Interfering with the Dimerization of the ErbB Receptors by

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Interfering with the Dimerization of the ErbB Receptors by Transmembrane Domain Derived Peptides Inhibits Tumorigenic Growth in Vitro and In Vivo Erez M Bublil, Tomer Cohen, Christopher J. Arnusch, Adi Peleg, Gur Pines, Sara Lavi, Yosef Yarden, and Yechiel Shai Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00450 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 12, 2016

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Biochemistry

Interfering with the Dimerization of the ErbB Receptors by Transmembrane Domain Derived Peptides Inhibits Tumorigenic Growth in Vitro and In Vivo

Erez M. Bublil1*, Tomer Cohen2*, Christopher J. Arnusch2, Adi Peleg2, Gur Pines1, Sara Lavi1, Yosef Yarden1 and Yechiel Shai2# *

Both authors contributed equally

1

Department of Biological Regulation and the 2Department of Biological Chemistry, the Weizmann Institute of Science, Rehovot, Israel.

Address correspondence to Yechiel Shai, Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel. Tel: 972-8-9342711; Fax: 972-8-9344112, e-mail: [email protected] Running title: Interfering with the Dimerization of the ErbB Receptors

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Key Words: Epidermal growth factor receptor (EGFR), Transmembrane Domain, Helix-helix interaction, membrane function, molecular biology, membrane protein, biophysics, cancer therapy, inhibition mechanism, GXXXG dimerization motif.

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ABSTRACT The ErbB family of tyrosine kinase receptors is a key element in preserving cell growth homeostasis. This family is comprised of four members of a single transmembrane domain proteins designated ErbB-1 through 4. Ligand binding initiates dimerization followed by tyrosine phosphorylation and signaling, that when uncontrolled lead to cancer. Accordingly, extensive research is devoted to find ErbB-intercepting agents, directed against ErbB-1 and ErbB-2, but so far no inhibitor has targeted the transmembrane domain (TMD), which is instrumental for receptor dimerization and activation. Moreover, no antitumor agents targeted ErbB-3, which although cannot generate signals in isolation, its hetero-dimerization with ErbB-2 leads to the most powerful and oncogenic signaling unit in the ErbB family. Here, to further elucidate a role for the interactions between the TMDs of the ErbB family in cancer, we investigated peptides derived from the TMDs of ErbB-1 and ErbB-2. We then focused on the C-ter domains (B2C) and their analog, named B2C-D, that contains both D- and L-amino acids. Both peptides incorporated the distal GXXXG dimerization motif to target the TMD of ErbB-3. Our results revealed that B2C-D is highly active both in vitro and in vivo. It significantly inhibits neuregulinand EGF-induced ErbB activation, impedes the proliferation of a battery of human cancer cell lines, and retards tumor growth in vivo. Notably, combining low concentrations of B2C-D and gemcitabine chemotherapy completely arrested proliferation of pancreatic cancer cells. Biochemical and in-vitro interaction studies suggest direct interference with the assembly of the wild-type ErbB-2: ErbB-3 hetero-dimer as the underlying mode of action. To our knowledge, this is the first agent to target the TMD domains of ErbB to delay tumor growth and signaling.

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Along the line of advances in understanding and combating cancer, the ErbB family of tyrosine kinase receptors became important target molecules

1-6

. In charge of conveying a

repertoire of growth-supporting signals into the intracellular environment, the ErbB receptors are key elements in preserving cell growth homeostasis. Positioned at the critical border between signal input and biological output, ErbB-mediated signaling requires rigorous regulation

7-9

,

which when compromised may contribute to a plethora of human diseases, including cancer

10

.

The ErbB family is comprised of four members designated ErbB-1 through 4, embedded in the plasma membrane. ErbB-activation is governed by ligand binding, to initiate a combinatorial ecto-mediated dimerization 11, 12 that is then complemented by kinase dimerization and activation 13, 14

. The latter results in tyrosine phosphorylation of the cognate cytoplasmic tails, which serve

as docking sites for signaling molecules. This activation paradigm, however, is not fully shared by ErbB-2 and ErbB-3. ErbB-2 binds no soluble ligand 15 and ErbB-3 cannot generate signals in isolation,

since it

harbors

an

impaired

kinase domain

16

.

