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. 2014 Feb;16(2):105–114. doi: 10.1593/neo.14182

Efficacy of an EGFR-Specific Peptide against EGFR-Dependent Cancer Cell Lines and Tumor Xenografts1,2

Aarif Ahsan *,3, Susmita G Ramanand *,3, Ingrid L Bergin †, Lilli Zhao ‡, Christopher E Whitehead §, Alnawaz Rehemtulla *, Dipankar Ray *, William B Pratt , Theodore S Lawrence *, Mukesh K Nyati *
PMCID: PMC3978391  PMID: 24709418

Abstract

We have recently synthesized a peptide called Disruptin, which comprised the SVDNPHVC segment of the epidermal growth factor receptor (EGFR) that inhibits binding of heat shock protein 90 (Hsp90) to the EGFR and EGF-dependent EGFR dimerization to cause EGFR degradation. The effect is specific for EGFR versus other Hsp90 client proteins [Ahsan et al. (2013). Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization. J Biol Chem 288, 26879–26886]. Here, we show that Disruptin decreases the clonogenicity of a variety of EGFR-dependent cancer cells in culture but not of EGFR-independent cancer or noncancerous cells. The selectivity of Disruptin toward EGFR-driven cancer cells is due to the high level of EGF stimulation of EGFR in EGFR-dependent tumor cells relative to normal cells. When administered by intraperitoneal injection into nude mice bearing EGFR-driven human tumor xenografts, Disruptin causes extensive degradation of EGFR in the tumor but not in adjacent host tissue. Disruptin markedly inhibits the growth of EGFR-driven tumors without producing the major toxicities caused by the Hsp90 inhibitor geldanamycin or by cisplatin. These findings provide proof of concept for development of a new Disruptin-like class of antitumor drugs that are directed specifically against EGFR-driven tumors.

Introduction

Many tumors have oncogenic addiction, and the epidermal growth factor receptor (EGFR) is among the best established oncogenes [1]. The EGFR is a member of the ErbB family of transmembrane tyrosine kinases [EGFR/erythroblastic leukemia viral oncogene 1 (ErbB1)/human epidermal growth factor receptor 1 (HER1), ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4], which play critical roles in regulating cell proliferation, differentiation, and migration [2]. The major clinically approved approaches for treating EGFR-driven tumors have been the use of the EGFR-specific kinase inhibitor erlotinib or gefitinib and of the monoclonal anti-EGFR antibodies cetuximab and panitumumab. Although useful, inhibition of EGFR tyrosine kinase activity is limited by the development of resistance due to acquired mutations, such as threonine790methionine (T790M) EGFR [3,4]. Moreover, there is evidence that EGFR may contribute to the progression of cancer independent of its kinase activity [5] and that EGFR degradation is an important mechanism that regulates chemotherapyinduced cytotoxicity [6–9]. Such findings support the notion that receptor degradation is more effective in producing cytotoxicity of EGFR-driven tumors than inhibition of kinase activity alone.

Heat shock protein 90 (Hsp90) is a protein chaperone that maintains the stability of many oncogenic kinases [10,11], including ErbB2 [12,13], wild-type EGFR [14], and erlotinib/gefitinibresistant mutant EGFRs [15–17]. The kinase domain of ErbB2 forms a more stable complex with Hsp90 than with the EGFR, which undergoes more dynamic cycling with the chaperone and is less stringently regulated by the Hsp90 chaperone machinery [18]. On Hsp90 inhibition by geldanamycin (GA), ErbB2 is polyubiquitinated and rapidly degraded, whereas the EGFR is modestly ubiquitinated and more slowly degraded [12,13]. This difference in GA sensitivity is accounted for by an eight-amino acid segment that lies within the αC-β4 loop region of many protein kinases [19–21]. Swapping the eight-amino acid segments between the EGFR and ErbB2 yields the appropriate exchange of dynamic versus stable cycling with Hsp90 and the corresponding change in GA sensitivity [19]. Scrambling the sequence of the first six amino acids within this segment results in a mutant EGFR that degrades rapidly [22].

