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Molecular Cancer Research 3:32-41 (2005)
© 2005 American Association for Cancer Research

Signaling and Regulation

AND-34 Activates Phosphatidylinositol 3-Kinase and Induces Anti-Estrogen Resistance in a SH2 and GDP Exchange Factor–Like Domain-Dependent Manner 1

Kyriacos N. Felekkis2, Radha P. Narsimhan1, Richard Near1, Ariel F. Castro3, Yi Zheng4, Lawrence A. Quilliam3 and Adam Lerner1,2

1 Department of Medicine, Section of Hematology and Oncology, Boston Medical Center, and 2 Department of Pathology, Boston University School of Medicine, Boston, Massachusetts; 3 Department of Biochemistry and Molecular Biology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana; and 4 Division of Experimental Hematology, Children's Hospital Research Foundation, Cincinnati, Ohio

Requests for reprints: Adam Lerner, Department of Medicine, Section of Hematology and Oncology, Boston Medical Center, Evans Biomedical Research Center, Room 427, 650 Albany Street, Boston, MA 02118. Phone: 617-638-7530. E-mail: alerner{at}medicine.bu.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor–like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser473 phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ER{alpha} levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ER{alpha}-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 µmol/L LY294002 or 10 µmol/L NSC23766 a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.

Key Words: AND-34 • BCAR3 • PI3K • Akt • breast cancer • anti-estrogen


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
AND-34 is a member of a family of three related proteins: NSP-1, AND-34/BCAR3/NSP-2, and CHAT/SHEP1/NSP-3 (1-5). All three proteins bind by a COOH-terminal domain with homology to Cdc25 Ras subfamily GDP exchange factors (GEF) to the COOH terminus of the focal adhesion complex adapter protein p130Cas (4, 6). In a study by van Agthoven et al. in which random retroviral integration into an estrogen-dependent human breast cancer cell line was done, 6 of 80 subsequently isolated anti-estrogen-resistant clones were shown to derive from independent retroviral integration into the AND-34/BCAR3 promoter, with resulting AND-34/BCAR3 overexpression (1). In a second report from the same group, four further clones were shown to arise from independent retroviral integration into the p130Cas (BCAR1) locus, with p130Cas overexpression (7). These seminal studies by Dorssers et al. suggest that elucidation of the signaling pathways activated by expression of AND-34 may shed light on the mechanism by which initially estrogen-dependent human breast carcinomas invariably with time develop resistance to anti-estrogen therapy.

Efforts to understand the signaling role of AND-34 and its family members NSP-1 and CHAT/SHEP1/NSP-3 have focused on their modest homology to the Cdc25 Ras GEF domain. Initial studies of AND-34 overexpression in Cos7 cells detected GEF activity toward Ral, Rap1, and R-Ras by a technique in which glutathione S-transferase (GST)-GTPase chimeras are transfected transiently, cells labeled with 32P, followed by isolation of the GTPase and quantitation of the associated guanine nucleotide by TLC (6). Similarly, SHEP1 was found to bind R-Ras and Rap1A in a GDP-dependent manner (5). Using another assay for GEF activity, so-called pull-down analysis, Sakakibara et al. showed Rap1 activation in CHAT-overexpressing cells (8). However, in these latter studies, SHEP1 was not found to have in vitro activity as a GEF toward Rap1 or R-Ras, and the ability of CHAT to activate Rap1 was suggested to be indirectly mediated through its association with p130Cas as dominant-negative mutants of p130Cas, Crk, or the Rap1 GEF C3G abrogated the ability of CHAT to induce Rap1 activation (5, 8). Studies of AND-34/BCAR3 in other cell lines using pull-down assays have not confirmed GEF activity toward Ral or Rap1 (9-11). Most recently, following the observation that AND-34 induced striking morphologic changes in an inducible cell line, we determined that overexpression of AND-34 induces both Cdc42 and Rac activation, a somewhat surprising finding given that AND-34 does not have the pleckstrin homology and Dbl GEF domains characteristic of Rac and Cdc42 GEFs (10, 11). Rac activation was linked to AND-34-mediated anti-estrogen resistance in that inhibition of signaling by Rac itself or the Rac effector kinase PAK1 blocked AND-34-mediated cyclin D1 up-regulation in MCF-7 cells, whereas stable transfection of estrogen-dependent ZR-75-1 cells with a constitutively active form of Rac1 resulted in acquisition of resistance to the estrogen antagonist ICI 182,780 (Faslodex).

