Development of a prosaposin-derived therapeutic cyclic peptide that targets ovarian cancer via the tumor microenvironment

Abstract

The vast majority of ovarian cancer-related deaths are caused by metastatic dissemination of tumor cells, resulting in subsequent organ failure. However, despite our increased understanding of the physiological processes involved in tumor metastasis, there are no clinically approved drugs that have made a major impact in increasing the overall survival of patients with advanced, metastatic ovarian cancer. We identified prosaposin (psap) as a potent inhibitor of tumor metastasis, which acts via stimulation of p53 and the antitumorigenic protein thrombospondin-1 (TSP-1) in bone marrow-derived cells that are recruited to metastatic sites. We report that more than 97% of human serous ovarian tumors tested express CD36, the receptor that mediates the proapoptotic activity of TSP-1. Accordingly, we sought to determine whether a peptide derived from psap would be effective in treating this form of ovarian cancer. To that end, we developed a cyclic peptide with drug-like properties derived from the active sequence in psap. The cyclic psap peptide promoted tumor regression in a patient-derived tumor xenograft model of metastatic ovarian cancer. Thus, we hypothesize that a therapeutic agent based on this psap peptide would have efficacy in treating patients with metastatic ovarian cancer.

Fig. 1.. Stimulation of TSP-1 and its effects on ovarian cancer cell growth and survival

(A) Western blot of TSP-1 and β-actin in WI-38 lung fibroblasts that were untreated (–) or treated with the native DWLP L-amino acid psap peptide (WT), dWlP psap peptide (d1,3), or DwLp psap peptide (d2,4) (n=5); (B) Western blot of TSP-1 and β-actin in pooled mouse lung tissue harvested from mice that were untreated (–), treated with metastatic prostate cancer cell conditioned medium alone (CM) or in combination with DWLP psap peptide (WT) or dWlP psap peptide (d1,3 peptide) at doses of 30 mg/kg/day intraperitoneally (i.p) for 3 days (n=3 mice per group); (C) Western blot of CD36 and β-actin in 9 patient-derived ovarian cancer cell lines (DF); (D) Plot of cell number as measured by Wst-1 assay of patient-derived ovarian cancer cell line DF14 treated with 0.2 nM recombinant human TSP-1 (rhTSP-1) for 8, 24, 48, or 72 hours (p-values were calculated by ANOVA) (n=3) (error bars indicate SEM); (E) FACS analysis of Annexin V and PI staining in patient-derived ovarian cancer cell line DF14 treated with saline (control, left), 0.2 nM recombinant human TSP-1 (rhTSP-1, middle), and 10 μg/ml cisplatin (right) for 48 hours (cells staining positive for both markers are apoptotic) (n=3).

Fig. 2.. Regression of primary ovarian tumors induced by the psap peptide

(A) Plot of luciferase intensity over time in 1D8 tumors treated with saline (control) or psap peptide (peptide). Mice were treated daily with 40 mg/kg of dWlP peptide on days 31–51 and 83–104 (n=8 mice/group). Green arrows indicate initiation of treatment, red arrow indicates cessation of treatment (mean ± SEM); (B) Images of luciferase intensity of 1D8 tumors in mice that were treated with saline (control) or psap peptide 51 and 104 days after injection (n=12); (C) Plot of the average mass of 1D8 tumors at day 104 from mice that were treated with saline (control) or psap peptide (peptide) (mean ± SEM); (D) Plot of the average ascites volume of mice bearing 1D8 tumors that were treated with saline (control) or psap peptide (peptide) (n=8) (mean ± SEM); (E) Immunofluorescence staining for GR1 (red), TSP-1 (green), and DAPI (blue) in paraffin-embedded sections of 1D8 tumors treated with saline (control) or psap peptide (peptide) (yellow scale bars=100 μm, white scale bar=25 μm); (F) Immunofluorescence staining for TUNEL (green) and DAPI (blue) in paraffin-embedded sections of 1D8 tumors treated with saline (control) or psap peptide (peptide) (yellow scale bars=100 μm, white scale bar=25 μm); (G) Plot of average percentage of TUNEL+ cells in 1D8 tumors treated with saline (control) or psap peptide (peptide) (mean ± SEM); (H) Western blot of CD36 and β-actin expression in 1D8 and DF14 cells.