The EGFR is activated by ligand-induced dimerization [23], forming either an asymmetrical homodimer [24] or a heterodimer with another member of the ErbB2 family. Hsp90 forms a dynamic heterocomplex with the monomer [22] to restrain signaling by limiting dimer formation, as was shown for ErbB2 [19]. The eight-amino acid segment determining GA sensitivity lies in close association with the EGFR ATP-binding cleft [25] and the receiver dimerization face [24], and the unstablemutant EGFR with the scrambled sequence does not undergo EGFR-dependent dimerization and phosphorylation [22].

We postulated that this segment might participate in an intermolecular or intramolecular interaction that could be competed with a peptide containing the eight-amino acid segment (Figure W1). Accordingly, we synthesized two peptides representing the eight-amino acid segment of the EGFR, one with the wild-type sequence, which we call Disruptin, and a second eight-amino acid peptide with the sequence of the first six amino acids scrambled, which we call Scram-Peptide [22]. Both peptides were synthesized along with 11 amino acids selected from the human immunodeficiency virus-transactivator of transcription (HIV-TAT) gene to enable cellular uptake [26]. We found that Disruptin, but not the scrambled peptide, inhibits Hsp90 binding to the EGFR and causes slow degradation of the receptor in EGFR-dependent cancer cell lines in vitro [22]. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin inhibits EGFR dimerization and causes rapid degradation of the receptor. In contrast to the Hsp90 inhibitor GA, Disruptin appears to decrease the survival fraction of cancer cells in culture by a nonapoptotic mechanism.

In this work, we show that Disruptin is active against EGFR-dependent cancer cells in culture but not against EGFR-independent cancer or noncancerous cells. Disruptin causes a marked degradation of EGFR in human head and neck tumor xenografts but not in host cells outside the tumor periphery. The selectivity of Disruptin toward EGFR-driven cancer cells is due to the presence of EGF in EGFR-driven tumor cells and the lack of activation of EGFR in normal cells. Disruptin markedly inhibits tumor growth without producing the major tissue toxicities associated with the Hsp90 inhibitor GA or with cisplatin, an important drug employed against tumors of the lung and the head and neck. These findings provide proof of concept for development of a new Disruptin-like class of antitumor drugs that specifically cause EGFR degradation.

Materials and Methods

Materials

The human head and neck squamous cell carcinoma cell lines UMSCC1, UMSCC10B, UMSCC17B, and UMSCC74B were kindly provided by Dr Thomas Carey (University of Michigan, Ann Arbor, MI). The lung cancer cell lines NCI-H1975 and NCI-H3255 were provided by Dr J. A. Engelman (Massachusetts General Hospital, Boston, MA). EGFR-nullCHOcells and other cell lines were purchased from American Type Culture Collection (Manassas, VA). GA was acquired from Enzo Life Sciences (Farmingdale, NY). EGFR (sc-03) antibody was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for glyceraldehyde-3-phosphate dehydrogenase and cleaved poly(ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Danvers, MA), whereas antibodies to detect ErbB2 and ErbB3 were purchased from Neomarkers (Kalamazoo, MI). Anti-Hsp90 was from BD Pharmingen (San Diego, CA). Streptavidin and Alexa Fluor 568 and 488 reagents were from Life Technologies (Grand Island, NY).

Methods

Cell culture and clonogenic survival assay. All cell lines were grown in RPMI-1640 supplemented with 10% calf serum (Fisher Scientific, Pittsburgh, PA; Catalog No. SH3008704IR). For all in vitro experiments, cells were released from flasks using phosphate-buffered saline (PBS) containing 0.25% trypsin and 0.2 mM EDTA, and cells were plated onto culture dishes 2 days before any treatment.

Clonogenic assays were performed using a standard technique described previously [27]. Briefly, 500 cells were plated onto 60-mm dishes in triplicate, and the next day, cells were treated with Disruptin or the scrambled peptide, dissolved in distilled water. Eight to 10 days later, cells were fixed with acetic acid/methanol (1:7, vol/vol), stained with crystal violet (0.5%, wt/vol), and counted using a stereomicroscope. The fraction surviving each treatment was normalized to the survival of the control cells. Cell survival curves were fitted using the equation SF = (C50)m/[(C50)m + Cm], where SF is the surviving fraction, C is the peptide concentration, C50 is the concentration of peptide that produces a 50% cell survival, and m is the slope of the sigmoid curve.

