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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(2):244-251; doi:10.1093/carcin/bgm245
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Grb2 binding to Tyr284 in TβR-II is essential for mammary tumor growth and metastasis stimulated by TGF-β

Amy J. Galliher-Beckley and William P. Schiemann*

Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, CO 80045, USA

* To whom correspondence should be addressed at Department of Pharmacology, University of Colorado Health Sciences Center, RC1 South Tower, Room L18-6110, 12801 East 17th Avenue, PO Box 6511, Aurora, Colorado 80045, USA. Tel: +1-303-724-1541; Fax: +1-303-724-3663; Email: bill.schiemann{at}uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
We demonstrated previously that growth factor receptor-bound protein 2 (Grb2) associates with the transforming growth factor-β (TGF-β) type II receptor [TβR-II] upon its phosphorylation on Tyr284 by Src. Although this phosphotransferase reaction is critical in mediating TGF-β stimulation of epithelial-mesenchymal transition (EMT) and invasion in mammary epithelial cells (MECs), the necessity of Grb2 in promoting these TGF-β-dependent events remain purely correlative. Herein, we further evaluated the role of Grb2 in mediating the oncogenic activities of TGF-β and show that the binding of Grb2 to TβR-II paralleled the induction of EMT in MECs stimulated by TGF-β. Introducing siRNAs against Grb2 or expression of a TβR-II mutant that cannot bind Grb2 (i.e. Y284F-TβR-II) had no effect on the ability of TGF-β to activate Smad3, but significantly impaired its stimulation of p38 mitogen-activated protein kinase (MAPK) in MECs. Importantly, these same cellular conditions also prevented the ability of MECs to undergo EMT in response to TGF-β, and to invade synthetic basement membranes when stimulated by β3 integrin and TGF-β. Finally, we show that the ability of TGF-β to stimulate breast cancer growth and pulmonary metastasis in mice required TβR-II to be phosphorylated on Tyr284, which activated p38 MAPK in developing and progressing mammary tumors. Collectively, our findings have established the necessity of Grb2 in mediating TGF-β stimulation of EMT and invasion in MECs, as well as demonstrated the essential function of the {alpha}vβ3 integrin:Src:phospho-Y284-TβR-II:Grb2:p38 MAPK signaling axis to promote breast cancer growth and metastasis in vivo.

Abbreviations: bFGF, basic fibroblast growth factor; EMT, epithelial-mesenchymal transition; GFP, green fluorescent protein; Grb2, growth factor receptor-bound protein 2; MAPK, mitogen-activated protein kinase; MEC, mammary epithelial cell; RTK, receptor tyrosine kinase; TGF-β, transforming growth factor-β; TNF-{alpha}, tumor necrosis factor-alpha; TβR-II, transforming growth factor-β type II receptor; VEGF, vascular endothelial growth factor; WT, wild-type


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates all stages of mammary gland development; it also suppresses mammary tumorigenesis by prohibiting mammary epithelial cell (MEC) proliferation, and by creating a cell microenvironment that inhibits MEC motility, invasion and metastasis (1,2). Importantly, mammary tumors frequently acquire the ability to undermine the tumor-suppressing function of TGF-β, and in doing so, transform TGF-β from a suppressor of breast cancer formation to a promoter of its growth and metastasis (3,4). An important component of oncogenic signaling mediated by TGF-β lies in its ability to stimulate epithelial-mesenchymal transitions (EMTs) in malignant MECs (5), thereby enabling normal and malignant MECs to acquire highly motile and invasive mesenchymal phenotypes. Despite considerable advances in our understanding of the biological and pathological actions of TGF-β, science and medicine still lack the knowledge and means necessary to effectively antagonize the oncogenic activities of TGF-β in developing and progressing breast cancers.

