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[Cancer Research 65, 6891-6900, August 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

A Bifunctional Targeted Peptide that Blocks HER-2 Tyrosine Kinase and Disables Mitochondrial Function in HER-2-Positive Carcinoma Cells

Valeria R. Fantin1, Marcelo J. Berardi2, Holger Babbe1, Montserrat V. Michelman1, Charlene M. Manning1 and Philip Leder1

1 Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute and 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts

Requests for reprints: Valeria R. Fantin, Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, Room 356, New Research Building, 77 Avenue Louis Pasteur, Boston, MA 02215. Phone: 617-432-7578; E-mail: vfantin{at}genetics.med.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The HER-2 oncoprotein is commonly overexpressed in a variety of human malignancies and has become an attractive antitumor target. A number of strategies to inhibit the HER-2 receptor tyrosine kinase are currently the focus of intensive preclinical and clinical research. In the present study, we have engineered a bifunctional peptide, BHAP, which consists of two modular domains: a HER-2-targeting/neutralizing domain and a mitochondriotoxic, proapoptotic domain. The chimeric peptide is biologically active and capable of selectively triggering apoptosis of HER-2-overexpressing cancer cells in culture, even those previously described as Herceptin resistant. Furthermore, BHAP slows down growth of HER-2-overexpressing human mammary xenografts established in SCID mice. This approach can be extended to the development of tailored targeted chimeric peptides against a number of overexpressed cellular receptors implicated in the development and progression of cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Conventional chemotherapy and tumor-targeted therapy are two complementary approaches currently employed for the treatment of cancer. Most chemotherapeutic agents are aimed to preferentially affect tumor cells due to their faster rate of cell proliferation. However, despite the unquestionable benefits of systemic traditional anticancer therapies, treatment-related toxicities normally arise from death of rapidly dividing normal cells. As a consequence of the identification of essential molecular players involved in tumorigenesis in recent years, tumor-selective targets are beginning to emerge. The epidermal growth factor (EGF) receptor family member HER-2, also known as erbB-2/neu, is among those genes frequently altered in human cancers (1, 2). HER-2 overexpression, often as a consequence of gene amplification, is observed in 20% to 30% of breast cancers and correlates with poor prognosis (3, 4). The elevated levels of HER-2 detected in many breast tumors (up to 100-fold higher than in normal mammary tissue) and the accessibility of the receptor from the extracellular space makes HER-2 a suitable candidate for the development of targeted therapies. Herceptin, the humanized recombinant monoclonal antibody against HER-2, is currently used for the treatment of HER-2-positive breast cancers (5). In preclinical studies, Herceptin has been shown to neutralize HER-2 activity as well as elicit antibody-dependent cell cytotoxicity (6). However, it has been noted that HER-2 neutralization does not always translate into inhibition of tumor growth. The mechanisms by which many HER-2-positive tumors escape anti-HER-2-directed therapy are still not fully defined. Growth factor receptor signaling redundancy created by the activity of the insulin-like growth factor-I receptor and EGF receptor family members may help to explain, in part, resistance to HER-2 neutralization (7–9). More recently, resistance to Herceptin has been associated with decreased levels of p27kip1 and PTEN deficiency (10, 11). Thus, it has been proposed that combination-targeted therapies may be required to bypass resistance to anti-HER-2 monotherapy (12). In the present work, we asked whether it would be possible to overcome these barriers by engineering a "bifunctional peptide" capable to neutralize HER-2 and to deliver a toxin to disable mitochondrial function in tumors overexpressing HER-2. To this end, we created a hybrid peptide, BHAP (for bifunctional, HER-2-blocking and apoptosis-inducing peptide), composed of the previously described anti-HER-2 peptide (AHNP) fused to a mitochondriotoxic, proapoptotic peptide (PAP). AHNP is an exocyclic peptide designed to mimic the CDR3 loop of Herceptin (13, 14). Previous work has established that AHNP exhibits cytostatic effect towards HER-2-overexpessing tumors in vivo, similar to that of Herceptin. The mitochondriotoxic PAP domain of sequence (KLAKLAK)2 is a synthetic peptide originally developed to enhance the activity of a natural antimicrobial peptide (15). This class of polypeptides that preferentially permeabilizes bacterial membranes rich in anionic phospholipids have also been shown to affect mitochondrial function in vitro (16, 17). The PAP selected for this study cannot efficiently permeate across eukaryotic plasma membranes and consequently exhibits low mammalian cell cytotoxicity. However, when coupled to selective targeting domains, (KLAKLAK)2 is internalized by cells, induces mitochondrial damage, and triggers apoptosis (18). This approach has been successfully employed to target angiogenic endothelial cells and the vasculature of white fat (18, 19). It has been proposed that the cationic and amphipatic nature of PAPs drives the alignment of the positively charged peptide surface with the negatively charged mitochondrial membrane, affecting its electro-elastic properties and compromising the organelle's biological function (20). A unique feature of BHAP is that the anti-HER-2 peptide domain serves both to selectively target the toxin and to simultaneously neutralize HER-2 in cells overexpressing the protein.

