A peptide derived from adaptor protein STAP-2 inhibits tumor progression by downregulating epidermal growth factor receptor signaling

Abstract

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anticancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.

Keywords: EGFR; STAT3; adaptor protein; antitumor peptide; prostate cancer.

Figure 1

STAP-2–derived 2D5 peptide inhibits cancer cell proliferation.A, HEK293T cells were transfected with expression vectors for the FLAG-STAP-2 PH domain and GST-EGFR cytosol domain, and then lysates were pulled down using glutathione beads at 48 h posttransfection and blotted. B–D, DU145 cells and MCF7 cells were treated with STAP-2–derived peptides at the indicated concentrations for 48 h, and then relative cell viability was measured by Cell Titer glo assay. E, DU145 cells were treated with 50 μM 2D5 peptide for 72 h. Percentage of the cells that were Annexin V and/or PI positive was analyzed by flow cytometry. F, DU145 cells were treated with 0.3 μM FITC-conjugated 2D5 and ΔR8-2D5 peptides for 30 min, and then FITC-positive DU145 cells were analyzed by flow cytometry. G, DU145 cells were treated with truncated 2D5 peptides, and then cell viability was measured as described in (B). H, various cell lines were treated with 2D5 peptide, and then cell viability was measured as described in (B). T47D cells and MDA-MB-453 cells were treated with 2D5 peptide for 96 h, and then cell viability was measured. I, DU145 cells were treated with 1, 10, 20, 30, 40, and 50 μM 2D5 peptide for 48 h, and then relative cell viability was measured by Cell Titer glo assay. n = 3. Mean values and SDs are shown. EGFR, epidermal growth factor receptor; PH, Pleckstrin homology; PI, propidium iodide.

Figure 2

2D5 peptide suppresses EGFR signaling.A, HEK293T cells were transfected with expression vectors for GST-STAP-2 and EGFR-HA, and then lysates were incubated with 1 or 50 μM ΔR8-2D5 peptide for 1 h. GST-STAP-2 was pulled down and blotted. B–D, DU145 cells were serum starved for 1 h and then treated with 10 μM 2D5 peptide for 30 min. The cells were stimulated with 100 ng/ml EGF for 0, 10, and 30 min and then lysates were blotted. E, DU145 cells were treated with 10 μM 2D5 peptide for 0, 30, 60, and 120 min, and then lysates were blotted. F, DU145 cells were treated with 1 or 10 μM 2D5 peptide for 30 min, and then lysates were blotted. G, shCtrl- and shEGFR-expressing DU145 cells were treated with 2D5 peptide same as (B) and stimulated with 100 ng/ml EGF for 15 min, then lysates were blotted. H and I, SW620 cells were treated with 2D5 peptide and 100 ng/ml EGF same as (B), and then lysates were blotted. J, HEK293T cells were transfected with expression vectors for Myc-STAP-2 and EGFR-HA WT or Y1068F/Y1173F (YY/FF), and then lysates were immunoprecipitated and blotted. K, DU145 cells were treated with 10 μM 2D5 peptide, and then Ccnd1 and Survivin mRNA levels were quantified by qPCR. L, DU145 cells were treated with 25 μM 2D5 peptide and 0.1 or 1 μM gefitinib for 48 h, and then cell viability was measured. M, shCtrl- and shSTAP-2-expressing DU145 cells were treated with 25 μM 2D5 peptide for 48 h, and then cell viability was measured. N, THP-1 cells were treated with 20 ng/ml PMA for 3 days. THP-1–derived macrophages were treated with 10 μM 2D5 peptide for 1 h and then stimulated with 1 μg/ml LPS for the indicated times, and then lysates were blotted. n = 3, Mean values and SDs are shown. ∗p < 0.05, ∗∗p < 0.01 (paired Student’s t test). EGFR, epidermal growth factor receptor; PMA, phorbol 12-myristate 13-acetate; qPCR, quantitative PCR.

Figure 3

2D5 peptide decreases EGFR stabilization.A, DU145 cells were treated with 10 μM 2D5 peptide for 1 h under serum starvation and then stimulated with 100 ng/ml EGF for 20 min. The cells were fixed and stained with anti-EGFR (green) and anti-LAMP1 (red) antibodies. B, localization of EGFR and LAMP-1 was observed by confocal microscopy. We calculated the area of total EGFR and EGFR-LAMP-1 colocalization by an ImageJ software, and the percentage of EGFR-LAMP-1/total EGFR area in each 20 cells were shown. n = 20. C, DU145 cells were treated with 10 μM cycloheximide in serum-free medium for 1 h and then treated with 50 μM 2D5 peptide for 1 h together with 10 μM cycloheximide in serum-free medium. The cells were then stimulated with 100 ng/ml EGF, and lysates were blotted. D, band intensity of EGFR/Actin in (C) (n = 3). Mean values and SDs are shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (paired Student’s t test). EGFR, epidermal growth factor receptor.

Figure 4

2D5 peptide inhibits tumor growth in tumor-bearing mice.A, DU145 cells were subcutaneously injected into BALB/c-nude mice and then saline, and 2 mg/mouse 6E4 or 2D5 peptides were administered by intratumoral injection at day 14, 17, and 20. Tumor volume at days 14 to 32 and (B) tumor weight at day 32 are shown. n = 6. C, A549 cells were subcutaneously injected into BALB/c-nude mice, and then peptides were administered as described in (A). D, A549 tumor weight was measured at day 29. n = 5. E, SW620 cells were subcutaneously injected into BALB/c-nude mice, and then peptides were administered at day 9, 12, and 15 same as (A). Tumor volume at days 9 to 21 and (F) tumor weight at day 21 are shown. n = 4. G, BALB/c-nude mice were administered saline or 2 mg/mouse 2D5 peptide by subcutaneous injection at day 0, 3, and 6. Mouse body weights are shown. n = 3. Mean values and SDs are shown. ∗p < 0.05 (paired Student’s t test).