Targeting disease-causing proteins for ubiquitination and degradation by chimeric molecules represents a promising alternative therapeutic strategy in cancer. Here, several Cbl-based chimeric ubiquitin ligases were recombined to achieve effective down-regulation of HER2. These chimeric molecules consisted of the Cbl NH2-terminal tyrosine kinase binding domain, linker, and RING domain, with the Src homology 2 domain replaced with that from growth factor receptor binding protein 2 (Grb2), Grb7, p85, or Src. The chimeric proteins not only interacted with HER2 but also enhanced the down-regulation of endogenous overexpressed HER2. After the chimeric proteins were introduced into HER2-overexpressing breast cancer SK-BR-3 cells or ovarian cancer SK-OV-3 cells, they effectively promoted HER2 ubiquitination and degradation in a RING finger domain–dependent manner. Consequently, expression of these chimeric molecules led to an inhibition of colony formation, increased the proportion of cells in the G1 cycle, and suppressed tumorigenicity. Collectively, our findings suggest that the Cbl-based chimeric ubiquitin ligases designed in the present study may represent a novel approach for the targeted therapy of HER2-overexpressing cancers. [Cancer Res 2007;67(18):8716–24]

Controlled degradation of activated receptor tyrosine kinases (RTK) represents a primary mechanism by which cells negatively regulate receptor signaling and maintain normal cellular homeostasis (1). Ubiquitination plays a crucial role in this process. Increasing evidences suggest that Cbl, a 120-kDa protein product of the proto-oncogene c-Cbl, can bind to activated RTKs and promote their ubiquitination, endocytosis, and subsequent lysosomal degradation (29). Cbl is composed of a conserved tyrosine kinase binding (TKB) domain and a RING finger domain in its NH2-terminal region, as well as other protein-protein interaction motifs at its COOH-terminal half (4, 9). The TKB domain, consisting of a four-helix bundle (4H), a calcium-binding EF hand (EF), and a Src homology 2 (SH2) domain, is responsible for recognizing and interacting with phosphorylated tyrosines on RTKs or non-RTKs. The RING finger domain can recruit the ubiquitin-conjugating enzyme E2 and transfer ubiquitin onto the substrates bound to Cbl (911). Cbl, therefore, belongs to one of ubiquitin ligase E3 family and functions as a negative regulator for many RTKs (4, 9, 11).

Consistent with its role in RTKs signaling, Cbl-deficient mice exhibit hyperplastic changes in mammary ducts and lymphoid tissues (12). Cbl can also act as an oncoprotein when its ability to down-regulate RTKs is disrupted (13). On the other hand, some oncogenic proteins, such as mutant RTKs, can escape from Cbl-mediated negative regulation (3). Taken together, these findings highly suggest that restoration of Cbl ubiquitin ligase activity may help target oncoprotein degradation and therefore inhibit the associated tumor growth.

The epidermal growth factor receptor (EGFR) family of RTK, which includes the EGFR (EGFR/HER-1 or ERBB-1), HER-2 (Neu or ERBB-2), HER-3 (ERBB-3), and HER-4 (ERBB-4), has been found to be hyperactivated and to contribute to the growth and progression of numerous tumor types (1420). Importantly, overexpression of HER-2 often correlates with poor prognosis in multiple malignancies, including breast and ovarian cancer. Such findings make these family members as attractive therapeutic targets. Many approaches for EGFR and HER2 inhibition, such as anti–receptor monoclonal antibodies (mAb) against the extracellular domain of the receptors and tyrosine kinase inhibitors, are already in clinical use or preclinical trials and have shown promising results (1923). However, their efficacy and long-term use in patients are quite limited because of resistance to these inhibitors or other severe side effects (22, 23). Therefore, novel therapeutic strategies in this field are still required.

Targeting diseases-causing proteins to the ubiquitin-mediated proteolysis pathway may be an alternative therapeutic strategy (24, 25). In this study, we created several HER2-targeting, Cbl-based chimeric ubiquitin ligases composed of the Cbl NH2-terminal TKB backbone (4H and EF), linker, RING domain, and SH2 domains from growth factor receptor binding protein 2 (Grb2), Grb7, phosphatidylinositol 3-kinase (PI3K) subunit p85 or Src, which were immediate downstream adaptors in EGFR and/or HER2 signaling pathway and capable of associating with and transmitting signals from the activated receptors (2631). Our results indicate that these chimeric constructs can effectively promote the ubiquitination and degradation of HER2, and consequently inhibit colony formation and tumorigenicity of breast cancer SK-BR-3 and ovarian cancer SK-OV-3 cells that overexpress HER2.