Immunoblot analysis. Cells were scraped into PBS containing a sodium orthovanadate and protease inhibitor mixture (Roche Diagnostics Co, Indianapolis, IN). Cells were incubated for 15 minutes on ice in Laemmli buffer [63 mM Tris-HCl, 2% (wt/vol) sodium dodecyl sulfate, 10% (vol/vol) glycerol, and 0.005% (wt/vol) bromophenol blue] containing 100 mM NaF, 1 mM Na3Vo4, 1 mM PMSF, and 1 µg/ml aprotinin. After sonication, particulate material was removed by centrifugation at 13,000 rpm for 15 minutes at 4°C. The soluble protein fraction was heated to 95°C for 5 minutes, then applied to a 4% to 12% bis-tris precast gel (Life Technologies) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated for 1 hour at room temperature in blocking buffer consisting of 3% BSA and 1% normal goat serum in TBS [137 mM NaCl, 20 mM Tris-HCl (pH 7.6), and 0.1% (vol/vol) Tween 20]. Membranes were subsequently incubated overnight at 4°C with 1 µg/ml primary antibody in blocking buffer, washed, and incubated for 1 hour with HRP-conjugated secondary antibody (Cell Signaling Technology). After three additional washes in TBS, bound antibody was detected by Enhanced Chemiluminescence Plus reagent (GE Healthcare Life Sciences, Piscataway, NJ).

Detection of internalized peptide and immunostaining of tumor tissue. The UMSCC1 xenografts were cryopreserved in ornithine carbamoyltransferase (Electron Microscopy Sciences, Hatfield, PA). Five-micrometer sections were cut from tissues prepared on day 3 after peptide treatment. Sections were fixed in cold methanol for 20 minutes and then were blocked with 1% BSA. The sections were incubated overnight with anti-EGFR antibody. After washing, the sections were incubated with anti-rabbit Alexa Fluor 488 (Life Technologies) and streptavidin-conjugated Alexa Fluor 568 (Life Technologies). Coverslips were mounted with one drop of ProLong Gold antifade reagent containing 4,6-diamidino-2-phenylindole (Life Technologies) to visualize the nuclei. Fluorescence images were acquired using a DP70 camera fitted on an Olympus IX71 microscope (Center Valley, PA).

For immunofluorescence analysis of EGFR and Hsp90 expression, 5-µm thick sections were prepared from formalin-fixed, paraffin-embedded tissue. After slides were deparaffinized in xylene and rehydrated using serial ethanol dilutions, antigen site unmasking was performed by immersing slides in 100 nM citrate buffer for 20 minutes at high pressure and temperature inside a pressure cooker. Slides were then washed in PBS, blocked for 1 hour, and incubated in primary antibody at 4°C overnight. Slides were then washed again in PBS, incubated in secondary antibody for 1 hour, rewashed, and prepared with a coverslip after a drop of ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (Life Technologies) was added to each sample. Fluorescence images were acquired as mentioned above. For immunohistochemical analysis of CD31 antibody (clone JC70A; Dako, Carpinteria, CA), slides were incubated with anti-CD31 antibody overnight at 4°C (1:50). The EnVision peroxidase detection kit (Dako) was used to visualize the signal. Slides were counterstained with hematoxylin and then dehydrated to xylene before the coverslip was placed.

In vivo tumor growth studies. The in vivo studies were approved by and run in accordance with the policies of the University Committee for the Use and Care of Animals. To generate tumor xenografts, 2 x 106 UMSCC1 or NCI-H1975 cells were transplanted into the flanks of athymic nude Foxn1nu mice (Harlan Laboratories, Indianapolis, IN). When the tumors reached a volume of ∼50 mm3 (approximately 5–6 days for NCI-H1975 and 10–12 days for UMSCC1 cells), the mice were randomized into three groups (one untreated control and two experimental groups for Disruptin and scrambled peptide) containing from 15 to 26 tumors, and treatment was initiated. All the agents (except erlotinib; Genentech, San Francisco, CA) were injected through intraperitoneal (i.p.) route. Erlotinib was given orally for 5 days.