In attempting to fill this important knowledge gap, we recently discovered that the expression and activity of {alpha}vβ3 integrin and Src are essential for TGF-β stimulation of MEC proliferation, invasion and EMT (6,7). Moreover, the ability of these molecules to couple TGF-β to oncogenic signaling proceeds via the formation of β3 integrin:transforming growth factor-β type II receptor (TβR-II) complexes that enable TβR-II to become phosphorylated on Tyr284 by Src, a phosphotransferase reaction essential for TGF-β stimulation of breast cancer cell invasion and p38 mitogen-activated protein kinase (MAPK) activation (7). In addition, phosphorylated Tyr284 also functions as a SH2 domain-binding site for Shc in vitro, and for growth factor receptor-bound protein 2 (Grb2) both in vitro and in vivo (7), thereby associating the oncogenic activities of TGF-β with the docking of Grb2 to TβR-II. The aim of the present study was to establish the function of Grb2 in mediating oncogenic signaling by TGF-β in MECs, and to determine the consequences of preventing the docking of Grb2 to TβR-II on mammary tumor growth and metastasis in vivo. In doing so, our findings have established Grb2 as an essential component of the {alpha}vβ3 integrin:Src:pY284F-TβR-II:p38 MAPK-signaling axis that mediates the oncogenic activities of TGF-β in MECs both in vitro and in vivo. Our findings also lend credence to the notion that chemotherapeutic targeting of this non-canonical signaling axis may one day improve the clinical course of patients with metastatic breast cancer.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Retroviral plasmids and transgene expression
Retroviral vectors encoding for human wild-type (WT)- or D119A-β3 integrins, and for human WT-, Y284F- or Y470F-TβR-II molecules were described previously (6,7). WT- and mutant TβR-II retroviral supernatants were infected into NMuMG cells with a multiplicity of infection of 2, and subsequently were sorted, collected and expanded to yield stable polyclonal populations of transgene-expressing cells as described previously (6,7). NMuMG and 4T1 cells engineered to stably express β3 integrin variants or TβR-II variants, respectively, were reported previously (6,7).

Grb2 deficiency and immunoprecipitation assays
Grb2-deficient NMuMG cells were created by transient transfection of SMARTpool siRNAs against Grb2 (catalog number L-040166; Dharmacon, Lafayette, CO) using the procedures described previously (6). Seventy-two hours after transfection, the cells were stimulated with TGF-β1 (R&D Systems, Minneapolis, MN) for 0–240 min as indicated. Afterward, detergent-solubilized whole-cell extracts were prepared (200 µg per tube) for immunoprecipitation with anti-Grb2 antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA), followed by western blotting with antibodies against TβR-II (Santa Cruz Biotechnologies) as described previously (7). Differences in protein loading were monitored by reprobing stripped membranes with anti-β-actin antibodies.

Cell biological assays
The effect of Grb2 deficiency or Y284F-TβR-II expression on various TGF-β-stimulated activities in NMuMG cells were determined as follows: (i) phospho-specific antibodies were used on whole-cell extracts to monitor the activation status of Smad3 (Cell Signaling, Beverly, MA) and p38 MAPK (Cell Signaling) as described previously (6,7), (ii) cell invasion induced by 10% serum using 350 000 cells per well in a modified Boyden chamber coated with Matrigel matrices (diluted 1:25 in serum-free DMEM) as described (6,7) and (iii) cell cytoskeletal architecture was visualized by direct rhodamine-phalloidin (0.25 µM; Sigma, St Louis, MO) immunofluorescence as described previously (6,7). All images were captured on a Nikon Diaphot microscope.

Tumor growth and metastasis studies
Control green fluorescent protein (GFP), WT-, Y284F- or Y470-TβR-II-expressing 4T1 cells were resuspended in sterile PBS and subsequently were injected (50 000 cells per mouse) orthotopically into the mammary fat pads of 6-week-old female syngeneic Balb/C mice (five mice per condition; Jackson Labs, Bar Harbor, ME). Mice were monitored daily and primary tumors were measured with digital calipers (Fisher Scientific, Pittsburg, PA) on days 9, 13, 17 and 20. Tumor volumes were calculated using the following equation: tumor volume = (x2)(y)(0.5), where ‘x’ is the tumor width and ‘y’ is the tumor length. Twenty days after inoculation, the mice were killed and their primary tumors were excised, weighed and processed for histopathological analysis in the Pathology Core at the University of Colorado Cancer Center.

