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[Cancer Research 59, 5315-5322, October 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 5315-5322, October 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Ribozyme-mediated Down-Regulation of ErbB-4 in Estrogen Receptor-positive Breast Cancer Cells Inhibits Proliferation Both in Vitro and in Vivo1

Careen K. Tang2, Xiao-Zheng Wu Concepcion, Melissa Milan, Xiaoqi Gong, Elizabeth Montgomery and Marc E. Lippman

Lombardi Cancer Center, Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007-2197


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ErbB-4 is a recently discovered member of the class I receptor tyrosine kinase family (ErbB). Little is known about its expression and its importance in human malignancy. To delineate the biological function of ErbB-4 receptors in breast cancer, we used a hammerhead ribozyme strategy to achieve down-regulation of ErbB-4 receptors in various breast cancer cell lines. We observed that down-regulation of ErbB-4 in estrogen receptor-positive (ER+) human breast cancer cell lines (MCF-7 and T47D), which express relatively high levels of ErbB-4, significantly inhibited colony formation. No effects were observed in estrogen receptor-negative (ER-) MDA-MB-453 cells, which express low levels of endogenous ErbB4 and high levels of ErbB-2 and ErbB-3. This occurred despite the fact that fluorescence-activated cell sorter analysis of these latter cells revealed that the expression of the ErbB-4 receptor was completely abrogated by ribozyme treatment. Furthermore, down-regulation of ErbB-4 in T47D and MCF-7 cells significantly inhibited tumor formation in athymic nude mice (P < 0.03 and P < 0.001, respectively). In addition, NRG-stimulated phosphorylation of ErbB-4- and NRG-induced colony formation was significantly reduced in ribozyme-transfected T47D cells. These data provide the first evidence that elevation of ErbB-4 expression plays a role in the proliferation of some ER+ human breast cancer cell lines (T47D and MCF-7) that express high levels of ErbB-4.

We have also investigated the expression of ErbB-4 in human primary breast carcinoma specimens, using immunohistochemical staining with an anti-ErbB-4 monoclonal antibody. ErbB-4 expression was found in 60% of the 50 primary breast tumors examined, and high intense immunoreactivity of ErbB-4 was detected in 18% of these primary breast tumors. ErbB-4 receptor expression appeared to correlate with ER+ primary breast tumors. A similar correlation was also observed in the human breast cancer cell lines.

These results provide a better understanding of the biological significance of ErbB-4 receptor in breast cancer. Our data suggest that elevation of the ErbB-4 receptor plays a role in ER+ breast cancer cell proliferation. Moreover, ribozyme technology provides a useful tool to delineate the role of a particular gene product.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Members of the class I receptor tyrosine kinase family (ErbB) are most frequently implicated in human cancers (1, 2, 3) . These receptors include the EGFR, ErbB-2, ErbB-3, and ErbB-4 proteins (4, 5, 6, 7, 8) . ErbB-4 is the most recently discovered member of the ErbB family. More than a dozen different agonists have been reported for the ErbB family receptors. These growth factors exert their function by binding cell surface receptors with intrinsic protein tyrosine kinase activity and are implicated in the autocrine/paracrine growth of breast epithelial cells. The neu differentiation factors/NRG, NRG2 (also known as heregulins, neu differentiation factors, glial growth factors, and acetylcholine receptor inducing activity) bind to ErbB-3 and ErbB-4 (9, 10, 11, 12, 13) but can only activate ErbB-4 or ErbB-2/ErbB-3 heterodimer and cannot activate ErbB-3 (14, 15, 16) . In addition, multiple isoforms of NRG and NRG2 arising from alternative transcriptional splicing are ligands of ErbB-3 and ErbB-4 (14, 15, 16) and can also transmodulate ErbB-2 and EGFR through heterodimers with ErbB-3 and ErbB-4 (17, 18, 19, 20, 21) . Recently, it has reported that HB-EGF and BTC, as well as EGF, can activate ErbB-4 signaling pathways (22, 23, 24, 25, 26) . Sequencing of full-length human ErbB-4 cDNAs revealed the existence of two ErbB-4 isoforms (27) . The second c-ErbB-4 was found with deletion of 48 bp, which encodes a consensus phosphatidylinositol 3-kinase binding site (27) . This implies that the two forms of ErbB-4 might interact with different intracellular signaling pathways (27) . Both ErbB-4 transcripts are found to be expressed in normal breast and in most breast cancers (27) .

Amplification or overexpression of the erbB-2 proto-oncogene has been detected in 30% of breast cancers and is associated with a poor prognosis (2) . Overexpression of ErbB-4 in NIH 3T3 cells can transform these cells (28) . A recent report has indicated that amplification of ErbB-4 was found in 13% of human breast cancers and directly correlated with the tumor size (29) . Coexpression of ErbB-2, ErbB-4, and NRG is significantly related to the presence of metastases in human medulloblastoma (30) . ErbB-4 mRNA was significantly overexpressed in gastric cancer (31) . In addition, the physiological relevance of transmodulation is supported in gene-targeting experiments in transgenic mice. Mice that are homozygous for disruptions in the erbB-4 gene die in utero at day 10.5 and lack trabecular extensions of the developing ventricular myocardium (32) .

