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Cell Growth & Differentiation Vol. 10, 61-71, January 1999
© 1999 American Association for Cancer Research

Shc Dominant Negative Disrupts Cell Cycle Progression in Both G0-G1 and G2-M of ErbB2-positive Breast Cancer Cells1

Lisa E. Stevenson, Kodimangalam S. Ravichandran and A. Raymond Frackelton, Jr.2

Departments of Pathology and Laboratory Medicine and Medicine, Brown University, and Roger Williams Medical Center [L. E. S., A. R. F.], Providence, Rhode Island 02908, and Beirne Carter Center for Immunology Research and Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908 [K. S. R.].


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Shc protein helps to transmit signals from receptor and cytoplasmic tyrosine kinases to Ras. We have shown that several breast cancer cell lines (MDA-MB-453, BT-474, MDA-MB-361, and SKBR3), which overexpress the ErbB2 receptor tyrosine kinase, contain constitutively tyrosine phosphorylated Shc. To investigate the role of Shc in these cells, we transfected them with a Shc-Y317F dominant-negative mutant defective in signaling to Ras. The transfectants were unable to form stable colonies, suggesting a critical role for Shc in the proliferation of these cells. In contrast, dominant-negative Shc transfectants of the nontransformed breast epithelial cell line HBL-100 grew normally. Surprisingly, cell cycle analysis of transfected SKBR3 cells suggested that the cells were blocked not only in G0-G1, but also in G2-M. The G2-M block was unexpected because Shc-Y317 is downstream of receptor tyrosine kinases that drive the early events in the cell cycle. Both the G0-G1 and G2-M arrest were rescued by transfection with wild-type Shc or oncogenic Ras 12V. Rescue by Ras suggests that Shc Y317 signals upstream of Ras, and that Shc to Ras effector pathways are involved in G2-M, although confirmation awaits a detailed molecular analysis. Most importantly, this work provides the first evidence for Shc involvement in G2-M.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Overexpression of ErbB2 (HER2/neu), a member of the EGF3 receptor family of receptor tyrosine kinases (1, 2, 3, 4, 5, 6) is frequently associated with poor prognosis in patients with breast cancer (Ref. 7 ; reviewed in Refs. 8, 9, 10) . Overexpressed ErbB2 tyrosine kinase is often constitutively activated, either by spontaneous homodimerization or by heterodimerization with other EGF receptor family members (EGF receptor, HER3, and HER4; Refs. 9 and 11 ; reviewed in Refs. 12 and 13 ). Active ErbB2 autophosphorylates itself or its heterodimerized partner, thereby creating docking sites that are recognized by the SH2 and PTB domains of many second-messenger proteins (14, 15, 16, 17, 18) . Several of these proteins, including Shc (19 , 20) , Grb2-SOS (21) , RasGAP (22) , and SH2-containing tyrosine phosphatase-2 (23, 24, 25, 26, 27) , help regulate Ras, with Ras activation being a key step both for driving cells from the nonproliferating G0 to the G1 phase of the cell cycle (28) and for driving cells through a mid- to late-G1 restriction point (29, 30, 31) , thereby allowing cell cycle progression and DNA synthesis. Thus, active ErbB2 may contribute to breast cancer malignancy by activating Ras and thereby helping to drive cell proliferation.

We have focused our attention on the role in breast cancer of the adapter protein, Shc. Activated ErbB2 and other growth factor receptors phosphorylate Shc on tyrosine 317, which in turn is recognized by the SH2 domain of Grb2, typically found complexed with SOS (reviewed in Refs. 19 and 20 ). As a result, the complexes are translocated to the cellular membrane, where SOS may facilitate GDP release from Ras, thereby allowing Ras to bind GTP and return to its active state (32, 33, 34) . Indeed, signaling pathways to Ras appear activated in some breast cancer cells (8 , 15 , 35, 36, 37, 38, 39, 40) . In this regard, Janes et al. (15) have reported that Grb2 and SOS are constitutively associated with ErbB2 in the breast cancer cell lines, SKBR3 and BT474, and we have reported recently that several breast cancer cell lines that overexpress ErbB2 contain tyrosine-phosphorylated Shc, complexed to Grb2 (35) . However, Grb2 can dock to growth factor receptors by at least two different mechanisms: directly through the SH2 domain of Grb2, and indirectly through docking to tyrosine phosphorylated Shc, which through its own SH2 domain is docked to other phosphorylated tyrosines in the receptor (19 , 33 , 41) .

Although the relative contributions of each Grb2 docking mode to signaling by growth factor receptors is largely unknown, several studies using microinjected antibodies to Shc, Shc antisense, and various Shc dominant-negative constructs have suggested that signaling from many growth factor receptors, including the receptors for EGF (42, 43, 44, 45, 46) , insulin-like growth factor-1 (47) , nerve growth factor (42 , 44) , PDGF (48) , and hepatocyte growth factor (49) depends on functional Shc. However, the role of Shc signaling in ErbB2-positive breast cancers is unknown. To begin to address this question, we have asked whether Shc signaling is required for anchorage-dependent proliferation of ErbB2-positive breast cancer cell lines. Our approach has been to express in these cells a Shc dominant-negative mutant that interferes with Shc signaling to Ras.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Mutant Shc Inhibits Colony Formation by a c-ErbB-2-positive Cell Line.
A dominant-negative mutant of Shc, Shc-Y317F, retains the SH2 and PTB domains of Shc, but unlike normal Shc, cannot be tyrosine phosphorylated at residue 317, and thus is defective in binding Grb2-SOS and signaling to Ras (50 , 51) . However, although it is defective in activating Ras, the mutant Shc still retains its ability to compete with the normal cellular Shc for docking to activated receptor tyrosine kinases, presumably thereby inhibiting the ability of the endogenous normal Shc to signal to Ras (43 , 44 , 49 , 51, 52, 53, 54) .