BHAP is the first hybrid peptide of its class, intentionally designed to perturb two essential cellular functions such as growth factor receptor signaling and mitochondria activity. Our results indicate that the engineered peptide is selectively internalized by human breast cancer cells through HER-2-mediated endocytosis and induces apoptosis in vitro and in vivo, and that HER-2 overexpression is sufficient to render tumor cells sensitive to the fusion peptide. BHAP was effective against the HER-2-overexpressing human breast cancer cell lines tested, even those like MDA-MB-453 and MDA-MB-361 that have been previously described as Herceptin resistant (21), illustrating the potential therapeutic application of BHAP. At the molecular level, the chimeric peptide is capable of inactivating HER-2 as well as inducing mitochondria damage, yet the broad spectrum efficacy of BHAP against HER-2-positive tumor cells primarily correlates with its mitochondriotoxic effect. In addition, to increase the avidity of the peptide for HER-2, we have created tetramers through streptavidin-binding of biotin-labeled BHAP with improved in vitro efficacy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Linear peptides were synthesized as carboxy-terminus amides and purified to 90% to 95% in the W.M. Keck Facility (Yale University, CT). The anti-HER-2 peptide, AHNP, of sequence YCDGFYACYMDV (from NH2 to COOH terminus), and the PAP of sequence KLAKLAKKLAKLAK were linked through a diglycine linker to produce BHAP of sequence (AHNP)-GG-(PAP). The PAP domain was synthesized using D-amino acids to minimize proteolytic degradation (22). BHAP derivatives with fluorescein- and biotin-labeled COOH terminus were also synthesized. AHNP and BHAP were cyclized by air oxidation as described (14). Cyclized peptides were lyophilized and subjected to matrix-assisted laser desorption/ionization-mass spectrometry to determine their purity. Anti-phospho-HER-2, anti-HER-2, and anti-Src were purchased from Upstate Biotechnology, Inc. (Waltham, MA). Antibodies against mitogen-activated protein kinase (MAPK) and phospho-MAPK, PKB, and phospho-PKB, PLC-{gamma}1, and phospho-PLC-{gamma}1 were obtained from Cell Signaling (Lake Placid, NY). Anti-Src [pY418] was purchased from Biosource International (Camarillo, CA). Unless specified, reagents used for flow cytometry were obtained from BD Biosciences (San Jose, CA).

Cell culture and generation of stable cell lines. Human breast cancer cell lines SKBR-3, BT474, MDA-MB-453 (MDA-453), MDA-MB-361 (MDA-361), MDA-MB-231 (MDA-231), MCF-7, and human mammary epithelial MCF-10A cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM, 10% fetal bovine serum at 37°C/5% CO2. The HER-2 cDNA was subcloned into pcDNA-3 (Stratagene, La Jolla, CA) as follows. ApaI-digested pcDNA-3 was filled in with T4 DNA polymerase and subsequently digested with XhoI. The SalI/DraI fragment containing full length HER-2 cDNA was excised from pBR322/HER-2 (23) and was ligated into the linearized pcDNA-3. Transfection of MDA-MB-231 cells was done using Fugene Reagent according to manufacturer's protocol (Roche, Indianapolis, IN). Finally, stable clones were selected in medium containing G418 at 0.5 mg/mL.

Cell lysate preparation and immunoblotting. Cells were washed with PBS and lysed in buffer (40 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/L sodium pyrophosphate, 2% NP40, 10 mmol/L NaF, 2 mmol/L EDTA, 5 µmol/L Na3VO4.) containing Complete protease inhibitor cocktail (Roche). Insoluble material was removed by centrifugation. Protein concentration was determined on the supernatants using Bradford reagent (Bio-Rad, Hercules, CA). Protein lysates were resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA) in Towbin's transfer buffer (25 mmol/L Tris, 190 mmol/L glycine, 20% methanol, 0.005% SDS). Membranes were blocked with 1.5% bovine serum albumin (BSA) in TBST [150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), 0.3% Tween 20] and incubated with primary antibodies (1 µg/mL) and horseradish peroxidase–conjugated secondary antibodies in TBST/0.2% BSA. Membranes were subjected to chemiluminescence detection (Pierce, Rockford, IL). Quantification of band densities was done using the public domain NIH Image program (developed at the U.S. NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Effect of fusion peptide on cell proliferation. To monitor cell proliferation a few modifications were introduced to a previously described bromodeoxyuridine (BrdUrd) incorporation–based assay (24). Cells (5,000/40 µL) were seeded in a white 384-well plate (Costar plates; Corning Life Sciences, Acton, MA). Untreated or peptide-treated cells were incubated at 37°C with 5% CO2 for 24 hours. BrdUrd was added to a final concentration of 20 µmol/L to the cells 14 hours before fixation. Detection of BrdUrd incorporation was done with 0.5 µg/mL of mouse anti-BrdUrd antibody (Pharmingen-BD Biosciences, San Jose, CA) and 1:5,000 dilution of horseradish peroxidase–conjugated anti-mouse IgG, followed by addition of the enhanced chemiluminescence reagent (Pierce). Chemiluminescent signal from each plate was detected by autoradiograph and luminometer (Lmax; Molecular Devices, Sunnyvale, CA) to quantify results.