Although ectopic expression of recombinant ErbB receptors has provided important information on their signaling properties, the biological function and in vivo interplay of these receptors are still poorly understood. Little is known about the biological significance of ErbB-4 in breast cancer. To more fully understand the role of ErbB-4 in neoplastic transformation, we used a hammerhead ribozyme strategy to inactivate ErbB-4 and delineate its role in biological neoplastic transformation. In a previous study, it was demonstrated that we were able to generate biologically active ribozymes that target the ErbB-4 receptor. We demonstrated that the NRG-induced mitogenic effect was abolished in ribozyme-transfected 32D/ErbB-4 cells, a cell line dependent on signaling through ErbB-4. Inhibition of mitogenesis was proportional to ribozyme-mediated down-regulation of ErbB-4 expression (33) . In the present study, we have used these ribozymes to down-regulate endogenous levels of the ErbB-4 receptor in various breast cancer cell lines. We have observed that down-regulation of ErbB-4 in some of the ER+ breast cancer cell lines expressing relatively high levels of ErbB-4 dramatically reduces the ability of the cells to grow in an anchorage-independent assay. Furthermore, ribozyme-mediated down-regulation of ErbB-4 in these ER+ breast cancer cells exhibited inhibition of tumor formation in athymic nude mice. However, complete down-regulation of ErbB-4 expression in ER- breast cancer cell lines expressing low levels of ErbB-4 expression has no effect. These data suggest that ErbB-4 plays a proliferative role in cells expressing high levels of ErbB-4. In addition, a pilot study to determine the frequency of ErbB-4 expression in primary breast cancer specimens was conducted by immunohistochemistry. Sixty % of the primary breast cancer specimens were found to express ErbB-4. High intense immunoreactivity of ErbB-4 was detected in 18% (9 of 50) of these primary breast tumors. Interestingly, expression of ErbB-4 is directly correlated with ER+ human breast carcinomas. A similar correlation was also observed in human breast cancer cell lines. These results provide a better understanding of the biological significance of ErbB-4 receptors in breast cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Cell Culture.
T47D, MCF-7, MDA-MB-453, and MDA-MB-231 breast carcinoma cell lines and their derivatives were maintained in IMEM (Cellgro), supplemented with 10% FCS (Biofluids).

Transfection.
Cells (1 x 106) and 10–15 µg of plasmid DNA were used for each transfection. Transfections were performed using the Calcium Phosphate Transfection System (Life Technologies, Inc., Rockville, MD), according to the manufacturer’s protocol. The cells were then selected in a growth medium containing appropriate amounts of Geneticin (G418-sulfate; Life Technologies).

Autophosphorylation of ErbB Family Receptors.
Before cell lysis, the cells were serum starved overnight at 37°C. After incubation, cells were then treated with 100 ng/ml of NRG-symbol 97 {alpha} (R & D Systems) or 100 ng/ml of BTC (R & D Systems) for 5 min at 37°C. After a 5-min incubation, cells were lysed in HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton x - 100, 1.5 mM MgCl2, and 1 mM EGTA), and the cell debris was pelleted by centrifugation (33) .

The lysates were then immunoprecipitated with either anti-EGFR (Ab-1; Oncogene Science, Uniondale, NY), anti-ErbB-2 (Ab-3; Oncogene Science, Uniondale, NY), anti-ErbB-3 (C17; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-ErbB-4 (C18; Santa Cruz Biotechnology), in combination with protein A-agarose (Pharmacia, Piscataway, NJ) overnight at 4°C with gentle agitation. Immunoprecipitates were then separated by SDS-PAGE and transferred to nitrocellulose. Bound proteins were immunoblotted with anti-phosphotyrosine monoclonal antibody PY20 (United Biomedical, Inc., Lake Placid, NY), followed by blots with 0.5 µg/ml of secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected by with an enhanced chemiluminescence reagent (ECL; Amersham Corp.).

Fluorescence-activated Cell Sorter (FACStar) Analysis.
Cells (1 x 106) were harvested and then stained for 1 h with either anti-EGFR (Ab-1), ErbB-2 (Ab-2), ErbB-3 (Ab-4), or anti-ErbB-4 monoclonal antibody (Ab-1; NeoMarker, Fremont, CA) at 4°C. Stained cells were then washed with cold PBS. A secondary FITC-antimouse antibody was used, and the ErbB-4 level in each cell was quantitatively measured by flow cytometry.

Anchorage-dependent Growth Assays.
Cells were harvested using trypsin, and 1500 cells/well were plated in 24-well plates (Costar). All samples were prepared in triplicate. Cells were counted in a Coulter Counter (Coulter Electronics, Ltd., Hialeah, FL) on day 1 (the following day), day 3, and day 7. Values indicate the mean of triplicate determinations ±12 SD.

Anchorage-independent Growth Assays.
A bottom layer of 1 ml of IMEM containing 0.6% agar and 10% FCS was prepared in 35-mm tissue culture dishes. After the bottom layer solidified, cells (10,000/dish) were then added in a 0.8 ml top layer containing 0.4% Bacto Agar and 5% FCS. All samples were prepared in triplicate. Cells were incubated for ~12 days at 37°C. Colonies larger than 60 µm were counted in a cell colony counter (Ommias 3600; Imaging Products Int., Inc., Charley, VA).

In Vivo Studies.
Ovariectomized athymic nude mice were inoculated s.c. with either T47D/wt, T47D/poolA, T47D/pool20, MCF-7/wt, or MCF-7/vector, as well as ErbB-4 ribozyme-transfected clones, MCF-7/RzB1 and MCF-7/RzA4, in the presence of an estrogen source (0.72 mg). The slow-release pellets (60-day release) were implanted s.c. into the cervical scapular space. Tumor growth was monitored twice weekly for 10–12 weeks. Tumor size was measured twice weekly and calculated by measuring tumor volume (length x width x thickness). When tumors reached up to 2 cm in diameter, mice were sacrificed.

