thesis doctor of philosophy duffy ciara evelyn lilly 2020 part 2

Chapter 3 Enhancing the breast cancer selectivity of melittin with RGD, and assessing the mechanisms of the suppression of EGFR and HER2 phosphorylation by melittin... Modulating the seq

Chapter 3

Enhancing the breast cancer selectivity of melittin with RGD, and assessing the mechanisms of the suppression of EGFR and HER2 phosphorylation by melittin

different net charge at varying pH 7 A PEP-1 melittin peptide was also previously described, in which the KRKRQQ domain of melittin was cleaved from the C-terminus 8 This was similar to another C-terminally truncated analogue of melittin which had significantly reduced binding to phospholipid bilayers compared to parental melittin 9 These large alterations to the natural structure of melittin demonstrate that the amphipathic nature is not sufficient to explain the ability of melittin to lyse cells 8 Modulating the sequence of melittin by manipulating the cationic (positively charged C-terminal) region could modulate cancer cell membrane selectivity and reduce or enhance the lytic capacity of the peptide

In the field of peptide drug delivery, cell-penetrating peptides (CPPs) are also used in order to facilitate the entry of peptides into the cell and thus enhance the bioavailability of drugs 10 CPP peptides are positively charged, cationic and/or amphipathic peptides able to interact with the cell membrane surface proteoglycans that have a high negative charge Attaching a CPP motif to another peptide may enhance the entry of the peptide into a cell either through direct membrane penetration, or by endocytosis followed by endosomal escape 11,12 Common examples of CPP sequences include the trans-activator of transcription (TAT), penetratin, and Simian Virus 40 (SV40) 10,13,14 Exchanging the C-terminal sequence 21 to 26 of melittin with a CPP sequence may enhance the cellular penetration and thus potency of melittin, or simply retain the cytotoxicity by maintaining the overall positive charge of the peptide

Melittin can be modified in silico with the goal of improving its therapeutic effect, tumour

targeting and stability Previous studies have evaluated the effects of different methods of delivering melittin to cancer cells A pH-sensitive melittin has been successfully developed by introducing a pH-sensitive amide bond between 2,3-dimethyl maleimide (DMMA) and the lysine of melittin in order to mask the positive charge of melittin 6 The original structure

of melittin was restored when this modified peptide reached tumour tissues with a slightly acidic microenvironment, achieving lower toxicity and a more controlled tumour release in HeLa cervical cancer cells Another method of targeting melittin to cancer cells involved conjugating a matrix metalloproteinase-2 (MMP2) target sequence to melittin coupled with avidin 15 The melittin-avidin conjugate was cleaved at the site of MMP2 overexpressed by melanoma, prostate and ovarian cancer cells, reducing the viability of the cancer cells but not of normal fibroblast cells Much further research and validation is required to ensure the safe and effective delivery of melittin using these targeted peptide engineering approaches

Immunoconjugates linking melittin with antibodies specific for cancer cells have also been investigated for the targeted delivery of melittin Melittin was conjugated to a monoclonal antibody specific to human myeloma and lymphoma cells, with greater cytolytic activity against the cancer cells compared to native melittin alone 16 Another synthetic analogue of melittin (peptide 101) was developed with a truncated C-terminus that did not require internalisation for activity and retained a positive charge 17 Peptide 101 was cross linked to mouse monoclonal antibodies that recognise human prostate cancer cells, and these immunoconjugates successfully inhibited tumour growth and increased survival in nude mice bearing subcutaneous human prostate cancer xenografts While melittin immunoconjugates are beneficial in cancer treatment, further refinement and sequence modulation is required to enhance the cytotoxicity delivered at the tumour site