Tumor volume doubling was determined for each xenograft by identifying the earliest day on which it was at least twice as large as on the first day of treatment. A cubic smoothing spline method, which provides an estimate of the growth curve from the observed data [28], was used to obtain the time of tumor doubling. The Kaplan-Meier method was used to analyze the doubling times derived from the smoothed growth curves, and the log-rank test was used for comparisons between any two treatment groups. Results for continuous variables are presented as means ± SEM, and Student's t test was used to assess the statistical significance of differences. A significance level threshold of P < .05 was used.

Toxicity analysis and pathologic evaluation. Disruptin was evaluated for acute toxicity in immunocompetent 5-week-old C57BL/6 mice (Charles River Laboratories, Wilmington, MA), and the scrambled peptide was used as control. Cisplatin (University of Michigan Hospital, Ann Arbor, MI) and GA were used as agents that have known toxicity profiles as positive control for this experiment. Mice were injected i.p. with 10 or 30 mg/kg Disruptin, scrambled peptide, GA, or cisplatin. Three days after injection, the brain, lung, liver, heart, kidney, stomach, small intestine, cecum, colon, ovaries, uterus, bladder, spleen, mesenteric lymph nodes, thymus, left and right eyes, and sternal bone marrow were processed for histologic evaluation. The histologic evaluation was performed without knowledge of the experimental groups by a board-certified veterinary pathologist at the Pathology Cores for Animal Research in the Unit for Laboratory Animal Medicine at the University of Michigan.

Results

Selectivity of Disruptin for EGFR-Driven Cells in Culture

We began by assessing the concentration dependence (0–364 µM) of the effects of Disruptin on UMSCC1 and NCI-H1975 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumb bromide (MTT) assay (Figure W2). Both cell lines demonstrated growth inhibition at or below 10 µM concentration. Therefore, 10 µM concentration of Disruptin was selected for further experiments. The effect of Disruptin on clonogenic survival was assessed in several cell lines shown in Table W1. EGFR-driven cell lines were chosen to represent a spectrum of EGFR alterations. The three human head and neck cancer cell lines are driven by wild-type EGFR, and the two lung cancer cell lines are driven by the erlotinib-resistant T790M and erlotinib-sensitive leucine858arginine (L858R) EGFR mutants. Disruptin treatment decreased the clonogenic survival of all five of the EGFR-dependent cell lines, and the scrambled peptide had no effect. Disruptin did not affect the survival of the three EGFR-independent cell lines (Table W1).

To assess the selectivity of Disruptin on EGFR turnover, three cancer and two noncancer cell lines were treated with Disruptin or the scrambled peptide, and the cell lysates were immunoblotted for EGFR. The cells were also treated with GA as a positive control to demonstrate EGFR degradation when cycling with Hsp90 is inhibited. As illustrated in Figure 1, treatment with GA produced EGFR degradation and decreased clonogenic survival in all five cell lines. GA kills cells through an apoptotic mechanism [29], and GA induced PARP cleavage (an indicator of apoptosis) in all five cell lines, whereas Disruptin did not (Figure 1A). As we have previously reported for the UMSCC1 and NCI-H1975 cell lines [22], Disruptin caused EGFR degradation and decreased clonogenic survival in UMSCC10B and UMSCC17B cells, which are driven by overexpression of wild-type EGFR. In contrast, in SK-BR-3 breast cancer cells, which are driven by amplification of ErbB2/HER2 [30] and are not capable of inducing EGFR dimerization even in the presence of EGF [31], Disruptin did not degrade endogenous EGFR or decrease the survival fraction (Figure 1). Het1A cells are derived from human esophageal autopsy tissue by transfection with pRSV-T, and the MRC5 cell line was derived from normal human fetal lung tissue. Disruptin affected neither EGFR level nor clonogenic survival in these noncancerous cell lines (Figure 1A).

Figure 1.