Interestingly however,

heterodimerization of ErbB-2 with ErbB-3 gives rise to the most powerful and oncogenic signaling unit in the ErbB family of receptors 17. A major question is whether direct interfering with ErbB-2:ErbB-3 dimerization can take place and whether it can be of value for the purpose of cancer therapy 18. There are many ErbB-based cancer therapeutics including small molecule tyrosine kinase inhibitors (TKIs), which penetrate through the plasma membrane to silence the kinase domain (i.e. gefitinib, erlotinib, and lapatinib)

19

, and anti-ErbB monoclonal antibodies that bind to the

ecto domain (i.e. trastuzumab and cetuximab) 20 21-23. However, all clinically available agents are directed against ErbB-1 and ErbB-2 but not ErbB-3, and although the transmembrane domains

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(TMDs) are involved in the process of dimerization, only the extracellular or kinase domains are being targeted. The TMDs of ErbB proteins

24

similarly to e.g. toll-like receptors

25, 26

, although they were

thought originally to represent a passive anchor to the membrane, actively participate in the assembly of receptor dimers. It was previously shown that homodimerization of ErbB-1 relies on TM-TM interactions 24 and that TMDs of ErbB members can spontaneously homodimerize in the membrane

27-29

. Moreover, a point mutation in the TM domain of rodent ErbB-2 was found to

enhance its transforming properties by provoking dimerization and constitutive activation

30, 31

.

Of special interest are the GXXXG-like motifs embedded within the TMDs of ErbB receptors (Fig. 1A). Earlier studies conducted on the helix dimer of the bacterial glycophorin A (GPA) have indicated a role for this motif in TMD-dimerization

32, 33

. Further studies have attributed a

role for this motif in ErbB-mediated dimerization as well 27, 28, 34, and showed that GXXXG-like motifs facilitate promiscuous ErbB TMDs interactions 35. Here, we synthesized and investigated the biological activity in vitro and in animal model of cancer, as well as a plausible mode of action of a peptide derived from the TMD of ErbB-2, which incorporated the distal GXXXG motif to target the TMD of ErbB-3. This peptide was active both in vitro and in vivo, and showed synergy with chemotherapy. To our knowledge, this is the first agent to target the TMD domains of ErbB to delay tumor growth and signaling.

EXPERIMENTAL PROCEDURES Reagents and antibodies. Rink amide MBHA resin and 9-fluorenylmethoxycarbonyl (F-moc) amino acids were purchased from Nova-biochem AG (Laufelfinger, Switzerland). N,Ndiisopropylethylamine (DIEA), dimethylformamide, dichloromethane were purchased from

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Sigma Chemical Co. (St. Louis, MO, USA). Piperidine was purchased from Biolab (Jerusalem, IL). For the in vivo experiments B2C-D peptide was purchased from Peptide 2.0 Inc. (Chantilly, VA, USA). The following antibodies were used: An antibody to the phosphorylated form of ErbB3 and another antibody to the phosphorylated form of EGFR (tyrosine 1068 of EGFR/ErbB1) were purchased from Cell Signaling (Beverly, MA, USA). We also used a general anti-phosphortyrosine antibody (denoted PY). Antibodies against phosphotyrosine and ERK were obtained from Santa Cruz Biotechnology (Santa-Cruz, CA, USA). Gemcitabinehydrochloride was purchased from Eli Lilly (Indianapolis, IN, USA). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Peptide synthesis. Peptides were synthesized with a free N-terminal and purified as previously described 27 and identified using amino acid and mass spectrometry analysis. The stock solutions of the concentrated peptides were maintained in dimethyl sulfoxide (DMSO). The final concentration of DMSO in each experiment was lower than 0.25% vol/vol in the in vitro assays or 2% vol/vol in the in vivo.

Construction of the ToxR Chimeras (see details elsewhere

36-38

). A NheI-BamHI TM-DNA

cassette encoding 16 residues corresponding to the ErbB2 C-terminal TM region (660GILLVVVLGVVFGILI675) or the ErbB2 N-terminal TM region (649SPLTSIISAVVGI LLV664) were inserted between the ToxR transcription activator and the E. coli Maltose Binding Protein (MalE) within the ToxR-MalE plasmid. The TM domain of interest is flanked by

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hydrophobic amino acids (Alanins) to generate a hydrophobic sequence of 22 residues, typical for a TM domain. Sequences were verified by DNA sequencing.

Cell viability assays. Performed as indicated in the Figure legends. All cell lines were taken from American Type Culture Collection and used within six months after resuscitation.

Phosphorylation inhibition assays. SKBR-3 cells were incubated for 4 hours in the presence or absence of the peptides (20 or 40 µM) dissolved in 2% DMSO, or with DMSO as a control. Cells were then stimulated without or with NRG (10 ng/ml) and washed with cold PBS. Thereafter, cells were detached and their lysates cleared by centrifugation, resolved by electrophoresis and then transferred onto a nitrocellulose membrane. Anti-phopsho-ErbB-3, and anti-pY were used to monitor phosphorylation and anti-ERK antibodies verified equal loading. SKBR-3 cells were plated onto a 96-well plate (6 wells for each of the control and experimental groups) 24 hours prior to six hour incubation with the B2C-D peptide (40 µM). Cell viability was then assessed using a MTT assay according to manufacturer protocol

39, 40

. EGFR phosphorylation inhibition

by B2C-D: A431 cells were pre-treated with the B2C-D peptide (40 µM) or with DMSO as solvent control for 4hrs. Then, cells were either treated with EGF (10 ng) for 10 minutes or left untreated. Cell lysates were run on an acrylamide gel and Western Blot was performed using the indicated antibodies. Band intensities were measured and the graphs represent the phosphorylated to total EGFR signal ratio.