In terms of improving breast cancer cell selectivity, melittin can be conjugated to peptides containing an Arginine-Glycine-Aspartic acid (RGD) amino acid sequence These peptides target RGD motifs (specifically TGF-β3) which are overexpressed in breast cancer tumours and their associated vasculature 18–20 Integrins regulate cell growth and differentiation, and play a role in epithelial-to-mesenchymal transition 18,21 Previous studies have attempted to enhance the cancer targeting capacity of melittin using disintegrin (which contains an RGD

site for integrin interaction) and urokinase plasminogen activator (uPA)-cleavable linkers (uPA is overexpressed and plays an important role in tumour cell invasiveness) 22–24 However, these peptide conjugates are large, and their clinical translation is hampered by challenges such as reduced binding activity and poor site-specific delivery In this chapter, engineered melittin peptides will be assessed in multiple cell lines including T11 (murine claudin-low TNBC) cells, because these cells will be used for animal studies in Chapter 4

modulating downstream oncogenic signalling pathways in breast cancer cells

The HER2 and EGFR receptors are key regulators of normal cellular processes, but also have a role in the progression and development of cancer, and confer oncogenic signalling often dependent on the PI3K/Akt/mTOR signalling pathway 25 Anti-HER2 therapies such as trastuzumab (Herceptin) have been successful clinically, however targeting EGFR in TNBC has limited success due to a lack of target accessibility and therapeutic activity, and there are issues with drug resistance to both HER2 and EGFR inhibitors 26–28 Therefore, drugs which can successfully target HER2 and EGFR without the induction of drug resistance and side effects would significantly aid in the elimination of TNBC and HER2-enriched breast cancers

HER2 and EGFR are from the human epidermal growth factor receptor (ErbB) family of receptor tyrosine kinases (RTKs) that play a critical role in cell proliferation, differentiation and motility 29–31 RTKs are single-pass transmembrane proteins with extracellular domains involved in ligand (growth factor) binding Signal transduction is mediated by RTKs across the plasma membrane through lateral dimerization in the membrane plane 29 Upon ligands binding to the extracellular domains, RTKs cross-link together into a dimeric complex in the membrane plane to form either a homodimer (with two of the same type of ErbB receptors) or heterodimer (with two different types of ErbB receptors) 30,32 The receptors cross-

phosphorylate the catalytic domains of each other within the dimer, leading to signalling cascades 33, such as through PI3K which is important in regulating the cell cycle

Receptors in the ErbB family play a crucial role in breast cancer 34, and the overexpression of these receptors is associated with a more aggressive phenotype and poor patient prognosis 35 The PI3K/Akt pathway is over-active in TNBC cells and is a major driver of reduced apoptosis, increased cellular proliferation, tumour progression and drug resistance 25,34 PI3K phosphorylates and activates Akt, activating mTOR, which is important in cell survival and proliferation Further, the over-active mTOR pathway is associated with a high rate of cellular proliferation and drug resistance 36

The capacity of honeybee venom and melittin to suppress signalling downstream of ErbB receptors has been previously demonstrated in a limited number of cancer models Melittin induced caspase-3 dependent apoptosis in human leukemic cells by downregulating Akt

signalling pathways in vitro 37 The viability, invasion and migration of MDA-MB-231 and MCF7 breast cancer cells were reduced by honeybee venom and melittin by suppressing the EGF-induced mTOR phosphorylation 38 In Lewis lung carcinoma cells, honeybee venom reduced cell viability, and inhibited the phosphorylation of Akt and MAPK thereby

suppressing tumour angiogenesis in vivo 39 It is therefore possible that honeybee venom and melittin interfere with the dimerization of ErbB receptors in order to suppress downstream signalling, and this warrants further investigation

the interaction of melittin with growth factor receptors

Bioluminescence resonance energy transfer (BRET) is a naturally occurring phenomenon present in marine organisms such as jellyfish, that has been adapted into a light based assay to be used as a pharmacological research tool 40 BRET involves the non-radiative transfer

of energy (dipole-dipole) between two proteins or molecules of interest labelled with either a donor luciferase or an acceptor fluorophore after substrate oxidation by the luciferase and subsequent emission of light 40 NanoLuc was developed from engineered luciferase found in deep sea shrimp, and is a bright luminescent protein that can be used as a bioluminescent donor molecule genetically fused to EGFR 41,42 NanoLuc can be utilized in BRET assays to monitor receptor-ligand binding 43,44 Transfer of energy from the bioluminescent donor to the fluorescent acceptor occurs over distances less than 10 nm and is indicative of interactions between the tagged molecules of interest 40 The BRET signal is determined by monitoring the ratio of light emission from the acceptor over the emission from the donor