The current study is an effort to better understand the mechanism by which AND-34 induces Rac activation in human breast cancer cell lines. Our work suggests that such Rac activation is a result of the ability of AND-34 to induce phosphatidylinositol 3-kinase (PI3K) activation in a manner that requires both the SH2 domain and the GEF-like domain of AND-34. Both PI3K and Rac activation are also required for AND-34-mediated breast cancer cell line resistance to ICI 182,780.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
AND-34-Induced Rac Activation and Anti-Estrogen Resistance Are SH2 and GEF-Like Domain Dependent
Pull-down analysis with a GST-PAK RBD protein showed that transient transfection of full-length NH2-terminal hemagglutinin (HA)–tagged AND-34 into the estrogen-dependent human breast cancer cell line MCF-7 induces substantial activation of endogenous Rac GTPase (Fig. 1A). In contrast, overexpression of {Delta}NTer (an AND-34 construct in which the NH2-terminal 255 amino acids have been deleted), R171K [an AND-34 construct in which the SH2 domain is inactivated by point mutation of the arginine that interacts with phosphorylated tyrosine residues (FLVRES motif)], or {Delta}GEF (an AND-34 construct in which the COOH-terminal 284 amino acids have been deleted) failed to induced Rac activation in MCF-7 cells (Fig. 1A; ref. 12). Immunoblotting of whole cell lysates of the transfected cells confirmed robust expression of the wild-type and mutant AND-34 proteins. These studies suggest that both the SH2 domain and the COOH-terminal GEF-like domain are required for the ability of AND-34 to induce Rac activation in breast cancer cells on overexpression.



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  		FIGURE 1. AND-34-induced Rac activation and growth in the presence of the anti-estrogen ICI 182,780 requires functional SH2 and GEF domains. A. MCF-7 cells were transfected with vector-only (CT), HA-tagged full-length AND-34 (FL), HA-{Delta}NTer AND-34 ({Delta}NTer), HA-R171K AND-34 (R171K), or HA-{Delta}GEF AND-34 ({Delta}GEF). At 72 hours after transfection, the level of GTP-bound Rac was measured by incubating whole cell lysates with GST-PAK. After isolation of the GST-PAK with glutathione beads, eluted proteins were immunoblotted with anti-Rac antibody. To confirm equal expression of endogenous Rac, whole cell lysates were immunoblotted with anti-Rac antibody. To detect expression of wild-type or mutant AND-34, whole cell lysates were immunoblotted with anti-HA antibody. B. Cumulative numbers of vector-only (CT), full-length BCAR3 (FL), {Delta}NTer BCAR3 ({Delta}NTer), and {Delta}GEF BCAR3 ({Delta}GEF) ZR-75-1 transfectants 11 days after addition of 100 nmol/L ICI 182,780 to tissue culture medium. Columns, mean from two clones for each transfectant; bars, SE.

 
In prior studies, we suggested that AND-34-induced Rac activation was required for AND-34-induced anti-estrogen resistance (10). To confirm that AND-34 mutants that lack the ability to activate Rac in transient transfection assays also lose the ability to confer anti-estrogen resistance, we generated stable transfectants of ZR-75-1 cells, an estrogen-dependent human breast cancer cell line, with either control vector or wild-type, {Delta}NTer, or {Delta}GEF AND-34. Whereas full-length AND-34 allowed growth of ZR-75-1 cells in the presence of 100 nmol/L ICI 182,780, overexpression of {Delta}NTer or {Delta}GEF had no significant effect on ZR-75-1 cell growth relative to vector-only transfected cells (Fig. 1B). A comparison between the four groups of transfectants by single-factor ANOVA analysis showed a significant difference in cell growth in the presence of ICI 182,780 (P < 4.92 x 10–7). A secondary pair-wise comparison using Scheffe's test showed a significant difference in cell growth between the ZR-75 cells transfected with full-length AND-34 and each of the other three constructs but no significant differences between control vector, {Delta}NTer, or {Delta}GEF transfectants. Thus, both the SH2 domain and the GEF-like domain of AND-34 are required for the ability of AND-34 to confer anti-estrogen resistance.

