Background

Breast cancer is the most common cancer in women around the world [1, 2]. About 75% of the cases are estrogen receptor alpha (ERα)-positive breast cancer [3]. These patients undergo adjuvant endocrine therapy to increase disease-free survival (DFS) and overall survival (OS) [4]. According to the National Comprehensive Cancer Network (NCCN) guideline, patients with invasive breast cancer who are ERα-positive or progesterone receptor (PR)-positive are eligible for tamoxifen, the selective estrogen receptor modulator (SERM) [5]. Tamoxifen, or its active metabolite 4-hydroxytamoxifen (4-OHT), are reported to induce breast cancer cell apoptosis [6]. However, recurrence within 15 years occurs in one third of patients treated with tamoxifen for 5 years [4].

The underlying mechanisms of develo** resistance, especially acquired resistance, to tamoxifen are complex and numerous, including ligand-independent activation of ERα or its co-activators through phosphorylation, and the inhibition of apoptosis through constitutive activation of survival signaling [7]. Clinical evidence shows that patients with human epidermal growth factor receptor 2 (HER2) overexpression and lower ERα levels are more likely to become tamoxifen-resistant [8]. Preclinical studies implicate the contribution of growth factor receptor signaling pathways, such as EGFR and HER2, to tamoxifen resistance [9, 10].

Nogo isoforms, including Nogo-A, Nogo-B and Nogo-C, are members of a reticulon protein superfamily. Nogo-B is mainly expressed in peripheral tissues [11]. NgBR was identified as a Nogo-B receptor specific for the amino terminus of Nogo-B (AmNogo-B). NgBR is necessary for angiogenesis by mediating chemotaxis of endothelial cells [12], and is essential for vasculature development [13, 14]. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for receptor tyrosine kinase (RTK)-mediated activation of Ras signaling in human breast cancer cells and tumorigenesis [15]. Greater expression of NgBR in ERα-positive breast tumor tissues is significantly correlated with expression of survivin [16], which is an apoptosis inhibitor [17]. A proteomic study also showed that NgBR is essential for promoting epithelial-mesenchymal transition (EMT) in breast cancer cells [18]. However, the involvement of NgBR in tamoxifen resistance of ERα-positive breast cancer is still unknown. In this study, we showed that NgBR knockdown attenuates tamoxifen resistance in MCF-7 and T47D breast cancer cells by inhibiting EGF-stimulated phosphorylation of ERα. Also, NgBR knockdown restored the sensitivity of ERα-positive breast cancer cells to tamoxifen through decreasing p53-mediated expression of survivin. Our results suggest that NgBR is a potential therapeutic target for increasing the efficacy of tamoxifen and overcoming the resistance to tamoxifen in ERα-positive patients with breast cancer.

Methods

Antibodies, reagents and plasmids

A peptide (AHHRMRWRADGRSLEK, residues from 81 to 96 of NgBR) was used to immunize rabbits (Epitomics, Burlingame, CA, USA). Antiserum was purified using the same peptide-conjugated SulfoLink Coupling Gel (Pierce, Rockford, IL, USA). Purified NgBR rabbit polyclonal antibody was used for immunostaining. The peptide recognizing epitope 14–30 of human Nogo-B was used to immunize rabbits (IMG-5346A, Imgenex, San Diego, CA, USA). Antibodies for NgBR (#ab168351) and phosphorylated ERα (phos-S118) (#ab32396) were purchased from Abcam (Cambridge, MA, USA). Antibodies for phos-EGFR (#3777), phos-Akt (#S473), phos-p42/44 extracellular signal-related kinase (ERK) (#9101), total Akt (#4691), total ERK (#4595), ERα (#8644), HER2 (#4290) and survivin (#2808) were purchased from Cell Signaling Technology (Beverly, MA, USA). We also used antibodies for p53 (#10442–1-AP), β-actin (#60008–1) and Hsp90 (13171–1-AP) from Proteintech (Rosement, IL, USA). EGF (#E5036) was purchased from Sigma-Aldrich (St. Louis, MO, USA). HER2 plasmid DNA was a gift from Mien-Chie Hung (Addgene plasmid # 16257).

