S100P-derived RAGE antagonistic peptide reduces tumor growth and metastasis

Abstract

Purpose: The receptor for advanced glycation end products (RAGE) contributes to multiple pathologies, including diabetes, arthritis, neurodegenerative diseases, and cancer. Despite the obvious need, no RAGE inhibitors are in common clinical use. Therefore, we developed a novel small RAGE antagonist peptide (RAP) that blocks activation by multiple ligands.

Experimental design: RAGE and its ligands were visualized by immunohistochemical analysis of human pancreatic tissues, and siRNA was used to analyze their functions. Interactions between RAGE and S100P, S100A4, and HMGB-1 were measured by ELISA. Three S100P-derived small antagonistic peptides were designed, synthesized, and tested for inhibition of RAGE binding. The effects of the peptide blockers on NFκB-luciferase reporter activity was used to assess effects on RAGE-mediated signaling. The most effective peptide was tested on glioma and pancreatic ductal adenocarcinoma (PDAC) models.

Results: Immunohistochemical analysis confirmed the expression of RAGE and its ligands S100P, S100A4, and HMGB-1 in human PDAC. siRNA silencing of RAGE or its ligands reduced the growth and migration of PDAC cells in vitro. The most effective RAP inhibited the interaction of S100P, S100A4, and HMGB-1 with RAGE at micromolar concentrations. RAP also reduced the ability of the ligands to stimulate RAGE activation of NFκB in cancer cells in vitro and in vivo. Importantly, systemic in vivo administration of RAP reduced the growth and metastasis of pancreatic tumors and also inhibited glioma tumor growth.

Conclusion: RAP shows promise as a tool for the investigation of RAGE function and as an in vivo treatment for RAGE-related disorders.

Fig. 1. Pancreatic cancer cells expressed and were stimulated by RAGE and its ligands S100P, S100A4, and HMGB-1

(A) Immunohistochemical analysis of RAGE, S100P, S100A4, and HMGB-1 in normal and PDAC tissues showed the specific expression of S100P and S100A4 in cancer cells but not in normal cells. RAGE and HMGB-1 were expressed in both normal and cancer cells, but their expression was more prominent in cancer cells. (B). Silencing efficiency of SiRNAs for RAGE, S100P, S100A4 and HMGB-1 were confirmed by western blotting. (Full-length gels are shown in supplementary figure (S1). (C) Silencing of S100P or S100A4 but not HMGB-1 reduced the growth of Mpanc96, MOH, and HPAF II pancreatic cancer cells. Results shown are means +/− SEM for 3 independent experiments. *=p<0.05

Fig. 1. Pancreatic cancer cells expressed and were stimulated by RAGE and its ligands S100P, S100A4, and HMGB-1

(A) Immunohistochemical analysis of RAGE, S100P, S100A4, and HMGB-1 in normal and PDAC tissues showed the specific expression of S100P and S100A4 in cancer cells but not in normal cells. RAGE and HMGB-1 were expressed in both normal and cancer cells, but their expression was more prominent in cancer cells. (B). Silencing efficiency of SiRNAs for RAGE, S100P, S100A4 and HMGB-1 were confirmed by western blotting. (Full-length gels are shown in supplementary figure (S1). (C) Silencing of S100P or S100A4 but not HMGB-1 reduced the growth of Mpanc96, MOH, and HPAF II pancreatic cancer cells. Results shown are means +/− SEM for 3 independent experiments. *=p<0.05

Fig. 1. Pancreatic cancer cells expressed and were stimulated by RAGE and its ligands S100P, S100A4, and HMGB-1

(A) Immunohistochemical analysis of RAGE, S100P, S100A4, and HMGB-1 in normal and PDAC tissues showed the specific expression of S100P and S100A4 in cancer cells but not in normal cells. RAGE and HMGB-1 were expressed in both normal and cancer cells, but their expression was more prominent in cancer cells. (B). Silencing efficiency of SiRNAs for RAGE, S100P, S100A4 and HMGB-1 were confirmed by western blotting. (Full-length gels are shown in supplementary figure (S1). (C) Silencing of S100P or S100A4 but not HMGB-1 reduced the growth of Mpanc96, MOH, and HPAF II pancreatic cancer cells. Results shown are means +/− SEM for 3 independent experiments. *=p<0.05

Fig. 2. RAGE binding of S100P, S100A4, and HMGB-1 was inhibited by an S100P-derived RAGE antagonistic peptide (RAP)

(A-C) In ELISA-based assays, S100P, S100A4 , and HMGB-1 each bound with RAGE in a concentration-dependent manner. (D) The effects of S100P-based small antagonistic peptides (10µM) on the binding of S100P with RAGE were examined. Peptide #1 Elkvlmekel (renamed RAP) inhibited the binding of S100P nearly to control levels and was superior to peptides #2 and #3. (E). RAP (10µM) inhibited the binding of S100P, S100A4, and HMGB-1 with RAGE. Results shown are means +/− SEM for 3 independent experiments. *=p<0.05.

Fig. 3. RAP blocked RAGE induced cell growth, migration and NFκB activity in vitro

(A) Treatment of Mpanc96, MOH, or HPAF II PDAC cells with RAGE siRNA or with RAP each significantly reduced cell proliferation to a similar extent. (B) Inhibition of RAGE activity by either siRNA or RAP treatments inhibited the migration of PDAC cells. Mpanc96 cells are shown as representative. (C) RAP treatment blocked the ability of S100P to induce NFκB activity in PDAC cells. BxPC-3 cells stably labeled with an NFκB luciferase reporter are shown as representative. Results shown are means +/− SEM for 3 independent experiments. *=p<0.05.

Fig. 3. RAP blocked RAGE induced cell growth, migration and NFκB activity in vitro

(A) Treatment of Mpanc96, MOH, or HPAF II PDAC cells with RAGE siRNA or with RAP each significantly reduced cell proliferation to a similar extent. (B) Inhibition of RAGE activity by either siRNA or RAP treatments inhibited the migration of PDAC cells. Mpanc96 cells are shown as representative. (C) RAP treatment blocked the ability of S100P to induce NFκB activity in PDAC cells. BxPC-3 cells stably labeled with an NFκB luciferase reporter are shown as representative. Results shown are means +/− SEM for 3 independent experiments. *=p<0.05.

Fig. 4. RAP treatment reduced RAGE mediated NFκB activity in vivo

Constitutive NFκB activity in PDAC xenografts was inhibited by reducing RAGE activity with either (A) liposomal-coupled siRNA against RAGE, or with RAP treatments (100 µg/day) intratumoral (B) or intraperitoneal (C). NFκB activity was monitored by imaging the level of luciferase expressed downstream from an NFκB promoter stably transfected into BxPC-3 cells. *=p<0.05

Fig. 5. RAP treatment reduced tumor growth and metastasis

(A) Intraperitoneal delivery of RAP (100 µg/day) reduced C6-glioma tumor growth. (B) PDAC Mpanc96 tumor growth and metastasis to the liver (C) were also significantly inhibited by RAP treatments. (D) RAP treated animals did not showed any toxicity as evidenced by un-altered body weight. *=p<0.05

Fig. 5. RAP treatment reduced tumor growth and metastasis

(A) Intraperitoneal delivery of RAP (100 µg/day) reduced C6-glioma tumor growth. (B) PDAC Mpanc96 tumor growth and metastasis to the liver (C) were also significantly inhibited by RAP treatments. (D) RAP treated animals did not showed any toxicity as evidenced by un-altered body weight. *=p<0.05