AND-34 Overexpression Activates Rac and Akt through PI3K
Because AND-34 lacks a Dbl domain, we hypothesized that AND-34 overexpression might induce Rac activation indirectly through up-regulation of PI3K activity. PI3K has been reported to activate Rac at least in part as a result of Rac GEF pleckstrin homology domain interactions with phosphatidylinositol-3,4,5-triphosphate (reviewed in ref. 13). To test such a hypothesis, we did PI3K assays on p85 immunoprecipitates from MCF-7 cells 72 hours after transient transfection with AND-34 wild-type or deletion mutant constructs. As a positive control, we treated MCF-7 cells with 25 ng/mL heregulin ß for 5 minutes, as Her2/3 signaling has been reported previously to induce PI3K activation in MCF-7 cells (14). Transfection with full-length AND-34 or treatment with heregulin augmented p85-associated PI3K activity as judged by a clear increase in the amount of phosphatidylinositol-3,4,5-triphosphate product detected, whereas transfection with {Delta}NTer AND-34, R171K AND-34, or {Delta}GEF AND-34 did not alter the amount of phosphatidylinositol-3,4,5-triphosphate detected relative to p85 immunoprecipitates from control vector–transfected cells (Fig. 2A).



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  		FIGURE 2. AND-34 overexpression induces PI3K and Akt activation in MCF-7 cells. A. MCF-7 cells were transiently transfected with control vector, HA-tagged full-length AND-34, HA-{Delta} NTer AND-34, HA-R171K AND-34, or {Delta}GEF AND-34. PI3K was immunoprecipitated using anti-p85 antibody and subjected to an in vitro lipid kinase assay. Equal expression of p85 among transfectants was confirmed by immunoblotting whole cell lysates with anti-p85 antibody. As a positive control, MCF-7 cells transfected with control vector were treated for 5 minutes with 25 ng/mL heregulin ß (Her 5min). B. MCF-7 cells were transfected with HA-tagged full-length AND-34 or HA-tagged AND-34 deletion mutant constructs. Whole cell lysates were immunoblotted with an antibody specific for Akt phosphorylated at Ser473. Equal expression of Akt among transfectants was confirmed by immunoblotting with anti-Akt antibody. C. Endogenous Akt was immunoprecipitated from cell lysates of HA-AND-34 transfectants and subjected to an in vitro kinase assay using GSK3 {alpha}/ß fusion protein as a substrate. A comparable analysis of MCF-7 cells treated for 5 minutes with 25 ng/mL heregulin ß was used as a positive control.

 
PI3K activation results in phosphorylation of the serine/threonine kinase Akt at Thr308 by 3-phosphoinositide-dependent protein kinase-1 and at Ser473 by a still undefined kinase (15). Such dual phosphorylation results in full activation of Akt kinase activity. Overexpression of wild-type but not {Delta}NTer, R171K, and {Delta}GEF AND-34 in MCF-7 cells by transient transfection resulted in a marked augmentation in phosphorylation of Akt Ser473 (Fig. 2B). To confirm that AND-34 overexpression results in Akt activation, we immunoprecipitated Akt from AND-34-transfected MCF-7 cells and did a kinase assay with a GSK3 fusion protein as substrate. Overexpression of wild-type but not {Delta}NTer, R171K, and {Delta}GEF AND-34 resulted in increased Akt activity as judged by markedly higher levels of phosphorylated GSK3 fusion protein (Fig. 2C). As a positive control, treatment of MCF-7 cells with heregulin (25 ng/mL) for 5 minutes also increased Akt-induced GSK3 fusion protein phosphorylation.

Although the experiments described above suggest that overexpression of AND-34 induces PI3K and Akt activation, such studies did not establish whether AND-34-induced Rac activation is upstream or downstream of PI3K. Several studies have suggested that Rac can activate PI3K activity by direct interactions with p85 (16-18). As expected, a myristolated p110 construct that has been shown previously to induce constitutive PI3K activity augmented both GTP-bound Rac and Akt Ser473 phosphorylation in MCF-7 cells (Fig. 3C). The chromone LY294002 is a frequently used inhibitor of PI3K (IC50 1.40 µmol/L; ref. 19). Treatment with 10 µmol/L LY294002 inhibited both AND-34-mediated Rac activation and Akt Ser473 phosphorylation in MCF-7 cells (Fig. 3A). p{Delta}85, a p85 construct that lacks a binding site for p110, has been reported to act in a dominant-negative fashion, blocking PI3K activation to a variety of stimuli (20). Cotransfection of AND-34 and p{Delta}85 blocked the ability of AND-34 to induce Rac activation and Akt Ser473 phosphorylation in MCF-7 cells (Fig. 3B). Analysis of whole cell lysates confirmed that expression of p{Delta}85 did not alter levels of HA-AND-34. These studies show that both AND-34-induced Rac activation and Akt Ser473 phosphorylation occur in a PI3K and SH2 and GEF-like domain-dependent manner.