Cell culture

Human breast cancer cell lines MCF-7 and T-47D were obtained from the American Type Culture Collection. The cells were grown in DMEM with L-Glutamine (MCF-7 Gibco) or RPMI-1640 with L-Glutamine (T47D, Gibco) supplemented with 10% FBS (Sigma, St. Louis, MO, USA) and 1% penicillin streptomycin glutamine (Gibco). The tamoxifen-resistant cell lines (MCF-7-TamR and T47D-TamR) were derived from the parental cell lines by continuous gradual exposure to 4-OHT (Sigma, St. Louis, MO, USA) to reach the final concentration of 1 μM in methanol in 6 months [19]. Culture medium was replaced every 2–3 days. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The cell lots used in this study were authenticated prior to in vitro experiments.

Small interfering RNA (siRNA) and plasmid transfection

NgBR siRNA (forward, GGAAAUACAUAGACCUACA; reverse, UGUAGGUCUAUGUAUUUCC) oligonucleotides with 3′ dTdT overhangs were synthesized by QIAGEN (Valencia, CA, USA). The specificity of NgBR siRNA has been validated in our previous publication [12]. Control siRNA in experiments refers to a non-silencing (NS) siRNA (NSF, UUCUCCGAACGUGUCACGU; NSR, ACGUGACACGUUCGG AGAA) designed and synthesized by QIAGEN. P53 siRNA (sc44218) and survivin siRNA (sc-29,499) were purchased from Santa Cruz (Dallas, TX, USA). Cells were transfected with siRNA using Lipofectamine RNAiMAX reagent (ThermoFisher Scientific). For transient NgBR overexpression experiments, MCF-7 cells were transfected with pIRES-NgBR-HA plasmid DNA using Lipofectamine 2000 reagent (ThermoFisher Scientific).

Quantitative real-time polymerase chain reaction

Total RNA was extracted from cells by using TRIzol reagent according to the manual (ThermoFisher Scientific) and complementary DNA (cDNA) was reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, USA) and was run on MyiQ Single Color Real-Time PCR Detection System (Bio-Rad). The relative messenger RNA (mRNA) expression of each gene was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA levels. The primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The forward and reverse primers for NgBR are 5′-tgccagttagtagcccagaagcaa-3′ and 5′-tgatgtgccagggaagaaagccta-3′, respectively. The forward and reverse primers for p53 are 5′-cctttctatcagccccagaggata-3′ and 5′-GGGACATCCTTAATTATCTGGGGT-3′, respectively. The forward and reverse primers for GAPDH are 5′-aacctgccaagtatgatgac-3′ and 5′-tctcttgctcagtgtccttg-3′, respectively. The forward and reverse primers for EGFR are 5′-aagccatatgacggaatccc-3′ and 5′-ggaactttgggcgactatctg-3′, respectively. The forward and reverse primers for ERα are 5′-cgactatatgtgtccagccac-3′ and 5′-cctcttcggtcttttcgtatcc-3′, respectively. The forward and reverse primers for survivin are 5′-caaggagctggaaggctg-3′ and 5′-ttcttggctctttctctgtcc-3′, respectively.

Clonogenic survival assay

Cells were seeded in triplicate into cell culture dishes (1000 cells/well). MCF-7-TamR or T47D-TamR cells were transfected with NgBR siRNA or non-silencing siRNA. At 12 h after transfection, cells were treated with or without 4-OHT. After 14 days, cells were washed with PBS, fixed with methanol for 15 min and stained with 0.1% crystal violet for 15 min. Colonies containing 50 or more cells were counted [Raf-pull-down assay

Ras activity was assessed using GST-Raf-1 RBD beads (RF02, Cytoskeleton, Denver, CO, USA) according to the manufacturer’s protocol: 500μg total cell lysate was incubated with 10 μL GST-Raf1-RBD beads overnight at 4 °C with gentle rocking. Samples were washed five times, then dissolved in 20 μL 2X SDS sample buffer. Activated H-Ras and K-Ras was determined by western blot using specific H-Ras (GTX116041, GeneTex, San Antonio, TX, USA) or K-Ras (12063–1-AP, Proteintech, Rosemont, IL, USA) antibodies, respectively.