Therefore, we transfected ErbB2-positive breast cancer cell lines and the nontransformed breast epithelial cell line, HBL-100, with either the Shc-Y317F mutant contained in a pEBG vector or the pEBG vector alone as a control, and cotransfected all of these cells with pNeoNut to confer G418 resistance. The Shc-Y317F mutant profoundly inhibited the ability of breast cancer cells to form colonies (Fig. 1Citation and Table 1Citation ). The colonies were both dramatically smaller (Fig. 1A)Citation and fewer in number than colonies from cells transfected with the vector alone (Fig. 1BCitation and Table 1Citation ). Only 3% of cells in the mutant colonies incorporated BrdU compared with >40% of the cells from the parental colonies (Fig. 1C)Citation , suggesting that the mutant Shc was inhibiting the ability of the breast cancer cells to proliferate. In support of this view, the TUNEL assay and cell morphology suggested comparably low levels of apoptosis (2–4%) in cells from both the mutant and parental colonies (data not shown). In contrast to the growth inhibition seen in the breast cancer cells, the nontransformed breast HBL-100 cells transfected with the Shc-Y317F mutant formed colonies equal in both size and number to the vector alone controls (Fig. 1BCitation and Table 1Citation ). We attempted to derive stable lines from SKBR3 Shc-Y317F transfectants by adding exogenous growth factors to the individual microcolonies, subcultured in 96-well plates. However, the colonies failed to grow in response to either EGF (30ng/ml and 5ng/ml), insulin (30 µg/ml and 5 µg/ml), lysophosphatidic acid (10 µM), 20% serum, or transferrin (5 µg/ml) (data not shown). The results of the colony-forming assays indicate that the Shc dominant negative was able to suppress proliferation in several ErbB2-positive cell lines and further imply that Shc interactions with Grb2 are necessary for the proliferation of the ErbB2-positive breast cancer cells but not for proliferation of the nontransformed breast epithelial cells.



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Fig. 1. Shc dominant-negative mutant, Shc-Y317F, inhibits colony formation in several breast cancer cell lines. A, colonies formed by SKBR3 cells transfected with the dominant-negative Shc mutant are greatly reduced in size. Photomicrographs of colonies obtained from: SKBR3 cancer cells that had transfected with the empty vector (SKBR3-pEBG) or dominant-negative Shc (SKBR3-pEBG-ShcY317F). x30. B, comparison of colony-forming ability of cells transfected with either the pEBG empty vector ({blacksquare}) or the dominant-negative Shc (). The assay of colony formation is described in Table 1Citation . Results are expressed as a percentage of colonies formed by cells transfected pEBG-Shc-Y317F compared with colonies transfected with vector alone. Bars, SD of three separate experiments. *, colony numbers statistically different (P < 0.001) by Student’s t test from cells transfected with the empty vector. C, Shc dominant-negative inhibits DNA synthesis in SKBR3 cells. SKBR3 cells were transfected with the indicated constructs and then cultured for 3 weeks under G418 selective pressure. Multiple colonies were harvested, pooled, and tested for their ability to incorporate BrdU. Panels show phase contrast photomicrographs of cells using bright-field illumination (left panels) or immunofluorescent staining nuclei that have incorporated BrdU (right panels). Arrow, the same cell in both panels, for orientation. The fields shown are representative of two separate experiments in which 88/210 SKBR3 cells transfected with the empty pEBG vector were scored as positive, whereas only 2 of 63 SKBR3 cells transfected with the pEBG-Shc-Y317F mutant were scored as positive for BrdU incorporation.

 

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Table 1 Dominant-negative Shc inhibits colony formation in ErbB2-positive breast cell lines

 
Stable Expression of Shc-Y317F in Nontransformed HBL-100 Mammary Epithelial Cells.
Unlike the Shc-Y317F transfectants of the breast cancer cell lines, sufficient numbers of cells from Shc-Y317F transfected HBL-100s could easily be obtained to permit analysis of Shc expression by immunoblotting. The Mr 80,000 Shc-Y317F-GST fusion protein was highly expressed in several expanded clones, in some cases at levels comparable with the expression of Mr40,000, Mr 52,000, and Mr 66,000 endogenous Shc proteins (Fig. 2)Citation . Expression of the mutant protein did not diminish, even after several passages in culture (data not shown). Thus, unlike SKBR3 and the other ErbB2-positive breast cancer cells, the nontransformed, HBL-100 cells do not appear to depend upon signaling from Shc-Y317 for proliferation.