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  		FIGURE 3. AND-34-induced Rac activation requires the activity of PI3K. A. MCF-7 cells were transfected with control vector or HA-tagged full-length AND-34. Cells were left untreated or treated for 30 minutes with 10 µmol/L LY294002 followed by Rac pull-down analysis. Isolated GTP-bound proteins were immunoblotted with anti-Rac antibody. Equal expression of endogenous Rac was confirmed by immunoblotting whole cell lysates with anti-Rac antibody. Whole cell lysates were immunoblotted with anti-HA and anti-phospho-Akt (Ser473) antibodies to detect HA-AND-34 expression and Akt phosphorylation, respectively. B. MCF-7 cells were transfected with control vector or HA-tagged full-length AND-34 with or without HA-tagged dominant-negative p85 (p{Delta}85). Cells were subjected to pull-down analysis for endogenous Rac as described above. Whole cell lysates were immunoblotted with anti-HA antibody to detect expression of HA-AND-34 and HA-p{Delta}85. Level of Akt phosphorylated at Ser473 was detected by immunoblotting whole cell lysates with anti-phospho Akt [P-Akt(473)] antibody. C. MCF-7 cells were transfected with control vector or with varying quantities (in µg) of constitutively active p110 (Myr-p110) followed by Rac pull-down analysis. Equal expression of endogenous Rac was confirmed by immunoblotting whole cell lysates with anti-Rac antibody. Whole cell lysates were immunoblotted with anti-phospho-Akt antibody to determine the levels of phosphorylated Akt.

 
Transient Transfections with Constitutively Active Forms of R-Ras Induce Akt but not Rac Activation in MCF-7 Cells
Given the fact that R-Ras is known to activate PI3K and to promote estrogen-independent growth of breast cancer cells, we wished to examine whether AND-34 might activate PI3K and Rac indirectly by activating R-Ras (9, 21). To determine whether R-Ras activation induces Rac activation in MCF-7 cells, we transiently transfected such cells with either R-Ras 38V or 87L, two different constitutively active mutants of R-Ras (22). Interestingly, although both R-Ras constructs led to clear Akt activation as judged by Akt Ser473 phosphorylation, neither led to Rac activation. In contrast, when MCF-7 cells were instead transiently transfected with a myristolated p110 construct, both Akt Ser473 phosphorylation and Rac activation were observed (Fig. 4A).



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  		FIGURE 4. Assessment of the role of R-Ras in AND-34-mediated Rac activation in MCF-7 cells. A. MCF-7 cells were transfected with control vector or two different forms of constitutively active HA-tagged R-Ras (R-Ras 87L and R-Ras 38V) followed by pull-down analysis for endogenous Rac. As a positive control for Rac activation, MCF-7 cells were transfected with myristolated p110. Akt phosphorylation was determined by immunoblotting whole cell lysates with anti-phospho-Akt antibody. Whole cell lysates were immunoblotted with anti-HA antibody to detect expression of R-Ras. B. MCF-7 cells were cotransfected with HA-R-Ras and either control vectors, pMSCV AND-34, or pFLAG CMV2 GRP3 as indicated and the proportion of GTP-bound HA-R-Ras was determined by TLC analysis. Ratio of GTP / (GTP + GDP x 1.5) x 100%.

 
As AND-34 robustly activates Rac in MCF-7 cells, the results described above with R-Ras 38V and R-Ras 87L do not support the hypothesis that AND-34 activates PI3K by a R-Ras-mediated mechanism. To more directly address the role of R-Ras in AND-34-mediated PI3K activation, we did R-Ras GEF assays in MCF-7 cells following transfection with either AND-34 or GRP3, a GEF known to activate R-Ras (23). In this assay, cells were cotransfected with an epitope-tagged form of R-Ras and incubated with free 32Pi followed by lysis of the cells and immunoprecipitation of R-Ras. The radiolabeled R-Ras-associated guanine nucleotides were then eluted and separated by TLC followed by quantitation of the relative levels of GDP and GTP with a ß-scanner. Whereas transfection with GRP3 led to a doubling in the proportion of GTP R-Ras in two independent experiments (23-48%), transfection with AND-34 did not alter R-Ras GTP levels (Fig. 4B). Thus, these combined results would suggest that AND-34 activates PI3K by a R-Ras-independent mechanism.