Detection of apoptosis by flow cytometry. Untreated and peptide-treated human breast cancer cells were fixed and subjected to terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assay with ApoBrdU following manufacturer's procedure. Peptide detection was done with anti-fluorescein conjugated to AlexaFluor 488 (Molecular Probes-Invitrogen, Eugene, OR). Analysis of labeled cells was done using the FACSCalibur and Cell quest software (Becton Dickinson, Franklin Lakes, NJ).

Detection of cytochrome c release. Mitochondria were isolated from SKBR-3 and MDA-MB-231 cells by differential centrifugation in ice-cold mito buffer [0.25 mol/L sucrose, 10 mmol/L Tris-HCl (pH 7.4), 0.1 mmol/L EGTA] as described (25). In vitro release of cytochrome c from mitochondria was done as follows (26). Mitochondria (50 µg) were left untreated or were treated for 1 hour with BHAP or AHNP (10 and 50 µmol/L) in 0.1 mL mito buffer, and pelleted at 14,000 rpm for 5 minutes. Quantification of cytochrome c in supernatant fraction was done by anti-cytochrome c ELISA assay following manufacturer's procedure (R&D Systems, Minneapolis, MN). Release of cytochrome c from mitochondria to the cytosol from untreated or SKBR-3 and MDA-MB-231 cells treated as indicated was done as previously described (27). The mitochondria- and cytosol-containing fractions were analyzed by Western blot. Immunodetection was done with mouse monoclonal anti-cytochrome c antibody (PharMingen) and anti-manganese superoxide dismutase (anti-MnSOD; Stressgen Biotechnologies, San Diego, CA).

Immunofluorescent localization of cytochrome c and peptide. Untreated or peptide-treated cells were fixed with ice-cold 3.7% paraformaldehyde at room temperature, permeabilized with ice-cold 0.01% (v/v) NP40 for 20 minutes, and incubated with 0.5% BSA/PBS for 15 minutes. Fluorescein-labeled peptide was detected with rabbit anti-fluorescein-AlexaFluor 488 (Molecular Probes) and cytochrome c with monoclonal anti-cytochrome c antibody (PharMingen) and TRITC-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA) in PBSTB. Cells were photographed under Axioskop microscope using a Spot Camera (Diagnostic Instruments, Inc., Sterling Heights, MI).

Detection of peptide internalization and mitochondrial accumulation by electron microscopy. Untreated or peptide-treated cells were harvested and fixed for 4 hours at room temperature in 0.1 mol/L sodium phosphate buffer (pH 7.4) containing 4% paraformaldehyde. Ultrathin cryosections were then stained with anti-fluorescein antibody followed by protein A conjugated to 10-nm gold particles as previously described (28). The grids were examined using a JEOL 1200EX transmission electron microscope.

Determination of body distribution and toxicity of the fusion peptide. Mice were treated with fusion peptide at doses ranging from 1 to 50 mg/kg. Liver, heart. kidney, lung, spleen, skeletal muscle, and blood were collected from mice 24 hours after injection. Evidence of toxicity was determined by examination of tissue sections. Concentration of peptide in plasma was determined by liquid chromatography-mass spectrometry (LC/MS, Waters-MicroMass) against peptide standards. Plasma samples were processed with Montage albumin depleting kit and concentrated with ZipTip containing C18 reverse-phase media (Millipore). Peptide was eluted in 50% acetonitrile/0.1% formic acid solution and analyzed. Peptide accumulation in tumors was analyzed by flow cytometry. Cell suspensions were prepared from tumors dissected from vehicle or BHAPF-treated mice 1 hour post-injection. Samples were fixed and coincubated with anti-HER-2-APC conjugate and anti-fluorescein-AlexaFluor 488 conjugate. Cells gated on HER-2, which represent the human carcinoma cell population within the tumor cell samples, were analyzed for the presence of labeled peptide using the FACSCalibur (Becton Dickinson).