Plasmid construction. DNA fragments encoding the NH2-terminal half of Cbl (CblN, Cbl 1-440) were amplified by PCR from the pEFHACbl plasmid (a kind gift from Y.C. Liu, La Jolla Institute for Allergy and Immunology, La Jolla, CA) and cloned into the KpnI/XhoI sites of the pcDNA3.1(+) vector. BamHI and EcoRV restriction sites were introduced into SH2 flanks by site-directed mutagenesis using PfuUltra High-Fidelity DNA Polymerase (Stratagene). The genes encoding SH2 of Grb2, Grb7, p85 (C-SH2), and Src corresponding to the sequence of amino acids of Grb2 45 to 164, Grb7 428 to 532, p85 614 to 724, or Src 144 to 254 were amplified by PCR from cDNA derived from human breast cancer SK-BR-3 cells. The Flag-tagged expression constructs were obtained by replacing the SH2-encoding sequence of CblN with the corresponding amplified SH2 fragments. CblNWA-SH2Grb2 was obtained by site-directed mutagenesis. Cbl4H-EF-SH2Grb2 was subcloned into the KpnI/XhoI sites of the pcDNA3.1 (+) vector through changing the restriction site. Flag-tagged wild-type CHIP plasmids were constructed by amplifying DNA fragments of CHIP from pcDNA3.0-CHIP, provided by W.P. Xu (National Cancer Institute, Rockville, MD), and inserting the products into the EcoRI/XhoI sites of pcDNA3.1(+). Three copies of ubiquitin tagged with hemagglutinin (HA) in the pcDNA3.1(+) vector [pcDNA3.1(+)-3 × HA-Ub] were gifts from David Dornan (Genentech, Inc., South San Francisco, CA). The pGEX4T-2 bacterial expression constructs encoding the CblN-SH2Grb2, Cbl4H-EF-SH2Grb2, CblNWA-SH2Grb2, and CHIP proteins fused to the carboxyl terminus of glutathione S-transferase (GST) were generated by amplifying the corresponding DNA fragments from their expression plasmids by PCR and cloning them into the vectors. All construct sequences were verified by DNA sequencing.

Cell culture and transfection. Breast cancer SK-BR-3 and ovarian cancer SK-OV-3 cell lines were maintained in DMEM (Life Technologies) supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO2 humidified air. Cells were transfected with LipofectAMINE2000 (Invitrogen), according to the manufacturer's protocol. To obtain stably transfected clones, cells were selected with 400 μg/mL G418 (Life Technologies) for 3 to 4 weeks and single clones were isolated.

Immunoprecipitation and Western blotting. Cells were lysed in a lysis buffer as described elsewhere (32). For in vivo ubiquitination assay of HER2, the transfected cells were treated with MG-132 for 4 h before harvesting. The protein concentration was quantified using a BCA kit (Bradford). For immunoprecipitation, cell lysates containing 1 to 1.5 mg total proteins were incubated with anti-Flag M2 (Sigma) or anti-ErbB2 (Ab-11; Neomarkers) antibody for 4 h at 4°C, followed by protein A (or protein G) Sepharose beads (Pierce) for 2 h at 4°C. The precipitates were resolved by 7.5% to 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Life Science). For Western blotting, cell extracts containing 30 μg total protein were directly subjected to SDS-PAGE and transferred. The membranes were blocked and probed with primary antibodies that recognize Flag (Sigma), HER2 (Ab-1), EGFR (Ab-12; Neomarkers), Hsp90 (SPA-835; StressGen Biotechnologies), HA, or β-actin (Neomarkers). Secondary antibodies were chosen according to the species of origin of the primary antibodies and detected by enhanced chemiluminescence (Pierce) or by using the Odyssey Imaging System (Li-Cor Biosciences). The band intensity of HER2 was quantified by densitometry and normalized to β-actin using Kodak one-dimensional image analysis software.

GST pull-down assay. One-milligram aliquots of SK-BR-3 cell lysates were incubated with the purified GST, GST-CblN-SH2Grb2, GST-Cbl4H-EF-SH2Grb2, GST-CblNWA-SH2Grb2, or GST-CHIP fusion protein immobilized on glutathione-Sepharose beads (Amersham Biosciences) at 4°C for 3 h. The beads were washed five times with cold lysis buffer, and the bound proteins were detected by Western blotting with the anti-HER2 antibody.

Establishment of chimeric ubiquitin ligase inducibly expressing system in SK-BR-3 cells. To establish a stable ecdysone-inducible system, Flag-tagged CblN-SH2Grb2 (or CHIP) fragments were inserted into the KpnI/XhoI (or EcoRI/XhoI) sites of the pIND vector (Invitrogen). The SK-BR-3 cell lines inducibly expressing both the heterodimeric ecdysone receptor VgEcR and CblN-SH2Grb2 (or CHIP) were obtained by cotransfecting the cells with the pVgRXR vector and one of the pIND constructs, followed by dual selection with Zeocin (400 μg/mL; Invitrogen) and G418 (400 μg/mL). An ecdysone analogue ponasterone A (Invitrogen) was used to induce expression of CblN-SH2Grb2 or CHIP, which were under control of the ecdysone-inducible promoter.

PCR amplification analysis. Genomic DNA and total RNA were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Two micrograms of RNA were subjected to reverse transcription. The PCR primers used were as follows: for CblN-SH2Grb2, 5′-GGCGGATCCGACGGCTTCATTCCCAAGAAC-3′ (forward), 5′-GGCCTCGAGCTAGCCACTCCCTCTAGGATCAAA CGG-3′ (reverse), creating PCR products of 627 bp; For CHIP, 5′-GAATTCCCATGAAGGGCAAGGAGGAGAAGG-3′ (forward), 5′-CTCGAGCTAGTAGTCCTCCACCCAGCCATTCT-3′ (reverse), creating products of 981 bp; for HER2, 5′-CTGTTTGCCGTGCCACCCTGAGT-3′ (forward), 5′-CTTCTGCTGCCGTCGCTTGATGAG-3′ (reverse), creating products of 366 bp; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCCTCAAGATCAGCAAT-3′ (forward), 5′-AGGTCCACCACTGACACGTT-3′ (reverse), creating products of 310 bp. The PCR products were analyzed by agarose gel electrophoresis.