AND-34/BCAR3-Induced Anti-Estrogen Resistance Requires PI3K Activity
To determine the role of PI3K in AND-34-induced resistance to ICI 182,780 in ZR-75-1 cells, we used a pharmacologic experimental strategy. Vector-only or HA-AND-34 ZR-75-1 stable transfectants were grown in tissue culture with or without 100 nmol/L ICI 182,780 and with or without 2 µmol/L LY294002. At 2 µmol/L, LY294002 did not significantly inhibit the proliferation of either cell type in the absence of ICI 182,780 (Fig. 5A). As published previously, the AND-34/BCAR3 stable transfectant but not the vector-only transfectant grew in the presence of ICI 182,780. Despite the lack of significant growth inhibitory effect of LY294002 in normal medium, the same drug blocked growth of the AND-34/BCAR3 transfectant when such cells were cultured in 100 nmol/L ICI 182,780 (P < 0.05; Fig. 5A). Our results suggest that AND-34/BCAR3-induced anti-estrogen resistance requires PI3K activity.



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  		FIGURE 5. Ability of AND-34 stable transfectants to grow in the presence of the anti-estrogen ICI 182,780, a drug that induces degradation of ER in such transfectants, is blocked by inhibitors of PI3K. A. Vector-only and full-length BCAR3 ZR-75-1 transfectants were plated in a six-well tissue culture plate at a density of 130,000 cells per well. Cells were grown in the presence (right) or absence (left) of 100 nmol/L ICI 182,780 and left untreated or treated with 2 µmol/L LY294002 as indicated. At the 11th day after plating, cells were trypsinized and counted. Columns, mean; bars, SE. B. Three vector-only and three AND-34 transfectants were assessed for expression of ER following culture in normal medium. Equal loading of total protein was confirmed by immunoblotting for {alpha}-tubulin. C. A vector-only (CT #1) transfectant and a full-length AND-34 (A34 #6) stable ZR-75-1 transfectant were grown in the presence or absence of 100 nmol/L ICI 182,780 for 6 days followed by Western analysis for ER{alpha} expression.

 
Unlike partial agonists, such as tamoxifen, binding of ICI 182,780 results in estrogen receptor (ER) {alpha} degradation by a proteosomal pathway (24). Interestingly, under normal tissue culture conditions, the mean basal level of ER{alpha} in three AND-34/BCAR3 transfectants was 2.5-fold higher than that observed in three vector-only transfectants (Fig. 5B). However, treatment with 100 nmol/L ICI 182,780 substantially reduced ER levels in both ZR-75 vector-only and AND-34/BCAR3 stable transfectants (Fig. 5C). Given that ER{alpha} levels were reduced to low levels in ICI 182,780–treated AND-34/BCAR3 transfectants, these data support the hypothesis that AND-34 induces resistance to the growth inhibitory effects of ICI 182,780 by an ER{alpha}-independent mechanism.

Role of Rac in AND-34/BCAR3-Induced Anti-Estrogen Resistance
Although the experiments described above show that AND-34-induced anti-estrogen resistance requires PI3K activity, the effector responsible for this effect remained unclear. As the studies above have also shown that AND-34-induced Rac activation is PI3K dependent, we next sought to establish the contribution of this Rac activation to ICI 182,780 resistance. Efforts to obtain stable ZR-75-1 double transfectants with dominant-negative forms of Rac (Rac17N) were unsuccessful due to the drastically reduced growth rate of such double transfectants in normal medium (data not shown), a finding that has been reported previously by other investigators (25).

As an alternate approach, we took advantage of NSC23766 a recently developed small molecule inhibitor of Rac (26). In initial dose titration experiments, we determined that 10 µmol/L NSC23766was the highest concentration of the Rac inhibitor that had no significant effect on growth of ZR-75 vector-only or AND-34/BCAR3 stable transfectants in normal medium over 6 days (Fig. 6A). Treatment of MCF-7 cells transiently transfected with AND-34 with 10 µmol/L NSC23766markedly reduced AND-34-mediated Rac activation (Fig. 6B). Importantly, however, 10 µmol/L NSC23766completely blocked the ability of the AND-34/BCAR3 transfectants to grow in the presence of 100 nmol/L ICI 182,780 (Fig. 6C). This result supports the hypothesis that AND-34-mediated activation of PI3K induces anti-estrogen resistance at least in part through PI3K-induced Rac activation.