Immunohistochemistry of ErbB-4.
Paraffin-embedded sections of primary breast tumors were deparaffinized in Xylene (Fisher, Pittsburgh, PA) for 5 min, dehydrated in reagent alcohol (Fisher) for 5 min, air-dried for 5 min, dehydrated in reagent alcohol (Fisher) for 5 min, and rehydrated in PBS for 10 min. Endogenous peroxidase activity was blocked by 5-min incubation with 3% hydrogen peroxide (Fisher). A monoclonal anti-erbB-4 antibody (1:50 dilution; NeoMarker) was incubated with the section for 2 h at room temperature. After washing with PBS, a horseradish peroxidase-conjugated goat antimouse IgG (H+L) secondary antibody (Kirkegaard & Perry Lab, Gaithersburg, MD) was used at a dilution of 1/100, incubated for 30 min, and washed with water. Subsequently, color was developed using diaminobenzidine (Sigma) as a substrate. Sections were then counterstained with hematoxylin (Fisher) for 5 min. Sections were then placed on a coverslip using Permount. Before the analysis of the levels of ErbB-4 staining in clinical breast cancer specimens, the proficiency of the optimized immunocytochemical assay was established by detection of ErbB-4 expression, using a series of breast cancer cell monolayers. MDA-MB-231 cells were used as a negative control, and T47D cells were used as a positive control. In addition, assay performance was monitored by the inclusion of a breast cancer-positive control section of known immunostaining percentage and intensity.

Statistical Analysis.
The frequencies of ER/PR status among primary human breast cancer tumor samples were compared among the varying levels of ErbB-4 expression using the exact Kruskal-Wallis test (34) . ErbB-4 expression was categorized as weak/negative (-/+), positive (++), and strong (+++). ER/PR status was categorized as ER+/PR+, ER+/PR-, ER-/PR+, and ER-/PR-.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Biological Active Ribozyme Targeting ErbB-4 Receptors.
To investigate the biological significance of ErbB-4 in human breast cancer, we used molecular targeting of the ErbB-4 mRNA by ribozymes. In a previous study, we described that we had generated three ribozymes (Rz6, Rz21, and Rz29) targeted to specific sites within the ErbB-4 mRNA open reading frame. We demonstrated that all three ErbB-4 ribozymes cleaved ErbB-4 mRNA precisely and efficiently under physiological conditions in this cell-free system (33) . Point mutation of G to A in the catalytic domain of these ribozymes resulted in a loss of catalytic activity as expected (35) . We also illustrated the intracellular efficacy and specificity of the ErbB-4 ribozymes in a model system (32D cell system). 32D cells are a murine hematopoietic interleukin 3-dependent cell line that does not express detectable levels of endogenous EGF family receptors (36) . Overexpression of ErbB-4 receptors in 32D cells (32D/ErbB-4) abrogated interleukin 3 dependence by stimulation with NRG (33) . We showed that two of the ErbB-4 ribozymes (Rz6 and Rz29) were able to down-regulate ErbB-4 expression and were capable of abolishing the NRG-induced mitogenic effect in 32D/ErbB-4 cells. Rz29 is more efficient at down-regulation of ErbB-4 expression than Rz6. In contrast, Rz21 had no effect on responsiveness to NRG stimulation (33) . These results demonstrated that ribozymes Rz29 and Rz6 are biologically functional ribozymes and that Rz21 is an inactive ribozyme in 32D cells (33) .

Ribozyme-mediated Down-regulation of Endogenous ErbB-4 in Human Breast Cancer Cells.
In this study, we used these biologically active ribozymes to down-regulate endogenous levels of ErbB-4 in various human breast cancer cell lines with different levels of ErbB-4 expression. This was done to elucidate the biological significance of ErbB-4 in breast cancer. Four human breast cancer cell lines were selected as recipient cells: T47D, MCF-7, MDA-MB-453, and MDA-MB-231. In ER+ cell lines (T47D and MCF-7), there is a relatively high level of ErbB-4 receptor expression and a moderate level of other EGF family receptors, whereas ER- MDA-MB-453 cells express low endogenous levels of ErbB-4 and high levels of ErbB-2 and ErbB-3. MDA-MB-231 (ER-) expresses a high level of EGFR and a relatively low level of ErbB-2 but does not express a detectable level of ErbB-3 or ErbB-4. The functional ErbB-4 ribozymes (Rz29 and Rz6), as well as a control vector, were introduced into these cell lines by stable transfection. The sublines T47D/Rz, MCF-7/Rz, MDA-MB-453/Rz, and MDA-MB-231/Rz, as well as empty vector control cell lines, were established. However, we were unable to select the Rz29 transfected T47D clones. One possibility is that ErbB-4 plays a role in T47D cell proliferation, and a complete down-regulation of ErbB-4 would be lethal. The inability to select Rz29 clones could also be explained by a very efficient ErbB-4 ribozyme, which completely down-regulated the ErbB-4 expression and significantly inhibited cell proliferation; thus, the isolation of stably transfected T47D cells would be impossible. Therefore, we selected Rz6 transfectants for further characterization. We assessed the ribozyme-mediated down-regulation of ErbB-4 expression by FACS analysis. Fig. 1Citation illustrates that an ErbB-4 ribozyme is capable of down-regulation of endogenous ErbB-4 expression by 30 and 80% in two of the ribozyme-transfected T47D pooled population clones (T47D/Rz-poolA and T47D/Rz-pool20, respectively). These two pooled population clones were selected by geneticin resistance from two-independent transfections. We also found that ErbB-4 expression was almost completely down-regulated in some of the ErbB-4 ribozyme-transfected MCF-7 cells, such as MCF-7/RzA4 (Fig. 2)Citation , as well as in ribozyme-transfected MDA-MB-453 cells (Fig. 3)Citation . However, no effect was observed on other EGF family receptors in these ErbB-4 ribozyme-transfected cells, respectively (Figs. 1 and 2Citation ). Furthermore, ribozyme-mediated down-regulation of ErbB-4 receptor expression was confirmed by reduction of ErbB-4 mRNA by Northern blot analysis (data not shown).