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Fig. 2. Expression of the Shc-Y317F-GST fusion protein in stable HBL-100 transfectants. Cells were transfected with pEBG-Shc-Y317F using Lipofectin and then cultured for 3 weeks under G418 selective pressure. Cell extracts of G418-resistant clones (named AMT through VMT) were immunoprecipitated using anti-Shc polyclonal antibodies cross-linked to protein A-Sepharose beads, and the proteins were resolved on an SDS 7.5% polyacrylamide gel, blotted, and probed with anti-Shc antibodies as detailed in "Materials and Methods." *, location of the Mr 80,000 Shc-GST fusion protein; arrows, location the endogenous Shc isoforms.

 
Cell Cycle Changes Caused by Shc-Y317F.
Efforts to explore the biological effects of the Shc dominant-negative mutant on the ErbB2 positive breast cancer cells were hampered by the paucity of cells obtainable from stable transfections (see Fig. 1ACitation ). To circumvent this problem, we chose to examine SKBR3 cells that were transiently expressing the mutant Shc protein. Accordingly, SKBR3 cells were transfected with either normal Shc (pEBG-ShcWT) or mutant Shc (pEBG-Shc-Y317F) and then cultured in nonselective media for up to 82 h. We then analyzed expression of the Shc-Y317F-GST fusion protein at various times. Expression of both the Mr 80,000 wild type and Shc-Y317F proteins were optimal {approx}60 h after transfection (Fig. 3)Citation .



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Fig. 3. Expression of Shc-GST fusion protein in transiently transfected SKBR3 cells. Cells were transfected with pEBG-Shc-Y317F (numbered lanes) or pEBG-ShcWT (WT lane) using Lipofectin, cultured up to 84 h, and then analyzed for expression of Shc proteins as in Fig. 2Citation .

 
Shc is known to be important in the signaling of several growth factor receptors to Ras (42, 43, 44, 45, 46, 47, 48, 49) . Because of the well-known involvement of Ras in passage through the G1 phase of the cell cycle (28, 29, 30, 31) , we hypothesized that the dominant-negative Shc was inhibiting colony formation by the SKBR3 cells and other ErbB2-positive breast cancer cells by blocking passage through G1. To test this possibility, we transiently cotransfected wild-type Shc or Shc-Y317F together with the transfection marker, pGreen LanternTM. Transfected cells (typically 10–20% transfection efficiency) were identified by expression of the green fluorescent protein, which could be detected as early as 24 h after transfection by fluorescence microscopy (data not shown) or flow cytometry (Fig. 4)Citation . By staining cellular DNA with propidium iodide and instructing the flow cytometer to score only green fluorescent protein-positive cells, we were able to determine the cell cycle distribution of cells that were presumably coexpressing the Shc-GST fusion proteins. Analysis of DNA histograms indicated that neither wild-type Shc nor the pEBG vector alone affected the proportion of SKBR3 cells in G0-G1, S, and G2-M (Fig. 4, A, B, and D)Citation . This suggested that expression of the GST-fusion protein did not disrupt cell cycle progression. In contrast, as predicted by the colony formation assay, Shc-Y317F did appear to inhibit cell cycle progression of the SKBR3 cells (Fig. 4C)Citation . To our surprise, however, the cells appeared to be partially blocked in G2-M (an increase from 12 to 24% of cells in G2-M). This suggested that Shc may have a critical but previously unrecognized role in G2-M progression.



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Fig. 4. Cell cycle analysis of SKBR3 cells expressing Shc-Y317F. A, DNA histogram of asynchronously growing, untransfected SKBR3 cells; B–E, DNA histograms of SKBR3 cells that had been transfected as above with both 1 µg of pGreen LanternTM and 10 µg of the indicated construct(s) and then harvested 60 h later, stained with propidium iodide, and analyzed by a FACScan® flow cytometer, as detailed in "Materials and Methods." F, DNA histogram of SKBR3 cells exposed for 24 h to 60 ng/ml Taxol, known to arrest cells in G2. Jagged curve, the actual number of cells per channel; smooth curve, the best fit calculated by MODFIT software (Becton Dickinson). By deconvolutional analysis, Modfit then calculated the percentage of cells in each phase of the cell (shaded, labeled areas).

 
However, Shc-Y317F conceivably could have effects on cellular pathways that are not normally regulated by Shc. In one approach to address this concern, cotransfecting wild-type Shc along with Shc-Y317F rescued the arrested phenotype; the cotransfected cells progressed normally through the cell cycle (Fig. 4E)Citation .

Although not dramatic, cell cycle analysis of the SKBR3 cells that were transiently expressing mutant Shc hinted that Shc is involved in G2-M passage. To address this more definitively, we transfected large numbers of SKBR3 cells (8 x 106) with Shc-Y317F and the neo selectable marker, and after 3 weeks of culture with G418, we enumerated the colonies. As we observed previously (Fig. 1)Citation , colonies from SKBR3 cells transfected with Shc-Y317F were greatly reduced in size (data not shown) and number (Fig. 5)Citation , compared with colonies from SKBR3 cells transfected with the pEBG empty vector. With this large-scale transfection, we were able to harvest sufficient numbers of the microcolonies to obtain a pool of 1 x 105 Shc-Y317F cells for cell cycle analysis (Fig. 6A–D)Citation . DNA histograms show clearly that the Shc-Y317F cells are blocked in both G1 and G2-M. SKBR3 cells transfected with the mutant Shc appear only in the G0-G1 (61%) and the G2-M (39%) phases of the cell cycle (Fig. 6D)Citation , whereas cells transfected with the empty vector are found in all three phases of the cell cycle (63% in G0-G1; 28% in S phase; and 9% in G2-M). The BrdU incorporation studies (Fig. 1C)Citation are quite consistent with the flow data. Thus, the cells transfected with the mutant Shc are not synthesizing DNA, and therefore their cell cycles are clearly arrested.