In vivo efficacy in xenograft models. Human breast cancer cells were resuspended to 5 to 7 x 106 cells/100 µL in PBS/20% matrigel (BD Biosciences) and injected into mammary gland fat pads of 6- to 8-week-old CB.17-SCID female mice (Taconic Farms, Germantown, NY). To support growth of BT474 tumors, mice were implanted with 17ß-estradiol pellets (Innovative Research of America, Sarasota, FL; ref. 29). Tumors were measured with a caliper and tumor volume calculated using the formula: volume (mm3) = width2 x length / 2. Treatment began when tumors reached a volume of >200 mm3. Fusion and control peptides as well as vehicle were given as described in the text. Mice were monitored weekly for weigh loss and tumor progression. All animal studies were approved by the Institutional Animal Care and Use Committee at Harvard Medical School.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BHAP inhibits growth of HER-2-overexpressing human breast cancer cells. The effect of BHAP (Fig. 1A) on cell growth was tested on a panel of human breast cancer cell lines using a cell-based proliferation assay. Cells with diverse (high, medium, and normal) HER-2 expression profile were selected for this purpose (Fig. 1B). The panel included SKBR-3, BT474, and Herceptin-resistant MDA-361 and MDA-453 cells. MDA-231 breast cancer cells, like the nontransformed mammary epithelial MCF-10A cells, express low (normal) HER-2 protein levels and were included in experimental group for comparison. The effect of BHAP on cell proliferation was monitored by BrdUrd incorporation. A representative cytoblot assay for some of the cell lines tested is shown (Fig. 1C). Chemiluminescent signals were quantified and IC50 determined for BHAP responder cells among the panel examined (Fig. 1D). BHAP treatment resulted in a dose-dependent inhibition of cellular proliferation, and the degree of inhibition correlated with the level of HER-2 overexpression. Unlike AHNP-induced growth inhibitory effect that is limited to cells with the highest HER-2 protein level, BHAP blocked proliferation of all HER-2-overexpressing cell lines tested within the concentration range of the assay. Former studies have established that the low toxicity of the PAP against mammalian cells is a consequence of its inability to traverse the plasma membrane. Indeed, cells incubated with PAP alone were unaffected by the treatment. The fusion of the proapoptotic domain to AHNP improved its potency and expanded its spectrum of action towards MDA-453 and MDA-361, whereas retaining its selectivity for cells with higher than normal levels of HER-2. Furthermore, MDA-231 cells that were resistant to BHAP treatment due to low levels of HER-2 expression became sensitive to the peptide upon HER-2-overexpression (Fig. 1E). Altogether, this data implies that HER-2-overexpression is necessary and sufficient to confer sensitivity to BHAP.



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  		Figure 1. Determination of BHAP efficacy on a panel of human breast cancer cell lines. A, schematic representation and primary sequence of the BHAP peptide. Cysteines form an intramolecular disulfide bridge. B, whole cell lysates were prepared from a panel of human breast cancer cell lines and for comparison, from immortalized nontransformed mammary epithelial MCF-10A cells. HER-2 protein levels were assessed by Western blot. C, the growth inhibitory effect of BHAP was evaluated by cytoblot assay. Human breast cancer cell lines were treated with BHAP or control peptides as indicated for 24 hours, pulsed with BrdUrd for 15 hours, and subjected to immunodetection of incorporated BrdUrd. D, IC50 for the cell panel tested were determined by quantification of chemiluminescent signals. E, the effect of BHAP, AHNP, and PAP (50 µmol/L) on the proliferation of MDA-231 cells transfected with HER-2 cDNA-containing vector (clone H22), or transfected with empty vector (clone Hc11) was tested as in (B). Cytoblot (left) and anti-HER-2 Western blot (right).

 
BHAP is internalized by receptor mediated endocytosis and localizes to mitochondria. The results from the cytoblot assay confirmed that PAP alone is not toxic to HER-2-overexpressing human cells unless PAP is coupled to the HER-2-targeting peptide.

In control studies BHAP but not AHNP was detectable by LC/MS analysis in samples prepared from mitochondria of peptide-treated SKBR-3 and BT474 cells (data not shown). To examine the internalization of BHAP, we used a fluorescein-labeled form of this peptide, BHAPF. In the proliferation assay, the activity of the labeled peptide was identical to that of BHAP. Cells treated with BHAPF were analyzed by immunofluorescence microscopy, using anti-fluorescein-Alexa Fluor 488 conjugates to amplify the signal. A punctate fluorescent signal spreading throughout the cytoplasm was observed when HER-2-overexpressing cells were exposed to peptide for 30 minutes (Fig. 2A). Preincubation of cells with anti-HER-2 antibodies prevented internalization of BHAP (Fig. 2B). To assess whether BHAP entered by receptor-mediated endocytosis, cells were treated with receptor internalization inhibitors before incubation with the labeled peptide (30). Pretreatment with 5 µmol/L phenylarsenoxide for 30 minutes or 450 mmol/L sucrose for 5 minutes effectively blocked internalization of the peptide, and a weak fluorescent signal remained restricted to the external periphery of the cells (Fig. 2B). These results are consistent with BHAP entering the cells through HER-2-mediated endocytosis.



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  		Figure 2. Detection of BHAP internalization by immunofluorescence microscopy. A, sensitive SKBR-3 and resistant MDA-231 cells were treated with 10 µmol/L BHAPF. BHAPF immunofluorescence (top) and DNA (bottom). B, SKBR-3 cells were pretreated with anti-HER-2, phenylarsenoxide, and sucrose as indicated in the text and BHAPF was subsequently added to the medium.