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  		Fig. 1. ErbB-4 ribozyme-mediated down-regulation of endogenous ErbB-4 expression in T47D human breast cancer cells. The level of EGF family receptors in T47D/wt and T47D/Rz pool clones were quantitatively measured by flow cytometry. Cells (1 x 106 cells) were harvested and stained with specific monoclonal antibodies against ErbB-4 receptor, in combination with fluorescence-labeled antimouse IgG antibody, and analyzed by FACScan. · · · · (far left), nonspecific staining (primary antibody omitted). , expression of ErbB family receptors in T47D wild-type cells. (boldface), expression of ErbB family receptors in T47D/Rz-poolA. {blacksquare} {blacksquare} {blacksquare} {blacksquare}, expression of ErbB family receptors in T47D/Rz-pool20. Top left panel, expression of EGFR in T47D/wt, T47D/Rz-poolA, and T47D/Rz-pool20 cells. Top right panel, expression of ErbB-2 receptor in T47D/wt, T47D/Rz-poolA, and T47D/Rz-pool20 cells. Bottom left panel, expression of ErbB-3 receptor in T47D/wt, T47D/Rz-poolA, and T47D/Rz-pool20 cells. No effect was observed on the levels of EGFR, ErbB-2, and ErbB-3 receptor expression in these ErbB-4-ribozyme-transfected T47D pooled clones. Bottom right panel, expression of ErbB-4 receptor in T47D/wt, T47D/Rz-poolA, and T47D/Rz-pool20 cells. T47D/ErbB-4 expression was down-regulated by 30% in T47D/Rz-poolA cells and 80% in T47D/Rz-pool20 cells, respectively.

 


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  		Fig. 2. ErbB-4 ribozyme-mediated down-regulation of endogenous ErbB-4 expression in MCF-7 human breast cancer cells. The level of EGF family receptors in MCF-7 wild-type (MCF-7/Wt), vector-transfected only (MCF-7/Vector), and two of the ribozyme-transfected MCF-7 clones (MCF-7/B1 and MCF-7/A4) were quantitatively measured by flow cytometry. Cells (1 x 106) were harvested and stained with specific monoclonal antibodies against different receptors of the EGF family in combination with fluorescence-labeled antimouse IgG antibody and analyzed by FACScan. · · · ·, nonspecific staining (primary antibody omitted). , expression of ErbB-4. Top left panel, expression of ErbB-4 in MCF-7/wt cells. Top right panel, expression of ErbB-4 in MCF-7/vector cells. Bottom left panel, expression of ErbB-4 in MCF-7/RzB1 cells. Bottom right panel, expression of ErbB-4 in MCF-7/RzA4 cells. Ribozyme down-regulated ErbB-4 expression significantly in MCF-7/RzB1 and MCF-7/RzA4 cells.

 


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  		Fig. 3. ErbB-4 ribozyme-mediated down-regulation of endogenous ErbB-4 expression in MDA-MB-453 human breast cancer cells. The level of EGF family receptors in MDA-MB-453 wild-type (MDA-MB-453/wt) and one of the ribozyme-transfected MDA-MB-453 clones (MDA-MB-453/Rz) were quantitatively measured by flow cytometry. Cells (1 x 106) were harvested and stained with specific monoclonal antibodies against different receptors of the EGF family in combination with fluorescence-labeled antimouse IgG antibody and analyzed by FACScan. · · · ·, nonspecific staining (primary antibody omitted). , expression of ErbB-4. MDA-MB-453 expresses low levels of ErbB-4, and ErbB-4 expression was completely abrogated by ribozyme in MDA-MB-453/Rz cells.

 
Reduction of NRG- and BTC-induced ErbB-4 Autophosphorylation in T47D/Rz-Transfected Cells.
We next determined whether NRG- or BTC-induced ErbB-4 receptor tyrosine phosphorylation was affected by reduction of ErbB-4 expression in ribozyme-transfected cells. Phosphorylation experiments were performed on ribozyme-transfected clones. Fig. 4Citation demonstrates that the level of ErbB-4 intrinsic tyrosine kinase activity in T47D/Rz-pool20-transfected cells was markedly reduced when compared with control transfectants (T47D/wt and T47D/Vector) cells. Reduction of phosphorylation correlated with a reduction in ErbB-4 expression level. A similar effect was observed in BTC-induced ErbB-4 tyrosine phosphorylation. These experiments demonstrate that the reduction of ErbB-4 activation correlates with down-regulation of ErbB-4 protein production.



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  		Fig. 4. Reduction of NRG- and BTC-induced ErbB-4 autophosphorylation in T47D/Rz-transfected cells. Cells were treated with or without NRG1-a and BTC (100 ng/ml) for 5 min before lysis, and 400 mg of lysates were immunoprecipitated with a specific anti-ErbB-4 antibody. Precipitated proteins were then subjected to Western blotting with an anti-phosphotyrosine antibody (UBI). Lane 1, molecular weight (MW) standards. Lanes 2, 5, and 8, untreated samples. Lanes 3, 6, and 9, lysates from T47D/wt, T47D/vector, and T47D/Rz-pool20 cells treated with 100 ng/ml of NRG1-a. Lanes 4, 7, and 10, lysates from T47D/wt, T47D/vector, and T47D/Rz-pool20 cells treated with 100 ng/ml of BTC. Down-regulation of ErbB-4 in T47D cells dramatically reduced NRG- and BTC-induced ErbB-4 phosphorylation. WT, wild type; RZ, ribozyme.