Tissue microarray slides

Breast cancer tissue was collected from 22 patients at the First Hospital of Jilin University (Changchun, China).

We had consent from all patients for participating in this study. All of the patients received modified radical mastectomy and were diagnosed with infiltrating ductal carcinoma via pathological diagnosis. Immunohistochemistry was performed to examine NgBR, Nogo-B, and survivin expression levels following standard methodology described in our previous publication [16]. All of these breast cancer cases were histopathologically re-evaluated on hematoxylin and eosin-stained slides by two pathologists. The breast tissue specimens are anonymous. The study was approved by the ethical committee of the First Hospital of Jilin University.

Association between NgBR or survivin expression and survival in patients with breast cancer

All data were collected from a public online clinical database (http://kmplot.com). We analyzed the association between mRNA level of NgBR (NUS1, 225071_x at from Kaplan–Meier Plot database) or survivin (BIRC5, 202094_x at from Kaplan–Meier Plot database) and survival in patients with breast cancer. Kaplan-Meier survival curves according to NgBR expression status were used to analyze the relapse-free survival (RFS) and log-rank p values (SPSS 23.0 USA).

Statistical analysis

Data were analyzed from at least three independent experiments. The results were reported as the mean ± SD. Values of p < 0.05 were considered statistically significant. Student’s t test or analysis of variance (ANOVA) were performed as appropriate. Correlation between NgBR and survivin expression was analyzed using Fisher’s test. Statistical analyses were performed using Prism 6.0 software (GraphPad software, USA).

Results

NgBR expression is increased in tamoxifen-resistant breast cancer cells

Tamoxifen resistant MCF-7 (MCF-7-TamR) and T47D (T47D-TamR) ERα-positive breast cancer cells were established following the previously described method [19]. To validate tamoxifen resistance in established MCF-7-TamR and T47D-TamR cells, both normal and tamoxifen-resistant cells were treated with 0–5 μM 4-OHT. As shown in Fig. 1a–d, 5 μM 4-OHT cannot attenuate the colony formation capability of MCF-7-TamR and T47D-TamR cells. However, parental cells cannot survive treatment with 5 μM 4-OHT. CCK-8 cell viability assay was also used for determining the response of these breast cancer cells to tamoxifen (Additional file 1: Figure S1A and B). Similarly, both MCF-7-TamR and T47D-TamR can survive treatment with 5 μM 4-OHT. The levels of NgBR transcript and protein were determined by real-time PCR (Fig. 1e and f) and western blot analysis (Fig. 1g and h). The expression of NgBR was increased in both MCF-7-TamR (Fig. 1e, g and h) and T47D-TamR cells (Fig. 1f; Additional file 2: Figure S2) as compared to that in their parental cells. The alteration of other gene expression between MCF-7 and MCF-7-TamR cells is shown in Fig. 1g and h. Consistent with many previous studies [19, 21, 22], we also noted increased expression of EGFR, HER2, and survivin, and decreased expression of p53 and ERα in MCF-7-TamR (Fig. 1g and h).

Fig. 1
figure 1

Nogo-B receptor (NgBR) is highly expressed in the tamoxifen resistant MCF-7-TamR and T47D-TamR cells. a Colony formation assay was performed as described in “Methods”. Wild-type MCF-7 and tamoxifen-resistant MCF-7-TamR cells were treated with different concentrations of 4-OHT (0, 1 and 5 μM). b Quantification of colony number presented in colony formation assays of MCF-7 and MCF-7-TamR cells. c Colony formation assay of wild-type T47D and tamoxifen-resistant T47D-TamR cells treated with different concentrations of 4-OHT (0, 1 and 5 μM). d Quantification of colony number in colony formation assays of T47D and T47D-TamR cells. e, f mRNA level of NgBR was increased in MCF-7-TamR and T47D-TamR cells as compared to wild-type MCF-7 and T47D cells, respectively. The relative amount of NgBR mRNA level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). g NgBR protein level was increased in MCF-7-TamR cells. Protein levels of Nogo-B, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), estrogen receptor alpha (ERα), p53 and survivin in MCF-7 and MCF-7-TamR cells were determined using western blot analysis. h Quantitative analysis of protein levels using ImageJ and normalized to the housekee** gene β-actin. Data are presented as fold changes in MCF-7-TamR cells compared to MCF-7 cells. The data are from three separate repeat experiments, and are presented as the mean ± SD (*p < 0.05, n = 3)