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  		Figure 6. AND-34-induced Rac activation is required for AND-34-induced resistance to ICI 182,780 in the ZR-75 human breast cancer cell line. A. An HA-AND-34/BCAR3 ZR-75-1 stable transfectant was grown for 6 days in medium alone or in the presence of 10, 25, or 50 µmol/L NSC23766 a Rac inhibitor, followed by trypsinization and cell counting. B. MCF-7 cells were transfected with vector alone or full-length AND-34 and incubated for 72 hours with either medium alone or in the presence of the same concentrations of NSC23766as in A. Cells were then lysed and a Rac pull-down analysis was done to assess Rac activation under these different conditions. Levels of total Rac were also assessed in the whole cell lysates as indicated. C and D. Control vector or HA-AND-34/BCAR3 ZR-75-1 stable transfectants were grown for a total of 11 days in the presence or absence of 100 nmol/L ICI 182,780 and the presence or absence of 10 µmol/L NSC23766as indicated. During this period, they were counted and replated at equal densities on day 6. On day 11, cells were trypsinized and counted. Total amount of cell growth, taking into account the amount of growth by day 6.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Despite a 60% to 70% response rate to hormonal therapies, such as oophorectomy, tamoxifen, ICI 182,780, or aromatase inhibitors, in patients with metastatic ER-positive breast cancer, resistance to such treatment invariably develops (27, 28). Clinical resistance to mixed ER antagonists, such as tamoxifen, may result from restoration of ER-dependent transactivation through tumor-derived signaling pathways that regulate ER phosphorylation or ER-associated coactivators or corepressors (29, 30). In contrast, given that type II "pure" ER antagonists, such as ICI 182,780, induce destruction of ER{alpha} protein following ubiquitination, resistance to this class of anti-estrogens has been suggested to result from activation of signal transduction pathways that bypass the requirement for ER signaling altogether (24, 31). In this study, we show that overexpression of AND-34 in estrogen-dependent breast cancer cells induces resistance to ICI 182,780 at least in part as a result of the ability of AND-34 to activate PI3K and subsequently Rac.

A variety of recent studies implicate PI3K as a critical component of signaling pathways, the up-regulated activities of which result in breast cancer resistance to hormonal therapies. The estrogenic functions of epidermal growth factor and insulin-like growth factor-I have been attributed to their ability to activate PI3K and Akt, although this result remains controversial (9, 32). Akt2 phosphorylates Ser167 of the NH2-terminal AF-1 domain of ER{alpha}, promoting transcriptional activity in the absence of estradiol (33, 34). The ER has also been reported by several groups to form a complex with p85 of PI3K within both endothelial cells and MCF-7 epithelial cells (15, 35, 36). However, the ability of ICI 182,780 to induce degradation of ER{alpha} in stable transfectants, such AND-34, suggests that AND-34-induced PI3K activation may induce ER{alpha}-independent effects that are sufficient for growth in the absence of signaling by this receptor.

R-Ras is known to activate PI3K signaling (37). In a recent study by Yu and Feig, constitutively active R-Ras 38V was found to confer estrogen-independent proliferation in MCF-7 cells (9). Consistent with the hypothesis that this occurred through a PI3K-mediated mechanism, a dominant-negative Akt mutant (S197M) inhibited R-Ras 38V–induced anti-estrogen resistance in this study. Interestingly, however, stable transfection of constitutively active myristolated Akt was insufficient to allow estrogen-independent proliferation, suggesting that Akt was necessary but not sufficient for R-Ras 38V–mediated anti-estrogen resistance (9). In our current study, we find that, although both R-Ras 38V and another constitutively active R-Ras mutant, R-Ras 87L, activate Akt in MCF-7 cells, transfection with these constructs did not activate Rac. Such a result is a striking demonstration that PI3K activation by differing stimuli can lead to activation of differing effector pathways, perhaps as a result of variations in either subcellular localization or the effects of such stimuli on additional, non-PI3K-related signaling pathways. Further, in contrast to previous studies in which modest AND-34-induced R-Ras activation was detected in Cos7 cells, we did not find such activity in MCF-7 cells (6). The reason for this discrepancy is not clear. Although, in aggregate, our studies do not directly support a role for R-Ras in AND-34-mediated anti-estrogen resistance, given the conflicting data noted above, it would be of interest in the future to more definitively address this issue by inhibiting the expression of R-Ras with small interfering RNA techniques.

