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Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt am Main, Germany
Requests for reprints: Bernd Groner, Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt am Main, Germany. E-mail: groner{at}em.uni-frankfurt.de
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Abstract |
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 TopNotes Abstract IntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and tyrosine kinase inhibitors acting on the intrinsic enzymatic activity of the intracellular domain, have been developed as targeted drugs. Both have been shown to be beneficial for breast cancer patients. We targeted a third aspect of receptor function: its association with intracellular signaling components. For this purpose, we selected peptide aptamers, which specifically interact with defined domains of the intracellular part of the receptor. The peptide aptamers were selected from a random peptide library using a yeast two-hybrid system with the intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro and in vivo. The aptamers colocalized with the intracellular domain of ErbB2 within cells. We investigated the functional consequences of the aptamer interaction with the ErbB2 receptor within tumor cells. The aptamer sequences were either expressed intracellularly or introduced into the cells as recombinant aptamer proteins. The phosphorylation of p42/44 mitogen-activated protein kinase was nearly unaffected and the activation of signal transducers and activators of transcription-3 was only modestly reduced. In contrast, they strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells treated with heregulin, whereas AKT activation downstream of insulin-like growth factor I or epidermal growth factor receptor was not or only slightly affected. High AKT activity is responsible for the enhanced resistance of ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide aptamer interference with AKT activation resulted in the restoration of regular sensitivity of breast cancer cells toward Taxol. (Mol Cancer Res 2006;4(12):983–98) |
Introduction |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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After the discovery of the ErbB2 oncoprotein, research focused on the elucidation of the signaling pathways activated by this receptor and their role in cancer formation. The ErbB family of receptor tyrosine kinases are activated on binding of growth factors that belong to the EGF family (9). The binding of EGF-like ligands to ErbB receptor monomers induces a conformational change in the extracellular domain of the receptor. The conformational change of EGFR exposes a dimerization domain, which enables two monomers to interact with each other and form an activated receptor dimer (10). The ErbB2 receptor exhibits structural features that promote its oncogenic potential. In contrast to EGFR, this receptor is always present in the plasma membrane in an open conformation and the propensity to form dimers does not require the binding of a ligand (11); it heterodimerizes with other family members and can be activated (e.g., through heregulin and dimer formation with ErbB3). ErbB3 plays a crucial role in breast tumor cell division: it couples active ErbB2 to the phosphatidylinositol 3-kinase-protein kinase B/AKT pathway. ErbB2/ErbB3 dimers function as an oncogenic unit driving breast tumor cell proliferation (12).
Activated receptor dimers cause the subsequent activation of intracellular signaling pathways. This is achieved through the intrinsic tyrosine kinase activities present in the intracellular COOH-terminal tail of ErbB receptors. In a receptor dimer, the tyrosine kinase domain of one dimerization partner is able to phosphorylate specific tyrosine residues in the COOH-terminal part of the other monomer, a mechanism termed cross-phosphorylation (13). The phosphotyrosines then serve as binding sites for cytoplasmic signaling molecules and represent the vantage point of signaling pathways inducing proliferation, antiapoptotic responses, angiogenesis, and metastasis formation (reviewed in ref. 14).
Two pathways induced by ErbB2-containing heterodimers are the mitogen-activated protein kinase (MAPK) and AKT pathways, which are involved in cellular proliferation. AKT activation also provides a strong antiapoptotic response and protects cells against inducers of apoptosis (reviewed in ref. 15). AKT causes the direct inhibition of the apoptotic inducer Bad through its phosphorylation (16). AKT also phosphorylates and thereby inactivates the FOXO-1 transcription factor, which controls the expression of cell cycle inhibitors and proapoptotic proteins (17). AKT activation also causes the induction of nuclear factor 
B–mediated antiapoptotic responses and the repression of tumor suppressors (e.g., phosphatase and tensin homologue; ref. 18).
ErbB2 activation is therefore associated with an antiapoptotic phenotype mediated by multiple pathways. This is reflected in the inefficiency of chemotherapy in patients with ErbB2-overexpressing tumor cells and the insensitivity of cultured tumor cells in vitro toward such drugs (19). The strong antiapoptotic signaling in combination with the high metastatic potential of ErbB2-overexpressing cells is a major factor responsible for the unfavorable prognosis of patients. Drugs targeting the ErbB2 receptor not only interfere with cell proliferation but also result in an improvement of the therapeutic efficacy of chemotherapy (20, 21).