Colony formation assays, cell cycle analysis, and apoptosis assays. For colony formation assays, cells were seeded at 100 per 35-mm dish (or 200 per 60-mm dish) in triplicate and cultured for 14 days. Cell colonies were fixed, stained with 0.25% crystal violet in 50% ethanol for 20 to 30 min, air dried, and then counted, where a region containing >50 cells was considered one colony. The number of colonies was directly reported, or the colony formation ratio was calculated according to the following formula: colony formation ratio (%) = (colony number / seeded cells number) × 100%. For cell cycle analysis and apoptosis assay, cells were prepared and analyzed using a FACScan apparatus (Becton Dickinson) as described elsewhere (33). Early apoptotic cells were defined as Annexin V–positive, propidium iodide–negative cells.

Proliferation assays. Twenty-four hours after transfection, cells were seeded at 3,000 per well, in sextuple, in 96-well tissue culture plates in DMEM supplemented with 1% FCS and allowed to adhere to the plate. Then, the cells were daily treated with 500 nmol/L 17-allylamino-geldanamycin (17-AAG; A.G. Scientific, Inc.) or equivalent amount of DMSO for 3 to 5 days. Proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.

In vivo tumorigenicity assays. Four- to six-week-old athymic mice (three animals per group) were injected s.c. in the right limb with 1 × 107 cells. Tumor growth was monitored by measuring tumor size using Vernier calipers every week for a 5-week period and calculating tumor volume using a standard formula: tumor volume (mm3) = width (mm)2 × length (mm) × 0.5. At the end of the experiment, tumor weight was assessed by sacrificing the mice, and by removing and weighing the tumor.

Statistical analysis. Data are expressed as mean ± SD. Statistical analysis was done with the SPSS10.0 software package for Windows by using Student's t test for independent groups. Statistical significance was based on a value of P ≤ 0.05.

Generation of Cbl-based chimeric ubiquitin ligases. Construction of the Cbl-based chimeric ubiquitin ligases are shown in Fig. 1A. The NH2 terminus of c-Cbl (CblN, amino acids 1-440) confers and is sufficient for the functional E3 ubiquitin ligase activity (11, 34). The chimeric constructs were created by replacing the SH2 domain of CblN with various SH2 domains from Grb2, Grb7, p85, or Src. Cbl4H-EF-SH2Grb2 and CblNWA-SH2Grb2, which lack functional RING finger domain due to an entire deletion or a key point mutation W408A of the RING finger (11), served as negative controls. Flag-tagged CHIP (carboxyl terminus of Hsc70-interacting protein) is used as a positive control, which belongs to the U-box protein of E3 family and is capable of interacting with and mediating the degradation of HER2 (32, 35).

Figure 1.

Generation of Cbl-based chimeric ubiquitin ligases. A, schematic representation of Cbl-based chimeric ubiquitin ligases and CHIP. The indicated length of the NH2-terminal region of c-Cbl (CblN) contains TKB (4H, EF, and SH2), linker (L), and RING finger domains, with the SH2 domain being replaced by SH2 from Grb2, Grb7, p85, Src, or left intact. All constructs were tagged with Flag. B, interaction between chimeric ubiquitin ligases and HER2. SK-BR-3 cells were transfected as indicated and treated with 25 μmol/L MG-132 for 4 h before lysis. Lysates containing 1 mg total protein were immunoprecipitated (IP) with an anti-Flag antibody. HER2 or Hsp90 associated with CblN, CblN-XSH2, GST-Cbl4H-EF-SH2Grb2, GST-CblNWA-SH2Grb2, or CHIP was analyzed by Western blotting (IB) with an anti-HER2 (top) or anti-Hsp90 (bottom) antibody. The membrane was reprobed with an anti-Flag antibody (middle). CblN-XSH2 represents CblN-SH2Grb2, CblN-SH2Grb7, CblN-SH2p85, and CblN-SH2Src. C, interaction between GST-fused chimeric proteins and HER2. Purified GST, GST-CblN-SH2Grb2, GST-Cbl4H-EF-SH2Grb2, GST-CblNWA-SH2Grb2, or GST-CHIP immobilized on glutathione-Sepharose 4B beads was incubated with SK-BR-3 cell lysates that contained 1 mg total protein. Bound proteins were analyzed by Western blotting using anti-HER2 antibody. Left, a Coomassie blue–stained 10% polyacrylamide gel containing 1/3 input of the GST fusion proteins used in the experiment.

Figure 1.