 
Immunofluorescence microscopy did not provide sufficient detail to conclude the fate of the peptide once endocytosed. For that reason, the internalization of BHAPF was monitored by immunogold electron microscopy. HER-2-overexpressing breast cancer cells were left untreated or treated with the peptide and fixed in glutaraldehyde/paraformaldehyde-containing buffer at various times thereafter. Samples fixed upon 3 to 5 hours of incubation showed gold particles localizing to the plasma membrane, subcompartments of the endocytic pathway such as vesicles, endosome, and multivesicular bodies (31) and to a lesser extent to mitochondria (Fig. 3A, I-IV). Analysis of samples subjected to 8 to 16 hours of incubation showed extensive labeling of mitochondria, with gold particles frequently decorating areas of mitochondria fusion/fission (Fig. 3A, V). Electron microscopy analysis of glutaraldehyde-fixed samples, that better preserves membranes, reveals loss of internal organization and collapse of inner mitochondrial membrane following BHAP treatment (24 hours; Fig. 3B). On the other hand, no significant immunogold labeling or alterations of mitochondria morphology were detected in low HER-2-expressing MDA-231 cells. Thus, in sensitive HER-2-overexpressing cells an increase in peptide accumulation correlates with destabilization of mitochondria membranes.



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  		Figure 3. Detection of BHAP intracellular trafficking and mitochondrial alterations by electron microscopy. A, ultrathin cryosections prepared from cells treated BHAPF (10 µmol/L) for 3 hours were subjected to anti-fluorescein immunodetection followed by protein A-gold. Selective labeling of endosome (multivesicular bodies; I), vesicles (II), and mitochondria (III and IV) with gold particles (arrowheads) in HER-2-overexpressing cells. Increase labeling of mitochondria is observed upon 8 hours of incubation in peptide-containing medium (V). B, mitochondria morphological alterations observed in BHAP-treated cells subjected to glutaraldehyde fixation. Bars, 0.1 µm.

 
BHAP inhibition of HER-2 signaling may contribute but it is not sufficient to explain its broad spectrum anticarcinoma activity against HER-2-overexpressing tumor cell lines. Like Herceptin, the peptidomimetic AHNP exerts a growth inhibitory effect on some HER-2-overexpresing cell lines (13). The antitumor effect that follows Herceptin treatment has been attributed to antibody-mediated cell cytoxicity and to neutralization of HER-2 activity (6). In preclinical studies, inhibition of tumor cell proliferation by Herceptin has been associated with a variety of effects including receptor internalization and endocytic degradation, as well as direct blockage of HER-2 activity and downstream signaling. Therefore, we examined the effect of both AHNP and BHAP on HER-2 and on three major signal transduction pathways activated by this tyrosine kinase receptor. Treatment with the anti-HER-2 peptide AHNP for 3 hours resulted in a comparable decrease in HER-2 protein levels (~61-68%) and a uniform drastic tyrosine dephosphorylation (92-95% decrease) in all the cell lines tested (Fig. 4). However, AHNP could only suppress phosphorylation of the downstream signaling partners PKB/Akt, MAPK, PLC-{gamma}1, and Src in two of the four HER-2-overexpressing cell lines tested (i.e., SKBR-3 and BT474 cells). Moreover, AHNP anticarcinoma activity was also restricted to those two cell lines. As previously suggested, it is possible that MDA-361 and MDA-453 may represent examples of HER-2-overexpressing cell lines that do not solely rely on HER-2 signaling for proliferation due to, for instance, growth factor receptor signaling redundancy (7–9, 32). BHAP effect on signaling was comparable to that of AHNP (Fig. 4). However, as shown earlier BHAP is not only more potent than AHNP (lower IC50 in SKBR-3 and BT474 cells), but this peptide was effective against AHNP- and Herceptin-resistant MDA-453 and MDA-361 cells. Altogether, these results suggest that fusion of the toxic PAP domain to the HER-2-neutralizing AHNP may represent an effective way to overcome resistance to receptor neutralization strategies. Down-regulation of HER-2 signaling may result from BHAP treatment and may contribute to its anticarcinoma effect. However, the broad spectrum efficacy of BHAP is most likely a consequence of the mitochondrial damage in target cells.



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  		Figure 4. Effect of BHAP treatment on HER-2 and downstream signaling. Whole cell lysates were prepared from human breast cancer cell lines left untreated or treated for 3 hours with 10 µmol/L BHAP and AHNP for a comparison. Aliquots containing equivalent amounts of protein were subjected to immunoblot analysis using antibodies against HER-2, PLC{gamma}-1, PKB, MAPK, and Src as well as antibodies against the phosphorylated forms of the proteins.