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Fig. 5. Oncogenic Ras 12V overcomes the ability of the Shc mutant to inhibit SKBR3 colony formation. Colony formation assays were performed as described in "Materials and Methods." Results are expressed as percentages of colonies formed by cells transfected with the empty vector. Bars, SD.

 


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Fig. 6. Cell cycle analysis of colonies obtained from large-scale transfections of SKBR3. SKBR3 cells were transfected with the indicated construct and then were cultured for 4 weeks under G418 selective pressure. Multiple G418-resistant colonies were harvested and pooled, and cells were stained with propidium iodide similarly to Fig. 4Citation for analysis by flow cytometry.

 
Shc Is Phosphorylated on Tyrosine in SKBR3 Cells Arrested in G2 by Taxol or Nocodazole.
If the SKBR3 cells require Shc for passage through G2-M, then we might expect it to be tyrosine phosphorylated in these later stages of the cell cycle. To test this prediction, we arrested SKBR3 in G2 using Taxol (55) or nocodazole (56) and then examined Shc for tyrosine phosphorylation. SKBR3 cells arrested in G2 by either taxol or nocodazole showed very high levels of p52-Shc tyrosine phosphorylation, even higher than observed in asynchronously proliferating SKBR3 cells (Fig. 7)Citation .



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Fig. 7. Shc tyrosine phosphorylation in SKBR3 cells arrested in G2 by Taxol or nocodazole. Cells were cultured in 10% FBS-supplemented culture media with 0.06 µg/ml Taxol (T) for 20 h, with 0.5 µg/ml nocodazole (N) for 14 h, or with no drug (-), and then analyzed for expression of proteins as in Fig. 2Citation , but first probing with the 4G10 monoclonal antibody to phosphotyrosine, then stripping and reprobing with antibody to Shc protein. A very light exposure of the anti-PY blot is shown so that the increment caused by Taxol and nocodazole can be appreciated.

 
Active Ras Rescues the Shc-Y317F-induced G1 and G2-M Arrest.
As mentioned above, considerable evidence indicates that Shc tyrosine 317 signals through Grb2-SOS to p21Ras, and that active Ras is necessary for passage through the G1 phase of the cell cycle. Accordingly, we predicted that the Shc dominant negative block of G1 could be bypassed if the cells contained active Ras. Thus, although we expected that SKBR3 cells cotransfected with Shc-Y317F and active Ras would still form only a few, small colonies as seen before with Shc-Y317F alone, we did expect, however, that cell cycle analysis would show these double transfectants to be arrested only in G2-M. We were surprised, therefore, when SKBR3 cells that were cotransfected with Shc-Y317F and Ras-G12V formed stable colonies that were equal in both size (data not shown) and number (Fig. 5)Citation to colonies formed by SKBR3 cells transfected with Ras-G12V alone. We did observe a 20% reduction in the total number of colonies formed by the Ras-G12V transfected cells compared with the pEBG vector controls. However, this may be an artifact of the pUC expression vector that contains Ras-G12V, inasmuch as the pUC empty vector controls produced 20% fewer colonies as well.

If rescue by Ras-G12V were truly bypassing the need for Shc, then we would expect that Shc-Y317F should still be expressed in the rescued cells. To test this, we analyzed lysates from the Shc-Y317F:Ras-G12V clones for the presence of Shc proteins. Anti-Shc immunoblotting demonstrated that the Mr 80,000 Shc TM-Y317F GST fusion protein was expressed in these cells, although at a lower level than the endogenous Shc proteins (Fig. 8)Citation .



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Fig. 8. Expression of the Shc dominant negative in SKBR3 cells cotransfected with Ras-12V. Cells were transfected by Lipofectin (6 µl/ml) and cultured in complete medium under G418 selective pressure for 4 weeks. Detergent lysates of cells were clarified, mixed with Laemmli sample buffer, and resolved on a SDS 7.5% polyacrylamide gel as described previously. Proteins were blotted and probed with antibody to Shc. The molecular weight of the Shc-GST fusion protein is Mr 80,000, as indicated by the *. Arrows on the left, endogenous Shc isoforms.

 
The surprising ability of Ras-G12V to overcome the Shc-Y317F block in colony formation suggested that the Shc mutant functioned upstream of Ras-G12V both in G0-G1 and in G2-M. Consistent with this notion, cell cycle analysis of colonies from the Ras-G12V-rescued SKBR3 Shc-Y317F transfectants revealed that Ras-G12V rescued not only the G0-G1 but also the G2 arrest (Fig. 6F)Citation .