Generation of Cbl-based chimeric ubiquitin ligases. A, schematic representation of Cbl-based chimeric ubiquitin ligases and CHIP. The indicated length of the NH2-terminal region of c-Cbl (CblN) contains TKB (4H, EF, and SH2), linker (L), and RING finger domains, with the SH2 domain being replaced by SH2 from Grb2, Grb7, p85, Src, or left intact. All constructs were tagged with Flag. B, interaction between chimeric ubiquitin ligases and HER2. SK-BR-3 cells were transfected as indicated and treated with 25 μmol/L MG-132 for 4 h before lysis. Lysates containing 1 mg total protein were immunoprecipitated (IP) with an anti-Flag antibody. HER2 or Hsp90 associated with CblN, CblN-XSH2, GST-Cbl4H-EF-SH2Grb2, GST-CblNWA-SH2Grb2, or CHIP was analyzed by Western blotting (IB) with an anti-HER2 (top) or anti-Hsp90 (bottom) antibody. The membrane was reprobed with an anti-Flag antibody (middle). CblN-XSH2 represents CblN-SH2Grb2, CblN-SH2Grb7, CblN-SH2p85, and CblN-SH2Src. C, interaction between GST-fused chimeric proteins and HER2. Purified GST, GST-CblN-SH2Grb2, GST-Cbl4H-EF-SH2Grb2, GST-CblNWA-SH2Grb2, or GST-CHIP immobilized on glutathione-Sepharose 4B beads was incubated with SK-BR-3 cell lysates that contained 1 mg total protein. Bound proteins were analyzed by Western blotting using anti-HER2 antibody. Left, a Coomassie blue–stained 10% polyacrylamide gel containing 1/3 input of the GST fusion proteins used in the experiment.

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To test the interaction with HER2, all Flag-tagged chimeric constructs were transiently transfected into SK-BR-3 cells. As shown in Fig. 1B, Flag-tagged CblN-SH2Grb2, CblN-SH2Grb7, CblN-SH2p85, CblN-SH2Src, Cbl4H-EF-SH2Grb2, and CblNWA-SH2Grb2 were coimmunoprecipitated with endogenous HER2 as efficiently as CHIP-Flag, whereas Flag-CblN was not. Moreover, we found that Hsp90 was a component of the immunocomplex precipitated by CHIP-Flag, but not by other Flag-tagged constructs. These findings suggest that the Cbl-based chimeric molecules could bind to HER2 independently of chaperones. The interaction between these chimeric ubiquitin ligases and HER2 were further confirmed by a pull-down assay using GST-fused CblN-SH2Grb2, Cbl4H-EF-SH2Grb2, CblNWA-SH2Grb2, and CHIP (Fig. 1C).

Cbl-based chimeric ubiquitin ligases promote HER2 down-regulation through enhancing its ubiquitination and degradation. To assess the effect of these constructs on HER2, several SK-BR-3 cell lines that stably express chimeric molecules were chosen for the assay. We found that the stable clones expressing Flag-CblN-SH2Grb2, Flag-CblN-SH2Grb7, Flag-CblN-SH2p85, Flag-CblN-SH2Src, or CHIP-Flag exhibited a decrease in HER2 protein relative to the empty vector transfectants, whereas those expressing Flag-CblN did not show HER2 decrease (Fig. 2A,, left). Moreover, the data from stably transfected SK-OV-3 cells showed that the RING finger–defective forms of CblN-SH2Grb2, either Cbl4H-EF-SH2Grb2 or CblNWA-SH2Grb2, were incapable of down-regulating HER2 (Fig. 2A , right).

Figure 2.

Chimeric Cbl-based ubiquitin ligases promote the ubiquitination and degradation of HER2. A, SK-BR-3 or SK-OV-3 cells stably transfected with the indicated constructs were lysed and subjected to Western blotting with anti-HER2, anti–β-actin, and anti-Flag antibodies, respectively. B, total RNA was isolated from the cells, and the level of mRNA encoding GAPDH (top) and a partial HER2 sequence (bottom) were measured by RT-PCR. DNA marker: 2,000, 1,000, 750, 500, 250, and 100 bp. C, CblN-SH2Grb2 enhanced HER2 ubiquitination. SK-BR-3 cells were transfected with the indicated constructs together with pcDNA3.1(+)-3×HA-Ub and treated with 25 μmol/L MG-132 for 4 h before lysis. Anti-HER2 immunoprecipitates from 1.5 mg aliquots of lysates were resolved by 7.5% SDS-PAGE and analyzed by Western blotting with anti-HA antibody (top) or anti-HER2 antibody (middle). Cell lysates were subjected to Western blotting with an anti-Flag antibody (bottom). D, the stability of HER2 was analyzed using a cycloheximide (CHX) chase experiment. SK-BR-3 cells stably transfected with control vector or CblN-SH2Grb2 were treated with 50 μg/mL cycloheximide for 0, 4, 8, and 16 h, and cell lysates were subjected to Western blotting with an anti-HER2 antibody. Band intensity was quantified by using Kodak one-dimensional image analysis software and expressed as percentage of the corresponding value from time point 0 h.

Figure 2.

