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Cell Growth & Differentiation Vol. 11, 173-183, March 2000
© 2000 American Association for Cancer Research

Blocking HER-2/HER-3 Function with a Dominant Negative Form of HER-3 in Cells Stimulated by Heregulin and in Breast Cancer Cells with HER-2 Gene Amplification1

Tracy G. Ram2, Margaret E. Schelling and Howard L. Hosick

Schools of Biological Sciences and Molecular Biosciences, Washington State University, Pullman, Washington 99164-4236


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Amplification and overexpression of the HER-2 (neu/erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification. This indicates that inhibition of HER-2/HER-3 heterodimer function may be an especially effective and unique strategy for blocking the HER-2-mediated transformation of breast cancer cells. Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominant negative form of HER-3 in which most of the cytoplasmic domain was removed for introduction into cells. By using a bicistronic retroviral vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations. Breast carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/HER-3 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. Responses to exogenous HRG were strongly inhibited by dominant negative HER-3. In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and -independent growth assays in culture. Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The HER-2 (neu/erbB-2) gene encodes a Mr 185,000 protein tyrosine kinase that is highly homologous to the EGF3 receptor (HER-1/EGFR/erbB-1), HER-3 (erbB-3), and HER-4 (erbB-4; Refs. 1, 2, 3 ), which together comprise the type 1 family of receptor tyrosine kinases (4 , 5) . The HER family receptor tyrosine kinases all contain ectodomains with two cysteine-rich sequences. Despite this structural homology, these receptors differ in their ligand specificities (4) . Thus, HER-1 binds several ligands closely related to EGF, whereas HER-3 and HER-4 are the receptors for a number of different isoforms of neu differentiation factor/HRG (6, 7, 8) . Whereas no direct ligand for HER-2 has yet been cloned, it is now clear that HER-2 is capable of heterodimerization with HER-1 (9 , 10) , HER-3 (11 , 12) , or HER-4 (8) , and these HER-2-containing heterodimers form the highest affinity binding sites for their respective ligands (10 , 11) . HER-2 is amplified in 28% of primary breast carcinomas in vivo (13) , and another 10% of primary breast carcinomas overexpress HER-2 without amplification of the gene (14, 15, 16) . In addition, HER-2 gene amplification concordant with high-level overexpression is associated with increased tumor aggressiveness and the poor prognosis of breast cancer patients (13 , 14 , 17, 18, 19) . Other related genes, such as the HER-1 gene, are sometimes amplified in human breast cancers (13) . However, amplification of the HER-1 gene is much less common than that seen for HER-2 (2% versus 28%, respectively) in breast cancer. Whereas amplification of HER-3 or HER-4 has not been seen in various studies (2 , 3) , our own work and the studies of others (20, 21, 22, 23) have now shown that heterodimer interactions between HER-2 and HER-3 are constitutively activated in breast cancer cells with HER-2 gene amplification, and cotransfection of HER-3 with HER-2 greatly augments the transforming capability of HER-2 in genetically engineered cell lines (21) . HER-2/HER-3 heterodimer complexes are now thought to potently activate the PI 3-kinase and mitogen-activated protein kinase signal transduction pathways to a level that is effective for transformation. We are particularly interested in how the cooperative effects of HER-2 and HER-3 activate various mitogenic signal transduction pathways involved in cell growth.

Experimentally elevated HER-2 gene expression in various cell lines, including nontransformed human mammary epithelial cells, induces the complete transformation of cells injected into nude mice (24, 25, 26, 27) . The potent oncogenic potential of HER-2 is generally thought to be due to its ability to constitutively activate various key signal transduction pathways that are involved in the regulation of cell growth. However, whereas our current understanding of the oncogenic potential of HER-2 has expanded quite rapidly (for review, see Ref. 28 ), our knowledge of exactly how HER-2 induces the neoplastic transformation of human mammary epithelial cells still remains fragmentary. For example, although HER-2 was originally discovered as the neu transmembrane-mutated form of the gene in rat neuroblastoma cells (29) , the HER-2 gene found in human breast cancer has never shown such mutations (30) , but the level of tyrosine-phosphorylated HER-2 in primary human breast cancer in vivo always shows a direct correspondence with the overexpression of HER-2 (31) . This suggests that high-level overexpression of wild-type HER-2 alone is sufficient to constitutively activate its tyrosine kinase function. Furthermore, the protein encoded by the wild-type HER-2 gene was also previously shown to possess constitutive tyrosine kinase activity if sufficiently overexpressed in a variety of cell lines in culture in the absence of any identifiable ligand (24, 25, 26, 27 , 32 , 33) , and transfection of a gene encoding a chimeric receptor containing the HER-1 extracellular domain fused to the cytoplasmic domain of HER-2 results in the constitutive tyrosine kinase activity of the chimeric receptor in the absence of EGF (32 , 33) . This indicates that the tyrosine kinase domain of HER-2 exhibits a greater tendency toward ligand-independent activation than do the other HERs when overexpressed.