 
BHAP affects mitochondria in vitro and induces apoptosis in HER-2-overexpressing cells. The proapoptotic domain of our fusion peptide (KLAKLAK)2 belongs to a class of compounds that induces apoptosis via a direct effect on mitochondria. A key event following mitochondria permeabilization during the course of the apoptotic response is the release to the cytosol of proapoptotic proteins, such as cytochrome c, normally stored in the intermembrane space (33, 34). This event is in turn followed by the activation of cysteine aspartyl proteases (caspases) as well as endonucleases that execute the cleavage of specific protein substrates and of genomic DNA. PAP has been previously shown to induce release of cytochrome c from mitochondria and loss of mitochondrial potential in vitro (18). To determine if BHAP behaved similarly, cytochrome c release from isolated mitochondria in response to the fusion peptide was quantified by ELISA assay. A significant redistribution of cytochrome c from the mitochondria pellet to the supernatant was observed upon both PAP and BHAP treatment (Fig. 5A). In contrast, incubation of mitochondria in AHNP-containing buffer did not result in cytochrome c release. These results indicate that AHNP has no effect on mitochondria membrane unless fused to PAP. Death of HER-2-overexpressing cells in response to BHAP treatment exhibits the typical hallmarks of apoptotic demise (i.e., cell shrinkage and nuclear condensation partially blocked by the pan-caspase inhibitor z-VAD-fmk; data not shown). We also examined the localization of cytochrome c following BHAP treatment of tumor cell lines. Treatment of sensitive cells with BHAP for 8 hours resulted in release of cytochrome c from mitochondria to the cytosol as determined by anti-cytochrome c Western blot analysis (Fig. 5B). Quantification of the signals is consistent with a 60% reduction of cytochrome c in the mitochondria fraction of BHAP-treated SKBR-3 cells. Similar results were obtained upon treatment with the PKC inhibitor staurosporine (2 µmol/L; data not shown). On the other hand, no release of other mitochondrial proteins such as manganese superoxide dismutase (MnSOD) was detectable in the same samples. These results were further confirmed by immunofluoresence analysis of untreated cells or cells exposed to fluorescein-labeled BHAP followed by simultaneous detection of the peptide and cytochrome c (Fig. 5C). Moreover, following peptide treatment oligonucleosomal DNA fragmentation was only detectable by TUNEL assay in cells overexpressing HER-2 (Fig. 5D; top right-hand quadrants). Unlike the nonselective mitochondriotoxic effect of PAP and BHAP on in vitro isolated mitochondria, BHAP merely triggered cytochrome c release from mitochondria of tumor cells in a HER-2-dependent manner. These results along with the electron microscopy studies are consistent with the notion that following selective HER-2-mediated endocytosis of BHAP, peptide molecules reach the mitochondria and selectively trigger apoptosis in HER-2-overexpressing breast cancer cells.



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  		Figure 5. Determination of apoptosis in BHAP-treated cells. A, mitochondria (50 µg) isolated from SKBR-3 (gray columns) and MDA-231 (black columns) cells were incubated for 1 hour in 100 µL of mito buffer containing BHAP or control peptides and pelleted. Quantification of cytochrome c release was done by ELISA. B, SKBR-3 and MDA-231 cells were left untreated or treated with BHAP. Mitochondria- and cytosol-containing fractions were prepared as described in Materials and Methods and subjected to anti-cytochrome c Western blot. Loadings were normalized to cell number. C, subcellular localization of cytochrome c in BHAPF-treated cells assessed by immunofluorescence microscopy. Peptide immunofluorescence (left), cytochrome c (middle), and Hoechst nuclear staining (right). D, untreated or BHAPF-treated SKBR-3 and MDA-231 were subjected to TUNEL assay and analyzed by flow cytometry. Numbers refer to percentage of cells in each quadrant. The top right-hand quadrants represent the peptide-positive and TUNEL-positive (apoptotic) cell subpopulations.

 
BHAP inhibits tumor growth in vivo. The anticarcinoma effect of BHAP observed in vitro prompted us to test whether this fusion peptide exhibited efficacy in vivo. In preliminary studies, mice were injected i.p. with BHAP at various doses. At high doses (i.e. 50 mg/kg) of five mice treated with BHAP at 50 mg/kg one died ~24 hours post-injection. Histologic examination of tissues collected at necropsy showed signs of nephrotoxicity at this dose. No indication of rhabdomyalysis or hemolysis was detectable among samples from the treated group. Peptide accumulation in tubules and collecting ducts in the kidneys during excretion are most likely responsible for the toxic effect. Lower doses (10 mg/kg) were well tolerated. No toxic effects were detectable upon a comprehensive histologic examination of tissue sections. Blood samples were collected from mice tail veins at various times points post-injection and peptide levels in plasma were determined by LC/MS. A peak in plasma (17 µmol/L) was detected 30 minutes after injection.

The effect of BHAP on tumor growth was assessed in three tumor xenograft models. Tumors were initiated in CB.17-SCID mice by injection of HER-2-overexpressing human breast cancer MDA-453 and BT474 as well as MDA-231 cells into the fat pads of the fourth mammary glands. BHAP accumulation in tumors following i.p. injection of fluorescein-labeled fusion peptide was examined by flow cytometry. Analysis of cell suspensions prepared from tumors 1 hour post-injection showed that 35% to 75% of HER-2-positive BT474 and MDA-453 cells accumulated BHAP (Fig. 6A). BHAP was not detectable in MDA-231-derived tumors (data not shown). Thus, BHAP selectively homes to HER-2-overexpressing carcinoma cells. Peptides were given to mice bearing established tumors as follows: when tumor volume reached 200 to 300 mm3, mice were treated with 10 mg/kg daily every other day, for a total of three doses as indicated (Fig. 6B). Progression of tumor growth was monitored thereafter. For a comparison, mice were treated with AHNP and PAP alone as well. Administration of BHAP resulted in inhibition of growth of MDA-453- and BT474-derived tumors. In contrast, AHNP was only effective against BT474-derived xenografts. BHAP showed no antitumor effect on mice bearing MDA-231 initiated tumors. Thus, the selectivity of BHAP for HER-2-overexpressing tumor cells exhibited in vitro was also recapitulated in vivo. No body weigh loss in excess of 5.5% was observed. Histologic examination of sections prepared from MDA-453 and BT474-derived tumors excised 24 hours from the final BHAP dose revealed areas with condensed cells and extensive cell death (Fig. 6C). Apoptotic (TUNEL positive) cells were detected by flow cytometry in samples from BHAP-treated MDA-453 and BT474 xenografts (data not shown). The overall 4.3 and 3.8 fold decrease in tumor burden in BHAP-treated mice bearing BT474 and MDA-453 carcinoma xenografts respectively shows that the peptide effectively slowed down tumor growth. The mouse study also highlights the importance of the dual targeting effect. In the case of BT474, the 2-fold increase in the growth inhibitory activity of BHAP versus AHNP suggests that addition of the toxin further improves the outcome of the HER-2-targeted approach.