Two strategies have been exploited to block the function of ErbB receptors. Drugs can act either through interaction with the extracellular domain and prevent the activation or dimerization, or they can inhibit the tyrosine kinase activity in the intracellular part of the receptor. Antibodies against the extracellular ErbB2 domain were derived many years ago (22), but it took until 1998 for the first ErbB2 antibody trastuzumab (Herceptin) to be approved as a drug for the treatment of metastatic breast cancer. It binds specifically to extracellular domain IV of the ErbB2 receptor (11) and acts by initiating the endocytosis of ErbB2, leading to its degradation in the lysosome (23). Its activity is further enhanced by the induction of an antitumoral immune response (24). In the meantime, it has become clear that this monoclonal antibody shows considerable therapeutic benefit. It leads to reductions in the rates of recurrence of tumors and in the number of deaths of patients (20, 21, 25). To target the tyrosine kinase domain, chemical compounds have been developed that bind the ATP binding pocket (e.g., PKI-166) and act as competitive binding inhibitors. For this reason, tyrosine kinase inhibitors have to overcome the high concentrations of ATP in cancer cells (26).
Both strategies, based on Herceptin and tyrosine kinase inhibitor, showed encouraging results with considerable patient benefit. Still, only a fraction of the patients respond and the responses are largely limited in duration. The combination of drugs inhibiting ErbB2 function with conventional chemotherapeutic agents seems to be promising. ErbB2-targeted approaches might further be enhanced by the interference with ErbB2 properties related to its signal transduction functions. Such a strategy has been outlined when investigators tried to adapt antibody binding to intracellular conditions. A single-chain derivative of a monoclonal antibody was used to sequester the receptor from the cell surface (27). These single-chain antibodies are composed of heavy and light chain variable domains connected by a flexible peptide linker. When expressed in ErbB2-transformed cells, the single-chain antibodies bound to the receptor, prevented its transit through the endoplasmic reticulum, and resulted in the reversion of the transformed phenotype (27).
The use of intracellular antibodies has been shown to be limited by the restrictions on folding imposed by the reducing milieu of the cytoplasm. For this reason, we have employed a strategy based on peptide aptamers, rather than on intracellular antibodies, to interfere with intracellular ErbB2 functions. In these constructs, a variable peptide region is displayed by a scaffold protein (e.g., thioredoxin; ref. 28). This protein has a defined structure under intracellular conditions and peptide sequences can be presented in the active loop structure of the protein. Peptide aptamers represent random sequences of 12 to 40 amino acids in length (29). They can be selected for the interaction with a target protein in a yeast two-hybrid system (30). Peptide aptamers have been shown to specifically bind and functionally interfere with intracellular proteins, such as transcription factors E2F and signal transducers and activators of transcription 3 (Stat3), viral oncoprotein E6, and growth factor receptors like EGFR and others (31-35).
Delivery of proteins that inhibit intracellular targets requires the passage through the cellular membrane. Recent advances have shown that recombinant proteins can be provided with a protein transduction domain (PTD), which greatly enhances their exogenous uptake (36) and improves their therapeutic potential. The most frequently used PTD is derived from the HIV-TAT protein, a positively charged sequence (37). The positive charge allows the interaction of the fusion protein with negatively charged heparin sulfate glycosaminoglycans on the surface of plasma membranes (38) and leads to the uptake of the conjugated proteins by macropinocytosis (39, 40), an actin-mediated form of endocytosis.
In the present study, we isolated peptide aptamers that specifically interact with the intracellular domain of ErbB2 and are able to inhibit selected functions of this receptor. Peptide aptamers were either expressed after gene transfer into tumor cells or delivered as recombinant proteins fused with a PTD. The aptamers were able to inhibit the activation of AKT on heregulin induction of tumor cells and thereby sensitize chemoresistant breast cancer cells for the treatment with paclitaxel. These peptide aptamers might become useful in the combination therapy of ErbB2-dependent cancers.
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Results |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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In the yeast two-hybrid experiments, a total of 3.6 x 108 yeast transformants were screened. Thirteen different peptide aptamers were initially isolated that were able to interact with the kinase domain of ErbB2 and induce growth of the yeast cells in selective medium lacking histidine. The three aptamers (AI-1, AII-7, and Ag-11) that induced growth under stringent selection conditions are shown in Table 1 . These aptamers bind to different regions of the ErbB2 intracellular region. AI-1 was selected using fragment I of the kinase domain as a bait (see Fig. 1). Ag-11 selection was carried out with the entire kinase domain as a bait. These two baits include the active site (ATP-binding pocket) of the kinase domain. AII-7 interacts with fragment II of the kinase domain, which does not include the active site.