Another area of great importance concerns the heterodimeric associations that are now known to occur between the different HER proteins, including HER-1 and HER-2 (9 , 10) , HER-2 and HER-3 (11 , 12) , HER-2 and HER-4 (8) , and HER-1 and HER-3 (34 , 35) in response to ligands. Our own work and that of others (20, 21, 22) has now established that the heterodimer interactions between HER-2 and HER-3 are also constitutively activated in breast cancer cells with HER-2 gene amplification, and the cooperative interactions between HER-2 and HER-3 are associated with the constitutive activation of various signaling pathways in cancer cells with HER-2 gene amplification. However, the involvement of HER-2/HER-3 heterodimers in the constitutive activation of signaling pathways that transform cancer cells with HER-2 gene amplification has not yet been tested with perturbative analysis. One strategy that has been used successfully to block the function of other receptor tyrosine kinases uses dominant negative expression vectors in which the region coding for the cytoplasmic domain of the receptor is almost completely removed. Although the truncated receptor still contains the transmembrane domain and can thus dimerize within the cell, it lacks tyrosine kinase activity and inhibits the signal transduction docking function. This strategy has been used effectively to block HER-1 (36) , platelet-derived growth factor receptor (37) , and fibroblast growth factor receptor (38) . Recently, a dominant negative HER-2 vector was also used successfully to block HER-2 function in normal mouse development (39) . The use of such HER-2 vectors has apparently not yet been useful for blocking HER-2 function in cancer cells with HER-2 gene amplification, probably due to the stoichiometric problems associated with the inability to generate mutant/wild-type levels high enough for effective inhibition. However, the fact that HER-3 is not highly overexpressed in these cells and that activated HER-2 and HER-3 have a particularly high affinity interaction (40, 41, 42) suggests that dominant negative HER-3 may be especially effective in blocking HER-2/HER-3 function.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
H16N-2 and 21 MT-1 Cells Provide a Model System for Studying the Role of HER-2/HER-3 in HRG-induced Mitogenesis and the Autonomous Growth of Cancer Cells with HER-2 Gene Amplification.
For these studies, we used cell lines originally isolated from a patient with infiltrating and intraductal carcinoma of the breast with HER-2 gene amplification (43 , 44) . The 21 MT-1 metastatic breast carcinoma cell line was isolated from a pleural effusion collected during an advanced stage of the disease. H16N-2 cells are nonneoplastic cells isolated from normal mammary tissue of the same patient; thus, they serve as an ideal control for studying the 21 MT-1 cells as well as the effects of HRG in nontransformed cells. RFLP analysis had previously shown that these cell lines share common genetic polymorphisms (43) , and we have also verified that the H16N-2 and 21 MT-1 cells are derived from a single individual by DNA fingerprinting analysis of a hypervariable region of the BRCA-1 locus.4 We have previously shown that the amplification and high-level overexpression of HER-2 in the 21 MT-1 cells is associated with HER-2/HER-3-mediated activation of PI 3-kinase and growth factor independence (i.e., autonomous growth) in SF culture (22 , 45) . This system is ideal for studying receptor activation and signaling under well-defined conditions that allow us to distinguish constitutive from externally mediated growth factor responses in culture. To directly measure the activation of HER-2 and HER-3 in these cells, we starved the cells of growth factors for 48 h in SF medium and then directly extracted the cells (Fig. 1, A and B, Lanes 1 and 3Citation ) or stimulated the cells with HRG-ß for 10 min before extraction (Fig. 1, A and B, Lanes 2 and 4Citation ). Immunoprecipitation followed by immunoblotting directly showed the levels of HER-2 and HER-3 activated in these cells. HRG induced tyrosine phosphorylation of both HER-2 and HER-3 in H16N-2 cells (Fig. 1, A and B, Lane 2Citation ), whereas 21 MT-1 cells show high-level constitutive activation of both HER-2 and HER-3 in the absence of exogenous growth factors in culture (Fig. 1, A and B, Lane 3Citation ) due to the amplification and overexpression of HER-2 in these cells (22 , 45) .



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  		Fig. 1. Activation of HER-2/HER-3 by HRG and its constitutive activation in breast cancer cells with HER-2 gene amplification. Samples containing 2 mg of cell lysate protein were immunoprecipitated with anti-phosphotyrosine antibody, followed by immunoblotting with anti-HER-2 antibody (A) or immunoprecipitation with anti-HER-3 antibody followed by immunoblotting with anti-phosphotyrosine antibody (B) to show the level of HER-2 and HER-3 activated in cells with or without HRG stimulation in culture. H16N-2 nontransformed human breast epithelial cells (Lanes 1 and 2) and 21 MT-1 metastatic breast carcinoma cells with HER-2 gene amplification (Lanes 3 and 4) were starved of growth factors for 48 h in SF medium and then directly extracted (Lanes 1 and 3) or stimulated with 10 ng/ml HRG-ß for 10 min at 37°C before extraction (Lanes 2 and 4).