We also observed an increase in dead cells in the "stable" transfectants (see particularly Fig. 6, C–FCitation , the shaded area of the graph between 0 and 10 on the abscissa) compared with the SKBR3 cells transiently expressing the various recombinant proteins (see Fig. 4Citation ). This may be because the stable transfectants had been fighting for survival for 6 weeks under G418 selective pressure. The dead cells do not appear related to the expression of only Shc proteins, because cells transfected with the oncogenic Ras showed similar levels of nonviable cells (Fig. 6E)Citation .


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Several breast cancer cell lines that overexpress ErbB2 contain constitutively tyrosine-phosphorylated Shc (35) . Here we have asked whether Shc signaling is crucial to the proliferation of these cell lines. Our approach has been to express in these cells a Shc dominant-negative mutant, Shc-Y317F, that interferes with Shc signaling to Ras. The mutant Shc blocked colony formation by the ErbB2-positive breast cancer cell lines, apparently by inhibiting signaling to Ras and thereby inhibiting cell cycle progression through both G1 and, surprisingly, G2-M.

Shc-Y317F: a Highly Dominant-Negative Mutant in ErbB2-overexpressing Breast Cancer Cells.
It is perhaps surprising that no colonies expressing only G418 resistance emerged from the neo/Shc-Y317F transfectants. Although the Shc:neo DNA ratio was high (5:1), we might have expected that some cells would have stabily incorporated and expressed only the G418 resistance gene. These data suggest that the Shc mutant is acting in a highly dominant manner and is not merely competing with endogenous normal Shc for SH2/PTB binding sites. This dominance is unlikely to be an artifact of the GST-fusion protein, because not only did expression of GST-WT-Shc not adversely affect SKBR3 proliferation, but it also rescued SKBR3 cells that were cotransfected with the mutant Shc. Consistent with our results, Baldari et al. (57) reported that a Shc dominant-negative construct, the Shc-SH2 domain, potently inhibited CD4-mediated activation of NF-AT in Jurkat cells (57) . The mechanism of this highly dominant behavior might be better understood if one could compare: (a) the cellular molecules with which Shc-Y317F and normal Shc associate; and (b) the subcellular distributions of the normal and mutant Shc proteins.

Unlike SKBR3 cells, proliferation of the nontransformed breast epithelial cell line HBL-100 was clearly unaffected by the presence of SHC-Y317F (Figs 1Citation and 2Citation ). Shc-Y317F transfectants of HBL-100 cells formed colonies equal in size and number to HBL-100 cells that had been transfected with just the empty vector (Fig. 1)Citation . This suggests either that HBL-100 cells do not require Shc signaling, or that they are otherwise much less sensitive to interference by the mutant Shc. Inasmuch as autocrine fibroblast growth factor-2 reportedly drives HBL-100 proliferation (58) , fibroblast growth factor receptor signaling may be less dependent than ErbB2 signaling on Shc (59, 60, 61, 62, 63) . Alternatively, the SKBR3 and HBL-100 cells may differ in other signaling components or in regulators of the cell cycle.

Shc Is the Major Ras-activating Pathway in ErbB2-overexpressing Breast Cancer Cells.
Although Ras activity can be regulated by many mechanisms, often Ras appears to be activated when Grb2-SOS binds to tyrosine-phosphorylated growth factor receptors and is thereby translocated to the cellular membrane, where it gains access to Ras (32, 33, 34) . Grb2-SOS can bind to growth factor receptors either directly by docking to receptor phosphotyrosines, or indirectly by docking to the tyrosine-phosphorylated 317 residue of Shc (19 , 32, 33, 34 , 41) . On the basis of the ability of Shc-Y317F to inhibit proliferation of ErbB2-overexpressing breast cancer cells and on the ability of Ras to rescue the SKBR3 cells from Shc-Y317F-induced growth arrest, we conclude that the indirect pathway through Shc to Ras is dominant and required for growth of these cells (rigorous proof of this will require a molecular analysis of Shc, its associated proteins, and Ras activity in cells expressing Shc-Y317F; such analyses will require substantial numbers of cells expressing the mutant Shc, most readily accomplished by engineering the mutant Shc gene to be under the control of an inducible promoter). This is clearly not the case for all cells. Shc-Y317F does not inhibit the growth, as shown here, of the nontransformed breast epithelial cell line HBL-100 (Fig. 1Citation and Table 1Citation ). Furthermore, Shc-Y317F does not inhibit the growth of NIH3T3 cells (43 , 51) or Rat-1 fibroblasts (53) in serum-supplemented media. However, Shc-Y317F does inhibit EGF-induced DNA synthesis (43) and serum-free growth and transformation of NIH3T3 cells (51) . Another dominant-negative Shc construct consisting of only the Shc SH2 domain effectively inhibited EGF responses in NIH3T3 cells (45) , in 293T human kidney epithelial cells (64) , and in rat fibroblasts (46) . Similarly, the Shc SH2 domain inhibited PDGF-induced DNA synthesis in NIH3T3 cells (48) . Further consistent with a prominent role for Shc in EGF responses, a phosphotyrosyl-peptide that can bind to the Shc SH2 domain inhibits EGF-driven Ras activity in permeabilized PC12 cells (42) .