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. The potential phosphorylation site in aptamer Ag-11 is predicted to interact with the SH2 domain of Src. It is possible that the aptamers could serve as substrates for the ErbB2 tyrosine kinase and interact as phosphopeptides with the identified SH2 domains. Aptamer AII-7, which interacts with sequences outside the active site in the kinase domain, was chosen for further analysis. We hypothesized that a potential inhibitory function might be based on a mechanism distinct from interference with the tyrosine kinase activity.
Confirmation of the Specificity of Peptide Aptamer Interactions with the Intracellular Domain of the ErbB2 Receptor In vivo and In vitro
The peptide aptamers obtained in the yeast two-hybrid screening procedure were further analyzed and the specificity of their interaction properties was studied in in vitro and in vivo experiments. For this purpose, they were tested in vivo by reintroduction into yeast cells expressing bait constructs encoding fragments of different receptors. The specificity of the in vitro interactions was investigated in glutathione S-transferase (GST) pull-down experiments. These experiments were carried out to examine the cross-reactivity of the peptide aptamers with other receptor tyrosine kinases.
KF1 yeast cells were cotransformed with constructs encoding the selected aptamer sequences and the complete tyrosine kinase domains (KDg) of insulin-like growth factor receptor (IGF-IR) or EGFR. IGF-IR belongs to the type II receptor tyrosine kinases. The kinase domain shows only a moderate homology with the ErbB2 receptor kinase. EGFR is a member of the type I ErbB receptor tyrosine kinase family and its kinase domain shares 82% homology with the kinase domain of ErbB2. Growth of the yeast cells on medium lacking histidine indicates that aptamers Ag-11 and AII-7 interact with the tyrosine kinase domain of the ErbB2 receptor, but not with IGF-IR (Fig. 2 ). Only a very weak interaction of aptamer AII-7 with the kinase domain of EGFR was observed (Fig. 2).
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Recombinant Purified Peptide Aptamers Can Be Introduced into Cells via a PTD and Interact Intracellularly with ErbB2
To evaluate the function of peptide aptamers in cell culture or animal models, it is necessary to introduce them into cells from the outside. To achieve uptake into target cells, the peptide aptamers were fused with a PTD, consisting of a homopolymer of nine arginine residues. The fusion proteins were bacterially expressed and purified. PTD fusion proteins are taken up by macropinocytosis and subsequently released from early endosomes into the cytoplasm (39, 43).
The PTD-aptamer proteins were furnished with a Flag epitope to facilitate their detection (Fig. 4A ), expressed in bacteria and purified from bacterial lysates by fast protein liquid chromatography under denaturing conditions (Fig. 4B). The denatured proteins were refolded in an arginine-containing buffer and dialyzed, which is necessary for these recombinant proteins to maintain their specific binding characteristics. The purity and concentration of the proteins was investigated by gel electrophoresis and Coomassie staining (Fig. 4C). The correct refolding and the binding properties were investigated by coimmunoprecipitation experiments. Cell extracts were prepared from SKBR3 breast carcinoma cells, which overexpress the ErbB2 receptor. The extracts were incubated with 0.5 µmol/L recombinantly expressed, purified peptide aptamers (Fig. 4D). Subsequently, the receptor was bound to a specific ErbB2 antibody and protein complexes were isolated with Protein-A magnetic beads. The presence of the peptide aptamers in the immunopurified ErbB2 complex was detected by Western blot with a Flag-specific antibody. Peptide aptamer AII-7 was prominently detected, indicating its strong interaction with ErbB2. The empty thioredoxin protein and aptamer Ag-11 only showed weak interactions (Fig. 4D).
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We confirmed the specific intracellular interaction of the transduced peptide aptamers with the ErbB2 receptor by direct visualization in immunofluorescence microscopy experiments. For this experiment, we used NIH#3.7 cells, which stably express the ErbB2 receptor (44), and transduced them with thioredoxin or AII-7 for 4 h. The cells were treated with 100 µmol/L chloroquine to enhance the release from the endocytotic vesicles. They were subsequently washed with 0.2 mol/L acidic acid to remove proteins associated with the cell surface. ErbB2 and peptide aptamers were detected by confocal laser scanning microscopy using specific antibodies and secondary antibodies tagged with a fluorescent dye. The NIH#3.7 cells could be efficiently transduced and the thioredoxin protein, as well as peptide aptamer AII-7, was detected. The empty thioredoxin protein was mainly present in the cytoplasm of the cells (Fig. 5 ). In contrast, a significant fraction of peptide aptamer AII-7 was found at the cell membrane, where it colocalized with the ErbB2 receptor. Colocalization was further verified by comparing the fluorescent intensity values of the signals detected with a microscope (Fig. 5). These results confirmed the data obtained in the coimmunoprecipitation experiments and strongly suggest that the aptamers are able to enter the cell by protein transduction and interact with ErbB2 intracellularly.