 
Construction of H16N-2 and 21 MT-1 Cell Lines Expressing Dominant Negative HER-3.
We previously found that the introduction of standard monocistronic expression vectors (in which the antibiotic resistance gene and marker gene are driven by separate promoters) into many different cell lines did not lead to very efficient coordinate coexpression of antibiotic resistance with the gene of interest. Experiments were performed using retroviral control vectors containing the Neor and LacZ+ genes placed in either monocistronic or bicistronic configuration and then either transfected or infected into target cell lines to assess the LacZ+ gene expression in G418-selected cell colonies (Fig. 2A)Citation . The results showed that cells infected with the bicistronic retroviral vector (in which the LacZ+ and Neor genes form a single transcription unit driven by one promoter) coordinately coexpressed antibiotic resistance with LacZ+ expression in 100% of the G418-selected cell colonies. Thus, the infection of bicistronic retroviral vectors completely eliminated the occurrence of false positive clones in genetically engineered cells and resulted in greater efficiency of LacZ+ gene expression within cell clones as well (data not shown). These results attained using the H16N-2 cells are also similar to those seen for a number of different mammary epithelial cell lines, including the 21 MT-1 cells (data not shown). Therefore, we used the pCMV bicistronic retroviral vector to express dominant negative HER-3 in target cells. By using the pBK-CMV phagemid expression vector (Stratagene) as an intermediate, we cloned a dominant negative HER-3 fragment into the pCMV bicistronic retroviral expression vector using flanking restriction sites located within the extensive polylinker region of pBK-CMV. The human HER-3 cDNA was used to clone a 2.2-kb fragment of HER-3 lacking most of the cytoplasmic domain into pBK-CMV to generate pBK-CMV-dn3, and this ligation also introduced an in-frame stop codon 12 codons downstream of the point of ligation. The dominant negative HER-3 insert removed from pBK-CMV-dn3 was then cloned into pCMV to generate pCMV-dn3 (Fig. 2B)Citation . Restriction digest analysis confirmed the proper construction of the vectors (data not shown). H16N-2 and 21 MT-1 cells were then infected with the pCMV backbone (used as a control) or pCMV-dn3 using the {psi}CRIP packaging cell line. The cell lines infected with the control vector are referred to as H16N-2 and 21 MT-1 cells, whereas those infected with pCMV-dn3 are referred to as H16N-2-dn3 and 21 MT-1-dn3 cells. Immunocytochemistry was performed to confirm that the H16N-2-dn3 and 21 MT-1-dn3 cells express the dominant negative HER-3 (Fig. 3)Citation . Expression of the ectopic HER-3 protein was assessed using the H105 anti-HER-3 monoclonal antibody that binds specifically to an epitope within the extracellular domain of HER-3. Although H16N-2 and 21 MT-1 cells express wild-type HER-3, the wild-type HER-3 protein levels are below the level for immunodetection with HRP/DAB staining using immunocytochemistry (Fig. 3, A and C)Citation . Therefore, the easily detectable levels of HER-3 measured in H16N-2-dn3 and 21 MT-1-dn3 cells (Fig. 3, B and D)Citation readily confirmed the expression of dominant negative HER-3 in cells infected with pCMV-dn3.



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  		Fig. 2. Development of the dominant negative HER-3 bicistronic retroviral expression vector. A, to test for the efficiency of antibiotic resistance gene and marker gene coexpression in our cell lines, both monocistronic (double promoter) and bicistronic (single promoter) forms of a retroviral expression vector containing the Neor and LacZ+ genes were either transfected into or used to infect H16N-2 cells. Colonies selected on G418 for a month were then stained for ß-galactosidase activity and counted to determine the percentage of blue-stained colonies. The mean average ± SD for triplicate wells is shown. {square}, monocistronic; {blacksquare}, bicistronic. B, the pCMV-dn3 bicistronic retroviral expression vector was constructed containing a dominant negative form of the HER-3 gene in which most of the cytoplasmic domain of HER-3 was removed. This vector also contains an internal ribosome-binding site (IRES) between the HER-3 gene and the Neor gene located downstream, which together form a single transcription unit. The expression of the pCMV-dn3 bicistronic transcript in mammalian cells then allows for the efficient coordinate coexpression of the dominant negative HER-3 gene with antibiotic resistance in retroviral-infected cell populations.

 


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  		Fig. 3. Expression of dominant negative HER-3 in cells infected with pCMV-dn3. Immunocytochemistry with anti-HER-3 monoclonal antibody was used to measure the level of HER-3 protein in H16N-2 cells (A), H16N-2-dn3 cells (B), 21 MT-1 cells (C), and 21 MT-1-dn3 cells (D). The H105 antibody is known to be highly specific for HER-3 and binds to an epitope within the extracellular domain. Although control cells express wild-type HER-3 (Fig. 6)Citation , the levels are below the level for immunodetection with HRP/DAB staining. Therefore, the easily detectable levels of HER-3 measured in H16N-2-dn3 and 21 MT-1-dn3 cells confirmed the ectopic expression of dominant negative HER-3 in cells infected with pCMV-dn3.

 
Inhibition of HER-2/HER-3 Activation in Cells Expressing Dominant Negative HER-3.
We next measured the effects of dominant negative HER-3 on the activation of HER-2/HER-3 in anti-phosphotyrosine immunoblots (Fig. 4A)Citation . Dominant negative HER-3 potently inhibited the HRG-induced tyrosine phosphorylation of HER-2/HER-3 in H16N-2-dn3 and 21 MT-1-dn3 cells (Fig. 4A, Lanes 4 and 8Citation ) as well as the constitutive tyrosine phosphorylation of HER-2/HER-3 in the 21 MT-1-dn3 cells (Fig. 4A, Lane 7Citation ). We also separately measured the levels of tyrosine-phosphorylated HER-2 and HER-3 by immunoprecipitation followed by immunoblotting (Fig. 5)Citation , which showed that dominant negative HER-3 inhibited HER-2 recruitment in anti-phosphotyrosine immunoprecipitates (Fig. 5A)Citation and almost completely blocked HER-3 tyrosine phosphorylation (Fig. 5B)Citation in H16N-2-dn3 and 21 MT-1-dn3 cells. Furthermore, immunoblots probed for HER-2 or HER-3 showed no significant effect of dominant negative HER-3 on the level of the wild-type HER-2 or HER-3 in H16N-2-dn3 and 21 MT-1-dn3 cells (Fig. 6)Citation , indicating that the effects of dominant negative HER-3 in H16N-2-dn3 and 21 MT-1-dn3 cells do not involve other effects on the expression of wild-type HER-2 or HER-3.