 
Down-Regulation of ErbB-4 in Cell Lines Expressing a Relatively High Level of ErbB-4 Resulted in an Inhibition of Colony Formation.
To assess the biological significance of ErbB-4 in human breast cancer, we evaluated the in vitro growth of ErbB-4 ribozyme (Rz6) transfected T47D, MCF-7, MDA-MB-453, and MDA-MB-231 cells by anchorage-dependent as well as anchorage-independent growth assays. Down-regulation of ErbB-4 expression in ER+ human breast cancer cell lines expressing a relatively high level of ErbB-4 (T47D and MCF-7 cells) resulted in an inhibition of colony formation that was independent of colony size. Fig. 4Citation illustrates that colony formation was almost completely abolished in T47D/Rz-pool20 cells, which had an 80% down-regulation of ErbB-4, indicating a partial reversion of transformation. Down-regulation of ErbB-4 by 30% in T47D/Rz-poolA cells also displayed a 40% reduction in their ability to form colonies in soft agar (Fig. 5)Citation . Furthermore, inhibition of colony formation was independent of threshold colony size. These data demonstrated that inhibition of growth is correlated with the level of down-regulation of ErbB-4 in these ribozyme-transfected cells. A similar inhibition phenotype was observed in ribozyme-transfected MCF-7 cells (Fig. 6)Citation . However, growth inhibition was not observed in ER- MDA-MB-453/Rz cells (Fig. 7)Citation , which express low levels of ErbB-4 and high levels of ErbB-2 and ErbB-3. Interestingly, FACS analysis revealed that the expression of the ErbB-4 receptor was completely abrogated by the ErbB-4 ribozyme in these cells as well (Fig. 3)Citation . In a parallel experiment, we verified the specificity and efficacy of the anti-ErbB-4 ribozymes with MDA-MB-231 cells, which do not express detectable level of ErbB-4 (data not shown). Obviously, no effect was observed in ribozyme-transfected MDA-MB-231 cells, respectively (Table 1)Citation . These data suggest that the biological effect of ErbB-4 receptor expression is dependent upon its relative levels in a given cell line.



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  		Fig. 5. Growth effects of ErbB-4 ribozyme on T47D cells (ER+). Expression of the ErbB-4 ribozyme in T47D cells (T47D/Rz-poolA and T47D/Rz-pool20) inhibits colony formation, independent of colony size. For anchorage-independent growth assays, a bottom layer of 0.1 ml IMEM containing 0.6% agar and 10% FCS was prepared in 35-mm tissue culture dishes. After the bottom layer solidified, cells (10,000 cells/dish) were then added in a 0.8-ml top layer, containing 0.4% Bacto Agar and 5% FCS. All samples were prepared in triplicate. The cells were incubated for ~12 days at 37°C. Colonies larger than 60, 80, 100, and 120 µm were counted by a cell colony counter. Bars, SD.

 


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  		Fig. 6. Growth effects of ErbB-4 ribozyme on MCF-7 cells (ER+). The degree of reduction of colony formation was correlated with the level of ErbB-4 expression down-regulated by ErbB-4 ribozyme in MCF-7 cells. Clones (MCF-7/Rz B1 and MCF-7/Rz A4) that exhibited almost depletion of ErbB-4 expression by ribozyme appeared to reduce colony formation by >60% compared with MCF-7/wt and MCF-7/Vector cells. For anchorage-independent growth assays, a bottom layer of 0.1 ml of IMEM containing 0.6% agar and 10% FCS was prepared in 35-mm tissue culture dishes. After the bottom layer solidified, cells (10,000 cells/dish) were then added in a 0.8-ml top layer, containing 0.4% Bacto Agar and 5% FCS. All samples were prepared in triplicate. The cells were incubated for ~12 days at 37°C. Colonies >120 µm were counted by a cell colony counter. Values indicate the mean of triplicate determinations; bars, SD.

 


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  		Fig. 7. Growth effects of ErbB-4 ribozyme on MDA-MB-453 cells (ER-). Anchorage-dependent growth assays were performed to assess the growth effect on ribozyme-transfected MDA-MB-453 cells (MDA-MB-453/Rz) cells. Wild-type MDA-MB-453 cells and MDA-MB-453/Rz cells were plated in 24-well plates. Cells were counted on days 1, 3, and 7. All samples were prepared in triplicate. Values indicate the means of triplicate determinations; bars, SD. No significant effect was observed in MDA-MB-453/Rz cells.

 

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  		Table 1 Selective growth inhibition with ribozyme-mediated down-regulation of ErbB-4 in breast cancer cellsa

 
The Sensitivity of Biological Responses to Different EGF-like Ligands Is Dependent upon the Relative Levels of ErbB Family Receptor Expression.
The regulation of ErbB receptor family member activation is very complex. A large number of ErbB ligands have been described (37, 38, 39, 40) . We next compared the effects of EGF-like ligands on ribozyme-transfected T47D cells (T47D/Rz) versus T47D/wt. We observed that NRG-induced colony formation was significantly inhibited in T47D/Rz-transfected cells. Down-regulation of ErbB-4 in T47D cells reduced NRG-stimulated colony formation by 80%. In contrast, wild-type T47D cells exhibited an 11-fold increase in colony formation when treated with NRG, which appeared to have the most dominant effect among the six of EGF-like ligands tested (Fig. 8)Citation . These data demonstrate that NRG was significantly more active than other EGF-like ligands in T47D wild-type cells, whereas down-regulation of ErbB-4 in T47D cells revealed almost complete abrogation of the NRG activity, suggesting that NRG signaling occurs primarily through ErbB-4 in T47D cells. The lower transformation activity stimulated by BTC in ErbB-4-depleted T47D cells suggested that BTC predominantly binds to EGFR. The BTC effect most likely is attributable to the net sum of ErbB-4 remaining dimers and non-ErbB-4 homo- or heterodimers (Fig. 8)Citation .



