Luận án Duffy: Ức chế EGFR/HER2 ung thư vú bằng melittin & nọc ong

SKBR3 Vehicle Honeybee venom Melittin RGD1-melittin DEDE-melittin Hoechst α-HER2 α-MELITTIN Merge Zoom Figure 3.4 | Melittin was localised in the plasma membrane of breast cancer cells overexpressing HER2 growth factor receptors treated with honeybee venom, melittin, and RGD1-melittin. Immunofluorescence images of HER2-enriched breast cancer (SKBR3) cells treated with vehicle, honeybee venom, melittin, RGD1-melittin and DEDE-melittin for 30 minutes. In blue: cell nuclei, in red: anti-HER2, and in green: anti-melittin. Merged images are shown.

The white outlines in the merged images indicate the respective regions in the zoomed images. Scale bars indicate 25 µm for all images except the zoomed images, which have scale bars of 6. Representative images are shown for each treatment.4 Honeybee venom and melittin suppressed the ligand-induced phosphorylation of EGFR and HER2 in breast cancer and modulated downstream signalling pathways in a time dependent manner The molecular mechanisms by which melittin suppresses tumour cell proliferation were next assessed. Considering the highly predominant localisation of melittin in the plasma membrane in tumour cells overexpressing EGFR and HER2, the next step was to assess whether honeybee venom and melittin interfered with growth factor receptor signalling as a mechanism of reducing the proliferation and survival of breast cancer cells.

For this reason, the hypothesis was that both honeybee venom and melittin could disrupt RTK-associated signalling pathways in EGFR and HER2 overexpressing cells by rapidly blocking the ligand- dependent activation of these receptors in the plasma membrane of breast carcinoma cells. To assess this, Western blot analysis was conducted on SKBR3 (HER2+, EGFR+) and SUM159 (EGFR+) extracts of cells previously stimulated with epidermal growth factor (EGF) and treated with the IC50 of honeybee venom or melittin from 2.5 to 20 minutes (Figure 3. Importantly, the results reveal that honeybee venom and melittin down-regulated the phosphorylation of the RTKs and modulated associated PI3K/Akt and MAPK signalling pathways in a time-dependent manner. In particular, treating SKBR3 cells with honeybee venom and melittin strongly downregulated p-HER2 (Tyr1248), p-EGFR (Tyr1068), p-p44/42 MAPK (Thr202/Tyr204), and p-Akt (Ser473 and Thr308) from 5 minutes onwards (Figure 3.

In SKBR3, there was also a slight decrease in total HER2, EGFR, and Akt protein only after 10 minutes of honeybee venom treatment, which could relate to membrane degradation and/or endosome-mediated receptor degradation 50. 83 In SUM159, p-EGFR (Tyr1068) was down-regulated by honeybee venom from 10 to 20 minutes, and melittin from 15 to 20 minutes of treatment (Figure 3. Treating SUM159 with melittin also suppressed p-Akt (Thr308) at all time points, yet upregulated p-p44/42 MAPK (Thr202/Tyr204) from 10 to 20 minutes, whereas honeybee venom upregulated p- p44/42 MAPK (Thr202/Tyr204), and p-Akt (Ser473 and Thr308) from 10 to 20 minutes. The anti-melittin antibody indicated an increasing amount of melittin present in the lysates of both cell lines over time, with a higher signal for the melittin treatment compared to whole honeybee venom in both cell lines.

The effects of honeybee venom and melittin on JAK/STAT pathway inhibitors were also assessed in TNBC (SUM159) cells because melittin was previously shown to inhibit JAK2/STAT3 signalling in ovarian cancer 52. No modulatory effects were observed after a 60-minute treatment with the IC50 of honeybee venom or melittin with or without the stimulation of 20 ng/mL EGF for 5 minutes (Supplementary Figure 3. 84 a SKBR3 Honeybee venom Melittin of treatment 2.5 5 10 15 20 α-p-HER2 Tyr 1248 185 kDa α-Total HER2 185 kDa α-p-EGFR Tyr 1068 175 kDa α-Total EGFR 175 kDa α-p-44/42 MAPK Erk1/2 Thr 202 / Tyr 204 44, 42 kDa α-p-AKT Ser 473 60 kDa α-p-AKT Thr 308 60 kDa α-Total AKT 60 kDa α-Total MELITTIN 2.85 kDa α-TUBULIN 50 kDa b SUM159 venom Melittin Minutes of treatment 2.5 5 10 15 20 α-p-EGFR Tyr 1068 175 kDa α-Total EGFR 175 kDa α-p-44/42 MAPK Erk1/2 Thr 202 / Tyr 204 44, 42 kDa α-p-AKT Ser 473 60 kDa α-p-AKT Thr 308 60 kDa α-Total AKT 60 kDa 2.85 kDa α-Total MELITTIN α-TUBULIN 50 kDa Figure 3.5 | Honeybee venom and melittin suppressed the ligand-induced phosphorylation of EGFR and HER2 and modulated downstream signalling pathways in breast cancer in a time dependent manner. Western blots showing the phosphorylation kinetics of the receptor tyrosine kinases (EGFR and HER2), downstream signalling pathways (MAPK and Akt), and total melittin after treatment with honeybee venom or melittin in (a) SKBR3 and (b) SUM159 breast cancer cells.