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  		Fig. 4. Inhibition of HER-2 and HER-3 activation in cells expressing dominant negative HER-3. A, samples containing 100 µg of cell lysate protein were immunoblotted with anti-phosphotyrosine antibody. B, the same blot was then reprobed with anti-p85 antibody as a control to confirm equal loading of the gel. H16N-2 cells (Lanes 1 and 2), H16N-2-dn3 cells (Lanes 3 and 4), 21 MT-1 cells (Lanes 5 and 6), and 21 MT-1-dn3 cells (Lanes 7 and 8) were starved of growth factors for 48 h in serum-free medium and then directly extracted (Lanes 1, 3, 5, and 7) or stimulated with HRG-ß before extraction (Lanes 2, 4, 6, and 8).

 


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  		Fig. 5. Dominant negative HER-3 inhibition of HER-2 and HER-3 tyrosine phosphorylation. Immunoprecipitation followed by immunoblotting was used to separately determine the levels of tyrosine-phosphorylated HER-2 (A) and HER-3 (B) in the different cell lines with or without stimulation with HRG-ß performed exactly as that shown in Fig. 1Citation . The results shown here were attained from scanning densitometry of negatives exposed by chemiluminescent substrate. {square}, SF; {blacksquare}, SF + HRG.

 


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  		Fig. 6. Dominant negative HER-3 has no effect on the levels of endogenous HER-2 or HER-3. Samples containing 100 µg of cell lysate protein were immunoblotted with anti-HER-2 (A) or anti-HER-3 (B) antibody. H16N-2 cells (Lane 1), H16N-2-dn3 cells (Lane 2), 21 MT-1 cells (Lane 3), and 21 MT-1-dn3 cells (Lane 4) are shown.

 
Inhibition of HER-2/HER-3-mediated Signaling in Cells Expressing Dominant Negative HER-3.
We next measured the effects of dominant negative HER-3 on the recruitment and tyrosine phosphorylation of the signaling molecules, p85PI 3-kinase, p46SHC, and p52SHC in cells with and without HRG stimulation in culture. The dominant negative HER-3 was found to inhibit the recruitment of p85PI 3-kinase in anti-phosphotyrosine immunoprecipitates (Fig. 7)Citation . We have previously shown that this assay is a very reliable measure of the recruitment and activation of p85PI 3-kinase by HER-2/HER-3 (22) . We (22) and others (46, 47, 48) have also found previously that activation of PI 3-kinase by various receptor tyrosine kinases involves recruitment of PI 3-kinase but does not involve detectable tyrosine phosphorylation of p85PI 3-kinase under more physiological conditions where p85PI 3-kinase is not artificially overexpressed (46) . Therefore, the changes measured in the recruitment of p85PI 3-kinase in anti-phosphotyrosine immunoprecipitates reflect the level of PI 3-kinase recruited by activated receptor complexes (22) . Furthermore, dominant negative HER-3 inhibited the recruitment of p46SHC and p52SHC in anti-phosphotyrosine immunoprecipitates (Fig. 8)Citation . However, in the case of SHC proteins, which are known to be highly tyrosine-phosphorylated during activation, the level in anti-phosphotyrosine immunoprecipitates likely reflects the combined effects on the tyrosine phosphorylation of SHC proteins as well as the level recruited by activated receptor complexes. In summary, the cells expressing dominant negative HER-3 showed impaired HER-2/HER-3 function as well as significant reductions in the recruitment and tyrosine phosphorylation of signaling molecules for both the PI 3-kinase and mitogen-activated protein kinase signal transduction pathways.



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  		Fig. 7. Inhibition of PI 3-kinase recruitment by HER-2/HER-3 in cells expressing dominant negative HER-3. Samples containing 2 mg of cell lysate protein were immunoprecipitated with anti-phosphotyrosine antibody followed by immunoblotting with anti-p85 antiserum to show the level of p85PI 3-kinase recruited by tyrosine-phosphorylated receptor complexes. H16N-2 cells (Lanes 1 and 2), H16N-2-dn3 cells (Lanes 3 and 4), 21 MT-1 cells (Lanes 5 and 6), and 21 MT-1-dn3 cells (Lanes 7 and 8) were starved of growth factors for 48 h and then directly extracted (Lanes 1, 3, 5, and 7) or stimulated with HRG-ß before extraction (Lanes 2, 4, 6, and 8).