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NgBR knockdown attenuates the tamoxifen resistance

To determine the contribution of increased NgBR expression to tamoxifen resistance, we knocked down the expression of NgBR with specific NgBR siRNA (siNgBR), which has been validated in our previous reports [18]. The effects of NgBR knockdown on cell apoptosis and necrosis of MCF-7-TamR (Fig. 2a and b) and T47D-TamR (Additional file 3: Figure S3A and B) were determined by Annexin V/PI staining and flow cytometry. The results showed that NgBR knockdown increases the sensitivity of both MCF-7-TamR and T47D-TamR cells to 4-OHT. Compared to the non-treatment group, 4-OHT treatment alone did not induce significant death of tamoxifen-resistant cells. However, NgBR knockdown along with 4-OHT treatment significantly increased the percentage of cell death. Cell viability was determined by counting the number of negative trypan blue stained cells using a hemocytometer. Consistently, MCF-7-TamR and T47D-TamR cells were resistant to 4-OHT treatment. However, NgBR knockdown restored the sensitivity of MCF-7-TamR (Fig. 2c) and T47D-TamR (Additional file 3: Figure S3C) to tamoxifen. The clonogenic survival assay further demonstrated that NgBR knockdown attenuates the colony formation capability of MCF-7-TamR (Fig. 2d and e) and T47D-TamR cells (Additional file 3: Figure S3D and E) under the condition of 5 μM 4-OHT treatment. These results demonstrated that NgBR knockdown increases the sensitivity of tamoxifen-resistant ERα-positive breast cancer cells to tamoxifen.

Fig. 2
figure 2

Nogo-B receptor (NgBR) knockdown decreases the resistance of MCF-7-TamR cells to tamoxifen. a NgBR knockdown increases 4-OHT-induced apoptosis of MCF-7-TamR cells; 4-OHT, 5 μM. The apoptotic cells were detected by Annexin V-PI staining as described in “Methods”. The total number of cells in the Q2 and Q4 quadrant were counted as apoptotic cells. b Percentages of apoptotic MCF-7-TamR cells are presented in the bar graph. c NgBR knockdown decreases cell viability of MCF-7-TamR cells treated with 4-OHT. Cell viability was determined using trypan blue staining. MCF-7-TamR cells were treated with 4-OHT (5 μM) for 48 h. The viable cell number in the non-silencing (NS) group is set as 100%. d NgBR knockdown decreases the clonogenenicity of MCF-7-TamR cells treated with 4-OHT (5 μM). Clonogenic survival assay was performed as described in “Methods”. e Quantification of colony number in colony formation assays as described in Fig. 2d. The data were repeated in three separate experiments, and are presented as the mean ± SD (*p < 0.05, n = 3)

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As in our previous report [15], overexpression of NgBR in MCF-7 cells increased the membrane-associated H-Ras and K-Ras (Fig. 3a). Consequently, if we transfected plasmid DNA expressing human influenza hemagglutinin (HA) tagged NgBR (NgBR-HA) to MCF-7 cells, we appreciated that the overexpression of exogenous NgBR-HA increased the viability of MCF-7 cells treated with 4-OHT (Fig. 3b). Similarly, overexpression of HER2-HA also increased the resistance of MCF-7 cells to 4-OHT (Fig. 3b). However, NgBR knockdown attenuated the resistance of MCF-7 cells overexpressing HER2-HA (Fig. 3b). In MCF-7-TamR cells, overexpression of NgBR-HA restored the resistance of NgBR knockdown MCF-7-TamR cells to tamoxifen (Fig. 3c). As shown in Fig. 3d, transfection of NgBR-HA plasmid DNA restored the expression of NgBR in MCF-7-TamR cells transfected with NgBR siRNA (siNgBR), which targets the 3′-untranslated region (UTR) of NgBR as described in our previous publication [12]. It suggests that NgBR is one of driving forces for tamoxifen resistance. However, knockdown of Nogo-B does not affect the sensitivity of MCF-7-TamR to tamoxifen (Fig. 3c). The efficacy of Nogo-B siRNA was confirmed by western blot analysis (Fig. 3d).