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MCF7 cells are breast carcinoma cells that express moderate levels of ErbB2 and ErbB3. When the cells are induced for a short period of time (10 min) with heregulin, ErbB2/ErbB3 dimers are formed and phosphorylation of the AKT kinase can be observed. This cell system was used to analyze the functional consequences of peptide aptamer binding to the ErbB2 receptor. Initial experiments were done in which the peptide aptamers were intracellularly expressed on transfection of a gene construct. Although the transfection efficiency was only 30% (data not shown), a clear reduction in AKT phosphorylation was detectable when MCF7 cells transfected with aptamer AII-7 were compared with cells transfected with the control thioredoxin construct (Fig. 6
). Aptamer AI-1 showed no reduction in AKT phosphorylation and aptamer Ag-11 showed only a weak reduction of AKT phosphorylation. No AKT phosphorylation was detected in cells not treated with heregulin (Fig. 6). These results are in accordance with the relative binding affinities for the different aptamers observed above.
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We carried out experiments to investigate the potential effects of the ErbB2-specific peptide aptamers on other signaling pathways induced in MCF-7 on treatment with heregulin. The kinase domain is able to phosphorylate multiple tyrosine residues in the COOH-terminal tail of the receptor. These residues then form docking sites for adaptor proteins such as growth factor receptor binding protein 2, which leads to the activation of the Ras/Raf/MAPK pathway on growth factor stimulation. The transcription factor Stat3 is found associated with the receptor and, in this case, tyrosine phosphorylation leads to the direct activation of Stat3, which then dimerizes and translocates to the nucleus. We analyzed the effects of the peptide aptamer AII-7 on the activation of these signaling molecules. MCF-7 cells were incubated with AII-7 (1.5 µmol/L) for 4 h and induced with heregulin for 15 min. Cell extracts were prepared and the amounts of p42/44-MAPK, its activated form phospho-p42/44-MAPK, Stat3, and its activated form phospho-Stat3 were compared in Western blots with specific antibodies. The phosphorylation of p42 (extracellular signal–regulated kinase 1) was only slightly affected when extracts from heregulin-induced cells treated with AII-7 and control cells were compared, whereas phosphorylation of p44 (extracellular signal–regulated kinase 2) was clearly unaffected. The induction of Stat3 phosphorylation was slightly reduced. These results indicate that aptamer AII-7 inhibits receptor function in a pathway-specific manner. It interferes only with a subset of signaling molecules and most likely does not lead to a complete loss of tyrosine kinase activity of the ErbB2 kinase domain. Our experiments show that this effect is dependent on the endogenous or exogenous provision of the peptide aptamer. Transfected cells, virally transduced cells, or cells into which the peptide aptamers had been introduced as recombinant proteins showed a similar behavior on ligand activation of the ErbB2 receptor.
EGFR- and IGF-IR–Mediated Induction of AKT Is Not Affected by Peptide Aptamer AII-7
The results presented above indicate that aptamer AII-7 inhibits AKT signaling induced by the activation of the ErbB2 receptor. We carried out additional experiments to confirm the specificity of AII-7 action with respect to the inhibition of signaling downstream of the ErbB2 receptor. Figures 2 and 3 show the preferential binding of aptamer AII-7 to the ErbB2 receptor in vitro and in vivo. In vitro GST pull-down experiments showed that the aptamer strongly binds the ErbB2 receptor, but not the EGFR. In yeast cells, the AII-7 aptamer binds preferentially to the ErbB2 receptor kinase domain and only very weakly to EGFR. In both experiments, additional sequences, Gal4-AD or GST, are present in the aptamer fusion proteins, which might have influenced the observed specificity. To further confirm the functional specificity, we investigated if the recombinant purified peptide aptamer AII-7, not containing Gal4-AD or GST sequences, affects the activation of AKT induced through IGF-IR or EGFR.
In this experiment, we induced MCF-7 cells with IGF-I (Fig. 9A ). AKT phosphorylation was low when the cells were kept in low serum overnight and could be induced after addition of IGF-I to the medium (Fig. 9A, lanes 1-3). Increasing amounts of aptamer AII-7 were added to the medium of the cells (lanes 4-6), but no effects on the induction of AKT phosphorylation were observed. This confirms the interaction studies shown in Fig. 2. The aptamer AII-7 does not interfere with AKT activation downstream of IGF-IR.