Chimeric Cbl-based ubiquitin ligases promote the ubiquitination and degradation of HER2. A, SK-BR-3 or SK-OV-3 cells stably transfected with the indicated constructs were lysed and subjected to Western blotting with anti-HER2, anti–β-actin, and anti-Flag antibodies, respectively. B, total RNA was isolated from the cells, and the level of mRNA encoding GAPDH (top) and a partial HER2 sequence (bottom) were measured by RT-PCR. DNA marker: 2,000, 1,000, 750, 500, 250, and 100 bp. C, CblN-SH2Grb2 enhanced HER2 ubiquitination. SK-BR-3 cells were transfected with the indicated constructs together with pcDNA3.1(+)-3×HA-Ub and treated with 25 μmol/L MG-132 for 4 h before lysis. Anti-HER2 immunoprecipitates from 1.5 mg aliquots of lysates were resolved by 7.5% SDS-PAGE and analyzed by Western blotting with anti-HA antibody (top) or anti-HER2 antibody (middle). Cell lysates were subjected to Western blotting with an anti-Flag antibody (bottom). D, the stability of HER2 was analyzed using a cycloheximide (CHX) chase experiment. SK-BR-3 cells stably transfected with control vector or CblN-SH2Grb2 were treated with 50 μg/mL cycloheximide for 0, 4, 8, and 16 h, and cell lysates were subjected to Western blotting with an anti-HER2 antibody. Band intensity was quantified by using Kodak one-dimensional image analysis software and expressed as percentage of the corresponding value from time point 0 h.

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HER2 mRNA levels, as analyzed by semiquantitative reverse transcription-PCR (RT-PCR), were not significantly altered (Fig. 2B), suggesting that HER2 down-regulation occurred at a posttranscriptional level. The results of in vivo ubiquitination assay showed that overexpression of CblN-SH2Grb2 or CHIP was associated with an increase in the ubiquitination of HER2 relative to control cells. However, the deletion or point mutation W408A of the RING finger domain disrupted the ability of CblN-SH2Grb2 to enhance HER2 ubiquitination (Fig. 2C). Next, we examined whether the increase in HER2 ubiquitination correlated with a decrease in its stability. The cells stably transfected with CblN-SH2Grb2 or the control vector were treated with cycloheximide for various times to block new protein synthesis and HER2 protein half-life was investigated. It was confirmed that HER2 protein in vector-transfected cells was more stable than in CblN-SH2Grb2 transfectants (Fig. 2D).

Cbl-based chimeric ubiquitin ligases decrease colony-forming ability of HER2-overexpressing cells. Because HER2 overexpression contributes to malignant transformation whereas silencing HER2 gene leads to an inhibition of HER2-positive tumor growth (33, 36), we examined the colony formation of the cells stably expressing the chimeric ubiqutin ligases in which HER2 had been down-regulated. As shown in Fig. 3A, stable transfection with the chimeric molecules led to an inhibition of colony-forming ability in all four cell lines compared with the control vector and CblN transfectants. The colony formation ratio of these cell lines were 78.1 ± 9.87% (control vector), 74.1 ± 10.13% (CblN), 28.8 ± 7.11% (CblN-SH2Grb2), 37.6 ± 8.43% (CblN-SH2Grb7), 30.7 ± 6.12% (CblN-SH2P85), 32.5 ± 6.11% (CblN-SH2Src), and 41.3 ± 3.11% (CHIP), respectively. In addition, the colony-forming ability of SK-OV-3 cells stably expressing CblN-SH2Grb2 was significantly reduced compared with control cells (70 ± 13.61% versus 44.2 ± 3.19%; P < 0.05), whereas that of the cells stably expressing Cbl4H-EF-SH2Grb2 or CblNWA-SH2Grb2 was not (56.7 ± 3.85% or 67.5 ± 9.57%; Fig. 3B).

Figure 3.

Chimeric Cbl-based ubiquitin ligases decrease colony-forming ability of HER2-overexpressing cells. SK-BR-3 stable clones (A) or SK-OV-3 stable clones (B) were plated at 200 cells in 60-mm dishes and cultured for 14 d to allow colonies to develop. The colonies were fixed, stained, and counted as described in Materials and Methods. The colony formation ratio was calculated according to the formula, colony formation ratio (%) = (colony number / seeded cells number) × 100%. Columns, mean of three independent experiments; bars, SD.

Figure 3.

Chimeric Cbl-based ubiquitin ligases decrease colony-forming ability of HER2-overexpressing cells. SK-BR-3 stable clones (A) or SK-OV-3 stable clones (B) were plated at 200 cells in 60-mm dishes and cultured for 14 d to allow colonies to develop. The colonies were fixed, stained, and counted as described in Materials and Methods. The colony formation ratio was calculated according to the formula, colony formation ratio (%) = (colony number / seeded cells number) × 100%. Columns, mean of three independent experiments; bars, SD.

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Inducible expression of the chimeric ubiquitin ligase in SK-BR-3 cells leads to HER2 down-regulation, colony formation inhibition, and cell cycle arrest. To exclude the possibility that stable clones obtained by screening with G418 may differ from their parental cells in some biological behaviors, we generated SK-BR-3 cell lines that express CblN-SH2Grb2 or CHIP under the control of the ecdysone receptor–inducible promoter. PCR analysis of genomic DNA from these cells showed integration of the introduced gene (Fig. 4A,, left). Expression of the chimeric proteins was induced with 5 μmol/L ponasterone A (Fig. 4A,, right). As expected, HER2 was decreased at the protein level but not at the mRNA level in ponasterone A–induced cells (Fig. 4B and data not shown), and the colony-forming ability of these cells was significantly reduced (Fig. 4C; CblN-SH2Grb2 ponasterone A− versus ponasterone A+, P < 0.001; CHIP ponasterone A− versus ponasterone A+, P < 0.001). This growth inhibition was also accompanied by an increase in the proportion of cells in G1 phase (Fig. 4D). By contrast, HER2 protein level and colony formation in the pIND-transfected cells were not affected by ponasterone A treatment (Fig. 4B–D). We also compared the percentage of apoptotic cells in these stable clones treated with or without ponasterone A. Unexpectedly, expression of CblN-SH2Grb2 or CHIP did not cause an increase in cell apoptosis (Supplementary Fig. S1).