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  		Fig. 8. Down-regulation of endogenous ErbB-4 expression in T47D cells strongly inhibited NRG-induced colony formation. For anchorage-independent growth assays, a bottom layer of 1 ml of IMEM containing 0.6% agar and 10% FCS was prepared in 35-mm tissue culture dishes. After the bottom layer solidified, cells (10,000 cells/dish) were then added on a 0.8-ml top layer containing 0.4% Bacto Agar, 5% FCS, and 100 ng/ml of EGF-like ligands. All samples were prepared in triplicate. The cells were incubated for ~15 days at 37°C. Colonies >60 µm were counted in a cell colony counter. {square}, T47D wild-type cells. {blacksquare}, ErbB-4 ribozyme-transfected T47D cells (T47D/Rz-pool20). Down-regulation of ErbB-4 expression decreases the spectrum and potency of EGF-like, ligand-stimulated colony formation. NRG-{alpha}-stimulated colony formation was reduced by 70%, and HB-EGF-stimulated colony formation was completely abolished.

 
Inhibition of Tumor Formation in Nude Mice.
Down-regulation of ErbB-4 led to dramatic effects on anchorage-dependent and anchorage-independent growth in MCF-7 and T47D cells. We next explored the in vivo effects of down-regulation of ErbB-4 in MCF-7 and T47D cells. Wild-type cells (5 x 106 MCF-7 or T47D), as well as the ribozyme-transfected cells, were implanted in ovariectomized mice. With estradiol treatments, the T47D wild-type cells grew to a mean tumor size of 500 ± 20 mm3 (Fig. 9)Citation . In contrast, tumor growth of ribozyme-expressing T47D cells was significantly inhibited (P < 0.03; Student’s t test) with a mean tumor size of 80 ± 14 mm3 (Fig. 9)Citation . Moreover, tumor growth of T47D cells transfected with the catalytically inactive ribozyme (Rz21) was not significantly different from control cells (data not shown). Similar experiments were performed with ribozyme-transfected MCF-7 cells. Fig. 10Citation demonstrated that the down-regulation of ErbB-4 expression in MCF-7 cells dramatically reduced the tumor formation. With estradiol treatment, the MCF-7 wild-type (MCF-7/wt) and empty vector-transfected MCF-7 cells (MCF-7/vector) grew large tumors with a mean tumor size of 2400 ± 270 mm3. In contrast, tumor growth of ribozyme-expressing MCF-7 cells was drastically inhibited with a mean tumor size of 580 ± 74 mm3 (P < 0.001; Student’s t test). Table 1Citation summarizes the in vitro and in vivo effects of down-regulation of ErbB-4 in human breast cancer cell lines.



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  		Fig. 9. ErbB-4-ribozyme-mediated down-regulation of ErbB-4 in T47D cells resulted in reduction of tumor growth in vivo. Wild-type cells (5 x 106 T47D), as well as the ribozyme-transfected cells (T47D/Rz-poolA and T47D/Rz-pool20), were implanted in ovariectomized mice. With estradiol treatments, the T47D wild-type cells grew to a mean tumor size of 500 ± 20 mm3 (•). In contrast, tumor growth of ribozyme-expressing T47D cells was significantly reduced (P < 0.03; Student’s t test) with a mean tumor size of 80 ± 14 mm3 ({blacktriangleup}, {blacksquare}).

 


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  		Fig. 10. ErbB-4-ribozyme-mediated down-regulation of ErbB-4 in MCF-7 cells resulted in reduction of tumor growth in vivo. Wild-type cells (5 x 106 MCF-7), as well as the ribozyme-transfected cells, were implanted in ovariectomized mice. With estradiol treatments, the MCF-7 wild-type cells, as well as the empty vector-transfected cells, grew large tumors to a mean tumor size of 2000 ± 200 mm3 (•, {circ}). In contrast, tumor growth of ribozyme-expressing MCF-7 cells was significantly inhibited (P < 0.001; Student’s t test) with a mean tumor size of 600 ± 74 mm3 ({blacktriangledown}, {triangledown}). Bars, SD.