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Because AKT phosphorylation could not be down-regulated in these cells, we also investigated the effects of AII-7 on the induction of EGFR in Renca-EGFR cells. These murine renal caricinoma cells have been stably transfected with the human EGFR gene and express high amounts of the human EGFR, whereas ErbB2 expression is very low. Starvation reduced the phosphorylation of AKT to very low levels (Fig. 9C, lane 1) and the addition of EGF to the medium resulted in AKT phosphorylation and activation (lane 2). The addition of increasing amounts of aptamer AII-7 only slightly decreased pAKT. These results confirm that IGF-I- or EGF-dependent activation is not, or only very slightly, affected by aptamer AII-7; this is in contrast to heregulin-mediated AKT activation via the ErbB2/ErbB3 heterodimer.
Aptamer AII-7 Sensitizes Chemoresistant MCF-7 (her2) Cells toward Paclitaxel Treatment
AKT activity is a well-recognized survival signal in cancer cells. Because aptamer AII-7 is able to suppress AKT activation, we tested if the sensitivity of ErbB2-expressing tumor cells toward chemotherapeutic drugs could be modulated by this peptide aptamer. It has recently been shown that the overexpression of ErbB2 in MCF-7 cells changes its sensitivity toward chemotherapeutic drugs. Stable transfection of the ErbB2 gene and its concomitant overexpression increases the resistance of MCF7 cells toward paclitaxel, doxorubicin, and 5-fluorouracil (47). These agents induce apoptosis in proliferating cells. The drug resistance in ErbB2-overexpressing cells (MCF7-her2 cells) is dependent on AKT signaling. The antiapoptotic effect of AKT dampens the induction of apoptosis. We used MCF7-her2 cells to investigate if the prevention of AKT phosphorylation by aptamer AII-7 changes their sensitivity toward paclitaxel treatment. MCF7-her2 and MCF7-neo control cells were infected with lentiviral vectors encoding the empty thioredoxin, aptamer Ag-11, and aptamer AII-7. These cells were then subjected to paclitaxel treatment and cytotoxicity was assayed. The cells were treated with increasing concentrations of paclitaxel for 5 h. The percentage of living cells was determined with a 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay 3 days later (Fig. 10
).
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Recombinant aptamer proteins were used to determine if the induced sensitivity toward paclitaxel could also be conferred by protein transduction. For this purpose, MCF7-her2 and MCF7-neo cells were incubated with 2 µmol/L of purified peptide aptamers 2 h before and during the paclitaxel treatment (Fig. 10C and D). PKI-166 (1 µmol/L) was used as a control. Consistent with the results obtained in the experiments in which virally infected MCF7-her2 cells were used, we observed that transduced aptamer AII-7 sensitizes these cells to paclitaxel. About 37% of the MCF7-her2 cells are viable on treatment with AII-7 and 0.4 µmol/L paclitaxel, compared with 70% viable MCF7-her2 cells treated with paclitaxel alone. It did not affect proliferation and paclitaxel sensitivity of MCF7-neo cells. Aptamer Ag-11 again shows moderate effects on MCF7-her2 cells (55% viable MCF7-her2 cells treated with 0.4 µmol/L paclitaxel) and the percentage of viable cells after incubation with empty thioredoxin (65% of viable MCF7-her2 cells treated with 0.4 µmol/L paclitaxel) was similar to that of MCF7-her2 cells incubated without recombinant proteins. The incubation of PKI-166 also sensitized the cells toward paclitaxel treatment (50% viable MCF7-her2 cells treated with 0.4 µmol/L paclitaxel) and had a similar effects as aptamer Ag-11.
The reduction of AKT phosphorylation by aptamer AII-7 sensitizes the relatively more resistant MCF7-her2 cells to paclitaxel treatment. This observation was made after infection of the cells with a viral expression vector encoding aptamer AII-7 and on protein transduction of recombinant AII-7 protein. The weaker sensitizing effect was exerted by aptamer Ag-11. The effects caused by the aptamers are most likely mediated via relatively subtle influences on the function of the ErbB2 receptor; no consequences on the growth of MCF7-her2 cells were observed. PKI-166 was able to sensitize the cells similarly to Ag-11.
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Discussion |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Up to now, only the ATP binding pocket in the tyrosine kinase domain of the ErbB2 receptor has been targeted to inhibit its intracellular function. This prevents the phosphorylation of multiple tyrosine residues in the COOH-terminal tail of the receptor and therefore affects multiple signaling pathways at once. This, however, could be one of the reasons for the side effects observed on usage of such inhibitors. In the present study, we show that not only the enzymatic activity but also the individual signaling aspects of the ErbB2 receptor can be targeted to influence tumor cell properties. We employed a peptide aptamer that interacts with the intracellular domain of the ErbB2 receptor but does not involve the ATP binding site directly. The peptide aptamer AII-7 recognizes a region between amino acids 853 and 987, which lies outside the ATP binding site (amino acids 726-734). Aptamer Ag-11 was identified using amino acids 678 to 986 as bait. The exact binding positions for these aptamers have not been localized yet.