Figure 1

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Selective effect of Disruptin on EGFR-driven tumor cells. (A) Effect of Disruptin on EGFR and cell survival. The indicated cancer or noncancerous cells were treated with vehicle (C), 50 nM GA, 10 µM Disruptin (Dis), or 10 µM scrambled peptide (Scr) for 72 hours. Lysates were prepared and immunoblotted for EGFR, Hsp70, cleaved PARP, and glyceraldehyde-3-phosphate dehydrogenase. Cell survival was assessed using a colony formation assay. Survival fraction data are means ± SE from the three experiments. *, significantly different from control at P < .05. (B and C) Effect of EGF stimulation on EGFR degradation in noncancer cells. (B) Het1A or (C) MRC5 cells were treated with vehicle (control), 30 ng/ml EGF, 10 µM Disruptin, or both EGF and Disruptin, and at the indicated times, lysates were prepared and immunoblotted for EGFR. The blots were scanned, and data are means ± SE for three experiments. **, significantly different from control at P < .01.

When we observed that Disruptin induced EGFR degradation only in the EGFR-driven tumor cells rather than the normal cells, we hypothesized that the well-established production of EGF by the tumor cells played a key role [32,33]. To test this hypothesis, EGFR was stimulated by EGF treatment in noncancer cells that were also treated with Disruptin. The effect on EGFR levels was determined over time (Figure 1, B and C). Disruptin treatment caused rapid loss of EGFR in the EGF-treated noncancer cells similar to that seen in EGFR-driven cancer cells. Taken together, the data of Table W1 and Figure 1 suggest that Disruptin is selective against cells that are capable of inducing EGFR dimerization in the presence of EGF.

Disruptin Effect on EGFR in Tumor Xenografts

Disruptin or scrambled peptide were injected i.p. into nude mice bearing established UMSCC1 xenografts, and after 3 days, cryosections of tumor tissue were prepared and immunostained for EGFR (green) and the biotin-containing peptide (red). As shown in Figure 2A, the biotin signal from both peptides is present in the tumor tissue, and there is a marked decrease in immunostained EGFR in the Disruptin-treated tumor versus tumor treated with the scrambled peptide. Immunoblot analysis of tumor tissue lysate shows a marked reduction of EGFR level that is not seen with the classic Hsp90 client proteins ErbB2 and ErbB3 (Figure 2B).

Figure 2.

Figure 2

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I.p. administration of Disruptin causes EGFR degradation in UMSCC1 xenografts. (A) EGFR immunostaining. Mice bearing UMSCC1 xenograft tumors were injected i.p. with 8 mg/kg scrambled peptide or Disruptin. Three days after treatment, tumors were removed, cryosections were immunostained for EGFR (green), and the internalized biotin-conjugated peptides (red) were visualized by streptavidin-Alexa Fluor 568 reagent. (B) Effect and selectivity of Disruptin treatment were evaluated by immunoblotting tumor cell lysates for EGFR and two members of the ErbB family that are classic Hsp90 client proteins. (C) Differential effect of DisruptinonEGFRin tumor versus adjacent normal tissue. One week after two injections, tumors (white arrow) along with adjacent normal tissue (yellow arrow) were harvested and immunostained for EGFR (red) and Hsp90 (green). The dotted white line in the upper panels demarcates the border between tumor and normal tissue above it.

As we found with cells in culture (Figure 1), the EGFR in the EGFR-driven tumor tissue is markedly reduced by Disruptin treatment, whereas the EGFR in normal tissue is not. In the experiment shown in Figure 2C, slices containing both UMSCC1 tumor and adjacent normal tissue were prepared 1 week after two injections of Disruptin. The EGFR immunostaining (red in top row of panels) in Disruptin-treated tumor (below the white dotted line) is markedly reduced compared to tumor tissue from control mice or mice treated with the scrambled peptide. In contrast, EGFR immunostaining in the normal tissue (above the dotted white line) has the same intensity in Disruptin-treated and control samples. A number of reports published during the 1990s noted that a variety of Hsps were expressed at higher levels in various cancers than in normal tissues, probably as a stress response to conditions of hypoxia and low pH generated in tumors [34]. The overexpression of Hsp90 in tumor versus normal tissue is clearly apparent in Figure 2C (middle row of panels).