The capacity of 4T1 cell variants to metastasize to the lungs was determined via two complementary procedures based on the clonogenic assay described by Shan et al. (8). In brief, the lungs of 4T1 tumor-bearing mice were removed at the time of necropsy, and subsequently were minced and digested proteolytically in PBS supplemented with 1 mg of Blendzyme (Roche Applied Science, Indianapolis, IN). Enzymatic reactions were allowed to proceed for 3 h at 37°C under continuous rotation, and subsequently were filtered through a 70-µm nylon cell strainers. The resulting single-cell suspensions were washed twice in PBS prior to culturing the cells (8 x 106 cells per plate) onto in 10-cm plates in DMEM/10% FBS media supplemented with 60 µM 6-thioguanine to select for metastatic 4T1 cells, which are resistant to 6-thioguanine treatment. After 14 days of growth in selection media, the resulting metastatic foci were fixed in 10% MeOH/10% acetic acid and stained with crystal violet. Alternatively, the resulting single-cell suspensions (2 x 106 cells) were immediately analyzed for GFP fluorescence on a Becton Dickinson FACSCaliber to detect the presence of metastatic 4T1 cell variants in the lungs of tumor-bearing mice.

All animal studies were performed three times in their entirety and were performed according to animal protocol procedures approved by the Institutional Animal Care and Use Committee of University of Colorado.

Immunohistochemistry
Excised primary 4T1 tumors were sectioned for histopathological analyses in the Pathology Core at the University of Colorado Cancer Center. Afterward, formalin-fixed, paraffin-embedded primary 4T1 tumor tissue sections (4 µm) were deparaffinized in xylene and rehydrated through a graded series of alcohols prior to inactivating endogenous peroxidase activity by incubation in 3% hydrogen peroxide for 5 min at room temperature. Antigen retrieval was performed by pressure cooking the sections in 10 mM sodium citrate/0.5% Tween 20 (pH 6.0) for 10 min at 120°C, at which point non-specific binding sites were blocked by incubation of the sections in PBS containing 0.1% Tween-20/1% FBS for 1 h at room temperature. Afterward, anti-phospho-Smad2 (1:100; Cell Signaling, Boston, MA), -phospho-p38 (1:100; Cell Signaling) or -GFP (1:100; Clontech, Mountain View, CA) antibodies were applied to the tissue sections for 1 h at room temperature, followed by an additional 1-h incubation with biotin-conjugated anti-rabbit secondary antibodies (1:200; Jackson Immuno Research, West Grove, PA). The resulting immunocomplexes were visualized using the VectaStain ABC kit (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine (DAB; Chemicon, Temecula, CA) and subsequently were counterstained with hematoxylin prior to tissue section dehydration and mounting with Permount Mounting Media (Fisher Scientific). Negative staining controls for these analyses comprised the use of adjacent tissue sections that were processed in parallel in the absence of primary antibody.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Formation of Grb2:TβR-II complexes facilitates activation of p38 MAPK by TGF-β in NMuMG cells
We recently demonstrated that Src phosphorylates TβR-II at Y284, which then serves as a SH2 domain-binding site for Shc in vitro, and for Grb2 both in vitro and in vivo (7). Although expression of Src-resistant Y284F-TβR-II mutants in murine 4T1 breast cancer cells prevented TGF-β from stimulating their activation of p38 MAPK and invasion through synthetic basement membranes (7); these studies failed to establish the necessity of Grb2 in mediating these cellular events. In an attempt to better define the role of Grb2 in mediating oncogenic signaling by TGF-β, we transiently transfected NMuMG cells with siRNAs directed against Grb2. Figure 1A shows that TGF-β treatment of NMuMG cells induced a rapid and transient interaction between Grb2 and TβR-II, a binding reaction that was lost in Grb2-deficient cells. Interestingly, extended TGF-β treatment of NMuMG cells restored the formation of Grb2:TβR-II complexes in a manner consistent with that observed for EMT stimulated by {alpha}vβ3 integrin and TGF-β [data not shown; (6)], suggesting that Grb2 may play an essential role in mediating oncogenic signaling by TGF-β. We explored this possibility by transiently transfecting NMuMG cells with siRNAs directed against Grb2 and subsequently monitored their ability to activate Smad3 and p38 MAPK in response to TGF-β. As expected, Grb2 deficiency failed to alter the ability of TGF-β to activate Smad3 in NMuMG cells (Figure 1B); however, this cellular condition significantly inhibited p38 MAPK activation stimulated by TGF-β (Figure 1C). Additionally, the formation of Grb2:TβR-II complexes preceded the activation of p38 MAPK by TGF-β, indicating that the docking of Grb2 to TβR-II is a key event during TGF-β stimulation of p38 MAPK in MECs. Taken together, our findings reveal an essential function for Grb2 in coupling TβR-II to activation of p38 MAPK in MECs.