Establishment of Gastric tumor xenografts (N87) in athymic nude mice. Female athymic nude mice were injected subcutaneously with 5*106 N87 cells to their right flank. Once tumor

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reached the volume of ~250 mm3 (for intra-tumor peptide injection) or ~45 mm3 (for intraperitoneal peptide injection) mice were randomized into a treatment and a control group (day 0, 6 mice per group). Mice in the intra-tumor treatment group were injected with 2 mg/kg body weight of the B2C-D peptide dissolved in PBS + 2% DMSO at days 0, 3, 7, 9, 15, 18 and 21. Mice in the intra-peritoneal treatment group were injected with 10 mg/kg body weight of the B2C-D peptide dissolved in PBS + 2% DMSO at days 0, 3, 6, 10, 12,18 and 21. Mice in the control group were injected with 2% DMSO alone. Tumor size was measured using a caliper according to the formula length*width*height. SEM values are indicated. Pancreatic tumor xenografts (BXPC-3): Tumors were established by injection of 1*106 cells BXPC-3 subcutaneously into the right flank of athymic female nude mice and were randomized into treatment and control groups (n=25). Only mice that showed tumor establishment and growth were used, and treatment was started once tumor reached the volume of 50-100 mm3, (day 0, n=12-16 mice per group). Mice were treated by intra-peritoneal injection with gemcitabine alone (4 mg/kg) or the B2C-D peptide alone (25 mg/kg, 200 µL, PBS + 3.5% DMSO) on days 0, 5, 8, and 12, then (12.5 mg/kg, 200 L, H2O) on days 15, 19, 21, 26, 29, 30, 33, 35, and 37. Combination treatment was a pre-mixed solution of above doses, and the control group received injections of PBS + 3.5% DMSO or H2O as described above. Tumor size was measured using a caliper according to the formula length*width*height twice per week. All animal experiments were conducted at the Weizmann Institute of Science and approved by the Weizmann Institutional Animal Care and Use Committee (IACUC) according to the Israel law and the National Research Council guide (Guide for the Care and Use of Laboratory Animals 2010).

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RESULTS An ErbB-2 TM fragment incorporating the C-terminal (Ct), but not N-terminal (Nt), GXXXG-like motif, can homodimerize in the membrane. ErbB-2 encompasses two distinct GXXXG-like motifs embedded within its TM domain (Fig. 1A). To evaluate the contribution of each site for TM-mediated ErbB-2 dimerization, we employed the ToxR system, in which dimerization via the inserted TM component leads to ToxR activation and as a result, βgalactosidase expression (Fig. 1B). Two sequences derived from the TM domain of ErbB-2, integrating the Nt or Ct motifs (denoted ErbB-2 Nt and ErbB-2 Ct, respectively), were introduced into the ToxR construct. As shown in Figure 1C, ErbB-2 Ct induced a TM dimerization comparable to the positive control (Glycophorin A, GPA) and was superior to dimerization induced by the ErbB-2 Nt and the negative control (15 consecutive alanines, A15). These results imply that ErbB-2 TM dimerization necessitates the Ct, but not the Nt GXXXG motif. This statement seems to contradict previous data showing that the ErbB2 but not the ErbB1 constructs formed homodimers

28, 37

. Note however, that in the present study a longer

fragment was used in the ToxR assay compared with the previous one

37

. In particular, two

additional N-terminal residues, Ser-Pro, were inserted into the hydrophobic TM part of the ToxR chimera. This motif is common in kinked TM helices 41 and therefore could have been the reason for the observed lack of/slight dimerization.