However, interpretation of the inhibitor studies that used Shc PTB and SH2 domains or the SH2-binding site peptide has recently been complicated by the discovery of several additional Shc sites involved in molecular interactions. These include two additional tyrosine phosphorylation sites on Shc, at the vicinal residues 239 and 240 located in the Shc CH1 domain (43 , 44 , 54 , 65, 66, 67) . These sites may act in concert with tyrosine 317 in binding Grb2, but in addition to binding several as yet unidentified cellular proteins (44 , 67 , 68) , appear to drive Ras-independent activation of Myc in NIH-3T3 cells responding to EGF and in BaF3 mouse myeloid cells responding to IL-3 (43 , 54) . Thus, a dominant-negative Shc construct consisting of the Shc SH2 domain (or of the Shc PTB domain) might be not only a more potent inhibitor of Ras activation than Shc-Y317F but also an inhibitor of Ras-independent pathways as well. In contrast, we have no reason to suspect that Shc-Y317F interferes with ShcY239,240 signaling in breast cancer cells:

(a) In BaF3 cells Shc-Y317F interfered with IL-3 signaling through Shc to Ras but did not interfere with IL-3 survival signals through Shc(Y239,240) to Myc (54) . Similarly, the Shc-Y317F mutant did not interfere with Shc signaling through Shc(Y239,240) to Myc in NIH-3T3 cells responding to EGF (43) .

(b) We were able to rescue the SKBR3 Shc-Y317F cells with oncogenic Ras, and therefore do not need to invoke effects of Shc-Y317F on Ras-independent pathways to explain the effects of the mutant on the growth of the ErbB2-positive breast cancer cells.

Shc, Ras, and the Cell Cycle.
To the extent that Ras is activated via Shc-Y317, we expected the Shc-Y317F mutant to inhibit Ras activation. Active Ras is required for transit through two early phases of the cell cycle: G0 to G1 (28) and mid/late-G1 to S (29, 30, 31) , where Ras induces cyclin D1 and down-regulates the cyclin-dependent kinase inhibitor, p27KIP1 (31) . Therefore we predicted that SKBR3 cells transfected with the Shc-Y317F mutant would, if affected at all, arrest in G0-G1. Indeed, 60% of the Y317F SKBR3 cells did arrest in G0-G1, and this arrest could be rescued by oncogenic Ras, as expected (Fig. 6)Citation .

Unexpectedly, however, nearly 40% of the cells were blocked in G2-M, and this arrest too could be rescued by oncogenic Ras, implying that in SKBR3 cells (and by extension, in the other ErbB2-overexpressing breast cancer cells), Shc signaling to Ras is required for G2-M passage. (However, a caveat to the rescue experiments is that oncogenic Ras (or WT-Shc) could be affecting parallel pathways impacting cell growth; or coexpression of oncogenic Ras could be partially suppressing expression of the mutant Shc protein. The latter would be consistent with the low level of mutant Shc expression relative to endogenous Shc in the Ras-rescued cells. Alternatively, the mutant Shc may be a potent dominant negative. As discussed above, these sorts of questions could be addressed if the genes were engineered to be under control of inducible promoters.)

The differences in cell cycle distribution of SKBR3 cells that were transiently expressing (Fig. 4)Citation and stabily expressing (Fig. 6)Citation the Shc mutant are somewhat puzzling. For example, transiently expressing cells had the normal number of cells in S phase but fewer in G0-G1 and more in G2-M. In contrast, stabily expressing cells had no measurable cells in S phase, more cells in G2-M, but normal numbers of cells in G0-G1. A likely explanation derives from the observations that SKBR3 cell cycle time is long, {approx}40 h, and in the transiently expressing SKBR3 cells, the mutant Shc protein does not reach maximum expression until somewhere between 24–60 h after transfection. Because the transiently expressing cells were harvested for analysis 60 h after transfection, they would have experienced the effects of the mutant Shc for less than one full cell cycle. Thus, more pronounced changes would be observed in the number of cells in the rapid phases of the cell cycle (about 4 h for G2-M in SKBR3) than in the longer phases of the cell cycle (about 24 h for G0-G1 and about 12 h for S phase in SKBR3 cells). Additionally, it is possible that G2-M is more sensitive than G1 to low levels of the mutant Shc.

However, if Shc is activating Ras in G2 through Shc-Grb2-SOS complexes, we would expect to find Shc tyrosine phosphorylated in G2. Consistent with this prediction, SKBR3 cells arrested in G2 by either the microtubule-stabilizing drug, Taxol, or by the microtubule-disrupting agent, nocodazole, contained very high levels of tyrosine-phosphorylated Shc (Fig. 7)Citation . To date, only a few scattered reports have suggested that Ras activity is required in G2. G2-M passage in A549 lung adenocarcinoma cells is blocked by a farnesyl transferase inhibitor, FTI-277, which prevents Ras from localizing properly to the cell membrane (69) . However, one cannot rule out effects of the inhibitor on other farnesylated proteins, such as the nuclear lamins. More definitively, in elegant experiments using a temperature-sensitive Ki-Ras, normal rat kidney cells grown in the absence of serum were dependent upon Ras being active during G2 for progression through G2 (29) . Suggesting one mechanism for the Ras G2 requirement, active Ras and Myc cooperate in primary rat embryo fibroblasts to induce cdc2 (70) , a kinase that complexes with cyclins A and B and is well known to be required for G2 progression (71 , 72) .