Fig. 3.. Histological analysis of psap peptide-treated ovarian tumors

(A) Immunohistochemical analysis of CD31 staining to measure vascularity in saline (control) and d1,3 psap peptide (peptide) treated primary 1D8 ovarian tumors (scale bars=100 μm); (B) Graphical depiction of vessel density (vessel area as a percentage of total field area) as determined by CD31 staining of saline (control) and d1,3 psap peptide (peptide) treated 1D8 tumors (P=0.0011; by Mann-Whitney U-test); (C) Graphical depiction of vessel density (# vessels per field) as determined by CD31 staining of saline (control) and d1,3 psap peptide (peptide) treated 1D8 tumors (P=0.004; by Mann-Whitney U-test) (scale bars=100 μm); (D) Immunohistochemical analysis of Mac3 staining to measure macrophage infiltration in saline (control) and d1,3 psap peptide (peptide) treated primary 1D8 ovarian tumors, yellow arrows indicate Mac3 positive cells (scale bars=100 μm); (E) Graphical depiction of macrophage infiltration, measured as the number of macrophages per field as determined by Mac3 staining of saline (control) and d1,3 psap peptide (peptide) treated 1D8 tumors (P=0.00328 by Mann-Whitney U-Test) (n=8 mice per group); (F) H&E staining of liver surface implants (denoted by arrows) formed by 1D8 tumors in saline-treated mice (scale bar=100 μm); (G) H&E staining of a representative liver of a mouse bearing an 1D8 tumor treated with d1,3 psap peptide (scale bar=100 μm); (H) Graphical depiction of the number of liver metastases, both surface implants and parenchymal lesions in saline (control) and d1,3 psap peptide (peptide) treated mice bearing 1D8 tumors (P=0.033 by Mann-Whitney U-test) (n=5 mice per group).

Fig. 4.. Effects of a d-amino acid psap peptide on a PDX model of metastatic ovarian cancer.

(A) Plot of relative luciferase intensity of metastatic ovarian PDX tumors that were treated with saline (control), cisplatin 4 mg/kg every other day, and dWlP psap peptide (40 mg/kg daily). Red arrow indicates initiation of treatment (n=12 mice/group) (mean ± SEM); (B) Luciferase imaging of two control-treated mice and two dWlP psap peptide-treated mice at day 17 (treatment day 0) and day 48 (treatment day 31); (C) Photographs of the livers of mice bearing metastatic ovarian PDX tumors treated with saline (control) or dWlP psap peptide (peptide) (arrows indicate metastases); (D) H&E staining of the liver of a mouse bearing metastatic ovarian PDX tumors treated with saline (control) or dWlP psap peptide (peptide) (arrow indicate metastatic lesions, scale bars=100 μm); (E) FACS analysis of GR1⁺/Cd11b⁺ cells in the peritoneal fluid of control and dWlP psap peptide (peptide) treated mice bearing metastatic ovarian PDX tumors after 48 days of treatment.

Fig. 5.. Effects of a cyclic psap peptide on TSP-1 expression and a PDX model of metastatic ovarian cancer.

(A) Western blot of TSP-1 and β-actin in WI-38 lung fibroblasts that were untreated (–) or treated with cyclic DWLPK psap peptide (c) or d1,3 psap peptide (L) (line represents digital excision of bands not relevant to this study); (B) ELISA of TSP-1 expression in WI-38 lung fibroblasts that were untreated (–) or treated with dWlP psap peptide (d1,3) or with cyclic DWLPK psap peptide after up to 24 hours of incubation in human plasma at 37^oC (P-values calculated by ANOVA) (mean ± SEM); (C) Plot of relative luciferase intensity of metastatic ovarian PDX tumors that were treated with saline (blue line) or cyclic DWLPK psap peptide (red line) (10 mg/kg daily). Green arrow indicates onset of treatment; (D) Plot of average area of metastatic lesions in saline (control) treated mice and cyclic DWLPK psap peptide (peptide) treated mice. (P-values were calculated by ANOVA) (error bars indicate mean ± SEM); (E) H&E staining of metastatic lesions (indicated by red arrows) in the omentum of mice treated with vehicle (saline) (control) or cyclic psap peptide (peptide) (scale bar=100 μm); (F) Immunofluorescent staining of GR1 and TSP-1 expression in metastatic lesions of control-treated mice and cyclic DWLPK psap peptide (peptide) treated mice (scale bar = 50 μm; enlarged panel = 4-fold enlarged); (G) Immunohistochemistry (leftmost panels) of TSP-1 expression (scale bar = 100 μm) and immunofluorescent staining of TUNEL (green) and DAPI (blue), and merged images of TUNEL and DAPI for metastatic lesions in control and cyclic DWLPK psap peptide (peptide) treated mice (scale bar = 100 μm). (H) Plot of %TUNEL positive cells in saline (control) treated and psap peptide-treated tumors (P-values were calculated by Fisher’s Exact Test).

Fig. 6.. Expression of CD36 and psap in a TMA of human ovarian cancer patients.

(A) Expression of CD36 in a tumor tissue microarray (TMA) compiled from normal tissue, primary human ovarian tumors, human ovarian cancer visceral metastases (metastases), and human ovarian cancer lymph node metastases (LN mets) (Black scale bars=200 μm, yellow scale bars=50 μm); (B) Plot of CD36 staining indices for normal human ovarian and endometrial tissue, primary human ovarian tumors, human ovarian cancer metastases, and human ovarian cancer lymph node metastases (P-values were determined by Wilcoxon-Mann-Whitney analysis) (mean ± SEM); (C) Expression of psap in a tumor tissue microarray (TMA) compiled from normal tissue, primary human ovarian tumors, human ovarian cancer visceral metastases (metastases), and human ovarian cancer lymph node metastases (LN mets) (scale bars=50 μm); (D) Plot of psap (psap) staining indices for normal human ovarian and endometrial tissue, primary human ovarian tumors, human ovarian cancer metastases, and human ovarian cancer lymph node metastases (P-values were determined by Wilcoxon-Mann-Whitney analysis) (mean ± SEM).