The B2C-L and B2C-D synthetic peptides inhibit ErbB phosphorylation. Next, we tested whether the ErbB-2 Ct fragment could interfere with ligand-induced ErbB-3 phosphorylation. Thus, we measured Neuregulin-1 (NRG)-induced ErbB-3 activation in the breast cancer cell line SKBR-3, in the presence of selected synthetic TM peptides (Fig. 2A) derived from the TM

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domains of ErbB-1 or ErbB-2 (Fig. 1A). SKBR-3 cells were incubated without or with 40 µM of B2C-L peptide (ErbB-2 Ct fragment, consisting of all L amino acids) for 4 hours prior to stimulation with NRG, or left untreated. Control cells were incubated with 2% DMSO. ErbB-3 phosphorylation was monitored using an anti-phospho-ErbB-3 antibody. Preincubation with the B2C-L peptide, as well as with a peptide derived from the TM domain of ErbB-1 (B1-L), markedly inhibited NRG-induced receptor activation (Fig 2B). In the next step, we evaluated the capacity of a diastereomeric derivative of the B2C-L peptide (B2C-D, Fig. 2A), harboring two d-valines at positions 3 and 7, to interfere with NRG-induced ErbB-3 phosphorylation. Cells were incubated for 4 hours, with or without the B2C-L or B2C-D peptides, and stimulated with NRG as above. The B2C-D peptide was superior to B2C-L with respect to ErbB-3 inhibition (Fig. 2C). Moreover, the B2C-D peptide was also active at lower concentrations (20 µM) (Fig. 2D). The MTT assay was employed to measure cell viability in the presence of the B2C-D peptide to ensure that ErbB-3 inhibition was not due to cell death. Indeed, six hours incubation with the B2C-D peptide did not alter cell viability as compared to control cells (data not shown). We also tested the active B2C-D peptide on EGF-treated A431 cells, and an inhibition of EGFR (ErbB-1) phosphorylation was observed (Fig. 2E), probably through binding to the TM domain of ErbB-1 preventing its association with ErbB-2. Quantification showed that B2C-D strongly inhibited phosphorylation with EGF stimulation (Fig. 2F).

The B2C-D peptide significantly inhibits cell viability in vitro. In line with its capacity to effectively inhibit ErbB-mediated signaling, we evaluated the ability of the B2C-D peptide to retard cancer cell proliferation, utilizing a battery of sixteen human cancer cell lines of different origins, including breast, pancreatic, gastric, lung, prostate, ovary and epidermoid (Figs. 3A and

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3B). Cells were treated with 10 µM of the peptide for 72 hours, while replenishing the medium and inhibitors daily. Cell viability was measured utilizing the MTT assay. Of the breast cancer cell lines, BT474 and MDA468 exhibited marked growth inhibition (50%), whereas T47D and SKBR-3 were not affected (Fig 3A). Up to 40% inhibition was recorded for the RAS-mutated pancreatic cell lines, Miapaca and PancI, whereas the BXPC-3 cell line, harboring a wild-type RAS, was shown to be the most sensitive cell line, exhibiting 80% inhibition as compared to control. A mild effect was observed for the gastric N87 cell line. All cell lines of lung origin were sensitive to B2C-D with an inhibition range of 40-65%, which was comparable to the effect on PC-3 and DU145 prostate cell lines (Fig 3B). A431 cells of epidermoid origin exhibited a marked 70% inhibition. No growth inhibition was recorded for the TOV ovary cell line. Note that for most of the sixteen cell lines the levels of ErbB-3 expression are unknown and the involvement of ErbB receptors and ErbB-3 specifically was not documented. As such the use of the 16 cell lines is intended to serve as a general screen, and niether serves as a proof for ErbB-3 targeting, nor the involvement of ErbB-3 in these cancers.

The activity of the B2C-D peptide is specific and non-toxic. In order to test whether the activity of the B2C-D is due to specific targeting of growth-promoting cellular machineries, or due to non-specific membranolytic effects, we assessed the toxicity of the B2C-D peptide using an MTT assay in comparison to two peptides derived from the TAR-1’s TM region (Tar-1 F/G, KKMVLGVFALLFLIGGSLKK; TAR-1 P/S, KKMVLGVFALLPLISGSLKK). Peptide TAR-1 F/G exhibits membranolytic activity, whereas TAR-1 P/S lacks membranolytic capacity. The TAR-1 F/G, the TAR-1 P/S and the B2C-D peptides were separately added to the B2C-Dsensitive A431 and BXPC-3 cell lines, and to the resistant SKBR-3 and T47D cell lines. The

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non-specific membranolytic peptide TAR-1 F/G strongly affected the viability of all cell lines examined, whereas the TAR-1 P/S peptide did not affect the viability of any of the cell lines (Figs. 4A and 4B). The B2C-D peptide, in line with Figure 3, inhibited the growth of A431 and BXPC-3 cancer cell lines but did not inhibit the SKBR-3 and T47D cells (Fig. 4C). The selective activity of the B2C-D peptide, in contrast to the non-selective effect exhibited by the TAR-1 derivatives, suggests a specific mode of action, which is not mediated via membranolytic mode of action. To further characterize the activity of the B2C-D peptide, BXPC-3 pancreatic cancer cells were incubated with varying doses of B2C-D or TAR-1 F/G (10 nM to 1 µM), as described above. Figure 4D shows the effect of both peptides on BXPC-3 viability. The B2C-D peptide exhibited 70% and 40% inhibition using 1 µM and as low as 50 nM, respectively. In contrast, mild to no effect was recorded for the TAR-1 F/G peptide using the same concentrations, all of which were below its membranolytic threshold concentration.