Effect of Disruptin on Xenograft Growth

The effect of a single 8 mg/kg injection of Disruptin on UMSCC1 tumor growth is presented in Figure 3. Disruptin slowed the increase in relative tumor volume (Figure 3A) by increasing the median tumor doubling time from 4.5 to 16 days (Figure 3, B and C). A single injection of 8 mg/kg Disruptin is approximately equally effective to 50 mg/kg cetuximab or five oral doses of 100 mg/kg erlotinib against UMSCC1 xenografts (Table W2). When two 8 mg/kg injections are given 3 days apart, more profound effects on tumor growth and doubling time are obtained (Figure 4). A similar analysis of the effect of Disruptin on NCI-H1975 human lung tumor xenografts is shown in Figure W3. These tumors grew more rapidly than UMSCC1 xenografts, but the median tumor doubling time with Disruptin is nevertheless more than three-fold compared to either Scram-Peptide or 1 week of erlotinib treatment.

Figure 3.

Figure 3

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Effect of Disruptin on UMSCC1 xenograft growth. (A) The effect of Disruptin on tumor growth was assessed after a single i.p. injection of 8 mg/kg. (B) Time to tumor volume doubling was plotted for each treatment condition using the Kaplan-Meier method. (C) Comparisons between any two treatment groups were analyzed by log-rank tests. ***, significantly different from control at P < .001. Numbers in parentheses represent the lower and upper limits of doubling time.

Figure 4.

Figure 4

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Effect of a double dose of Disruptin on UMSCC1 xenograft growth. (A) The effect of Disruptin on tumor growth was assessed after two 8 mg/kg i.p. injections of Disruptin separated by 3 days. (B) Time to tumor volume doubling was plotted for each treatment condition using the Kaplan-Meier method. (C) Comparisons between any two treatment groups were analyzed by log-rank tests. ***, significantly different from control at P < .001.

To assess the effect of Disruptin on tumor histology, UMSCC1 and NCI-H1975 tumor slices were stained with hematoxylin and eosin (Figure 5A). The control and scrambled peptide samples had homogenous tumor architecture; whereas the Disruptin-treated UMSCC1 tumors had decreased tumor mass and more connective tissue, the Disruptin-treated NC1-H1975 tumors had decreased tumor cells with more necrosis. Xenograft slices were also stained with antibody against the endothelial cell adhesion molecule CD31 [platelet endothelial cell adhesion molecule (PECAM-1)] glycoprotein to assess tumor cell vascularization, which is markedly less in tumor samples from animals treated with Disruptin versus the scrambled peptide (Figure 5B).

Figure 5.

Figure 5

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Effect of Disruptin treatment on xenograft tumor histology and vascularization. (A) Histology. Mice bearing UMSCC1 or NCI-H1975 xenografts were treated with saline, scrambled peptide, or Disruptin. Tumors were harvested 1 week later and stained with hematoxylin and eosin. A representative field from each group is included. (B) Tumor slices were stained with antibody against CD31. **P < .01 and ***P < .001. Original magnification, x200.

Histopathologic Alterations in Mice Treated with GA, Cisplatin, or Disruptin

The effects of Disruptin on selected tissues are compared with those of GA and cisplatin in Figure 6. GA-treated animals developed hepatic necrosis and acute cataract formation (arrows indicate globular degeneration of lens fibers). Cisplatin-induced histologic changes were consistent with its activity against rapidly proliferating cells as evidenced by villus damage in the intestine (marked by arrows), depletion of hematopoietic elements in bone marrow (asterisk), and ovarian follicular degeneration (arrows indicate degenerating follicles). Neither Disruptin nor the scrambled peptide produced acute (3-day) histopathologic changes, and complete blood counts and liver cytosolic enzymes did not differ from control mice (data not shown).

Figure 6.