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  		Figure 6. Effect of BHAP on growth of established human breast cancer carcinoma xenografts. Tumors from HER-2-overexpressing BT474 and MDA-453 (Herceptin resistant) cells were initiated in CB.17-SCID mice as described in the text. A, cell suspensions prepared from tumors dissected from mice treated with vehicle or BHAPF 1 hour post-injection, were subjected to flow cytometry. BT474 (black) and MDA-453 (gray) samples were gated for HER-2-positive cells and analyzed for the presence of labeled peptide as indicated in Materials and Methods. B, when indicated, BHAP and control peptides were given by i.p. injection (arrows). Tumor volumes were calculated as indicated in Materials and Methods. Point, the average of five to seven mice. C, histology of xenografts isolated after 24 hours of last dose of vehicle or BHAP. Paraffin-embedded sections were stained with H&E. Photographs of representative fields of view were taken at x10 and x40. Arrows point to pyknotic nuclei, characteristic of apoptotic cells.

 
Development of BHAP tetramers with a lower IC50. Streptavidin has been widely used as an adaptor molecule to produce oligomers from a variety of proteins and peptides. Streptavidin is a tetramer with unique affinity for biotin (Kd = 4 x 10–14 M; ref. 35). We exploited the stability of the streptavidin: four biotin complex to test whether oligomerization of biotinylated-BHAP (BHAPB) could improve its activity. The BHAP tetramer can be visualized by Coomassie blue staining of samples resolved by nondenaturing polyacrilamide gel electrophoresis (Fig. 7A). The effect of the BHAP-streptavidin oligomer on the proliferation of breast cancer cells was compared with that of monomeric BHAP (Fig. 7B). For this experiment, we included MCF-7 cells that express intermediate (lower than MDA-453 and higher than MDA-231) levels of HER-2 in comparison to the rest of the panel. Like the monomer, the BHAP tetramer exhibited selectivity for the cells overexpressing HER-2. The IC50 of the oligomer for responsive cell lines ranged from 100 to 800 nmol/L, a 19- to 80-fold decrease when compared with BHAP monomer (Figs. 1D and 7C). Thus, this approach resulted in a significant improvement of the efficacy of BHAP against the HER-2-positive breast cancer cells tested.



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  		Figure 7. Determination of BHAPB tetramer IC50 for the human breast cancer cell panel. A, BHAP B and streptavidin (SAv) in the molar ratios indicated were incubated for 10 minutes. Samples were resolved in a reversed polarity native gel (pH 3.9; ß-alanine/HAc buffer) at 300 V (4°C) and stained with Coomassie blue. BHAPB tetramer is the predominant species at 4:1 ratio. B, dose-response curves correspond to cells treated with BHAPB tetramer (µmol/L). Cells were treated with SAv and BHAPB for comparison. BrdUrd incorporation was examined by cytoblot assay as described in Materials and Methods. Results were quantified and proliferation was expressed relative to the vehicle-treated cells. C, quantification of IC50 for BHAPB/SAv complex (3.7:1 molar ratio).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have linked two functional domains to produce a novel chimeric peptide BHAP. BHAP was designed as a "bifunctional peptide" both to block the HER-2 protein function and to affect mitochondria in target HER-2-overexpressing carcinoma cells. The choice of a mitochondriotoxic peptide over a mitochondriotoxic small molecule was based on the simplicity of peptide chemistry for conjugation purposes.

Mitochondria are unique organelles in that they play a central role in a plethora of biological functions essential for cell survival (36). A number of laboratories, including ours (24), have focused efforts to develop and to characterize novel small molecules and peptides that selectively obliterate mitochondrial function in tumor cells by directly targeting the organelle as a means to interfere with tumor progression (37, 38). The attractive perspective of this strategy lies in the potential to circumvent resistance to apoptosis that normally arises as a consequence of accumulation of mutations in apoptosis signaling intermediates upstream of mitochondria in a variety of cancers (39–41). It is well established that the selectivity of a toxin for tumor cell mitochondria over normal cell mitochondria can be dictated by an inherent differential trait of the organelle between the normal and transformed cellular states. Alternatively, a promiscuous and/or plasma membrane impermeable mitochondria toxin could be turned into a selective anticarcinoma reagent through targeted delivery. Our data shows that indeed it is feasible to target the plasma membrane impermeable mitochondriotoxic (KLAKLAK)2 peptide to cancer cells overexpressing the HER-2 tyrosine kinase receptor.

