lens. (A, B) To confirm the specificity of NgBR and Nogo-B IHC staining, we performed IHC staining in human IDC tissue sections and used primary antibodies preabsorbed with their corresponding epitope peptide-conjugated beads (+ peptide conjugated beads) as negative controls. (TIF) . All these three cell lines were treated with 10 nM estradiol for 24 hours. Survivin gene expression was determined by real-time PCR and is normalized with beta-actin. All groups are compared to MCF-7 no estradiol treatment group. (B) NgBR regulates estradiol-induced survivin gene expression in MCF-7 cells. NgBR was knocked down in MCF-7 cells using siRNA as described in methods. The cells were treated with 10 nM estradiol for 24 hours. Survivin gene expression was determined by real-time PCR and is normalized with beta-actin. All groups are compared to NS no estradiol treatment group. E2: estradiol. (TIF) Figure S4 (A) NgBR knockdown impairs estradiolstimulated growth of T47D breast tumor cells. Fifty thousand T47D cells were sub-cultured to each well of 12 wells plate. T47D cells were knocked down by siRNA targeting NgBR (siNgBR) and treated with 10 nM estradiol for 24 hours. Viable cell numbers were counted using the Bio-Rad TC10 TM Automated Cell Counter. Data is presented as mean6SEM (n = 3, # 24 hrs estradiol treatment vs baseline p,0.05; * siNgBR vs NS p,0.05).