The B2C-D peptide synergizes with gemcitabine in vitro. Currently, clinical intervention in cancer is through the use of chemotherapy as mono-therapy or in combination with other agents. Since gemcitabine is a clinically used chemotherapy for pancreatic cancer, we utilized the B2CD peptide in combination with gemcitabine, and measured the effect on BXPC-3 viability. To determine the IC10 and IC15 of gemcitabine, BXPC-3 cells were incubated for 72 hours in the presence of varying concentrations of the drug, and the IC10 and IC15 were determined as 0.244 ng/ml and 0.488 ng/ml, respectively (Fig. 5A). IC10 and IC15 of B2C-D were determined as 10 nM and 25 nM, respectively (Fig. 4D). BXPC-3 cells were incubated with IC10 and IC15 concentrations of gemcitabine and B2C-D, either separately or in combination for 72 hours.

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Medium containing the inhibitors was replenished daily and a MTT assay was used to measure cell viability. As shown in Figure 5B, treatment with low concentrations of gemcitabine or the B2C-D peptide alone, resulted in mild or no alteration of cell viability. In sharp contrast, combination of both reagents resulted in close to complete inhibition of cell viability (90%). We next performed the same experiment, however without the daily replenishment of drugs. IC10 concentrations of both reagents in this experimental scenario failed to exhibit inhibition of BXPC-3 viability, whereas combinations of the higher IC15 concentrations exhibited 75% inhibition of BXPC-3 viability (Fig. 5C).

Inhibition of human xenografts by the B2C-D peptide. We tested in vivo activity of the B2CD peptide in models of both human gastric and pancreatic cancers. We observed that the peptide alone inhibited growth of tumors xenografts of N87 (Fig. 6A, B), and BXPC-3 cancer cell lines (Fig. 6C). For the gastric cancer model (N87 cells), two different doses and administration routes were tested (7 injections over the first 21 days, intratumoral: 2 mg/kg, and intraperitoneal: 10 mg/kg), which resulted in inhibition of 45% and 60%, respectively. For the pancreatic cancer model (BXPC-3) either the peptide (12.5-25 mg/kg) or gemcitabine (4 mg/kg) alone resulted in a slight reduction in tumor growth (13 injections over 37 days, intraperitoneal). However, synergy was observed with the combined treatment resulting in a significant reduction of tumor growth (p=0.03) compared to the control group. Here, average tumor size was ca. 50% smaller on day 40, and rapid tumor growth was delayed by 1 week.

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DISCUSSION Targeting ErbB-1/EGFR and Her2/ErbB-2 has been at the forefront of scientific focus, and has led to the development of specific anti-cancer therapeutics. The aberrant expression and untimely activation of their kinase domains in the context of cancer marked these receptors as targets for interception and silencing. In contrast, the third member of the family, namely ErbB-3, has received less attention due to its silenced inactive kinase. Currently however, the role of ErbB-3 in tumor growth and progression is recognized and provides novel prospects for therapy. In mammary tumors, ErbB-3 is co-expressed with ErbB-2 17 and ErbB-3 down regulation was found to eradicate the transforming capacity of ErbB-2 in breast and lung cancer cell lines 42, 43. Furthermore, ErbB-3 expression was documented in 50% of ovarian cancers and correlated with reduced survival. Moreover, in approximately 25% of patients with advanced ovarian cancer, an operational NRG-1/ErbB-3 autocrine loop exists

44-46

. ErbB-3 correlates with androgen receptor

transcription as well, and contributes to androgen-independent growth in prostate cancer

47

.

Interestingly, ErbB-3 was found to be instrumental for the onset of resistance to hormone, TKI, and chemotherapy

48-50

. Accordingly, ErbB-3 seems to have an essential role in triggering and

maintaining malignant transformations. Thus, we envisioned that interfering with ErbB-3 by means of preventing its dimerization capacity, namely with ErbB-2, may potentially represent a therapeutic opportunity. The inactive kinase harbored by ErbB-3, renders kinase interception via TKIs highly unlikely to be clinically beneficial. Notwithstanding, preclinical studies have confirmed that antibodymediated interception of the extracellular domain of ErbB-3 to compete with NRG-binding, inhibits tumor growth and re-sensitizes TKI-resistant cells 46, 51. In the study presented herein we have utilized a different approach, unique due to both the target, namely the TM domain of

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ErbB-3, and the composition of the targeting agent, namely a synthetic diastereomeric (containing both D, and L-amino acids) TM-derived short peptide. Targeting the TM domain in other proteins has proven to be a powerful strategy for interruption of signaling cascades, with therapeutic benefit. Short synthetic peptides, derived from the TM domain of T-cell receptors (TCRα) were shown to inactivate T-cells, possibly by specific interactions with TCRα. In addition, short synthetic TM domains exhibited inhibitory effects in animal models of the autoimmune disease adjuvant arthritis, relieving the illness

52

.