Figure 1
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  		Fig. 1. Formation of Grb2:TβR-II complexes facilitates activation of p38 MAPK by TGF-β in NMuMG cells. Grb2-deficient NMuMG cells were generated by siRNA transfection. Afterward, control and Grb2-deficient cells were serum starved for 6 h prior to their stimulation with TGF-β1 (5 ng/ml) for 0–240 min as indicated. (A) Detergent-solubilized whole-cell extracts (200 µg per tube) were immunoprecipitated with anti-Grb2 antibodies, followed by western blotting (WB) with antibodies against TβR-II. The extent of Grb2 deficiency was monitored by probing whole-cell extracts (W.C.E.) with anti-Grb2 antibodies, whereas differences in protein loading were monitored using anti-β-actin antibodies. (B and C) Whole-cell extracts were prepared to monitor the activation status of Smad3 (B) or p38 MAPK (C) by using phospho-specific antibodies. The degree of Grb2 deficiency was determined by reprobing stripped membranes with anti-Grb2 antibodies. Accompanying graphs depict the mean (±standard error) increase of Smad3 (B) or p38 MAPK (C) stimulation relative to untreated control (cont) siRNA-transfected cells observed in three independent experiments (*P < 0.05; Student’s t-test).

 
Formation of Grb2:TβR-II complexes is essential for induction of EMT by TGF-β in NMuMG cells
Because Grb2 is necessary for TGF-β stimulation of p38 MAPK in NMuMG cells and because p38 MAPK activation is required for TGF-β stimulation of EMT in these same cells (9,10), we then determined whether Grb2 deficiency alters the ability of TGF-β to induce EMT in NMuMG cells. Figure 2A shows that when stimulated with TGF-β, Grb2-expressing NMuMG cells became apolar and lost their ability to form cell–cell contacts; they also acquired an elongated, fibroblast-like morphology within 24 h of TGF-β treatment, a response that became even more pronounced at 48 h. In stark contrast, Grb2-deficient NMuMG cells were resistant to these morphological alterations induced by TGF-β (Figure 2A), and unlike their control counterparts, these cells also failed to develop actin cytoskeletal rearrangements and the formation of actin stress fibers when stimulated with TGF-β (Figure 2B). Thus, these findings demonstrate the necessity of Grb2 in mediating EMT stimulated by TGF-β, presumably via its ability to bind phosphorylated Y284 in TβR-II [Figure 1; (7)].


Figure 2
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  		Fig. 2. Formation of Grb2:TβR-II complexes is essential for induction of EMT by TGF-β in NMuMG cells. (A and B) NMuMG cells were rendered Grb2 deficient by siRNA transfection and subsequently were stimulated with TGF-β1 (5 ng/ml) for 0–48 h as indicated. The ability of TGF-β to alter NMuMG cell morphology was monitored by phase contrast microscopy (A) or by direct rhodamine-phalloidin immunofluorescence (B). Shown are representative images from a single experiment that was repeated twice with identical results. (C and D) NMuMG cells-expressing WT-, Y284F- or Y470F-TβR-II were stimulated with TGF-β1 (1 ng/ml) for 0–24 h as indicated. The ability of TGF-β to alter NMuMG cell morphology and cytoskeletal architecture was monitored by phase contrast microscopy (C) and rhodamine-phalloidin immunofluorescence (D). Shown are representative images from a single experiment that was repeated twice with identical results.

 
To address whether the formation of Grb2:TβR-II complexes was necessary for EMT stimulated by TGF-β, we engineered NMuMG cells to stably express various TβR-II proteins including WT-, Y284F-TβR-II or Y470F-TβR-II, which is readily phosphorylated by Src and binds Grb2 (7), and subsequently monitored their ability to undergo EMT in response to TGF-β. As expected, TGF-β treatment of parental cells (i.e. yellow fluorescent protein expressing) induced a fibroblast-like morphology (Figure 2C) and the formation of actin stress fibers (Figure 2D). These TGF-β-dependent cellular events were clearly enhanced in NMuMG cells that expressed WT- or Y470F-TβR-II proteins, but were significantly suppressed in those that expressed Y284F-TβR-II (Figures 2C and D). Collectively, these findings demonstrate the essential function of phosphorylated Y284 in coordinating the docking of Grb2 to TβR-II and, consequently, in mediating TGF-β stimulation of EMT in MECs.