3.2 Aim

The aim of this chapter was to determine the effects of changing the amino acid sequence of melittin, in terms of the importance of the positive charge for membrane interaction and pore formation Furthermore, the aim was to enhance the specificity of melittin for breast cancer cells by conjugating melittin to a cancer targeting RGD motif Finally, the interaction of engineered melittin peptides and honeybee venom with plasma membrane receptors was assessed, particularly the mechanisms underlying the interaction of melittin with HER2 and EGFR phosphorylation and downstream signalling pathways in breast cancer cells.

3.3 Methods

Melittin, DEDE-melittin, RGD1-melittin and SV40-melittin (explained in Sections 3.4.1 and 3.4.2) were modelled using PEP-FOLD3, with images generated using PyMOL 45

Melittin, DEDE-melittin, RGD1-melittin, SV40-melittin, EN1-mutant, biotin-melittin and biotin-DEDE-melittin were synthesized and purified by China Peptides Corporation (Shanghai, China), suspended in phosphate buffered saline (PBS) and stored at -80 ºC For certain experiments as indicated in the methods, a fluorescein isothiocyanate (FITC) fluorescent tag was conjugated to the N-terminus of melittin (FITC-melittin), DEDE-melittin (FITC-DEDE-melittin), and EN1-mutant (FITC-EN1-mutant) CellTiter-Glo 2.0 from the Luminescent Cell Viability Assay, NanoLuc-EGFR, FuGENE, and furimazine were all obtained from Promega TAMRA-EGF, the Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit secondary antibodies, and the Dynabeads M-280 Streptavidin were obtained from Invitrogen (Thermo Fisher Scientific) Hoechst, EGF and the monoclonal antibody to α-Tubulin were obtained from Sigma-Aldrich Monoclonal antibodies against ErbB2 [CB11] and EGFR [EP38Y] were manufactured by Abcam Antibodies against phospho-HER2 (Tyr1248), phospho-EGFR (Tyr1068), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-Akt (Ser473), phospho-Akt (Thr308), Total AKT, and Cleaved Caspase-3 (Asp175) were manufactured by Cell Signaling Technology The polyclonal goat anti-mouse IgG γ chain specific secondary antibody was obtained from Millipore The 12-Tube Magnetic Separation Rack was obtained from New England BioLabs (NEB) The control antibody for the ELISA (mouse monoclonal IgG antibody 28/00 8C1-6 that reacts with human IL-12) was produced at the Monoclonal Antibody Facility at the Harry Perkins Institute of Medical Research The vehicle (negative control) was cell media

3.3.3 Bee venom collection

The honeybee venom used in these experiments was collected in Perth, Australia as described in Section 2.3.2

Cells were purchased from the American Type Culture Collection (ATCC) unless stated otherwise, incubated at 37 °C and 5% CO2 and supplemented with 1% Antibiotic-Antimycotic HDFa (normal primary adult human dermal fibroblasts) was cultured in DMEM with 10% fetal bovine serum (FBS) SUM159 (human claudin-low breast cancer) was obtained from Asterand Bioscience and cultured in F-12 with 5% FBS and supplements (5 µg/mL insulin and 1 µg/mL hydrocortisone) SKBR3 (human HER2 enriched breast cancer) was cultured in RPMI with 10% FBS and 1% sodium pyruvate HEK293FT (human embryonic kidney 293 cells stably expressing the SV40 large T antigen) were obtained from Invitrogen (Australia) and cultured in DMEM with 10% FBS and supplements (1% glutamine and 0.4 mg/mL G418 Geneticin; Gibco) T11 (murine p53- claudin-low breast cancer) cells were obtained from the University of North Carolina at Chapel Hill and maintained in RPMI 1640 medium with 10% FBS The media used were manufactured by the Harry Perkins Institute of Medical Research to ATCC specifications