Figure 4.

Inducible expression of CblN-SH2Grb2 in SK-BR-3 cells leads to HER2 down-regulation, colony formation inhibition, and cell cycle arrest. A, identification of inducible SK-BR-3 stable clones. Integration of the introduced genes in the corresponding stable clones was detected by genome-PCR (left) and the expression of introduced genes was detected by Western blotting with an anti-Flag antibody after the cells were treated with vehicle or ponasterone A (PonA) for 24 h (right). DNA marker: 2,000, 1,000, 750, 500, 250, and 100 bp. B, HER2 down-regulation in inducible SK-BR-3 clones. SK-BR-3 cells stably transfected with the indicated constructs were treated with vehicle or ponasterone A for 24 h, lysed, and subjected to Western blotting with anti-HER2 and anti–β-actin antibody. Relative protein levels normalized for β-actin. C, colony formation inhibition in inducible SK-BR-3 clones. SK-BR-3 clones were plated at 100 cells onto 35-mm dishes and cultured with vehicle or ponasterone A for 14 d to allow colonies to develop. The colonies were fixed, stained, and photographed (the representative dishes are shown). The numbers of colonies were counted as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. D, cell cycle arrest in inducible SK-BR-3 clones. SK-BR-3 stable clones were cultured with vehicle or ponasterone A for 48 h, and cell cycle distribution was assessed by flow cytometry. The respective populations of cells in G1, S, or G2-M phase are shown.

Figure 4.

Inducible expression of CblN-SH2Grb2 in SK-BR-3 cells leads to HER2 down-regulation, colony formation inhibition, and cell cycle arrest. A, identification of inducible SK-BR-3 stable clones. Integration of the introduced genes in the corresponding stable clones was detected by genome-PCR (left) and the expression of introduced genes was detected by Western blotting with an anti-Flag antibody after the cells were treated with vehicle or ponasterone A (PonA) for 24 h (right). DNA marker: 2,000, 1,000, 750, 500, 250, and 100 bp. B, HER2 down-regulation in inducible SK-BR-3 clones. SK-BR-3 cells stably transfected with the indicated constructs were treated with vehicle or ponasterone A for 24 h, lysed, and subjected to Western blotting with anti-HER2 and anti–β-actin antibody. Relative protein levels normalized for β-actin. C, colony formation inhibition in inducible SK-BR-3 clones. SK-BR-3 clones were plated at 100 cells onto 35-mm dishes and cultured with vehicle or ponasterone A for 14 d to allow colonies to develop. The colonies were fixed, stained, and photographed (the representative dishes are shown). The numbers of colonies were counted as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. D, cell cycle arrest in inducible SK-BR-3 clones. SK-BR-3 stable clones were cultured with vehicle or ponasterone A for 48 h, and cell cycle distribution was assessed by flow cytometry. The respective populations of cells in G1, S, or G2-M phase are shown.

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Chimeric ubiquitin ligase works additively with CHIP and 17-AAG. It has been known that heat shock protein (Hsp90) inhibitors such as geldanamycin and its analogue 17-AAG were able to induce ubiquitination and degradation of HER2 through recruiting cochaperone ubiqutin ligase CHIP to it and, similarly, overexpression of CHIP caused significant HER2 down-regulation (32, 35). Therefore, we examined whether there was an additive effect between Cbl-based chimeric constructs and CHIP or Hsp90 inhibitors. As shown in Fig. 5A, transient expression of both CblN-SH2Grb2 and CHIP in SK-BR-3 cells led to more HER2 reduction than expression of either alone, and these effects were enhanced in the presence of 17-AAG. Cells growth inhibition, as indicated in Fig. 5B, was also more significant when the cells were transfected with the two constructs simultaneously or combined with 17-AAG treatment, especially after incubation with the drug for 5 days. These results suggest that CblN-SH2Grb2 could cooperate with either CHIP or Hsp90 inhibitors.

Figure 5.

Chimeric ubiquitin ligase works additively with CHIP and 17-AAG. SK-BR-3 cells grown in six-well plates were transiently transfected with the indicated amounts of constructs. A, 36 h after transfection, cells were treated with 500 nmol/L 17-AAG (+) or DMSO (−) for 4 h, then lysed and subjected to Western blotting with anti-HER2, anti–β-actin, and anti-Flag antibodies, respectively. Relative HER2 levels normalized for β-actin. B, 24 h after transfection, cells were seeded in 96-well plates, serum-starved, and treated with 17-AAG or DMSO for 3 to 5 d as described in Materials and Methods. Cell proliferation was assessed using MTT assays. Columns, mean of three independent experiments; bars, SD. ++, 4 μg; +, 2 μg; −, 0 μg.