In contrast, our prior demonstration of AND-34-induced Rac activation in HEK 293 and Cos7 cells is here confirmed in transiently transfected MCF-7 cells (10). Our prior studies suggested that Rac activation was likely to be an important component of AND-34-mediated anti-estrogen resistance, as inhibition of either Rac or the Rac effector Pak1 in MCF-7 cells blocked AND-34-mediated cyclin D1 promoter activation and constitutively active RacV12-induced resistance to ICI 182,780 (10). In confirmation of these studies, we now report that in growth studies done over 11 days, the small molecule Rac inhibitor NSC23766at 10 µmol/L effectively blocks AND-34/BCAR3-induced ICI 182,780 resistance, although it has no significant effect on the growth of ZR-75 cells in control medium. Further studies will be needed to establish how AND-34-induced PI3K activation leads to Rac activation in these breast cancer cell lines. Activation of one well-studied Rac GEF, Vav1, is regulated not only by PI3K but also by tyrosine phosphorylation (38). Given that AND-34 and R-Ras activate PI3K and Akt but only AND-34 overexpression induces Rac activation, it will be of interest to determine whether AND-34 but not R-Ras activates a parallel pathway resulting in such tyrosine phosphorylation of a Rac GEF.

In this study, we show that the SH2 and GEF-like domains of AND-34 are required for AND-34 to induce Rac and Akt activation and anti-estrogen resistance in MCF-7 cells. The tyrosine-phosphorylated ligand(s) for the SH2 domain of AND-34 remains unknown. PI3K activation requires recruitment of p85/p110 PI3K heterodimers from the cytoplasm to the plasma membrane, resulting in both alleviation of autoinhibition of PI3K activity by p85 and access of p110 to its main substrate, phosphatidylinositol-4,5-bisphosphate (39). Our studies suggest two models for how AND-34 overexpression could induce PI3K activation. AND-34 could be recruited either directly or through an adapter protein to a tyrosine-phosphorylated growth factor receptor known to activate PI3K signaling, such as epidermal growth factor receptor or insulin-like growth factor-I receptor 1. Interaction of AND-34 itself or an AND-34-associated (or perhaps p130Cas-associated) protein would then result in a further tyrosine phosphorylation event that recruited p85/p110 heterodimers to the plasma membrane. Alternatively, given that p130Cas has been shown to form a stable complex with p85 in v-Crk-transformed 3Y1 cells, it is possible that AND-34 instead serves to recruit p130Cas-associated PI3K activity to a membrane location (40). Our demonstration that {Delta}GEF AND-34, a GEF-like domain deletion mutant that cannot bind to p130Cas, is incapable of inducing PI3K activation, Akt phosphorylation, Rac activation, or anti-estrogen resistance would be consistent with this latter model. Interestingly, Bouton et al. have reported that overexpression of AND-34 results in recruitment of p130Cas to a plasma membrane location, a process that requires the GEF-like domain of AND-34 (41). Identification of the target or targets of the SH2 domain of AND-34 should substantially advance our understanding of how AND-34 overexpression leads to PI3K activation and assist in elucidating the normal role of AND-34 in the physiology of cells.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Antibodies
The following antibodies were used in this study: mouse anti-Rac (BD PharMingen, San Diego, CA); rabbit anti-Akt, anti-phospho-Akt (Ser473), and anti-p85 (Cell Signaling, Beverly, MA); mouse anti-ER{alpha} (Chemicon, Temecular, CA); mouse anti-HA (Covance, Princeton, NJ); and mouse anti-tubulin (Sigma, St. Louis, MO).

Plasmid Constructs
The generation of full-length, HA-tagged AND-34-MSCV construct has been described previously (11). HA-tagged AND-34 {Delta}NTer, AND-34 R171K, and AND-34 {Delta}GEF were subcloned from their corresponding pcDNA1 constructs by PCR into the XhoI and R1 sites of the retroviral vector pMSCV-IRES-GFP (6). Myristoylated p110 construct was a generous gift of Dr. Thomas M. Roberts (Dana-Farber Cancer Institute, Boston, MA; ref. 42). The HA-tagged p{Delta}85 was described previously and was a kind gift from Dr. Alex Toker (Mayo Clinic, Rochester, MN; ref. 43). The pCGN R-Ras wild-type, 38V, and 87L constructs were a gift from Dr. Adrienne Cox (University of North Carolina, Chapel Hill, NC).

Transient Transfection
Plasmids were transfected into MCF-7 cells using FuGene 6 reagent (Roche Diagnostics, Indianapolis, IN). Briefly, MCF-7 cells were grown to 50% confluence in six-well cell culture plates. A total of 100 µL fetal bovine serum–free DMEM medium was mixed with 5 to 10 µL FuGene 6 reagent and left at room temperature for 5 minutes. Then, 1 or 2 µg DNA were added into the FuGene 6 solution and maintained at room temperature for an additional 15 minutes before adding the final mixture into cell cultures. Fresh medium was added into the cell culture on the second day and whole cell lysates were prepared 48 to 72 hours after transfection.