Furthermore, plasmids encoding the full TM domains of ErbB-1 or ErbB-2 that were expressed in human cell lines provoked specific inhibition of the respective receptors following EGF stimulation 53. In the same manner, a synthetic peptide encoding the TM domain of ErbB-1 was shown to inhibit EGF-induced ErbB-1 phosphorylation 53, and specific inhibition of a pathogenic receptor tyrosine kinase was induced by its transmembrane domain 54. In addition, a recent study showed that a short peptide derived from the transmembrane sequence of HER2/ERBB2 reduces receptor phosphorylation and exhibits anticancer properties in a transgenic mouse model

55

. In

the present study, insufficient size of the hydrophobic part of the B2C fragments can make them membrane-active, which could contribute to ErbB kinase inhibition. This assumption is corroborated by the fact that dV-substitution, which increased the solubility of the peptide also enhanced its anti-tumor activity. It might be therefore possible to interpret differently the finding that the membranolytic peptide TAR-1 F/G strongly affected the viability of all cell lines examined, although less specifically and effectively than B2C-D fragment. According to the recent structural studies of a presumed inactive state of the EGFR TM domain dimer

56

, the

agents which can perturb local lipid environment surrounding the ErbB receptor would shift the receptor into an inactive state, inhibiting tumor growth.

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Some hydrophobic peptides, which are positively charged behave like cell penetrating peptides, although the exact mechanism by which they penetrate the cell wall is not known 57. In addition, the side chains of these residues may snorkel, i.e. they may bury themselves with their aliphatic part in the hydrophobic region of the lipid bilayer while positioning the charged amino group in the more polar interface i.e., the cytoplasm

58

. Thus the length of the hydrophobic chain is also

increased. In this study, we have utilized the ToxR system to identify elements within the TM domain of ErbB-2 that may be involved in receptor dimerization (Fig. 1C). We then explored the capacity of respective short peptides to affect ErbB-3-mediated signaling through interference to formation of ErbB-3-containing dimers (Fig. 2). Assembly of ErbB-3 into dimers was instigated by NRG stimulation to promote association with ErbB-2, and with ErbB-1 59. We show that the B2C-L and the B2C-D peptides containing the Ct GXXXG TM motif of ErbB-2, can significantly reduce NRG-induced phosphorylation of ErbB3 and even EGF-induced phosphorylation of ErbB1 (Fig. 2), implying that blocking the dimerization site on the TM domain may alter the capacity of ErbB receptors to dimerize with other family members. Moreover, unlike the TAR-F/G peptide, which affected the four cell lines tested, and unlike the TAR-P/S peptide affecting none of the cell lines, the B2C-D peptide inhibited the proliferation of a subset of cancer cell lines, however failed to significantly inhibit others (Fig. 4). Taken together, these data imply that the B2C-D peptide operates via a non-membranolytic, and a specific mode of action. The response to ErbB-targeting TM peptides is hard to predict. Sensitivity to the treatment may stem from ErbB1-4 expression levels, their respective basal activation and phosphorylation, mutations downstream of the ErbB system (i,e. Ras mutated cell lines are less affected than those

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with wild type Ras (Fig. 4) and more. Notably, B2C-D reduced NRG- or EGF-mediated receptor phosphorylation in SKBR-3 and A431 cells, respectively (Fig. 2), whereas growth inhibition was recorded for A431 but not for SKBR-3 cells (Fig. 3). A lower peptide concentration was used in the growth inhibition assays. It appears thus, that SKBR-3 cells are less sensitive to partial inhibition of ErbB-3 exerted by the B2C-D peptide, in concert with the fact that even higher concentration of the B2C-D peptide (20 µM, Fig. 2D) did not significantly reduce the basal and NRG-induced ErbB-3 phosphorylation. Indeed, complete ErbB-3 inhibition, by downregulation of its expression, was previously shown to significantly reduce the proliferation of SKBR-3 cells 42, 43

.