 


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  		Fig. 8. Inhibition of SHC protein tyrosine phosphorylation and recruitment in cells expressing dominant negative HER-3. Samples containing 2 mg of cell lysate protein were immunoprecipitated with anti-phosphotyrosine antibody followed by immunoblotting with anti-SHC antiserum to show the level of p46SHC and p52SHC protein tyrosine phosphorylation and recruitment by tyrosine-phosphorylated receptor complexes. H16N-2 cells (Lanes 1 and 2), H16N-2-dn3 cells (Lanes 3 and 4), 21 MT-1 cells (Lanes 5 and 6), and 21 MT-1-dn3 cells (Lanes 7 and 8) were starved of growth factors for 48 h and then directly extracted (Lanes 1, 3, 5, and 7) or stimulated with HRG-ß before extraction (Lanes 2, 4, 6, and 8).

 
Dominant Negative HER-3 Inhibits HRG-induced Proliferation and the Autonomous Growth of Breast Cancer Cells with HER-2 Gene Amplification.
We routinely use the H16N-2 and 21 MT-1 cell lines for our studies because they were derived from the same patient and can be grown under completely defined SF conditions in culture. This well-defined system allows us to study growth factor responses as well as growth factor-independent (i.e., autonomous) proliferation in culture in a manner that is not possible for other cell lines derived in high serum-containing conditions. Anchorage-dependent monolayer growth assays with and without exogenous growth factors showed that dominant negative HER-3 inhibited the HRG-induced proliferation of both H16N-2-dn3 and 21 MT-1-dn3 cells in culture (Fig. 10A)Citation . In contrast, dominant negative HER-3 had no apparent effect on the insulin/EGF-induced proliferation of H16N-2-dn3 and 21 MT-1-dn3 cells in culture (Fig. 10A)Citation . Furthermore, the proliferation of the 21 MT-1-dn-3 cells was completely blocked in the absence of exogenous growth factors in culture (Figs. 9Citation and 10A)Citation . These results indicate that dominant negative HER-3 preferentially inhibits only proliferation induced by HRG or the growth factor-independent proliferation of cells that that overexpress HER-2. Finally, soft agarose growth assays were also performed to assess the potential effects of dominant negative HER-3 on the anchorage-independent growth of 21 MT-1-dn3 cells in culture. Dominant negative HER-3 strongly blocked the transformed growth of 21 MT-1-dn3 cells in soft agarose and inhibited growth even with maximal activation of HER-2/HER-3 in the presence of exogenous HRG (Fig. 10B)Citation .



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  		Fig. 10. Dominant negative HER-3 inhibits the HRG-induced proliferation of cells as well as the autonomous proliferation and transformed growth of 21 MT-1 cells in culture. A, anchorage-dependent growth assay showing the proliferation of H16N-2, H16N-2-dn3, 21 MT-1, and 21 MT-1-dn3 cells in monolayer culture for 9 days with SF medium plus insulin and EGF ({square}), without any growth factors (), or plus HRG-ß ({blacksquare}). The mean average ± SD for triplicate wells is shown. B, anchorage-independent growth assay of 21 MT-1 and 21 MT-1-dn3 cells cultured for a month in soft agarose with or without HRG-ß. The mean average ± SD for triplicate wells is shown. {square}, medium; {blacksquare}, medium + HRG.

 


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  		Fig. 9. Dominant negative HER-3 inhibits the autonomous proliferation of 21 MT-1 cells in monolayer culture. Phase-contrast microscopy of 21 MT-1 (A) and 21 MT-1-dn3 cells (B) cultured in SF medium in the complete absence of growth factors for 9 days is shown.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Amplification and overexpression of the HER-2 gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. In those individuals with HER-2 gene amplification, this dominant genetic event is likely the principle change that drives malignancy because HER-2 is such a potent oncogene when highly overexpressed in experimental systems. However, our understanding is still fragmentary concerning the exact mechanisms by which signals for cell growth are constitutively activated in breast cancer cells with HER-2 gene amplification, and methods for permanently inhibiting the constitutive activation of signaling in such cells have not yet met with great success. Our recent insights into the interaction between HER-2 and HER-3 offer exciting new opportunities for blocking the mechanism of autonomous growth in breast cancer cells with HER-2 gene amplification. By constructing cell lines that stably express a dominant negative form of HER-3, we have now successfully targeted the interaction between HER-2 and HER-3 in cells stimulated by exogenous HRG as well as that which is constitutively activated in breast cancer cells with HER-2 gene amplification. Also, the use of the pCMV-dn3 bicistronic retroviral vector results in highly efficient coordinate coexpression of antibiotic resistance and dominant negative HER-3 in target cell lines.