Cell viability was determined as described in Section 2.3.4

The anti-melittin monoclonal antibody was developed at the Monoclonal Antibody Facility at the Harry Perkins Institute of Medical Research as described in Section 2.3.5

3.3.7 Enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assays were conducted as described in Section 2.3.6

SUM159 cells were seeded onto 6-well plates at a density of 300,000 cells/well and incubated at 37 °C and 5% CO2 for 24 hours The cells were treated for 24 hours with either vehicle, the IC50 concentration of melittin or RGD1-melittin, and DEDE-melittin at the equivalent molar concentration as the IC50 for melittin Then the standard Western blot protocol was followed as described in Section 2.3.8

Glass coverslips (12 mm diameter; Menzel, Thermo Fisher Scientific) were placed in 24 well plates and coated with poly-L-lysine (Sigma-Aldrich) for 20 minutes and then washed twice with purified water TNBC (SUM159) and HER2-enriched breast cancer (SKBR3) cells were plated onto the glass slides at a density of 43,750 cells/well and incubated at 37 °C and 5% CO2 for 24 hours Cells were treated for 30 minutes with vehicle, or the IC50 of honeybee venom, melittin, RGD1-melittin, and the equivalent molar concentration as melittin for DEDE-melittin Cells were washed twice with PBS then fixed with 4% paraformaldehyde in PBS for 25 minutes, then washed again three times with PBS Non-specific antibody binding was blocked using 5% Normal Goat Serum (Thermo Fisher Scientific) in PBS for one hour at room temperature Primary antibodies were added to the cells including the mouse monoclonal anti-melittin antibody (5 µg/mL), and 1:500 of anti-EGFR for SUM159 cells, or 1:500 of anti-ErbB2 for SKBR3 cells The samples were incubated with gentle rocking at 4 ºC overnight The cells were washed three times with PBS, and then incubated with 1:500 of Alexa Fluor 488 goat anti-mouse, 1:500 of Alexa Fluor 594 goat anti-rabbit secondary antibody, and 1:5000 of Hoechst in PBS at room temperature for one hour The samples were washed three times with PBS and mounted onto glass coverslips with SlowFade

Diamond Antifade Mountant (Thermo Fisher Scientific) Slides were imaged using the confocal fluorescence Nikon Ti-E inverted microscope Images were taken using a 20x air objective (numerical aperture 0.75), and sequential excitation using wavelengths of 405 nm (Hoechst 34580), 488 nm (Alexa Fluor 488 secondary antibody), and 561 nm (Alexa Fluor 594 secondary antibody) Images were collected using NIS-C Elements Software and processed using FIJI (ImageJ) 46 at the Centre for Microscopy, Characterisation and Analysis at the Harry Perkins Institute of Medical Research

downstream pathway analysis

HER2-enriched breast cancer (SKBR3) and TNBC (SUM159) cells were plated onto 6-well plates at a density of 300,000 cells/well and incubated at 37 °C and 5% CO2 for 24 hours The cells were treated with either vehicle, or the IC50 of honeybee venom or melittin for 2.5, 5, 10, 15 and 20 minutes Except for vehicle control, all cells (including the EGF control) were treated with 20 ng/mL EGF for 2.5 minutes immediately prior to washing and lysing cells Then the standard Western blot protocol was followed as described in Section 2.3.8

Receptor-ligand interactions were assessed with BRET, using a method similar to that described previously 43,44 FITC tags were conjugated to the N-terminus of melittin (FITC-melittin) and DEDE-melittin (FITC-DEDE-melittin) HEK293 cells stably expressing the SV40 large T antigen (HEK293FT) were seeded onto 6-wells plates at a density of 550,000 cells/well for 24 hours HEK293FT cells were transfected with plasmids containing cDNA for NanoLuc-EGFR using FuGENE Briefly, plasmid cDNA was incubated for 10 minutes at room temperature with a mix of transfection reagent and serum free DMEM at a ratio of 10 ng/µL NanoLuc-EGFR : 4 µL FuGENE : 100 µL SFM The mix was added to the HEK293FT cells at a final concentration of 10 ng/µL NanoLuc-EGFR per well of the 6-