The isolated aptamers, initially selected in the yeast two-hybrid system, were found to retain their specificity for ErbB2 in other interaction assays (e.g., GST pull-down experiments and coimmunoprecipitation analysis). No cross-reactivity with the highly related EGFR or IGF-IR tyrosine kinase domain was detected in these assays. Specificity was confirmed by showing that AKT or MAPK signaling was unaffected if activated through these receptors. Although no binding constants have been established, these properties indicate high affinities. Aptamer AII-7 interacts stronger with the ErbB2 receptor when compared with aptamer Ag-11. To further develop these aptamers, we might have to further increase the binding affinity, which could be achieved through mutational analysis. For this purpose, the evaluation of the aptamer structure in conjunction with the binding site on the receptor will be important.
In our coimmunoprecipitation experiments, we evaluated interactions between the ErbB2 receptor and transduced peptide aptamers or by mixing cell lysates with purified peptide aptamers. The interaction with the receptor showed that we are able to refold recombinantly expressed peptide aptamers correctly. Transduction procedures and subsequent analysis of the localization of the peptide aptamers by confocal microscopy confirmed this conclusion. These experiments motivated us to pursue the functional evaluation of the aptamers and to investigate their intracellular effects. The efficiency of protein transduction is most likely a limiting factor if the application of peptide aptamers as protein therapeutics is being considered. The efficacy of protein transduction can be enhanced by chloroquine, which disrupts intracellular vesicles and thereby releases the PTD-fused proteins (43). Chloroquine was used in our protein transduction experiments; however, due to its toxicity, use might be restricted to cell culture experiments. The use of a HA2 peptide sequence has recently been proposed to circumvent this problem. This peptide was combined with a PTD and enhanced the efficiency of delivery (39, 49). HA2 peptides are able to form pores in vesicular structures at low pH and release enclosed proteins into the cytoplasm of the cell. The principle has been shown in cell culture (50), but its effective use still has to be confirmed in animal models.
In addition to the transduction of purified proteins, virally based gene transfer methods were used to evaluate the aptamer function. Both methods showed that aptamer AII-7 reduced the induction of AKT phosphorylation after ErbB2 activation in MCF7 cells. Although aptamer AII-7 is not binding to the active site, the aptamer interaction with the kinase domain interferes with the activation of a kinase substrate. The phosphatidylinositol 3-kinase-AKT pathway activates an antiapoptotic response in the cell (reviewed in refs. 15, 51) and its activation seems to be responsible for a drug resistance phenotype, which can be reversed by phosphatidylinositol 3-kinase inhibitors (52, 53). AKT activation can either be mediated by constitutive active mutations of phosphatidylinositol 3-kinase or AKT or by components of upstream signaling like the ErbB2/ErbB3 heterodimer. Therefore, the inhibition of this pathway, directly at the receptor level, could improve the efficacy of chemotherapy and reduce side effects.
MCF7-her2 cells show increased resistance toward chemotherapeutic agents such as paclitaxel, doxorubicin, 5-fluorouracil, etoposide, and camptothecin compared with the parental MCF7 cells (47). The correlation of ErbB2 expression and chemoresistance has also been observed in other breast and ovarian cancer cell lines (54, 55). Chemoresistance has also been correlated with the overexpression of other receptor tyrosine kinases (e.g., platelet-derived growth factor receptor) and could be prevented through inhibition of the tyrosine kinase activity (56). This enhanced drug resistance is dependent on the activation of AKT and can be blocked through dominant negative AKT variants or phosphatidylinositol 3-kinase inhibitors (47). Inhibition of this pathway sensitizes these cells for apoptosis induced by the chemotherapeutic agents. The aptamer-mediated AKT inhibition has been used to influence the efficacy of chemotherapy of ErbB2-overexpressing cancer cells and the regular chemosensitivity of these cells could be restored (Fig. 11 ). The presence of the aptamers did not cause other measurable effects on cell proliferation. This was confirmed by XTT assays and was also shown by the fact that the Stat3 and MAPK pathways are only moderately affected after aptamer treatment. Proliferation of cancer cells is important if chemotherapeutic agents are being used because they are only effective on dividing cells. This idea is supported by the finding that the inhibitor PKI-166 and aptamer AII-7 showed comparable inhibitory effects on the induction of AKT phosphorylation, but, interestingly, the sensitizing effect of the aptamer in the cytotoxicity assays was clearly more pronounced. PKI-166 inhibits the kinase domain more effectively, leading to the down-regulation of multiple pathways, including those involved in proliferation. These results suggest that the combination of the specific aptamer-mediated inhibition of AKT signaling and chemotherapy might become useful.