Drawbacks of TM-derived peptides that only contain L-amino acids are their low solubility and high susceptibility to proteolytic degradation, as well as their immunogenicity. Therefore, the diastereomeric B2C-D peptide is advantageous in these respects. Moreover, in this study, the B2C-D peptide was found to be more active than its L-amino acids-containing counterpart (B2CL). The BXPC-3 pancreatic cells, which exhibited the highest sensitivity to incubation with the B2CD peptide, are of special interest. Pancreatic cancer is the eighth most common cause of cancerrelated deaths, with very poor prognosis, reflecting the failure of current treatments to significantly delay disease progression

60, 61

. Gemcitabine chemotherapy is currently the most

effective treatment approved for advanced pancreatic cancer, albeit with limited efficacy

62

.

Unfortunately, combining gemcitabine with other chemotherapeutic agents showed no added value as compared to gemcitabine monotherapy

61

. However, combining gemcitabine with

erlotinib, a TKI directed against ErbB-1, showed superiority vs. gemcitabine treatment alone

63

and the combination is approved for treatment of advanced pancreatic cancer. Indeed, current

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treatments of hyper-proliferative diseases using combinations of chemotherapy and antibodybased therapies, have proven to show an advantage over monotherapies. Here we observed that the B2C-D peptide effectively synergizes with gemcitabine both in vitro and in vivo (Figs. 5B and 5C, Fig. 6C). Specifically, combination of low concentrations (IC10) of both agents resulted in almost complete inhibition of BXPC-3 proliferation in vitro and inhibition of tumor growth in vivo was also observed. Application of the B2C-D peptide as monotherapy resulted in a significant inhibition as well, in both the N87 and BXPC-3 cell lines (Fig. 6), however the effect was more profound in N87. The TMDs of ErbB receptors may have a tendency to homodimerize in the ToxR system, but in the context of the full ErbB-2 receptor expressed in cells homodimerization of ErbB-2 is rarely observed. In fact, even in the case of ErbB-2 overexpression, as shown in Holbro et al 42, it is the heterodimerization of ErbB-2 and ErbB-3 that drives tumor growth, rather than ErbB-2 homodimerization. However, it is important to note, that using the TMD of ErbB-2 is not exclusively targeting ErbB-3, but may target any dimerization partner of ErbB-2

35

, such as

ErbB-1 and ErbB-4. As shown in Figure 1, the peptides inhibit EGF-induced activation as well, which favors ErbB-1:ErbB-2 dimer formation rather than ErbB-2:ErbB-3. We conclude that the “isolated” TMD of ErbB-2 may indeed target the ErbB-2 TM domain in vivo (although we believe this has low probability), but this should not impact tumor growth nor signaling of ErbB receptors. To our knowledge, the presently described peptides are the first synthetic molecules to target the TM domain of ErbB to delay tumor growth and signaling, and the first TM peptides to inhibit NRG-mediated signaling. A significant in vivo tumor growth inhibition was observed both alone and in combination with chemotherapy. Due to the ever-growing resistance of cancers

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to chemotherapy, combined with the still unmet need for effective therapies for certain cancers, targeting the TM domains of ErbB-members may potentially be clinically beneficial.

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Footnotes Acknowledgement: This study was supported by Israel Cancer Association to Y.S and the following agencies to Y.Y.: National Cancer Institute (CA072981), the European Commission, the German-Israeli Project Cooperation, the Israel Cancer Research Fund, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, Kekst Family Institute for Medical Genetics, Kirk Center for Childhood Cancer and Immunological Disorders, the Women's Health Research Center funded by Bennett-Pritzker Endowment Fund, Marvelle Koffler Program for Breast Cancer Research, Leir Charitable Foundation, and the M.D. Moross Institute for Cancer Research. Y.Y. is the incumbent of the Harold and Zelda Goldenberg Professorial Chair, and Y.S is the incumbent of the Harold S. and Harriet B. Brady Professorial Chair in Cancer Research.

Author's contributions to the manuscript: TC and EB conceived and coordinated the study and wrote the paper. TC and EB designed, performed and analyzed the experiments shown in Figures 2,3,4,5 and 6. AP designed, performed and analyzed the experiments shown in Figure 1. CJA, GP and SL provided technical assistance and contributed to the preparation of the Figures. All authors reviewed the results and approved the final version of the manuscript. YS and YY coordinated the study and wrote the paper.

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FIGURES LEGENDS Figure 1. An ErbB-2 transmembrane fragment comprising the C-terminal, but not the Nterminal GXXXG motif can dimerize. (A) The amino acid sequences of TM domains of ErbB family members. GXXXG-like motifs are shaded grey. (B) The ToxR activator of transcription measures TM-TM interactions within the E. coli’s inner membrane. TM-TM interactions result in ToxR dimerization and the subsequent transcription of LacZ. Increased β-galactosidase activity indicates higher dimerization. (C) ToxR: β-galactosidase activity was monitored for sequences indicated in B and is normalized to the amount of the chimeric protein.