 
Expression of ErbB-4 in Primary Breast Carcinomas.
We conducted a pilot study to investigate the frequency of ErbB-4 expression in breast carcinomas using immunohistochemical analysis with an anti-ErbB-4 monoclonal antibody. The expression of ErbB-4 was analyzed in five benign tumors and 50 primary breast carcinomas. No expression was detected in four of five benign tumors, and one of five of these benign tumors was observed with weak ErbB-4 expression. We observed that 60% (30 of 50) of the primary breast carcinomas express ErbB-4. High intense immunoreactivity of ErbB-4 was detected in 18% (9 of 50) of these primary breast tumors. The ErbB-4 immunoreactivity was detected in both membrane and cytoplasmic locations in most of the cases. No nuclear staining and negligible of ErbB-4 immunostaining in stromal elements of the tumor specimens were observed in all cases. Fig. 11Citation illustrated a representative breast carcinoma sample stained for ErbB-4 expression. Interestingly, 87% (26 of 30) of the ErbB-4-positive samples were ER+ breast carcinomas and 55% (11 of 20) of the negative or weak ErbB-4 expressions were ER- breast carcinomas (Table 2)Citation . It appears that there was a statistically significant (P = 0.01) direct correlation between the expression of ERs and the expression of ErbB-4. In addition, we also surveyed the ErbB-4 expression in a panel of human breast cancer cell lines by FACS analysis. Surprisingly, most of ER+ cell lines expressed relatively high levels of ErbB-4, and ER- cell lines expressed low levels or nondetectable levels of ErbB-4 (Table 3)Citation . The ErbB-4 expression seems inversely correlated with EGFR expression.



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  		Fig. 11. Expression of ErbB-4 is correlated with ER+ primary breast carcinomas. Immunohistochemistry staining of ErbB-4 in paraffin sections of human primary breast tumor specimens (brown). All sections were counterstained with hematoxylin for viewing negatively stained cells (blue). Top left panel, benign ducts do not express ErbB-4. Top right panel, infiltrating ductal carcinoma. ER+ tumors express high levels of ErbB-4. Bottom left panel, intraductal and infiltrating ductal carcinoma. ER+ tumors express high levels of ErbB-4. Bottom right panel, intraductal and infiltrating ductal carcinoma. ER- tumors do not express detectable levels of ErbB-4. Expression of ErbB-4 is correlated with ER+ primary breast carcinomas. Stromal cells and the ductal epithelium were negative for ErbB-4 in most cases.

 

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  		Table 2 Correlation of ErbB-4 expression with hormonal receptor status in primary breast tumor

 

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  		Table 3 ErbB-4 expression correlated with ER expression in human breast cancer cell lines

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies from our laboratory have demonstrated that ErbB-4 ribozymes (Rz6 and Rz29) are biologically functional ribozymes and are capable of the down-regulation of ErbB-4 in 32D/ErbB-4 cells and the abolishment of NRG-induced mitogenesis (33) . In this study, we used ribozyme technology to achieve the functional gene "knockout" strategy to define the role and biological significance of ErbB-4 in human breast cancer. We demonstrated that ErbB-4 ribozymes are capable of down-regulating endogenous ErbB-4 expression in several human breast cancer cell lines. The ErbB-4 ribozymes had no effect on other members of the EGF receptor family. In stable mass-transfected T47D cells, ErbB-4 ribozyme expression depleted ErbB-4 mRNA and protein levels by 30 and 80%. This inhibition is even more remarkable when considering that mass-transfected cells (and not clonal subpopulations) were used. This substantial inhibition enabled us to begin a novel study of the effects of a functional ErbB-4 knockout on in vitro and in vivo tumor growth of breast cancer cells. We observed that down-regulation of ErbB-4 in ER+ breast cancer cell lines (T47D and MCF-7), which express relatively high levels of ErbB-4, significantly inhibited colony formation. In addition, down-regulation of ErbB-4 in T47D cells significantly impaired NRG-induced ErbB-4 phosphorylation. However, complete depletion of ErbB-4 did not affect the anchorage-dependent and anchorage-independent growth in ER- MDA-MB-453 cells, which express low levels of endogenous ErbB-4 and high levels of ErbB-2 and ErbB-3. Furthermore, down-regulation of ErbB-4 in T47D and MCF-7 cells significantly inhibited tumor formation in athymic nude mice with P < 0.03 and P < 0.001, respectively. These data provide the first evidence that an elevation of ErbB-4 expression plays a role in the proliferation of some ER+ human breast cancer cell lines (T47D and MCF-7) both in vitro and in vivo. These data suggest that there is an inhibition of growth when relatively high levels of expressed receptors are targeted. Furthermore, ErbB receptors undergo extensive heterodimerization. The inactivation or blocking of ErbB-4 signaling may also disrupt and diminish the EGFR or ErbB-2 signaling pathways, through heterodimerization with ErbB-4. Expressing low levels of ErbB-4 does not significantly affect breast cancer cell proliferation. A similar conclusion was reported by Jannot et al. (41) and Kataoka et al. (42) . Blocking cell surface expression of ErbB-2 and EGFR by intracellular expression of a single-chain antibody specific for ErbB-2 (scFv-5R) and EGFR (scFv-R1R) led to only a slight reduction in colony formation of T47D cells, which express low levels of ErbB-2 and EGFR. However, in MDA-MB-468 cells, scFv-5R and scFvR1R inhibited colony formation by 90 and 94%, respectively. In MDA-MB-468 cells, which express high levels of EGFR and transforming growth factor symbol 97 {alpha}, treatment with a monoclonal antibody competes with ligand binding and inhibits cell growth, indicating that these cells are dependent upon an autocrine loop for growth. Despite the fact that these cells have very low levels of ErbB-2, inhibition of colony formation by scFvR suggests that transforming growth factor symbol 97 {alpha}-activated heterodimers of EGFR and ErbB-2 provide the major growth stimulus to these cells (41 , 42) . The presence or absence of a specific EGF family receptor is not the only influence in the nature of cell proliferation, but the actual expression levels of the individual ErbB family receptors and their cognate ligands contribute a significant role to breast cancer cell proliferation. Overexpression of an ErbB receptor or coexpression of its cognate ligands plays an essential role in breast cancer proliferation. Conversely, the expression of low levels of ErbB receptors does not significantly contribute to breast cancer cell proliferation. These studies further imply that ErbB family receptors and their ligands play an important role in breast cancer cell proliferation. In general, the inhibition of growth in breast cancer cells was observed when overexpressed receptors were targeted.