Fig. 3
figure 3

Overexpression of Nogo-B receptor (NgBR) increases the resistance of MCF-7 cells to tamoxifen. a Overexpression of NgBR in MCF-7 cells increases the membrane-associated H-Ras and K-Ras. The plasma membrane proteins were isolated by the ultracentrifugation method. Protein levels of pan-cadherin, NgBR, H-Ras, K-Ras and Hsp90 in MCF-7 cells were determined using western blot analysis. b Viability of MCF-7 cells treated with 4-OHT (0, 1 or 5 μM) was determined using CCK-8 assay. Overexpression of either human influenza hemagglutinin (HA)-tagged NgBR or HER2 in MCF-7 cells decreases their sensitivity to 4-OHT. Knockdown NgBR in MCF-7 cells restores the sensitivity of MCF-7 cells overexpressing HER2-HA to 4-OHT. The number of viable cells in the untreated group is referred as 100% (*p < 0.05, n = 3). c Viability of MCF-7-TamR cells treated with 4-OHT (0, 1 or 5 μM) was determined using CCK-8 assay. Knockdown of NgBR in MCF-7-TamR cells increases their sensitivity to 4-OHT. Overexpression of NgBR decreases the sensitivity of NgBR-knockdown MCF-7-TamR cells to 4-OHT. Knockdown of Nogo-B in MCF-7-TamR cells does not affect their sensitivity to 4-OHT. The number of viable cells in the untreated group is referred to as 100% (*p < 0.05, n = 3). d NgBR regulates the expression of ERα, p53 and survivin independent of its ligand Nogo-B. MCF-7-TamR cells were transfected with control siRNA or NgBR siRNAs targeting either NgBR or Nogo-B. In MCF-7-TamR cells transfected with siRNA targeting the untranslated region of NgBR, NgBR expression was restored by the transfection of NgBR-HA plasmid DNA

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NgBR knockdown promotes apoptosis by regulating the expression of p53 and survivin

Our previous study showed that NgBR deficiency in ERα-positive breast cancer cells decreases the resistance to chemotherapy by increasing p53 and decreasing survivin [23]. To determine if and the extent to which NgBR is dependent on p53-mediated survivin expression to promote the resistance to tamoxifen, we examined the alteration of p53 and survivin expression in tamoxifen-resistant ERα-positive breast cancer cells before and after NgBR depletion. As shown in Fig. 4a and b, NgBR depletion in MCF-7-TamR cells increased the expression of p53 but decreased the amount of survivin at the protein levels. Overexpression of NgBR-HA restored the expression pattern of p53 and survivin in MCF-7-TamR cells transfected with siNgBR to levels similar to that in MCF-7-TamR cells transfected with control siRNA (Fig. 3d). When p53 was knocked down by siRNA, survivin was increased in MCF-7 cells (Fig. 4c and d). To determine if either p53 or survivin is involved in regulating the apoptosis of NgBR-deficient cells, we knocked down either p53 in MCF-7 cells or survivin in MCF-7-TamR cells using siRNA either targeting p53 (si-p53) or targeting survivin (si-survivin), respectively. As shown in Fig. 4e and f, knockdown of p53 increased the resistance of MCF-7 cells to 4-OHT, while survivin knockdown restored the sensitivity of MCF-7-TamR cells to 4-OHT (Fig. 4g and h). Similar results were also observed in p53 knockdown T47D cells (Additional file 4: Figure S4A–D) and survivin knockdown T47D-TamR cells (Additional file 4: Figure S4E–F). These results demonstrated the contribution of increased p53 expression and decreased survivin expression that occurs in NgBR knockdown cells for restoring the sensitivity of ERα-positive breast cancer cells to tamoxifen. The clonogenic survival assay further demonstrated that loss of p53 in NgBR knockdown MCF-7 cells attenuated the effects of NgBR deficiency on increased sensitivity of MCF-7 cells to tamoxifen (Fig. 4i and j). In addition, knockdown of both H-Ras and K-Ras in MCF-7-TamR cells also resulted in the increased amount of p53 and decreased amount of survivin (Fig. 5a). Overexpression of either NgBR-HA or HER2-HA in MCF-7 cells decreased the protein levels of p53 and ERα but increased the protein level of survivin (Fig. 5b). Interestingly, knockdown of NgBR in MCF-7 cells overexpressing HER2-HA restored the protein levels of p53, ERα and survivin levels to those occurring in control MCF-7 cells transfected with empty vector (Fig. 5b).