Formation of Grb2:TβR-II complexes is essential for NMuMG cell invasion stimulated by TGF-β
Our previous findings (7) and those presented thus far implicate Grb2 as an important intermediary operant in mediating oncogenic signaling by TGF-β. In addition, MECs that have undergone EMT not only exhibit fibroblast-like characteristics but also become highly motile and invasive (11), properties that enable breast cancer cells to disseminate to distant sites (5,12). We therefore determined the effects of Grb2 deficiency on the ability of TGF-β to induce the invasion of NMuMG cells through Matrigel matrices. Figure 3A shows that Grb2 deficiency completely abrogated NMuMG cell invasion stimulated by TGF-β. As noted previously (6,7), β3 integrin expression significantly enhanced the invasive function of TGF-β, a response that was lost in Grb2-deficient cells (Figure 3A). Quite interestingly, expression of inactive β3 integrin [i.e. D119A-β3 integrin, (6)] in NMuMG cells, which blocked their invasion stimulated by TGF-β [Figure 3A; (6)], partially rescued this response to TGF-β in Grb2-deficient NMuMG cells (Figure 3A). Although the molecular mechanism underlying this intriguing response remains to be determined, it is tempting to speculate that the loss of Grb2 in this cellular context alters β3 integrin and/or TGF-β receptor trafficking from the cell surface in such a way that these NMuMG cells become sensitized to TGF-β.


Figure 3
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  		Fig. 3. Formation of Grb2:TβR-II complexes is essential for NMuMG cell invasion stimulated by TGF-β. (A and B) Parental (i.e. GFP), β3 integrin- and D119A-β3 integrin-expressing NMuMG cells were rendered Grb2 deficient by siRNA transfection prior to inducing their invasion through Matrigel matrices for 36 h in the absence or presence of TGF-β1 (5 ng/ml) (A) or in the absence or presence of TNF-{alpha} (20 ng/ml), bFGF (30 ng/ml), VEGF (50 ng/ml), or TGF-β1 (5 ng/ml) (B). Data are the mean (±standard error) invasion relative to TGF-β-stimulated β3 integrin-expressing NMuMG cells observed in three independent experiments (*, **P < 0.05; Student’s t-test). (C) Control (i.e. yellow fluorescent protein), WT-, Y284F- or Y470F-TβR-II-expressing NMuMG cells were induced by TGF-β1 (1 ng/ml) to invade Matrigel matrices for 30 h. Data are the mean (±standard error) invasion relative to TGF-β-stimulated WT-TβR-II-expressing NMuMG cells observed in three independent experiments. (*, **P < 0.05; Student’s t-test).

 
It should be noted that epithelial cells also undergo invasion when stimulated by other growth factors and cytokines, such as tumor necrosis factor-alpha (TNF-{alpha}), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) (13–15). We therefore, compared the necessity of Grb2 in mediating NMuMG cell invasion stimulated by TGF-β with that induced by TNF-{alpha}, bFGF and VEGF. As expected, NMuMG cell invasion occurred readily in response to TNF-{alpha}, bFGF or VEGF treatment (Figure 3B). However, whereas Grb2 deficiency completely abrogated NMuMG cell invasion stimulated by TGF-β, this cellular condition failed to affect the invasive activity induced by TNF-{alpha}, bFGF or VEGF (Figure 3B). Thus, MEC invasion stimulated by TGF-β is exquisitely sensitive to the expression and activity of Grb2.

Finally, we determined whether the formation of Grb2:TβR-II complexes was necessary for invasion induced by TGF-β. Figure 3C shows that NMuMG cells readily invaded synthetic basement membranes when stimulated by TGF-β, a cellular response that was potentiated by the expression of WT- or Y470F-TβR-II proteins. Consistent with the effects of Grb2 deficiency, expression of Y284F-TβR-II in NMuMG cells prevented their invasion in response to TGF-β. Taken together, these results show that Grb2:pTyr284-TβR-II complex formation is essential and specific for NMuMG cell invasion induced by β3 integrin and TGF-β. Our findings also suggest that preventing the docking of Grb2 to TβR-II (i.e. by Y284F-TβR-II expression) may negate the oncogenic activities of TGF-β in developing and progressing mammary tumors, particularly their ability to undergo metastatic dissemination.