A second potential link between Shc and passage from G2 to M is suggested by recent findings that chicken embryo fibroblasts that had been transformed with v-erb-B contained complexes of tyrosine-phosphorylated Shc and caldesmon colocalized in the cells to areas of actin stress fiber disassembly (73) . Stress fiber disassembly is necessary to permit the rounding-up of cells prior to mitosis and is required for normal passage of adherent cells through the G2-M boundary (74 , 75) .

A role for Shc in G2-M raises several questions. What kinase is activating Shc during G2-M? Several lines of evidence suggest Src family proteins as candidate kinases of Shc in G2. Src family members, Src, Yes, and Fyn, are all activated in NIH3T3 cells responding to PDGF and are required for PDGF-driven passage through G2-M (48) . Furthermore, Shc is tyrosine phosphorylated in cells transformed by oncogenic v-src and in vitro kinase assays have also shown that Shc can act as a substrate of Src family members (19 , 76) . Particularly relevant here, Src family tyrosine kinases are activated in breast cancers and breast cancer cell lines, including SKBR3 (77, 78, 79) .

Alternatively, ErbB2 or other growth factor receptors could be responsible for the G2 Shc tyrosine phosphorylation in SKBR3 cells and the other ErbB2-overexpressing breast cells. These breast cancer cells display various fibroblast growth factor receptors, as well as receptors for insulin, insulin-like growth factor-1, EGF, heregulins, Cripto-1, hepatocyte growth factor, mammary-derived growth factor, and others (8 , 80, 81, 82) . In this regard, a tyrosine kinase inhibitor, tyrphostin AG879, reportedly has high specificity for ErbB2 relative to the EGF, PDGF, and nerve growth factor receptors (83) . AG879 markedly inhibits both ErbB2 autophosphorylation and Shc tyrosine phosphorylation in asynchronously cycling SKBR3 cells (35) , suggesting that at least in these cells, ErbB2 is the primary driver of Shc activity. However, it is not known to what extent Shc is phosphorylated directly by ErbB2 or indirectly by ErbB2 activating other cellular kinases such as Src.

In conclusion, our results imply that signaling from Shc tyrosine 317 to Ras is necessary for transit of both the G1 and G2-M phases of the cell cycle in several ErbB2-positive cell lines. This has important implications for growth control in breast cancer cells and suggests Shc-Y317 as a potential therapeutic target in human breast cancer.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Plasmids.
The Shc-Y317F dominant-negative mutant was prepared by PCR-directed mutagenesis to substitute phenylalanine for tyrosine at amino acid 317 (84) . The mutant construct was then subcloned into the plasmid, pEBG, which contains a mammalian promoter EF1{alpha} for expression of GST fusion proteins (84 , 85) . The resulting plasmid, pEBG-Shc-Y317F, produces a Mr 80,000 GST-Shc fusion protein. pNeoNut (a kind gift from A. Rosmarin, Brown University, Providence, RI) was cotransfected at a 1:5 ratio with pEBG-Shc-Y317F, pEBG, and/or pUC-Ha-Ras-G12V (Ref. 86 ; kindly provided by C. Der, University of North Carolina, Chapel Hill, NC) to confer resistance to G418 (500 µg/ml); Life Technologies, Inc., Gaithersburg, MD). For transient transfections, pGreen Lantern (Life Technologies), which expresses the green fluorescent protein, was used as a reporter gene at a 1:10 ratio with pEBG-Wild Type Shc, pEBG-Shc-Y317F, or the pEBG vector alone. The pUC-Ha-Ras-12V was transfected at a ratio of 1:1 with the pEBG-Shc-Y317F.

Cell Culture and Transfection.
The human breast cell lines BT-474, MDA-MB-361, MDA-MB-453, SKBR3, and HBL-100 (American Type Culture Collection, Rockville, MD) were cultured in Iscove’s modified Dulbecco’s media supplemented with low endotoxin 10% FCS, penicillin and streptomycin (1000 U/ml each), and 20 mM glutamine. Transfections were carried out in the presence of Lipofectin (6 µl/ml; Life Technologies, Inc.) for 12 h. Cells were then cultured with serum supplemented media with G418 (500 µg/ml) for selection of stable clones. After 3 weeks, positive clones were harvested by isolating visible colonies with cloning circles (Belco, Vineland, NJ) and trypsinization. Transient transfections were treated similarly but were cultured for 60 h after transfection in serum-supplemented medium.

Colony Formation Assay.
Transfections were carried out in duplicate as mentioned above using equal amounts of DNA for each 100-mm tissue culture plate. SKBR3 cells (1 x 106) were transfected by Lipofectin with the following plasmid combinations: pEBG Wild Type Shc, pEBG, pEBG-Shc-Y317F, pEBG-Shc-Y317F and pUC-Ha-Ras-G12V, and pUC-Ha-Ras-G12V alone. Two days after transfection, cells were split 1:4 and cultured for 3 weeks in complete medium supplemented with G418 (500 µg/ml). Resistant colonies were counted and harvested for cell cycle analysis. Data were obtained from at least two independent transfection assays performed in triplicate.