Regulation of the activation of ErbB receptor family members is very complex. ErbB receptors undergo extensive heterodimerization, which makes ligand-induced signaling even more complex. We show that NRG-stimulated phosphorylation of ErbB-4 was significantly reduced and NRG-induced colony formation was substantially reduced from 11-fold to only 2.5-fold in ribozyme-transfected T47D cells (T47D/Rz), indicating that the major NRG signaling was through ErbB-4. It implies that NRG signaling through ErbB-2/ErbB-3 heterodimers may play a minor role in T47D cells because of their low expression levels. Furthermore, down-regulation of ErbB-4 expression decreases the spectrum and potency of EGF-like, ligand-mediated proliferation. HB-EGF, the ligand for EGFR and ErbB-4- mediated proliferation, was completely abolished in ribozyme-transfected T47D cells. These results suggest that down-regulation of ErbB-4 expression might interrupt the ErbB-4 heterodimerization that interrupts the other EGF-like ligand signaling through EGF family receptors. With BTC, a ligand for both EGFR and ErbB-4 as well as ErbB-2/ErbB-3 heterodimers, down-regulation of ErbB-4 only partially affected the BTC signaling (Fig. 8)Citation . These results suggest that the BTC effect in ErbB-4-depleted cells is most likely attributable to the binding of BTC to EGFR homodimers or EGFR heterodimers. Thus, the sensitivity of a cell line to EGF-like ligands is correlated with the levels of expression of the ErbB receptors in the cell line. In addition, EGF-related growth factors show distinguishable biological activities, which most likely depend upon the subsets of ErbB receptors that become activated. Relative availability of ligands, receptors, and secondary pathways would appear to critically alter the proliferation/differentiation status of breast cancer.

In addition, we have also investigated the expression of ErbB-4 in primary breast carcinoma, using immunohistochemical analysis with an anti-ErbB-4 monoclonal antibody. No expression was detected in the majority (four of five) of benign tumors, and only one case (one of five) was observed with very weak ErbB-4 expression. ErbB-4 expression was found in 60% (30 of 50) of the 50 samples examined. High intense immunoreactivity of ErbB-4 was detected in 18% (9 of 50) of these primary breast tumors. Most of the staining was found in both cell membrane and cytoplasm. No nuclear staining and negligible ErbB-4 immunostaining in stromal elements of the tumor specimens were observed in most of cases (Fig. 11)Citation . These results are consistent with other investigators’ finds. Expression of ErbB-4 is common in breast cancer cells and was detected as often as in 75% of cases (43 , 44) . A very recent report has demonstrated that amplification of the erbB-4 oncogene was detected in 13% of primary human breast cancers (29) . Table 2 shows the frequencies of ER/PR status for varying levels or ErbB-4 expression. The correlation between the ErbB-4 expression and ER expression was assessed by the Kruskal-Wallis test (34) . ER/PR status does differ with varying levels of ErbB-4 expression. Expression of ErbB-4 appeared to correlate with ER+ primary breast tumors with P = 0.01. However, the correlation between ErbB-4 expression and PR+ expression is not significant with P = 0.0845 by Fisher’s exact test. In addition, Table 3Citation shows that most ER+ cell lines express relatively high levels of ErbB-4, and ER- cell lines express low levels or nondetectable levels of ErbB-4. The ErbB-4 expression seems inversely correlated with EGFR expression. Taken together, it is interesting that expression of ErbB-4 is associated with the prognostically favorable ER phenotype, unlike other EGF family receptors. As this report was being prepared for submission, it was reported that a direct association was found between ErbB-3 and ErbB-4 mRNA and ER marker status. Inverse associations were seen between ErbB-3 and ErbB-4 mRNA marker status and EGFR expression (40) . These data are consistent with our findings that ErbB-4 expression is associated with ER+/PR+ breast tumors. Elevated ErbB-4 expression in breast cancer cells, particularly in ER+ tumors could therefore represent a differentiated feature. In addition, its ligand NRG-1 has been shown to initiate cellular differentiation in breast cancer cells in vitro (45 , 46) . It will be intriguing to define the mechanisms that the expression of the ErbB-4 receptor may use in maintaining the ER expression in human breast cancer.

In conclusion, elevation of the ErbB-4 receptor plays a role in ER+ breast cancer cells proliferation. Furthermore, these results also indicate that the role of ErbB family receptors in breast cancer cells is not solely dependent on the absolute expression levels of any single ErbB family member but also depends on the relative expression levels of all ErbB family members.


   FOOTNOTES
 
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.

1 Supported in part by United States Army Medical Research and Materiel Command Grant DAMD17-96-1-6031 (to C. K. T.) and by Specialized Programs of Research Excellence Grant IP50-CA58185-04 from the National Cancer Institute. The FACS analysis data shown in Figs. 1Citation 2Citation 3Citation were supported in part by the Lombardi Cancer Research Center Flow Cytometry Core Facility. Xenografts in Figs. 9Citation and 10Citation were supported by the Lombardi Cancer Center Animal Shared Resource Facility, USPHS Grant P30-CA-51008. Back

2 To whom requests for reprints should be addressed, at Georgetown University Medical Center, E512 Research Building, 3970 Reservoir Road NW, Washington, DC 20007-2197. Phone: (202) 687-0361; Fax: (202) 687-7505; E-mail: Tangc{at}gunet.georgetown.edu Back

Received 4/21/99. Accepted 8/18/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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