α-Tubulin was used as the loading control.5 Melittin interacted with EGFR in the plasma membrane in a time and concentration dependent manner Considering TNBC and HER2-enriched breast carcinoma cells are highly dependent on the activation of EGFR and HER2 for signalling and growth, bioluminescence resonance energy transfer (BRET) experiments were performed to determine whether melittin directly interfered with the binding of EGF to EGFR leading to the observed suppressed growth factor receptor phosphorylation. The NanoLuc reporter has been extensively shown to be useful in monitoring receptor-ligand binding in real time BRET assays 43,44. In this experiment, NanoLuc was used as the bioluminescent donor molecule, and genetically fused to EGFR 41,42. Kinetic and saturation BRET experiments were used to monitor the proximity of NanoLuc- EGFR with the fluorescently tagged acceptor molecules TAMRA-EGF (positive control), FITC-melittin, and the inactive FITC-DEDE-melittin (negative control).

The experiments were conducted with HEK293FT cells previously transfected with NanoLuc-EGFR fusion plasmid. 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. First, cell viability was assessed by treating HEK293FT cells with FITC-melittin and FITC- DEDE-melittin to obtain IC50 values for the subsequent experiments (Supplementary Figure 3. Then, a range of concentrations of each peptide was selected including the IC50 of FITC-melittin, with the corresponding molar concentrations of FITC-DEDE-melittin.

The kinetics of the interaction of the fluorescent peptides with NanoLuc-EGFR were assessed with BRET using a range of peptide concentrations over time (Figure 3. The BRET signal increased in a dose-dependent manner for TAMRA-EGF, FITC-melittin and FITC- DEDE-melittin. Interestingly, FITC-DEDE-melittin displayed much higher BRET ratios 86 than FITC-melittin at the same concentrations, as well as reaching maximal BRET ratios at each dose very rapidly (Figure 3. In all cases, higher concentrations of peptide correlated with a higher BRET ratio.

As an additional negative control, a non-specific peptide designed against the Engrailed 1 (EN1) transcription factor 47 (FITC-EN1-mutant) exhibited similar BRET ratios and kinetics to the FITC-DEDE-melittin, supporting the specificity of the interaction of active forms of melittin with EGFR (Supplementary Figure 3. Further experiments were required to ascertain the specificity of the binding interactions of each peptide with EGFR. To determine this specificity of melittin binding to EGFR at the EGF binding site, saturation BRET assays were conducted to assess the competition of the endogenous ligand EGF with each of the peptides binding to NanoLuc-EGFR. The BRET signal of TAMRA-EGF with NanoLuc-EGFR was saturable and significantly reduced from 2.5 nM and all higher concentrations of TAMRA-EGF when it was combined with 1 µM EGF (Figure 3.6, b, left; two-way ANOVA, F 7,28 = 38, p<0.0001) confirming that TAMRA-EGF was binding to the EGF ligand binding site in the absence of endogenous EGF.

In contrast, the BRET signal of FITC-melittin and FITC-DEDE-melittin were not saturable and were not significantly different with or without 1 µM EGF (Figure 3.6, b, middle and right; F 7,32 = 0.999, respectively), suggesting that neither melittin nor DEDE- melittin bound to EGFR at the EGF binding site. The BRET ratio of the interaction of FITC- melittin and FITC-DEDE-melittin was higher than with TAMRA-EGF, likely due to the much higher concentrations of fluorescent peptide used which were required to reach concentrations above the IC5o.6 | Melittin interacted with EGFR in the plasma membrane in a time and concentration dependent manner. (a) Bioluminescence resonance energy transfer (BRET) kinetic analysis of TAMRA-EGF, FITC-melittin and FITC-DEDE-melittin interaction with NanoLuc- EGFR in HEK293FT cells. The peptides were added after the cells were equilibrated in the reader with the NanoLuc-substrate furimazine for 5 minutes.

