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

<div><p>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.</p></div

Details of antibody and dilution.

<p>Details of antibody and dilution.</p

NgBR is essential for estradiol-induced survivin expression and cell growth of MCF-7 breast tumor cells.

<p>MCF-7 is an estrogen-dependent breast cancer cell line. NgBR was knocked down in MCF-7 cells using siRNA. (A) NgBR knockdown diminished estradiol-induced survivin expression in MCF-7 breast tumor cells. MCF-7 cells were treated with 10 nM estradiol for 6 and 48 hours. Protein levels of NgBR and survivin were determined by Western blot analysis. Beta-Actin is applied as a housekeeping protein. The density of each band was measured using NIH ImageJ and presented as relative intensity of survivin after normalized with beta-actin housekeeping protein. (B) Folds of survivin increase were determined by measuring relative western blot intensity of survivin. Data is presented as fold changes of estradiol treatment group as compared to the non-treatment group (n = 3; 6 hrs estradiol treatment, NS vs siNgBR <i>p</i> = 0.178; * 48 hrs estradiol treatment, NS vs siNgBR <i>p</i><0.05). (C) NgBR knockdown impaired estradiol-stimulated cell growth of MCF-7 breast tumor cells. MCF-7 cells were treated with 10 nM estradiol for 24 and 48 hours. Viable cell numbers were counted using the Bio-Rad TC10™ Automated Cell Counter. Data is presented as mean±SEM (n = 3, # 24 hrs or 48 hrs estradiol treatment vs baseline <i>p</i><0.05; * siNgBR vs NS <i>p</i><0.05); E2: estradiol.</p

NgBR regulates estradiol-induced survivin expression in estrogen receptor positive breast tumor cells.

<p>T47D is an estrogen receptor positive breast tumor cell line. MDA-MB-468 is an estrogen receptor negative breast tumor cell line. NgBR was knockdown in both T47D and MDA-MB-468 cells using siRNA as described in methods. Both tumor cells were treated with 10 nM estradiol for 48 hours. Protein levels of NgBR, ER-alpha and survivin were determined by Western blot analysis. Beta-Actin is applied as a housekeeping protein. The density of each band was measured using NIH ImageJ and presented as relative intensity of survivin after normalized with beta-actin housekeeping protein. (A) NgBR knockdown diminished estradiol-induced survivin expression in T47D breast tumor cells. (B) Quantitative analysis of survivin protein level change in T47D cells by measuring intensity of survivin western blot band. Data is presented as fold changes of estradiol treatment group as compared to the non-treatment group (n = 3; * siNgBR vs NS <i>p</i><0.05). (C) NgBR knockdown has no effect on survivin expression in MDA-MB-468 breast tumor cells. (D) Quantitative analysis of survivin protein level change in MDA-MB-468 cells by measuring intensity of survivin western blot band. Data is presented as fold changes of estradiol treatment group as compared to the non-treatment group. (n = 3; siNgBR vs NS <i>p</i> = 0.092).</p

NgBR and survivin transcripts in breast tumor tissues determined by real-time PCR.

<p>Normalized human breast tumor qPCR panels were utilized (Origene). The copy number of NgBR and survivin was determined by real-time PCR. (A) NgBR RNA levels in ductal adenocarcinoma specimens were presented as fold changes as compared to the average NgBR RNA levels of all normal breast tissue (* tumor vs normal, <i>p</i><0.05). (B) Survivin RNA levels in ductal adenocarcinoma specimens were presented as fold changes as compared to the average survivin RNA levels of all normal breast tissue (* tumor vs normal, <i>p</i><0.05).</p

Correlation analysis of NgBR and survivin transcripts in different stages of ductal adenocarcinoma.

*<p><i>p</i><0.05; N: case number; (%): percentage of total case number.</p

Tip60 Expression during Heart Development & Maturation.

<p>Total RNA and protein was purified at the indicated stages of development and subjected to semi-quantitative (<b>A</b>) RT/PCR and (<b>B</b>) western blotting analysis. <b>A</b>, the 184 bp band in the upper panel is amplified from all known isoforms of Tip60, whereas the 402 and 558 bp bands in the lower panel are respectively amplified from the Tip60 β and α isoforms. <b>B</b>, the upper panel is a western blot sequentially probed with antibodies recognizing Tip60 (α & β isoproteins were detected with the same antibody) and GAPDH; protein from individual hearts was evaluated at each postnatal day, whereas pools of three hearts each were evaluated at each embryonic day. The lower panel shows densitometric analysis in which each point indicates the mean of three (N = 3) independent western blot determinations. Error bars indicate ±SEM.</p

Tip60-Haploinsufficiency Augments 4-OHT-Induced Induction of Cell-Cycle Activity in MycER Transgenic Hearts.

<p>Eight week old MycER transgenic Tip60 wild-type (WT) and heterozygous (Het) mice were induced with 4-OHT for seven days and assessed for cell-cycle activity by immunostaining phosphorylated histone H3 (H3P; arrows in <b>A</b>,<b>B</b>). This was verified by immunostaining BrdU-incorporated nuclei (arrows in <b>D</b>,<b>E</b>). Percentages of labeled cells were determined by evaluating at least 5,000 (<b>C</b>) or 2,500 (<b>F</b>) hematoxylin-stained nuclei for H3P or BrdU antigen, respectively. (N)  =  number of hearts; vertical bars  =  ±SEM; p-values were calculated using Student's t-Test (two-tailed, unpaired). The scale bar in all images  =  10 µm. (Note: A control utilizing WT-MycER mice demonstrated that 4-OHT had no effect on these parameters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031569#pone.0031569-Xiao1" target="_blank">[26]</a>).</p

Increased Cell-Cycle Activity in Tip60^+/− Neonatal Cardiomyocytes.

<p>Cardiomyocytes were isolated from 2 day-old neonatal hearts and cultured on gelatin-coated dishes. When the cells were ∼60% confluent (72 hrs later) they were double-immunostained for phosphorylated histone-H3 (H3P) to detect M-phase cells (arrows in <b>A</b> & <b>C</b>) and for sarcomeric α-actin (not shown) to verify cardiomyocyte identity. Nuclei were stained with DAPI (<b>B</b>,<b>D</b>); H3P-labeled nuclei are encircled because DAPI is obscured DAB-stained nuclei. E shows percentages of H3P-positive neonatal cardiomyocytes, based on enumerating 1,000-2,000 cells in each dish; error bars  =  +/−SEM. Scale bars in <b>A</b>–<b>D</b> = 10 µm.</p

Over-Expression of Tip60β, but not Tip60α, Inhibits Cell Proliferation in NIH/3T3 Cells.

<p>Cultured NIH/3T3 cells were transfected with plasmid p3xFLAG-CMV-7.1 (empty vector), or with the same vector containing cDNA encoding either Tip60α or Tip60β. <b>A</b> shows the average cell number in each well of a 12-well plate, five days after transfecting the cells with plasmid. Error bars  =  ±SEM; p-values were calculated using Student's t-test (two-tailed, unpaired). <b>B</b> shows western blots confirming exogenous expression of Tip60α and Tip60β isoproteins.</p