Figure 5.

Chimeric ubiquitin ligase works additively with CHIP and 17-AAG. SK-BR-3 cells grown in six-well plates were transiently transfected with the indicated amounts of constructs. A, 36 h after transfection, cells were treated with 500 nmol/L 17-AAG (+) or DMSO (−) for 4 h, then lysed and subjected to Western blotting with anti-HER2, anti–β-actin, and anti-Flag antibodies, respectively. Relative HER2 levels normalized for β-actin. B, 24 h after transfection, cells were seeded in 96-well plates, serum-starved, and treated with 17-AAG or DMSO for 3 to 5 d as described in Materials and Methods. Cell proliferation was assessed using MTT assays. Columns, mean of three independent experiments; bars, SD. ++, 4 μg; +, 2 μg; −, 0 μg.

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Chimeric ubiquitin ligase suppresses tumor growth of HER2-overexpressing SK-OV-3 cells in nude mice. Finally, we examined the effect of CblN-SH2Grb2, the most efficient form of our constructs, on the tumorigenicity of HER2-overexpressing ovarian cancer SK-OV-3 cells in nude mice. The mice injected with SK-OV-3 cells stably expressing CblN-SH2Grb2 developed tumors more slowly than the other groups (Fig. 6A). As a result, the average tumor weight of CblN-SH2Grb2–transfected cells was significantly less than that of other cells (versus vector, P < 0.01; versus Cbl4H-EF-SH2Grb2, P < 0.05; versus CblNWA-SH2Grb2, P < 0.01; Fig. 6B).

Figure 6.

Chimeric ubiquitin ligase suppresses tumor growth of SK-OV-3 cells in nude mice. Mice (three animals per group) were injected s.c. in the right limb with 1 × 107 SK-OV-3 stable clones. A, tumor size was measured over a 5-wk period and tumor volume was calculated by the formula (width2 × length × 0.5). B, tumor weight (g) was recorded at the end of the experiment. Points and columns, mean; bars, SD.

Figure 6.

Chimeric ubiquitin ligase suppresses tumor growth of SK-OV-3 cells in nude mice. Mice (three animals per group) were injected s.c. in the right limb with 1 × 107 SK-OV-3 stable clones. A, tumor size was measured over a 5-wk period and tumor volume was calculated by the formula (width2 × length × 0.5). B, tumor weight (g) was recorded at the end of the experiment. Points and columns, mean; bars, SD.

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Ubiquitin-dependent protein degradation is a primary tactic used by the cell to irreversibly inactivate proteins (1, 57, 37). This process provides an important mechanism through which the cell can destroy misfolded/damaged proteins or cycling ones involved in cell cycle and signaling, or can attenuate signaling from membrane receptors, so as to maintain its homeostasis (1, 38). Such features have prompted several groups to explore the Skp1-Cdc53-F-box (SCF) ubiquitin machinery as a possible approach for the targeted degradation of specific disease-associated proteins (24, 25). One example is the chimeric F box protein (CFP)–mediated eradication of pathogenic β-catenin, which eventually inhibits in vitro and in vivo colorectal cancer cell growth (25). Another separate study showed that an artificially designed “Protacs” (proteolysis-targeting chimeric molecule) could effectively ubiquitinate and degrade cancer-promoting estrogen or androgen receptors (24). However, the disadvantage of either CFP or Protacs is that their efficacy relies on the abundance of endogenous SKP1, CUL1, and ROC1, with which F-box proteins are able to form functional SCF ubiquitin ligase complexes to ubiquitinate the intended substrates (37). Considering that Cbl contains a catalytic RING domain and substrate-interacting domains in a single molecule, as well as its significant role in the negative regulation of epidermal growth factor RTKs (9, 13), we generated several Cbl-based chimeric constructs, to target HER2 oncoprotein for ubiquitination and degradation. The results obtained herein show that chimeric ubiquitin ligases, used to eliminate disease-causing proteins, represent a potential alternative strategy for targeted therapy.

In the present study, the ability of the Cbl-based chimeric ubiquitin ligase to bind to the target HER2 was crucial. SH2 domains are protein modules responsible for binding with high affinity to pTyr-X-Asn motif–containing target peptides, where specificity is conferred by three to six amino acid residues COOH-terminal to tyrosine (39). Several adaptors and kinases possess such SH2 domains and therefore can bind to different phosphorylated tyrosine residues on activated RTKs (40). For example, Grb2, Grb7, p85, Src, and other SH2-containing molecules are able to interact with and convey signals from activated EGFR/HER2 in this manner (2631). Although Cbl possesses a modified SH2 within its TKB domain through which it can be recruited to specific tyrosine residues of EGFR (41), this SH2 variant either cannot mediate a direct association with activated HER2 or such an association is too weak to be detected (Fig. 1B; ref. 42). Therefore, we generated the chimeric ligases by replacing the CblN SH2 domain with that of Grb2, Grb7, p85, or Src. As expected, the interaction between the chimeric proteins and HER2 can be detected both in vivo and in vitro (Fig. 1B and C), suggesting that the chimeric ligases can target activated HER2 and such interaction do not require molecular chaperones like Hsp90.