Figure 6

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Disruptin toxicity screen in mice. Female C57BL/6 mice were given i.p. injections of 10 mg/kg GA, 10 mg/kg cisplatin, 30 mg/kg scrambled peptide, or 30 mg/kg Disruptin. Three days later, histologic evaluation was carried out on brain, lung, liver, heart, kidney, stomach, small intestine, cecum, colon, ovaries, uterus, bladder, spleen mesenteric lymph nodes, thymus, eyes, and sternal bone marrow. Histologic evaluation was performed without knowledge of the experimental group by a certified pathologist at the Unit for Laboratory Animal Medicine facility at the University of Michigan. Original magnification, x600 (for lens and bone marrow) and x400 (for other tissues).

Discussion

In this study, we show that Disruptin, a 19-amino acid peptide designed to prevent Hsp90 binding to the EGFR monomer and EGFR dimerization, selectively promotes EGFR turnover and decreased survival in EGFR-dependent tumor cell lines versus EGFR-independent cancer cells and noncancerous cells. Disruptin also selectively increases the turnover of EGFR in an EGFR-driven tumor xenograft versus EGFR in adjacent normal tissue (Figure 3). To explain this extraordinary degree of selectivity, we must consider both how Disruptin works and the state of the EGFR in EGFR-dependent tumor cells.

As outlined in the model of Figure 7, in the absence of EGF, the EGFR is primarily present as the nonphosphorylated monomer, which is the form that cycles into heterocomplex with Hsp90 [22]. Hsp90 cycling with the monomer stabilizes the EGFR, and inhibition of Hsp90 binding with Disruptin causes very slow EGFR degradation [22]. Thus, in the absence of EGF, Disruptin is competing with the stabilizing effect of the Hsp90/Hsp70-based chaperone machinery in cycling Hsp90 with an otherwise unstable monomer. Hsp90 and Hsp70 are very abundant proteins, and, at the concentrations used in this work, Disruptin partly inhibits but does not block cycling with Hsp90. On EGF binding, the EGFR undergoes a conformational change in the region of the αC helix [35] adjacent to the SVDNPHVC segment comprising Disruptin such that the receptor can no longer cycle with Hsp90. In the presence of EGF, it is likely that Disruptin specifically affects the EGFR by competing with the eight-amino acid segment at the dimerization interface, which is also where Hsp90 binds. This would account for both Disruptin inhibition of Hsp90 binding to the EGFR monomer and inhibition of EGF-stimulated EGFR dimerization. Disruptin inhibits EGFR dimerization, but the EGF-bound EGFR monomer cannot be stabilized by Hsp90. Thus, in the presence of EGF, the EGFR is degraded very rapidly when Disruptin is also present [22].

Figure 7.

Figure 7

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Disruptin interaction with EGFR in presence or absence of EGF. In the absence of EGF, EGFR cycles with Hsp90. Disruptin inhibits EGFR cycling with Hsp90, but due to the abundance of the Hsp90 chaperone machinery in cells, EGFR cycling with Hsp90 is minimally affected, permitting only slow degradation of EGFR. In contrast, in the presence of EGF, EGFR forms a dimer, and the EGF-bound EGFR cannot bind with and be stabilized by Hsp90. Disruptin inhibits EGFR dimerization, leaving EGF-bound EGFR in the unstable monomeric form and resulting in rapid EGFR degradation.

In normal cells in the absence of EGF stimulation, the EGFR is predominantly present as the unphosphorylated monomer, whereas in EGFR-driven tumor cells, the receptor is activated and highly phosphorylated. This difference in EGFR phosphorylation has also been noted in paired tumor and normal tissue samples [36,37]. The EGFR-driven tumor cells produce EGF, which stimulates EGFR dimerization, whereas the non-EGFR-driven tumor cells, noncancer cells, and tumor-adjacent normal tissue are not exposed to high levels of EGF and remain relatively unaffected versus EGFR-driven cells in culture and in tumor xenografts. As we noted in our previous mechanistic study, when it comes to promoting EGFR turnover, it seems that Disruptin is more effective at inhibiting EGFR dimerization than cycling of the ligand-free EGFR with Hsp90.