Fig. 4
figure 4

Sensitivity of MCF-7 cells to tamoxifen is regulated by p53 and survivin. a Nogo-B receptor (NgBR) knockdown increases p53 protein level and decreases survivin in MCF-7-TamR cells. Protein levels were determined by western blot analysis. b Quantitative analyses of proteins presented in Fig. 3a were carried out using ImageJ and were normalized to the housekee** gene β-actin. Data are presented as fold changes in the siNgBR group compared to the non-silencing (NS) group. c Knockdown of p53 increases survivin level in MCF-7 cells. MCF-7 cells were transfected with siRNA specifically targeting p53 as described in “Methods”. The protein levels of p53, NgBR, survivin and β-actin were determined using western blot analysis. d Quantitative analyses of proteins presented in Fig. 3c were carried out using ImageJ and were normalized to β-actin. Data are presented as fold changes in the sip53 group compared to the NS group. e Knockdown of p53 increases the clonogenenicity of MCF-7 cells. Clonogenic survival assay was used for measuring clonogenicity of MCF-7 cells treated with 4-OHT (1 μM). f Quantification of colony number in colony formation assays is presented in Fig. 3e. g Survivin knockdown increases apoptosis of MCF-7-TamR cells induced by 4-OHT (5 μM). h Percentages of apoptotic cells in Fig. 3g are shown in the bar graph. i Knockdown of p53 decreases the sensitivity of NgBR-deficient MCF-7 cells to tamoxifen. NgBR knockdown restored the sensitivity to tamoxifen, which is attenuated by silencing p53. j Quantification of colony number in colony formation assays described in Fig. 3i. The data are from three separate repeat experiments, and are presented as the mean ± SD (*p < 0.05, n = 3)

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Fig. 5
figure 5

Nogo-B receptor (NgBR) regulates protein levels of p53 and survivin through Ras-mediated pathways. a Knockdown of both H-Ras and K-Ras increases the protein levels of p53 and decreases the protein level of survivin. Control siRNA and siRNAs targeting either H-Ras or K-Ras siRNA were transfected into MCF-7-TamR cells. b Overexpression of either NgBR-HA or HER2-HA in MCF-7 cells decreases the protein levels of p53, ERα, and increases the protein level of survivin. Knockdown NgBR in MCF-7 cells overexpressing HER2-HA restores the protein levels of p53, ERα and survivin to levels similar to those in control MCF-7 cells transfected with empty plasmid DNA vector. Change in NgBR or human epidermal growth factor receptor 2 (HER2) has no effects on the total protein levels of AKT and extracellular signal-related kinase (ERK)

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NgBR knockdown diminished EGF-stimulated phosphorylation of ERα