Our previous work showed that the elevated levels of HER-2 overexpression in 21 MT-1 cells are associated with high-level constitutive activation of PI 3-kinase and growth factor independence in culture (22 , 45) . In the present study, we sought to experimentally assess the importance of the cooperative interactions that occur between HER-2 and HER-3 in cells in response to HRG and during the growth factor-independent proliferation of breast cancer cells with HER-2 gene amplification. Our data now confirm the importance of the HER-2/HER-3 heterodimer interaction for recruiting key mitogenic signal transduction molecules involved in the growth of normal cells stimulated by HRG as well as in breast cancer cells with HER-2 gene amplification. Dominant negative HER-3 was able to block the HRG-induced proliferation of H16N-2 and 21 MT-1 cells as well as the growth factor-independent proliferation of 21 MT-1 cells in growth factor-free medium. In addition, dominant negative HER-3 potently inhibited the anchorage-independent growth of 21 MT-1 cells in soft agarose culture. These major effects of dominant negative HER-3 on cell proliferation do not necessarily preclude additional effects involving the rate of apoptosis in these cells, which remains to be determined. Also, preliminary in vivo studies have been performed using the 21 MT-1 and 21 MT-1-dn3 cells for injection into nude mice. However, to date, the 21 MT-1 control cell line has not been sufficiently tumorigenic in our nude mice to allow us to sufficiently test the effects of dominant negative HER-3 in vivo. The low tumor take and limited growth seen for 21 MT-1 cells in nude mice are apparently common problems for a significant number of highly malignant and metastatic breast carcinoma cell lines (49) , and earlier studies with 21 MT-1 cells also suggested some difficulty in using these cells for tumor studies at later passages (50) . Therefore, additional studies are under way using other breast carcinoma cell lines with HER-2 gene amplification as well as 21 MT-1 cells for transplantation into various immunodeficient mouse strains.

By itself, HER-3 is known to be almost completely kinase deficient (as would be expected from sequence analysis, which shows alterations in the enzymatic site) and is therefore unable to activate signaling in and of itself in genetically engineered cell lines that do not coexpress any of the other HER kinases (5 , 40, 41, 42) . However, whereas the other HERs have active kinase domains, HER-3 contains multiple additional docking sites for p85PI 3-kinase and SHC proteins not found in the other HERs (5) . Also, as mentioned above, HER-2 is known to be an especially active tyrosine kinase that exhibits ligand-independent activation when overexpressed (33) . These combined considerations (i.e., HER-3 docking sites combined with HER-2 kinase potential) may account for the especially potent activation of signal transduction induced by HER-2/HER-3 heterodimers in response to HRG and seen constitutively in breast cancer cells (20, 21, 22) . Interestingly, the blocking of HER-2/HER-3 function with dominant negative HER-3 was preferential in that the cells still proliferated in response to exogenous EGF, suggesting that the interaction between HER-1 and HER-3 is not necessary for mitogenesis in cells stimulated by EGF or that dominant negative HER-3 does not block HER-1/HER-3 function as well as HER-2/HER-3. In fact, it was this specificity of dominant negative HER-3 inhibition of HER-2/HER-3 that allowed us to use a constitutive promoter to express dominant negative HER-3, because the cells infected with pCMV-dn3 were still able to proliferate in response to EGF. Whereas there is evidence that HER-1 and HER-3 interact to some extent in these and other cell lines (34 , 35 , 51) , the HER-1/HER-3 heterodimer interaction is clearly very weak compared to that for HER-2/HER-3 (40, 41, 42) .

Human breast carcinoma cells sometimes overexpress HER-3, and it has been suggested that this may be important for malignancy (2) . However, whereas HER-3 is commonly expressed at a low but functional level in most nontransformed and transformed human mammary epithelial cells that we have tested, the HER-3 gene has never been found to be amplified or highly overexpressed, as is HER-2 (2) . Furthermore, when cell lines are genetically engineered to overexpress HER-3, this alone is not sufficient to constitutively activate HER-3 or to transform cells (21) . As mentioned above, HER-3 is a very weak kinase compared to the other HERs (5 , 40, 41, 42) , but HER-3 is constitutively activated in HER-2-overexpressing cell lines in which the cooperative interaction between HER-2 and HER-3 activates HER-2/HER-3 heterodimers (20, 21, 22, 23) . However, a number of the breast cancer cell lines with HER-2 gene amplification, such as 21 MT-1 cells, do not overexpress HER-3 in comparison with normal cells (Fig. 6)Citation . Therefore, it is our contention that low-level HER-3 cooperates with HER-2 to effectively transform breast carcinoma cells with HER-2 amplification, but this mechanism of cell transformation does not require concordant overexpression of HER-3.