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20 kDa. This is comparable to camel antibodies called nanobodies, which show excellent tumor-penetrating properties (57). These properties are not altered if an albumin-binding peptide is added to the compounds. However, the use of the albumin-binding peptide strongly reduces the clearance of the nanobodies and results in a prolonged blood circulation. The pharmacokinetic variables of the protein might also be affected by the addition of a PTD, a question that has not yet been addressed. The use of PTD-conjugated proteins in vivo has shown promising leads. After i.v. injection, the proteins can be found in every tissue including the brain (58). These results illustrate that it might be possible to deliver therapeutic proteins to a variety of organs and cell types within an organism. If delivery of protein therapeutics can be targeted to specific cell types via surface receptors, side effects of such drugs might be further minimized (59). Additional targeting domains in such recombinant proteins might improve their properties (60, 61). The same is true for albumin-binding sequences and HA2 peptides. Our results show that the combination of peptide aptamers with PTDs allows the interference with specific signaling events and that it is thus possible to target crucial protein-protein interactions in cancer cells. Further in vivo experiments will have to complement our results obtained with cell culture models.
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Materials and Methods |
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TopNotesAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Peptide Aptamer Screening
The screening was done in the yeast strain KF1 (MAT
Trp-901 Leu2-3112 His3-200 gal4
gal80 LYS2::GAL1-HIS3 GAL2ADE2 MET2::GAL7-lacZ SPAL10-URA3; ref. 31). As baits, parts of the ErbB2 intracellular domain (for cytoplasmic domain, amino acids 678-1,254; for KDg, amino acids 678-987; for KDI, amino acids 678-852; and for KDII, amino acids 853-987) were fused to the GAL4-DNA binding domain into the pPC-97 vector. Screenings were done with a randomized 12-mer and 20-mer peptide aptamer library and selection procedures were done as previously described (31).
GST Pull-Down Assay
For GST pull-down experiments, aptamers were inserted in the pGex 4T3 vector (Amersham Biosciences, Freiburg, Germany) and expressed in Escherichia coli Bl21 (DE3) lysS (Stratagene, Amsterdam, the Netherlands) cells as a GST fusion protein. Aptamer expression was induced with isopropyl-1-thio-ß-D-galactopyranoside (1 mmol/L) for 2 h at 30°C. Proteins were purified and used in the pull-down experiment as previously described (32).
Bacterial Expression and Purification of PTD Peptide Aptamers
For protein transduction experiments, a PTD of nine arginine residues was fused to the COOH terminus of the peptide aptamers, which were inserted into the pET30a+ vector (Novagen, Schwalbach, Germany). Aptamer expression was induced with isopropyl-1-thio-ß-D-galactopyranoside (1 mmol/L) for 4 h at 30°C. Proteins were purified under denaturing conditions as previously published (62).
In vitro and In vivo Coimmunoprecipitation Experiments
For detection of the peptide aptamer interaction with full-length ErbB2 in in vitro coimmunoprecipitation experiments, SKBR3 cell lysates were incubated with recombinantly expressed peptide aptamers (0.5 µmol/L) for 1 h at 4°C. Afterwards, ErbB2 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was added and incubated for 1 h at 4°C. Immunocomplexes were collected with Protein-A DynaBeads (Invitrogen, Karlsruhe, Germany) and washed with PBS. Bound proteins were eluated by boiling in sample buffer and subjected to Western blot analyses. For detection of protein transduction through in vivo coimmunoprecipitation experiments, SKBr3 cells were incubated with recombinantly expressed peptide aptamers (2 µmol/L) for 4 h at 37°C. Cells were solubilized with radioimmunoprecipitation assay (RIPA) lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% NP40, 0.5% sodium desoxcholate, 1 mmol/L EDTA, protease inhibitors] and incubated on ice for 12 min. Lysates were clarified by centrifugation at 16,000 x g for 10 min and incubated with anti-ErbB2 antibody (Santa Cruz Biotechnology) for 1 h. Immunocomplexes were collected as described above and subjected to Western blot analyses.