Phosphorylation of ERα serine 118 (S118) residue has been reported to be involved in resistance to tamoxifen [24, 25]. To examine the involvement of NgBR in regulating EGF-stimulated phosphorylation of ERα S118, we treated MCF-7-TamR cells with 100 ng/mL EGF for 5 min, which is the peak of the phosphorylation signal in response to EGF stimulation. As shown in Fig. 6a and b, EGF treatment increased the phosphorylation of AKT, ERK and MDM2 in MCF-7-TamR cells treated with control non-silencing (NS) siRNA. NgBR knockdown attenuated EGF-stimulated phosphorylation of AKT, ERK and MDM2. But NgBR knockdown did not affect the total protein levels of AKT, ERK and MDM2 or the phosphorylation of EGFR (Fig. 6a). EGF treatment not only activated the downstream signaling of the EGF pathway, but also increased the phosphorylation of ERα (S118), which is in accordance with previous studies [10, 15]. Therefore, NgBR is essential for the Ras-mediated signaling pathway [15]. Previous reports have shown that EGF induces the phosphorylation of ERα at the serine 118 residue [51], which is the confirmed signal involved in ERα-mediated resistance to endocrine therapy [25]. In this study, we demonstrated that NgBR knockdown impairs EGF-stimulated phosphorylation of ERα (Fig. 4a; Additional file 10: Figure S6A) but also attenuates resistance to tamoxifen (Fig. 2; Additional file 3: Figure S3). This finding indicates that NgBR is a potential therapeutic target for blocking concurrent endocrine-resistant signaling. However, we need further investigation to determine synergetic roles of NgBR in coordinating with other growth factor receptors, such as insulin-like growth factor 1 receptor (IGF1-R) and mERα, to promote the acquired resistance to tamoxifen.

Except for the contribution of growth factor receptor signaling pathways to tamoxifen resistance [9, 10], decreased ERα expression [7, 52] and increased human epidermal growth factor receptor 2 (HER2) [8, 53] are more likely to be attributed to tamoxifen resistance. Previous studies by others showed that hyperactivation of Raf kinase induces the loss of ERα in ERα-positive breast cancer cells [54] and inhibition of mitogen-activated protein kinase (MAPK) activity induced the expression of ERα in ERα-negative breast cancer cells [55]. As shown in Fig. 1g, ERα decreased and HER2 increased in MCF-7-TamR cells as compared to parental MCF-7 cells. Overexpression of either NgBR-HA or HER2-HA in MCF-7 cells decreased the protein level of ERα (Fig. 5b). Interestingly, NgBR knockdown in either MCF-7-TamR cells (Fig. 3d and c) or MCF-7 cells overexpressing HER2-HA (Figs. 5b and 3b) restored the protein level of ERα to a level similar to that in control cells as well as sensitivity to tamoxifen. The contribution of NgBR-regulated ERα expression to tamoxifen resistance needs further investigation in our future studies.

Conclusions

In summary, our results demonstrate that increased expression of NgBR in tamoxifen-resistant breast cancer cells promotes EGF signaling by increasing phosphorylation of AKT/ERK and MDM2, which attenuates the expression of p53 and increases the expression of survivin, which may lead to acquired resistance to tamoxifen as shown in Fig. 8. Higher levels of NgBR in patients with ERα-positive breast cancer are associated with poor RFS outcomes in patients with breast cancer because they lead to easier acquisition of tamoxifen resistance. Our data suggest that NgBR is a potential therapeutic target for restoring the sensitivity of tamoxifen-resistant breast cancer cells to conventional endocrine therapy.

Fig. 8
figure 8

Nogo-B receptor (NgBR) is required for epidermal growth factor (EGF)-acquired resistance to tamoxifen in patients with estrogen receptor alpha (ERα)-positive breast cancer. EGF binds to EGF receptor (EGFR) and recruits SOS1, an activator for Ras, to the plasma membrane. NgBR promotes the translocation of Ras to the plasma membrane and enhances EGF signaling, such as phosphorylation of Akt and extracellular signal-related kinase (ERK). Phosphorylated Akt further induces the phosphorylation of MDM2, which promotes p53 ubiquitination leading to its degradation and attenuates p53-mediated apoptosis. Phosphorylation of Akt and ERK also induce the phosphorylation of ERα, which induces the expression of survivin, an apoptosis inhibitor

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