Figure 2. The B1-L, B2C-L and B2C-D synthetic peptides inhibit ErbB phosphorylation. (A) Amino acid sequences of the B1-L, B2C-L and the diastereomeric B2C-D peptides. (B) SKBR-3 cells were incubated for 4 hours in the presence or absence of the B1-L or B2C-L peptides (40 µM) dissolved in 2% DMSO, or with DMSO as a control. Cells were then stimulated without or with NRG (10 ng/ml) and washed with cold PBS. Thereafter, cells were detached and their lysates cleared by centrifugation, resolved by electrophoresis and then transferred onto a nitrocellulose membrane. Anti-phopsho-ErbB-3 and anti-ERK antibodies were used to monitor phosphorylation or verify equal loading, respectively. (C) SKBR-3 cells were incubated with the B2C-L or B2C-D peptide (40 µM) and then stimulated with NRG and treated as in B. Anti-phopsho-ErbB-3 and anti-pY antibodies were used to monitor phosphorylation and anti-ERK was used to verify equal loading. (D) SKBR-3 cells were incubated with 20 µM and 40 µM of the B2C-D and then stimulated with NRG and treated as in B. Anti-pY antibodies were used to monitor phosphorylation and anti-ERK was used to verify equal loading. (E) A431 cells were pre-treated with the B2C-D peptide (40mM) or with DMSO as control for 4hrs. Cells were

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either treated with EGF (10 ng) for 10 minutes or left untreated. Cells treated as in B using the indicated antibodies. (F) Band intensities were measured and the graphs represent the phosphorylated- to total EGFR signal ratio.

Figure 3. The B2C-D peptide inhibits cell growth in vitro. (A, B) A battery of 16 human cancer cell lines were inoculated on day 1 and 24 hours later were treated with 2% DMSO as a control or 10 µM of the B2C-D peptide. Every 24 hours the medium was replaced with fresh medium while replenishing the peptide. Cell viability was determined on day 5 utilizing the MTT assay.

Figure 4. The B2C-D is not toxic nor exhibits membranolytic capacity. Four different cancer cell lines (A431, BXPC-3, SKBR-3 and T47D) were inoculated on day 1 and 24 hours later were treated with 10 µM of TAR-1 P/S (panel A, grey column), TAR-1 F/G (panel B, grey column) or B2C-D (panel C, grey column) or 2% DMSO as a control. Every 24 hours, the medium was replaced with fresh medium while replenishing the peptides. Cell viability was determined on day 5 utilizing the MTT assay; (Panel D) BXPC-3 pancreatic cancer cells were inoculated on day 1 and 24 hours later were treated with 10 nM, 25 nM, 50 nM, 100 nM or 1µM of the B2C-D (black squares) or TAR-1 F/G (grey squares) peptides as indicated. Every 24 hours, the medium was replaced with fresh medium while replenishing the peptide. Cell viability was determined on day 5 utilizing the MTT assay.

Figure 5. The B2C-D peptide synergizes with gemcitabine in vitro. (A) IC10 and IC15 determined for gemcitabine-mediated BXPC-3 growth arrest. BXPC-3 cells were inoculated on

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Biochemistry

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day 1 and 24 hours later were treated with increasing doses of gemcitabine (0.244 ng/ml to 7.8 ng/ml). Every 24 hours, the medium was replaced with a fresh medium while replenishing the drug. Cell viability was determined on day 5 utilizing the MTT assay. (B, C) BXPC-3 cells were inoculated on day 1 and 24 hours later were treated with IC10 or IC15 concentration of the B2C-D or gemcitabine alone, or in combination. Every 24 hours, the medium was replaced to a fresh medium while replenishing the peptide (B) or left unchanged (C). Cells viability was determined on day 5 utilizing the MTT assay.

Figure 6. B2C-D peptide inhibits tumor growth in vivo. (A) Intratumor (i.t.) B2C-D treatment of mice with gastric tumor xenograft (N87 cell line). B2C-D peptide () given at a dose of 2 mg/kg, compared to vehicle control () (PBS + 2% DMSO), in N87 xenografts (n=6); (B) Intraperitoneal (i.p.) B2C-D peptide () given at a dose of 10 mg/kg, compared to vehicle control (), (PBS + 2% DMSO) (n=6); (C) Intraperitoneal (i.p.) B2C-D treatment of mice with pancreatic tumor xenograft BXPC-3. () Vehicle control. () Gemcitabine alone. () B2C-D peptide alone. () Combination B2C-D peptide and gemcitabine. (see materials and methods for treatment regime, n=12-16). * p=0.03. ** p=0.02, *** p=0.01.

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Biochemistry

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Biochemistry

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Tumor size (mm3)

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Biochemistry

2000

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Days Figure 6

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Interfering with the Dimerization of the ErbB Receptors by

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