Formation of Grb2:TβR-II complexes enhances mammary tumor growth and pulmonary metastasis through a p38 MAPK-dependent mechanism
We tested the above hypothesis by orthotopically injecting the mammary fat pads of Balb/C mice with syngeneic 4T1 breast cancer cells previously engineered to stably express WT-, Y284F- or Y470F-TβR-II proteins. Importantly, these same cell lines were used previously to demonstrate that (i) Grb2 binds TβR-II at Y284 in breast cancer cells and (ii) Y284 mediates TGF-β stimulation of breast cancer cell proliferation and invasion (7). Consistent with these in vitro findings, the growth of 4T1 tumors in Balb/C mice was increased significantly by their expression of either WT- or Y470F-TβR-II (Figure 4A). Importantly, Y284F-TβR-II expression in 4T1 cells prevented TGF-β from promoting their growth in mice (Figure 4A). We also performed immunohistochemistry on primary tumor sections to monitor the activation status of Smad2 and p38 MAPK. As shown in Figure 4B, the phosphorylation and activation of Smad2 in 4T1 tumors was unaffected by the TβR-II variant expressed in these malignant MECs. Interestingly, the phosphorylation and activation of p38 MAPK in parental (i.e. GFP-expressing) 4T1 tumors was quite low, whereas that observed in their WT- and Y470F-TβR-II-expressing counterparts was extremely high, particularly at the leading edges and invasive fronts of these tumors (Figure 4C). In stark contrast, Y284F-TβR-II-expressing 4T1 tumors failed to activate p38 MAPK as compared with tumors that expressed WT- or Y470F-TβR-II proteins (Figure 4C). Thus, the formation of Grb2:TβR-II complexes promotes mammary tumor growth in part via the activation of a p38 MAPK-dependent signaling cascade.


Figure 4
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  		Fig. 4. Formation of Grb2:TβR-II complexes enhances mammary tumor growth through a p38 MAPK-dependent mechanism. (A) Parental (i.e. GFP), WT-, Y284F- or Y470F-TβR-II-expressing 4T1 cells were injected orthotopically into the mammary fat pads of syngeneic Balb/C mice. Tumor volumes were measured at 9, 13, 17 and 20 days after injection. Data are the mean (±standard error) tumor volumes observed in a single experiment (five mice per condition) that was repeated twice with similar results. (B and C) Tumor sections were stained with phospho-specific antibodies against Smad2 (B) or p38 MAPK (C). Arrows denote the margins of normal mammary tissue in each tumor section. Shown are representative x10 and x40 images from a single experiment that was repeated once with identical results.

 
Although some success in detecting and treating localized breast cancer has occurred in recent years, little progress has been made in successfully treating metastatic disease, which today remains incurable (16,17). Because disrupting the formation of Grb2:TβR-II complexes inhibits breast cancer cell invasion through synthetic basement membranes (7) and 4T1 tumor growth in mice (Figure 4), we suspected that expression of Y284F-TβR-II would negatively impact the metastatic abilities of 4T1 cells. We tested this possibility in 4T1 tumor-bearing mice at the time of necropsy by harvesting their lungs, which were immediately minced and digested proteolytically to produce single-cell suspensions that were analyzed by flow-cytometry for GFP expression, and by clonogenic assay under 6-thioguanine selection. Figure 5A shows that GFP-positive 4T1 cells were readily detected in the lungs of mice-bearing WT- and Y470F-TβR-II tumors, but not in their Y284F-TβR-II-expressing counterparts. Reduced pulmonary metastasis of Y284F-TβR-II tumors also was observed following 6-thioguanine selection of lung-cell suspensions (Figure 5B). Interestingly, flow-cytometry analysis of GFP expression in the resulting 4T1 cell lung colonies showed that parental-, WT- and Y470F-TβR-II-expressing pulmonary metastases all retained significant transgene expression, whereas those arising from Y284F-TβR-II-expressing tumors exhibited little GFP expression and, consequently, little Y284F-TβR-II expression (Figure 5C). Importantly, high levels of GFP expression were detected immunohistochemically in primary tumor sections derived from each inoculated cell line (Figure 5C), suggesting that Y284F-TβR-II expression suppressed pulmonary metastasis of 4T1 cells driven by TGF-β. Taken together, these findings establish the necessity of phosphorylated Y284 in mediating Grb2:TβR-II complex formation, which promotes pulmonary metastasis by breast cancer cells in response to TGF-β.