Data are expressed as raw BRET ratios with mean ± SEM. (c) Illustration of the proposed BRET kinetics (top) and saturation (bottom) analysis for TAMRA-EGF (left), FITC- melittin (middle) and FITC-DEDE-melittin (right) with NanoLuc-EGFR. BRET signals were recorded when the fluorescent acceptor molecules were within 10 nm of NanoLuc-EGFR. 88 An illustration to explain the BRET kinetics and saturation results is proposed (Figure 3.

This illustration demonstrates that the kinetics results (top panel) for TAMRA-EGF and FITC-melittin were similar because increasing concentrations of both fluorescent acceptor molecules came into close proximity with NanoLuc-EGFR until the recording saturated for each concentration of peptide used. This is most likely because TAMRA-EGF bound directly to NanoLuc-EGFR, and melittin inserted into and moved laterally in the plasma membrane 53 to interact with NanoLuc-EGFR in a fluid manner within a distance of 10 nm. In contrast, FITC-DEDE-melittin was not expected to enter the cell membrane due to the previously observed lack of cell reactivity to this mutant peptide, and so the same BRET levels were recorded across the hour as the peptide came within 10 nm of NanoLuc- EGFR in the extracellular fluid in a concentration dependent manner. For the saturation results (Figure 3.6, c, bottom panel), there was a significantly reduced BRET signal when TAMRA-EGF was combined with unlabelled EGF, because the latter bound to and thus blocked the EGF binding site of NanoLuc-EGFR, which interfered with the interaction between TAMRA-EGF and NanoLuc-EGFR.

In contrast, there was no change in the BRET signal for both FITC-melittin and FITC-DEDE-melittin with or without unlabelled EGF, because the latter did not block the capacity of these fluorescent acceptor molecules to interact with NanoLuc-EGFR. Overall, the BRET data indicates that melittin may be positioned at a distance within 10 nm from the RTKs without interfering with the endogenous growth factor binding site.6 Melittin indirectly interfered with RTK phosphorylation Considering melittin did not bind to EGFR at the endogenous EGF binding site, it was still unclear whether melittin bound directly to EGFR elsewhere on the receptor in order to interfere with receptor phosphorylation. To assess this, a co-immunoprecipitation experiment was conducted between biotin-melittin and NanoLuc-EGFR (Figure 3. 89 HEK293FT cells were either untransfected (wild-type) or transfected with Empty Vector or NanoLuc-EGFR fusion plasmid for 24 hours.

The cells were then treated for 10 minutes with either vehicle, biotin-melittin or biotin-DEDE-melittin using the IC50 of biotin-melittin (3.512 µM), and the co-immunoprecipitation conducted with streptavidin coated beads to pull down biotinylated melittin. Considering no band was detected in the lane where the cells were transfected with NanoLuc-EGFR and treated with biotin-melittin (as indicated by the grey arrow in Figure 3.7), these results indicate that melittin does not bind directly to EGFR, at least by 10 minutes of treatment. In summary, it is possible that melittin interacts with the plasma membrane broadly, indirectly interfering with the phosphorylation of growth factor RTKs such as EGFR and HER2 in breast cancer cells.7 | Melittin indirectly interfered with growth factor RTK phosphorylation. A co-immunoprecipitation assay was conducted to assess the association of melittin with EGFR.

HEK293FT cells were treated with media (wild-type) or transfected with Empty Vector or NanoLuc- EGFR fusion plasmid for 24 hours, and then treated with vehicle, biotin-melittin or biotin-DEDE- melittin for 10 minutes. Cell extracts were collected and subjected to co-immunoprecipitation using streptavidin coated Dynabeads, followed by Western blot with an anti-EGFR antibody. Wild-type and Empty Vector transfected cells, and vehicle and biotin-DEDE-melittin, served as controls. In the case that melittin bound directly to EGFR, a band would have been detected in the lane where the cells were transfected with NanoLuc-EGFR and treated with biotin-melittin (as indicated by the grey arrow).5 Discussion As discovered in Chapter 2, further investigation was required into the molecular mechanisms underpinning the interaction of melittin with plasma membrane receptors and downstream signalling pathways, and whether the breast cancer specificity of melittin could be enhanced.