Western Blot Analyses
Cells were solubilized in Triton extraction buffer [50 mmol/L Tris (pH 7.5), 5 mmol/L EGTA, 150 mmol/L NaCl, 1% Triton X-100, protease inhibitors] or RIPA lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% NP40, 0.5% sodium desoxcholate, 1 mmol/L EDTA, protease inhibitors] and incubated on ice for 10 min. Lysates were clarified by centrifugation at 16,000 x g for 10 min, subjected to SDS-PAGE, and blotted on polyvinylidene difluoride membranes. After blocking in 5% TBS with Tween 20 [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 0.05% Tween 20], membranes were probed with specific antibodies and proteins were visualized with peroxidase-coupled secondary antibodies using the enhanced chemiluminescence system (Amersham Biosciences). Antibodies were used to detect ErbB2, EGFR, and IGF-IR (Santa Cruz Biotechnology), peptide aptamers (anti Flag) and ß-tubulin (Sigma-Aldrich, Hannover, Germany), and AKT, pAKT, Stat3, pStat3, p42/44-MAPK, and phospho-p42/p44-MAPK (Cell Signaling, Frankfurt, Germany).
Immunofluorescence Imaging
Cells were grown on coverslips and protein transduced (1 µmol/L) for 4 h at 37°C. Afterwards, untransduced proteins were removed from the cell surface through acid wash (0.2 mmol/L acidic acid for 5 min at 4°C), fixed with 95% methanol, and permeabilized with 0.1% Tween 20 in PBS. Cells were washed, blocked (0.5% cold fish gelatin, 0.1% ovalbumin in PBS), and incubated with primary antibodies at 4°C overnight. After intensive washing, incubation with fluorescence-labeled antibodies (Molecular Probes, Karlsruhe, Germany) was done for 1 h at room temperature in the dark. The stained proteins were visualized with a confocal laser scanning microscope (Leica, Bensheim, Germany).
Transfection of Eukaryotic Cells
Transfections were done with LipofectAMINE 2000 (Life Technologies, Karlsruhe, Germany) according to the manufacturer's protocol. For expression of peptide aptamers in eukaryotic cells, the thioredoxin cassette was subcloned into the eukaryotic expression vector pRc/CMV (Invitrogen) and fused 3' to the herpes simplex virus VP22 gene (pRC/CMV-VP22-Trx).
Encoding of Peptide Aptamers in Lentiviral Particles and Lentiviral Transduction in Eukaryotic Cells
For stable expression of peptide aptamers in eukaryotic cells, the Flag-Trx cassette was subcloned into the lentiviral expression vector pSiEW. Peptide aptamer expression is driven by a spleen focus-forming virus promoter, which also drives the expression of enhanced GFP (EGFP) through an internal ribosome entry site. Lentiviral particles were produced by triple transfection of pSiEW-Flag-Trx, R.8.1, and pM2 in 293T cells. Viral particles were collected in 5-mL culture supernatants 2 days after triple transfection. Virus titers were estimated by titration of 1:1,000, 1:100, and 1:10 culture supernatants of 293T cells. The percentage of infected cells was measured 3 days after infection by fluorescence-activated cell sorting analyses.
AKT Phosphorylation Assay
For AKT phosphorylation analyses, after lentiviral transduction, 1 x 106 MCF7 cells expressing thioredoxin or aptamers were plated and starved overnight. Specific ErbB2/ErbB3 heterodimer activation was induced by 5 ng/mL heregulin-1ß for 15 min and stopped with ice-cold PBS. MCF7 cells were lysed with radioimmunoprecipitation assay lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% NP40, 0.5% sodium desoxcholate, 1 mmol/L EDTA, protease inhibitors] and incubated on ice for 10 min. Lysates were clarified by centrifugation at 16,000 x g for 10 min and subjected to Western blot analysis. For protein transduction experiments, 1 x 106 MCF7 cells were incubated with 1.5 and 2 µmol/L of purified peptide aptamers, as well as with 100 µmol/L of chloroquine, 4 h before heregulin-1ß induction.
Taxol Treatment and Cytotoxicity Assays
Cytotoxicity assays were done by plating 5 x 103 MCF7-her2 or MCF7-neo cells in 24-well plates. After lentiviral transduction, MCF7-her2 or MCF7-neo cells expressing thioredoxin or aptamers were incubated with increasing paclitaxel concentrations (0.01, 0.04, 0.4, and 4 µmol/L) for 5 h in charcoal-stripped DMEM. For protein transduction, cells were incubated with 2 µmol/L purified PTD-aptamers 4 h before and during paclitaxel treatment. Cell viability was measured 3 days after paclitaxel treatment with the XTT-based proliferation kit II (Roche Molecular Biochemicals, Mannheim, Germany).
Proliferation and Viability Assays
Relative viable cell numbers were quantified using the XTT-based proliferation kit II according to the manufacturer's protocol, which assesses cell viability via bioreduction of a tetrazolium compound by measuring absorbance at 490 nm in a 96-well plate reader (Roche Molecular Biochemicals).
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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 Baehr and Groner, unpublished results. 
Received 2/15/06; revised 9/12/06; accepted 10/ 5/06.
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