Figure 5
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  		Fig. 5. Formation of Grb2:TβR-II complexes is essential for TGF-β stimulation of breast cancer pulmonary metastasis. The lungs of 4T1 tumor-bearing mice were removed at the time of necropsy and subsequently subjected to proteolytic digestion to yield single-cell suspensions. (A) Lung single-cell suspensions derived WT-, Y284F- or Y470F-TβR-II-expressing 4T1 tumors were analyzed for GFP expression by flow cytometry. GFP-positive cells (G; blue) are indicted in the lower right quadrant as the percentage of cells within isolated lung-cell suspensions. Data are representative flow-cytometry analyses from a single experiment that was repeated twice with similar results. (B and C) Lung single-cell suspensions were cultured onto 10-cm plates in growth media supplemented with 6-thioguanine (60 µM). After 14 days, the surviving metastatic colonies were fixed, stained with crystal violet and counted. Data are mean (±standard error) colonies per plate observed in a single experiment that was repeated twice with similar results. Alternatively, the surviving metastatic colonies were analyzed by flow-cytometry to monitor GFP expression (C, left panel). GFP expression in corresponding primary tumor sections was monitored immunohistochemically using anti-GFP antibodies (C, rightpanel). Arrows denote the margins of normal mammary tissue in each tumor section. Shown are representative data from a single experiment that was repeated twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Transmembrane signaling by growth factors is initiated by ligand-induced dimerization of receptor tyrosine kinases (RTKs), which promotes their activation and autophosphorylation on cytoplasmic Tyr residues. Once phosphorylated, these Tyr residues coordinate the recruitment and docking of phosphotyrosine-binding proteins, particularly class I (i.e. enzymatic molecules, such as Src and PLC-{gamma}) and class II (i.e. non-enzymatic adapter molecules, such as Shc and Nck) SH2 domain-containing proteins (18). Grb2 is a class II SH2 adapter molecule whose recruitment to activated RTKs mediates their activation of the Ras/MAP kinase pathway (19), as well as regulates their coupling to AKT activation (20), focal adhesion disassembly (21) and RTK internalization (22). In addition, amplification of the Grb2 gene locus occurs in human cancers, particularly those of the breast (23,24). Indeed, ~50% of human breast cancers exhibit upregulated Grb2 expression, suggesting that amplified Grb2 signaling is selected for duringmammary tumorigenesis (20,25). Accordingly, heterozygosity at the Grb2 gene locus in mice significantly delays the formation of mammary tumors induced by the polyoma middle T antigen (26). Thus, preventing the ability of Grb2 to amplify RTK signaling in human breast cancers may afford novel avenues to prevent the development and progression of breast cancers.

Although the receptors for TGF-β are transmembrane Ser/Thr protein kinases, their activation and that of their downstream effectors exhibit a number of mechanistic analogies reminiscent of those observed during the stimulation of RTKs by growth factors. For instance, transmembrane signaling by TGF-β commences by its binding to TβR-II, which oligomerizes with transphosphorylates and activates TβR-I. Phosphorylation of TβR-I not only functions in activating its Ser/Thr protein kinase but also serves in recruiting and docking the transcription factors, Smads 2 and 3 (2,27), leading to their phosphorylation and activation by TβR-I (2,27). Once activated, Smads 2 and 3 form heterocomplexes with Smad4 that translocate into the nucleus where they, together with a variety of transcriptional activators or repressors, regulate the expression of TGF-β-responsive genes (2,27). Activation of this canonical Smad2/3/4 pathway by TGF-β plays an essential role in limiting the uncontrolled growth of MECs by preventing their progression through the cell cycle (2,27–30). However, similar to RTKs, TGF-β also governs MEC physiology through its stimulation of MAP kinases, including ERK1/2, p38 and JNK, and of phosphoinositol-3 kinase (6,7,9,27,31–33). Importantly, inappropriate or amplified activation of these non-canonical signaling pathways by TGF-β, together with altered Smad2/3 signaling inputs, plays an important role in promoting oncogenic signaling by TGF-β (9,10,31,34–36).