We then showed that the four designed constructs could down-regulate HER2 in a posttranslational manner (Figs. 2 and 4). Among them, CblN-SH2Grb2 and CblN-SH2p85 seemed to be more effective than the others. It is possible the SH2 domain in these two ligases exhibits a preferred conformational match with HER2. The results of ubiquitination assay and protein stability assay further suggest that it was by triggering ubiquitination-mediated protein degradation that the chimeric constructs down-regulated HER2. Moreover, as being required for E3 activity of Cbl (911, 13), the RING finger domain is also indispensable for our Cbl-based chimeric ubiquitin ligase to promote ubiquitination and degradation of the oncogenic receptor (Fig. 2).

As a result of HER2 reduction, a strong inhibition of colony formation and a significant retardation of tumorigenicity were observed both in our experiment and others (33). We also found that the decrease of HER2 associated with an increase in percentage of cells in G1 (Fig. 4D). This is consistent with the study of Yang et al. (33, 43, 44), owing to the fact that down-regulation of HER2 can lead to decreases in Akt and phosphorylated Akt, which regulate the molecules involved in G0-G1 arrest regulation, such as cyclin D1 and p27. However, we did not detect an increase in cell apoptosis concomitant with HER2 loss as they showed (33), probably due to different extent of HER2 reduction in their study and ours. Finally, we showed that CblN-SH2Grb2 exerts an inhibitory effect in an E3 activity–dependent manner. As indicated, although Cbl4H-EF-SH2Grb2 and CblNWA-SH2Grb2 can associate with HER2 by Grb2 SH2 domain and may compete with endogenous Grb2 or Grb7 for binding to HER2 and switch off oncogenic signals, we could not detect any significant growth-inhibitory effect of these two mutant forms on SK-OV-3 cells. A possible explanation is that their expression levels are not high enough to interfere with other downstream adaptors for recruitment to HER2, such as Src homology and collagen (Shc), Src, Nck, and PI3K, which are sufficient to convey oncogenic signals in HER2-overexpressing tumor cells (19, 20).

Another significant finding in our report is that the Cbl-based chimeric ubiquitin ligase degrades HER2 in concert with CHIP and anti-Hsp90 drug 17-AAG. It is well known that chaperone protein Hsp90 plays an essential role for the maturation or proper activation of many cancer-related protein kinases (45). Therefore, Hsp90 inhibitors, by causing inactivation, destabilization, and eventual degradation of the client proteins, exhibit potent antitumor activity (46). Growing evidences showed that such effects are mostly mediated by chaperone-dependent ubiquitin ligase CHIP, which is independent of Cbl ubiquitin ligase and does not require the kinase activity of oncoproteins (32, 35, 4749). More importantly, nascent kinases and some mutant forms of RTKs, which either escape from Cbl-negative regulation or resist to their inhibitors, still remain sensitive to CHIP overexpression– or geldanamycin/17-AAG–induced degradation (32, 4850). These data, together with our results, convincingly support the feasibility of combinatorial strategies that harness distinct ubiquitin ligase–mediated degradation pathway to achieve more effective down-regulation of oncoproteins or to overcome resistance for better anticancer therapy.

Apart from HER2, we also investigated the effect of the chimeric molecules on EGFR (Supplementary Fig. S2). Our results showed that the chimeric ligases could down-regulate exogenous EGFR in an activation-dependent manner. Considering that many tumors express multiple ERBB receptors, the distinguishing advantage of our chimeric constructs relative to a small interfering RNA strategy is that they can target at least dual, activated rather than inactive receptors simultaneously (i.e., EGFR and HER2). This may also overcome the resistance of HER2-overexpressing tumors to HER2-targeted antibody (trastuzumab), which were partially resulting from the existence of compensatory pathways initiated by HER2-containing heterodimers (e.g., EGFR/HER2; ref. 20). Although HER3, HER4, and other non-HER family tyrosine kinases have not been evaluated in this study, these receptors, as long as harboring tyrosine sites for SH2 used in our chimeric constructs, also might be recognized and degraded to some extent. Nonetheless, as HER2 predominates over and frequently cooperates with other receptors in promoting cancer cell survival in HER2-overexpressing tumors, mainly targeting HER2 combined with targeting other oncogenic signaling pathways should be of more therapeutic benefit.

Taken together, the Cbl-based chimeric ubiquitin ligases designed in the present study were able to promote the ubiquitination and degradation of HER2 and consequently down-regulate HER2, which, in turn, inhibited tumor cell growth. This may represent a novel strategy for the targeted therapy of HER2-overexpressing tumors.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

X. Li and L. Shen contributed equally to this work.

Grant support: Chinese National Key Research and Development Program 2002CB513007 (L. Yao), National Natural Science Foundation of China (NSFC) grant for Distinguished Young Scholars 30228012 (L. Yao), and NSFC grant 30571734 (X. Li).

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.

We thank Dr. Yuncai Liu at La Jolla Institute for Allergy and Immunology for his useful suggestions and generously supplying the plasmids, and Dr. David Dornan and Wanping Xu for kindly providing pcDNA3.1(+)-3 × HA-Ub and pcDNA3.0-CHIP constructs.

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