Characterization of the BHAP-mediated effects at the cellular level shows that the fusion peptide is selectively endocytosed by cells with high levels of HER-2. As shown by electron microscopy, once internalized the peptide traffics through the endocytic compartment. Although still not fully understood at the molecular level, the ability of polycationic and amphipatic "membrane active" peptides like (KLAKLAK)2 to escape the acidic endosome environment has been previously described (42, 43). Our results indicate that BHAP translocates endosomal/lysosomal membranes and reaches the mitochondria. The organelle's functional and structural integrity is compromised as a consequence of the PAP mitochondrial membrane-disrupting ability, triggering apoptosis in the target cells.

Besides the mitochondrial effects, BHAP directly affects HER-2 signaling. AHNP and BHAP have a similar effect on HER-2 and downstream cascades. However, unlike the limited spectrum of action of AHNP, BHAP showed anticarcinoma activity against the four HER-2-overexpressing cell lines tested. Therefore, neutralization of HER-2 and downstream proliferation promoting signaling pathways may contribute to the growth inhibitory effect of BHAP. But it is likely that the direct disrupting effect on mitochondria membrane is the general underlying mechanism of action of the fusion peptide.

We have also begun to explore the use of oligomeric forms of BHAP as a means to increase the avidity of the chimeric peptide for HER-2. For instance, tetramers with higher ligand avidity have been successfully produced from biotinylated class I MHC monomers (44, 45). Likewise, we have produced tetrameric BHAP by streptavidin binding of biotinylated monomers. The "BHAP tetramer" exhibits 19- to 80-fold lower IC50 (per mol of monomer) than BHAP against the panel of cell lines overexpressing HER-2. Further studies will determine the efficacy of the oligomeric form of BHAP in vivo.

In addition to the effects observed in vitro, the fusion peptide exhibits antitumor effect in HER-2-positive human breast carcinoma xenografts. The fact that HER-2 overexpression is sufficient to render cancer cells sensitive to fusion peptide treatment has important therapeutic implications. Our work shows that BHAP is effective against the Herceptin-resistant MDA-453 and MDA-361 cancer cell lines. Thus, the mitochondrial injury imposed by a reagent like BHAP may help to overcome the barrier encountered by therapies focused on HER-2 neutralization or by insufficient antibody-dependent cell cytotoxicity elicited by HER-2-directed antibodies. The benefits of BHAP treatment in two xenograft transplant models clearly provide proof-of-concept in vivo. The results illustrate that overexpression of HER-2 can be used as a selective entry gate for the designed peptide-based toxin. Under the dose regimen chosen for the in vivo experiments, BHAP did not completely eradicate tumors. In the future, we will test different BHAP dose schedules as well as BHAP tetramer in xenograft models.

Finally, the BHAP paradigm suggests that the development of tailored peptides with anticarcinoma activity is feasible. Like HER-2, a number of receptors overexpressed in an array of solid tumor types have already been defined. In the future, it may be possible to develop personalized medicines by creating peptide libraries that will enable customized treatment of tumors according to their molecular profile. Stable and high-affinity peptides against a number of cell surface targets continue to emerge by phage display methodology and by protein-based peptide library screening or, like AHNP, by molecular modeling after receptor-directed antibodies. Recent advances in conjugation technology may allow to reintroduce extremely potent cytotoxic small molecules that have been found too toxic for therapeutic purposes. To date, a number of antibody-immunoconjugates have been developed (46–48). In comparison with peptides, antibodies are in general more stable molecules and display exquisite affinity for their cognate antigens. However, peptides still exhibit some attractive features. The small molecular mass of a peptide increases the molar ratio of toxin to targeting agent. In addition, stable peptides with reasonable dissociation constants exhibit superior ability to diffuse across tissues, improving tumor exposure to the drug. In comparison with recombinant antibodies, the more cost-effective large-scale production of peptides may provide a viable alternative to produce peptide-based prodrug conjugates. This approach would minimize bystander toxicities providing an effective alternative to increase the therapeutic index of cytotoxic compounds for the treatment of cancer.


   Acknowledgments
 
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 Maria Ericsson for assistance with electron microscopy, Kerry Pierce for help with mass spectrometry, Janet Crawford and Jim Elliott for peptide synthesis, Dr. Roderick Bronson for providing expert advice with histopahology, and Dr. Nicholas Chester for helpful discussions.


   Footnotes
 
Note: V.R. Fantin and M.J. Berardi contributed equally to this work.

C.M. Manning is currently at the Genzyme Corporation, Framingham, MA 01701.

Received 2/ 7/05. Revised 5/ 8/05. Accepted 5/23/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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