Antibodies and Reagents.
Antibodies used in this study were: anti-Shc polyclonal antibody for immunoblotting and immunoprecipitation (Upstate Biotechnology, Lake Placid, NY), the 4G10 phosphotyrosine monoclonal antibody for immunoblotting (Upstate Biotechnology), the anti-GST polyclonal antibody for immunoprecipitation (Santa Cruz Biotechnology, Santa Cruz, CA), and monoclonal antibody to BrdU (Becton Dickinson, San Jose, CA) and goat anti-mouse ALEXA-conjugated antibody (Molecular Probes, Eugene, OR) for the fluorescence cell-based assay of DNA synthesis. Taxol (Bristol Myers-Squibb, Princeton, NJ) and nocodazole (Sigma, St. Louis, MO) were used to arrest cells in G2.

Immunoprecipitation and Immunoblotting.
Cells (1 x 107) were extracted at 4°C in 10 ml of cold extraction buffer consisting of 1% v/v Triton X-100, 10 mM Tris Base, 5 mM EDTA, 50 mM NaCl, 30 mM Na4P2O7, 50 mM NaF, and 100 µM sodium vanadate (pH 7.6), containing freshly added protease inhibitor, 1 mM phenylmethyl sulfonyl fluoride (Sigma). The extracts were clarified, and their protein content was determined by the Lowry method (DC protein quantitation; Bio-Rad, Hercules, CA). Clarified extracts (600 µg of protein) were then incubated for 8 h at 4°C with specific antibodies that had been previously cross-linked by dimethyl pimelimidate to protein A-Sepharose (Pharmacia). Immune complexes were washed three times in cold extraction buffer and eluted by boiling in nonreducing Laemmli buffer. Eluted proteins were then boiled with 2-mercaptoethanol and resolved by SDS-PAGE. The resolved proteins were transferred to nitrocellulose membranes (Hybond, Amersham, Arlington Heights, IL), probed with specific antibodies, and detected by enhanced chemiluminescence (36) .

BrdU Incorporation.
SKBR3 cells (1 x 106) were transfected by Lipofectin with the pEBG and pEBG-Shc-Y317F plasmids, and subjected to G418 selection pressure for 3 weeks, as described above. Sixteen h before assay, the G418-resistant colonies were harvested, pooled, and seeded at 1 x 104 per well on an eight-chambered culture slide. Fourteen h after plating, cells were incubated with BrdU (10 µM; Becton Dickinson) for 2 h and then fixed in 70% cold ETOH for 30 min (87) . After allowing the slides to air dry, the cells were treated with 0.7 NaOH for 2 min, neutralized in cold 1x PBS, and probed with anti-BrdU (20 µl/chamber; Becton Dickinson) in 50 µl of 0.01 MTRIS, 0.14 M NaCl, 0.1% Tween-20, pH 7.6 (TBST) for 1 h at room temperature. This was followed by three washes, 2 min each, in TBST. Secondary antibodies (Goat-anti-mouse IgG, ALEXA conjugated, diluted 1:100) were added to slides for 30 min at room temperature. The cells were coverslipped in 10% glycerol mounting media, and positive cells were visualized with a fluorescence microscope (AO Ultrastar) using 450–495 nm bandpass excitation and 520 nm longpass emission filters, along with sufficient white light to just allow visualization of non-fluorescing cells.

Cell Cycle Analysis.
Asynchronous SKBR3 cells were transiently transfected with 10 µg of pEBG Wild Type Shc or 10 µg of pEBG-Shc-Y317F in the presence of pGreen Lantern (1 µg). The transfected cells were harvested 60 h after transfection by trypsinization, fixed in 70% cold ethanol for 30 min, washed in PBS, and then stained with 5 µg/ml propidium iodide, 0.1 mg/ml RNase A, 0.1% NP40, and 0.1% trisodium citrate for 30 min. Fluorescence was determined using a FACScan flow cytometer (Becton Dickinson, San Jose, CA); pGreen Lantern fluorescence in the FITC channel and propidium iodide fluorescence in its channel were compensated for overlap using cells stained with only one or the other dye. Data were gated to exclude unstained cells and cell debris and were also gated on the pGreen Lantern fluorescence to include in the analysis of propidium iodide fluorescence only those cells that were expressing the transgene. Data were analyzed by MODFIT software (Becton Dickinson). Cells that were treated with Taxol (60 ng/ml) were harvested 24 h after treatment.


    Acknowledgments
 
We thank Nicola Kouttab for technical advice and flow cytometry analysis, Alan Rosmarin for helpful discussions and supplying pNeoNut, John Sedivy for helpful discussions, and Channing Der for providing pUC-Ha-Ras-G12V.


    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 This work was made possible by generous support from the Department of Medicine, Roger Williams Medical Center. L. E. S. was supported in part by NIH Training Grant T32ES07272-05. Back

2 To whom requests for reprints should be addressed, at Department of Medicine, Roger Williams Medical Center, 825 Chalkstone Avenue, Providence, RI 02908. Phone: (401) 456-2320; Fax: (401) 456-2016; E-mail: A_Frackelton_Jr{at}brown.edu Back

3 The abbreviations used are: EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PTB, phosphotyrosine binding; RasGAP, Ras GTPase activating protein; GST, glutathione S-transferase; IL, interleukin. Back

Received for publication 12/ 2/97. Revision received 11/ 3/98. Accepted for publication 11/19/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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