Despite these recent advances, the molecular mechanisms that enable malignant MECs to circumvent the tumor-suppressing activities of TGF-β remain to be elucidated fully. We recently established the importance of a novel {alpha}vβ3 integrin:TβR-II:Src-signaling axis whose activation results in Src-mediated phosphorylation at Y284 in TβR-II, which coordinates its docking of Grb2 and the subsequent activation of p38 MAPK (6,7). Importantly, measures that abolished the expression or function of β3 integrin or Src (6,7) or those that prevented phosphorylation of Y284 (7) abrogated oncogenic signaling by TGF-β in normal and malignant MECs (6,7). On the basis of these findings, we proposed that oncogenic signaling by TGF-β in MECs is evolutionarily and functionally redundant with that mediated by growth factor receptors (6,7).

The findings of present study significantly bolster this supposition by establishing the essential role of Grb2 in mediating the tumor-promoting activities of TGF-β (Figure 6). Indeed, the formation of Grb2:TβR-II complexes in NMuMG cells was essential for TGF-β to stimulate p38 MAPK activation (Figure 1), as well as for the ability of TGF-β to induce EMT (Figure 2) and invasion in normal MECs (Figure 3). Moreover, measures that prevent Grb2 docking to TβR-II (i.e. Y284F-TβR-II expression) not only impaired these same cellular events (Figures 2 and 3) but also inhibited the ability of TGF-β to promote the growth and pulmonary metastasis of mammary tumors produced in mice (Figures 4 and 5). Based on these findings, it appears that the key molecular event in this process probably is the phosphorylation of Y284 in TβR-II, which MECs presumably misinterpret as arising from RTK activation, and as such, results in the inappropriate docking of Grb2 on TβR-II and the initiation of oncogenic signaling by TGF-β. Interestingly, the clear demarcation between TGF-β stimulation of Smad2/3 versus Src:Grb2:p38 MAPK signaling suggests that measures capable of preventing phosphorylation of Y284 in TβR-II, or those operant in targeting its downstream effectors may offer new avenues in restoring the tumor-suppressing function of TGF-β in developing breast cancers (37). Our study provides the first preclinical test and validation of this hypothesis and shows that the ability of TGF-β to stimulate breast cancer growth and pulmonary metastasis in mice is intimately linked to the phosphorylation of Y284 in TβR-II. In light of our findings and those implicating upregulated Grb2 expression in promoting breast cancer progression (20,23–25), future studies need to identify which Grb2-regulated pathways mediate (i) oncogenic signaling by TGF-β [e.g. MAP kinase activation (19)], (ii) RTK internalization (22), (iii) Gab1/2 and AKT activation (38,39) and (iv) altered cytoskeletal architecture (21). Additionally, our murine studies fail to distinguish whether Y284F-TβR-II expression represents a specific defect in the ability of breast cancer cells to metastasize to the lung or a more generalized defect in their initiation of global metastasis. Future studies need to address this issue, as well as to determine whether therapeutic targeting of Grb2 effectors may function in alleviating the development of metastatic phenotypes in breast cancers stimulated by TGF-β. Experiments designed to examine these intriguing possibilities are currently underway.


Figure 6
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  		Fig. 6. Schematic depicting the role of Grb2 in mediating oncogenic signaling by TGF-β in MECs. Mammary tumorigenesis or TGF-β stimulation of EMT in MECs induces upregulated expression of {alpha}vβ3 integrin, an event that enables β3 integrin to interact physically with TβR-II, leading to its phosphorylation on Y284 by Src. Once phosphorylated, Y284 coordinates the binding of Grb2 to TβR-II, which mediates the ability of TGF-β to activate p38 MAPK and to induce breast cancer cell invasion. Recruitment of Grb2 to TβR-II in normal MECs converts TGF-β from a cytostatic to oncogenic cytokine, and as such, enables TGF-β to stimulate p38 MAPK and to induce EMT and invasion in normal MECs.

 

   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (CA095519 [GenBank] , CA129359 [GenBank] ); Komen Foundation to W.P.S.


   Acknowledgments
 
Members of the Schiemann Laboratory are thanked for critical reading of the manuscript. The technical expertise and support from members of the Flow Cytometry and Pathology Cores at the University of Colorado Cancer Center also is gratefully acknowledged.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received August 18, 2007; revised October 24, 2007; accepted October 26, 2007.


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