GTPase Activation Assays
Levels of activated Rac were determined by pull-down analysis as described previously (11). Forty-eight to 72 hours after transfection, MCF-7 cells were harvested in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 200 mmol/L NaCl, 5 mmol/L MgCl2, 1% NP40, 15% glycerol, and protease inhibitors]. Whole cell lysates were incubated for 2 hours with 10 µL glutathione-Sepharose 4B beads preincubated with 5 µg GST-PAK. The GST-PAK-RBD construct was a kind gift of Dr. Zhijun Luo and has been described previously (Section of Endocrinology, Boston Medical Center, Boston, MA; ref. 10). The beads were washed thrice with cell lysis buffer and GTP-bound Rac was released by boiling for 5 minutes in 2x SDS sample buffer. Rac was then detected by Western blot analysis.

In vivo R-Ras GTP/GDP Binding Assay
MCF-7 cells were transfected with 1 µg pCGN R-Ras wild-type along with 1 µg empty pMSCV-IRES-GFP or pFLAG-CMV2 vectors or vectors encoding AND-34 or GRP3 as indicated. After 24 hours, cells were serum starved overnight before incubation for 4 hours in serum- and phosphate-free medium supplemented with 150 µCi 32Pi. Cells were lysed in radioimmunoprecipitation assay buffer, HA-tagged R-Ras was immunoprecipitated with anti-HA monoclonal antibody, and associated GTP and GDP were separated by TLC essentially as described (44). GTP and GDP spots were quantitated using an Ambis (Muskegon, MI) ß scanner and gels were subsequently exposed overnight to autoradiography film.

PI3K Assay
The PI3K assay was carried out as described previously (33). PI3K was immunoprecipitated from lysates of MCF-7 cells transfected with HA-AND-34 constructs with an anti-p85 polyclonal antibody. The immunoprecipitates were washed twice with PBS-1% NP40 and twice with 20 mmol/L HEPES (pH 7.4)-5 mmol/L MgCl2. PI3K activity in immunoprecipitates was determined by incubating the beads with reaction buffer containing 20 mmol/L HEPES (pH 7.4), 5 mmol/L MgCl2, 10 µmol/L cold ATP, 10 µCi [{gamma}-32P]ATP, and 10 µg phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate for 15 minutes at 37°C. The reactions were terminated by adding 75 µL of 1 mol/L HCl. Phospholipids were extracted with 180 µL CHCl3/methanol. Phosphorylated products were separated by TLC and detected by autoradiography.

Akt In vitro Kinase Assay
Kinase reactions were carried out in the presence of 10 µCi [{gamma}-32P]ATP (Amersham, Piscataway, NJ) and 10 µmol/L unlabeled ATP in 50 µL buffer containing 20 mmol/L HEPES (pH7.4), 10 mmol/L MgCl2, 10 mmol/L MnCl2, and 1 mmol/L DTT. GSK-3 fusion protein (Cell Signaling) was used as an exogenous substrate. The GSK-3 fusion protein was prepared by fusing the GSK-3{alpha}/ß cross-tide (CGPKGPGRRGRRRTSSFAEG) to the NH2 terminus of paramyosin. After incubation at 37°C for 30 minutes, the reaction was stopped by adding 2x SDS loading buffer and boiling for 5 minutes. The mixture was separated by SDS-PAGE electrophoresis followed by autoradiography.

Growth Assay
ZR-75-1 cells were transfected with pBKCMV (allowing selection with G418) along with pcDNA1 or pcDNA1 HA-BCAR3, HA-BCAR3 {Delta}NTer, and HA-BCAR3 {Delta}GEF. For growth assays, stable transfectants were trypsinized, and single cell suspensions were plated in 100 mm tissue culture plates at a density of 750,000 cells per plate in 10% fetal bovine serum and 100 nmol/L ICI 182,780 (Tocris Cookson Ltd., Ellisville, MD). Culture medium was changed twice per week. On the 11th day, cells were trypsinized and counted. A comparable strategy was used for the PI3K and Rac inhibition growth assays. For these experiments, cells were plated in a six-well plate at a density of 130,000 cells per well. Fresh ICI 182,780 and/or NSC23766was added to the cells every 48 hours and LY294002 (Cell Signaling) every 24 hours.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Diko Kazandjian for excellent technical assistance and Dr. Thomas M. Roberts for assistance in setting up the PI3K assay.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Department of Defense Breast Cancer Idea Award BC-00-0756, Logica Foundation, and Indiana University School of Medicine Biomedical Research. Back

Received May 11, 2004; revised November 12, 2004; accepted November 30, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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