The selectivity of Disruptin for EGFR-driven tumors is reflected in the lack of acute toxicity, as seen in Figure 6. As opposed to GA, Disruptin does not cause a change in the Hsp70 levels (Figure 1), demonstrating that the peptide is not an inhibitor of Hsp90 in general. This also explains the lack of acute toxicity and stands in contrast to the toxicity of the general Hsp90 inhibitor GA, which promotes the turnover of a wide variety of client proteins. We used GA as a “proof of principle” Hsp90 inhibitor to demonstrate that we could detect toxicity produced by Hsp90 inhibition, and we recognize that newer Hsp90 inhibitors such as ganetespib may be less toxic than first-generation agents [38]. However, all Hsp90 inhibitors, by their nature, affect dozens if not hundreds of normal as well as abnormal signaling molecules, which have produced unwanted toxicity and have hampered clinical development.

We and others have found that EGFR degradation increases tumor cell-specific cytotoxicity of chemotherapy and radiotherapy beyond that of EGFR inhibition alone [6,39,40]. These studies support the notion that down-regulation of EGFR, not just inhibition of EGFR tyrosine kinase activity, is an important target in cancer therapy [41,42]. As we have seen here, GA promotes EGFR degradation in both cancer cells and cells of noncancer origin, and GA and its analog 17AAG have been shown to significantly enhance both chemosensitivity and radiosensitivity [43,44]. However, as noted above, these Hsp90 inhibitors have proven to be toxic in clinical trials [45,46]. Our preliminary observations suggest that Disruptin enhances chemosensitivity of EGFR-driven tumor cells in culture, and it might prove useful in a single-injection or twoinjection (as in Figure 4) protocol at increasing tumor cell-specific cytotoxicity of chemotherapy and radiotherapy with less toxicity than the Hsp90 inhibitors.

We have shown here that Disruptin is active against an erlotinib-resistant lung cancer cell line, and we think that it should be effective against EGFR-driven cancers, including those that are driven by EGFR class III variant and are resistant to other anticancer drugs. Despite the fact that Disruptin is a peptide and there is a general aversion to peptide drugs by pharmaceutical manufacturers, its specificity and potential lack of toxicity might make it attractive as a short-term agent to provide synergistic enhancement of other therapies directed against EGFR-driven tumors. Recently, small peptides have shown efficacy in blocking the interaction of Bcl-XL with Bcl2-associated X protein (Bax) [47], p53 with mouse double minute 2 homolog (MDM2) [48], and Sal like 4 (SALL4) with its epigenetic histone deacetylase (HDAC) [nucleosome remodeling deacetylase (NuRD)] complex [49,50] in preclinical cancer therapy models. This suggests that the use of peptides, like Disruptin, that block protein-protein interactions in the cell may move into the clinic as a new approach to cancer treatment.

Supplementary Material

Supplementary Figures and Tables
neo1602_0105SD1.pdf (326.5KB, pdf)

Acknowledgments

We thank the Tissue and Histology Core of the Comprehensive Cancer Center and the Pathology Cores for Animal Research in the Unit for Laboratory Animal Medicine at the University of Michigan for help in safety analysis, necropsy, and hematological, immunohistochemistry, and histologic analyses. American Peptide Company, Inc performed the synthesis, solubility, and stability studies of the peptides used in this study.

Footnotes

1

This work was supported by National Institutes of Health grants R01 CA131290 (to M.K.N.), R01 CA160981 (to D.R.), P50 CA097248 [to Principal Investigator (PI): Wolf, Co-Investigator Project 4 to M.K.N.], P50 CA097248 (to PI: Wolf, Career Development Award under P50 CA097248 to A.A.), and a Cancer Center support grant P30 CA46592. Support was also received from the Michigan Institute for Clinical and Health Research (to M.K.N.), the James Stuart and Barbara Padnos Funds for Cancer Research (to M.K.N.), and an Alfred Taubman Scholarshi (to T.S.L.). This project is a component of the University of Michigan Medical School's Fast Forward Strategic Research Initiative. Disclosure of potential conflicts of interest: Provisional Application entitled “Inhibitors of the Epidermal Growth Factor Receptor-Heat Shock Protein 90 Binding Interaction,” filed on 20 December 2010.

2

This article refers to supplementary materials, which are designated by Tables W1 and W2 and Figures W1 to W3 and are available online at www.neoplasia.com.

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