Growth factor independence, as a phenotype, is a good indicator of progressive cell transformation in tumor cells with HER-2 gene amplification (22 , 45) . Normal human mammary epithelial cells require both IGF-I (or supraphysiological levels of insulin) and EGF to proliferate under SF conditions in culture (52) . The synergistic requirement for both IGF and EGF in the proliferation of normal mammary epithelial cells suggests that the attainment of growth factor-independent proliferation in mammary carcinoma cells involves genetic changes that subvert requirements for both IGF and EGF. We have previously shown that the 21 MT-2 and 21 MT-1 breast carcinoma cell lines have equivalently amplified HER-2 but show progressively elevated levels of HER-2 transcription associated with increasing IGF and EGF independence in culture (23 , 45) . We also found that HRG substitutes for both IGF and EGF in stimulating the proliferation of nontransformed human mammary epithelial cells (which express both HER-2 and HER-3, but not HER-4) in culture (45 , 52) . Therefore, we previously proposed that HER-2/HER-3 constitutive activation of signaling pathways in breast cancer cells substitutes for growth factor-mediated signaling, which usually requires the combination of IGF and EGF in normal cells (22 , 45 , 52) . Furthermore, the distinguishing properties of HER-2/HER-3 function may help explain the occurrence, and potent oncogenicity and selection of amplified HER-2 in cell types that normally express HER-3.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Vector Construction.
For experiments to test the efficiency of antibiotic resistance gene and marker gene coexpression in our cell lines, control retroviral expression vectors were constructed from the pCMV vector (originally derived from pSLH1001, which was derived from pLNCX) in which the Neor and the LacZ+ genes were placed in either monocistronic or bicistronic configuration. The dominant negative HER-3 retroviral expression vectors were made using full-length human HER-3 cDNA (Amgen, Inc.), from which a 2.2-kb fragment missing most of the intracellular domain was generated by cutting out the insert with SalI and BamHI. By using the pBK-CMV phagemid expression vector (Stratagene) as an intermediate, we subcloned the dominant negative HER-3 fragment into the pCMV bicistronic retroviral expression vector. We first ligated the dominant negative HER-3 fragment into the SalI and BamHI sites located within the extensive polylinker region of pBK-CMV to generate pBK-CMV-dn3. This ligation also introduced an in-frame stop codon 12 codons downstream of the BamHI site. The dominant negative HER-3 fragment was then subcloned from pBK-CMV-dn3 into pCMV by ligation of the dominant negative HER-3 insert cut with SalI and ClaI (which does not contain these sites internally) into the XhoI and ClaI sites (SalI and XhoI have compatible ends) within pCMV to generate pCMV-dn3 (Fig. 2B)Citation . Restriction digest analysis confirmed the proper construction of the vectors.

Culture Infection and Selection of Cell Lines.
The H16N-2 and 21 MT-1 cell lines were provided by Dr. Vimla Band through the Dana-Farber Cancer Institute (Boston, MA). For routine culture, the cells were grown in F-12 growth medium containing 10 mM N-2-hydroxyethylpiperazine-2-ethane sulfonic acid, antibiotic/antimycotic, 0.5 µg/ml fungizone, 5 mM ethanolamine, 50 ng/ml sodium selenate, 1 µg/ml hydrocortisone, 5 µg/ml transferrin, 5 µg/ml insulin, 10 ng/ml EGF, 0.1 mg/ml BSA, and 2% fetal bovine serum. The cells were cultured at 37°C with 5% carbon dioxide, and the medium was changed every other day. For subculture, the cells were rinsed in calcium magnesium-free HBSS and then rinsed in 0.05% trypsin plus 0.025% EDTA in calcium magnesium-free HBSS. After aspiration of the trypsin solution, the cells were incubated at 37°C for 5–15 min, and the released cells were immediately resuspended in growth medium for replating in 60- or 100-mm tissue culture plates. For routine culture, the cells were counted with a hemocytometer and plated at a density of 104 cells/cm2. For experiments to test the efficiency of antibiotic resistance (Neor) and marker gene (LacZ+) coexpression, monocistronic and bicistronic control vectors were introduced into H16N-2 and 21 MT-1 cells by either transfection or infection. For transfection, the DNA was introduced into cells by lipofection with Lipofectin according to the manufacturer’s instructions (Life Technologies, Inc.). For infection, the vectors were first transfected into the {psi}CRIP packaging cell line followed by infection of target cells with replication-defective virus. Colonies selected for a month on 200 µg/ml G418 (Life Technologies, Inc.) were then fixed and stained for ß-galactosidase activity, and the proportion of blue-stained colonies was determined from colony counts. H16N-2 and 21 MT-1 cells were then infected with either the pCMV vector (as a control) or the pCMV-dn3 vector. For these infections, {psi}CRIP cells were transiently transfected with either pCMV or pCMV-dn3 by lipofection, and medium conditioned for 24 h containing virus was collected and spun down at 1200 rpm for 10 min before adding to subconfluent H16N-2 and 21 MT-1 cell cultures. The H16N-2 and 21 MT-1 cells were then incubated with {psi}CRIP conditioned medium for 3 days, with fresh conditioned medium added daily. After an additional 2-day incubation in fresh medium, the infected cell lines were then selected on 200 µg/ml G418 for a month before use in further analysis.

Immumocytochemistry.
The cells were plated in 24-well plates at a density of 5 x 102 cells/well and cultured to confluence. The cells were rinsed in PBS, fixed in methanol at -20°C for 10 min, and then rinsed three times with PBS before immunostaining with the anti-HER-3 monoclonal antibody, H105 (Neomarkers). The cells were equilibrated in TBS [150 mM NaCl and 50 mM Tris (pH 7.5)], blocked in TBS plus 1% BSA at room temperature for 60 min with mild agitation, and then with 2 µg/ml H105 antibody in TBS plus 1% BSA at room temperature for 60 min with mild agitation. The cells were then rinsed in TBS three times (5 min each time) with moderate agitation, incubated with biotinylated antimouse IgG secondary antibody (Vector Laboratories) at a 1:750 dilution in TBS plus 1% BSA at room temperature for 60 min with mild agitation, rinsed in TBS three times (5 min each time) with moderate agitation, and then incubated with ABC strepavidin HRP reagents (Vector Laboratories) diluted in TBS + 1% BSA at room temperature for 60 min with mild agitation. After rinsing of the cells in TBS three times (5 min each time) with moderate agitation, the cells were stained with DAB as the substrate.

Immunoprecipitations.
Cells cultures were incubated in SF medium without insulin and EGF for 48 h before extraction (i.e., the constitutive condition) and stimulation with 10 ng/ml HRG-ß for 10 min at 37°C before extraction of lysate protein for immunoprecipitation and/or Western blot analysis. After the cells were lysed in immunoprecipitation buffer [150 mM NaCl, 50 mM Tris (pH 7.5), 0.5% NP40, 5 mM EDTA, 5 mM sodium orthovanadate, 10 mM Na PPi, and 2 mM phenylmethylsulfonyl fluoride], the lysates were clarified by centrifugation at 14,000 x g for 15 min and either used directly for electrophoresis or used for immunoprecipitation after normalizing the samples. Total cell lysate protein was assayed using the Bradford assay (Bio-Rad), and 2 mg of protein were used for immunoprecipitation, or 100 µg of protein were used directly for electrophoresis. For immunoprecipitation, cell lysates were then incubated with either 30 µl anti-phosphotyrosine monoclonal antibody-conjugated agarose (Oncogene) for 2 h at room temperature with moderate agitation or with 2 µg of 2F12 anti-HER-3 monoclonal antibody (Neomarkers) for 2 h at room temperature with moderate agitation followed by incubation with 50 µl of protein A-agarose (Oncogene) for 1 h at 4°C with moderate agitation. The pellets were then washed three times in immunoprecipitation buffer, and the beads were boiled in 100 µl of electrophoresis sample buffer for 10 min to release protein conjugates from the agarose before electrophoresis.

Western Blot Analysis.
Cell lysates or immunoprecipitated samples were electrophoresed in 7.5% SDS-PAGE gels for approximately 18 h at 15 mA constant current. The samples were then transferred to Immobilon-P membranes (Millipore) by overnight electrotransfer in standard transfer buffer at 125 mA followed by 2 h at 325 mA. The blots were equilibrated in TTBS, blocked in TTBS plus 3% milk at room temperature for 60 min with moderate agitation, and then incubated with either 2 µg/ml PY20 anti-phosphotyrosine monoclonal antibody (Oncogene), 1:500 Pab9.3 anti-HER-2 polyclonal antiserum (Berlex Biosciences), 2 µg/ml 2F12 anti-HER-3 monoclonal antibody (Neomarkers), 1:500 anti-p85 polyclonal antiserum (Upstate Biotechnology), or 1:500 anti-SHC polyclonal antiserum (Transduction Laboratories) in TTBS plus 3% milk at room temperature for 60 min with moderate agitation. The blots were then rinsed in TTBS three times (5 min each) with moderate agitation, incubated with biotinylated antimouse IgG or biotinylated antirabbit IgG secondary antibody (Vector Laboratories) at a 1:750 dilution in TTBS at room temperature for 60 min with moderate agitation, rinsed in TTBS three times for 5 min each with moderate agitation, and then incubated with ABC streptavidin HRP reagents (Vector Laboratories) diluted in TTBS at room temperature for 60 min with moderate agitation. After the final rinsing of the blots in TTBS (three times; 5 min each) with moderate agitation, the bands were visualized with enhanced chemiluminescent substrate (Pierce) according to the manufacturer’s instructions. Negatives exposed by chemiluminescent substrate were scanned and quantified using the IQ25 Intelligent Quantifier system (Bio Image).

Cell Growth Assays.
For the monolayer growth assay, the cell lines were plated in 6-well tissue culture plates at a density of 105 cells/well in medium containing all of the supplements listed above minus the insulin and EGF. After 24 h, the medium was replaced with SF medium without growth factors, medium with 5 µg/ml insulin and 10 ng/ml EGF, or medium with 10 ng/ml HRG-ß, and the media were changed every other day. Cell counts were taken after 24 h to measure the plating efficiency and at day 10 to measure the proliferation during 9 days in SF culture. For counting cells, the cells from triplicate wells for each condition were trypsinized and counted using a hemocytometer. For the soft agarose assays, the cells were plated in 24-well plates within 0.3% agarose at a density of 2.5 x 104 cells/0.25 ml atop a 0.25-ml layer of 0.6% agarose in growth medium with or without 10 ng/ml HRG-ß and cultured for a month before counting colonies of at least 50 µm in diameter.


   Acknowledgments
 
We thank Dr. Stephen Ethier for his support, Dr. Eric Radany for the use of the pSLH1001 vector, and Dr. Vimla Band for developing the H16N-2 and 21 MT-1 cell lines.


   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 by Department of Defense Grant DAMD17-96-1-6196. Back

2 To whom requests for reprints should be addressed, at School of Biological Sciences, Washington State University, Pullman, Washington 99164-4236. Phone: (509) 335-5779; Fax: (509) 335-3184; E-mail: tram{at}mail.wsu.edu Back

3 The abbreviations used are: EGF, epidermal growth factor; BSA, bovine serum albumin; HRG, neu differentiation factor/heregulin; IGF, insulin-like growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; SF, serum-free; TBS, Tris-buffered saline; TTBS, Tween TBS; HRP, horseradish peroxidase; DAB, diaminobenzidine; TTBS, 150 mM NaCl, 50 mM Tris (pH 7.5), and 0.1% Tween 20. Back

4 Unpublished observations. Back

Received for publication 10/15/99. Revision received 2/11/00. Accepted for publication 2/14/00.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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