SSTP1, a Host Defense Peptide, Exploits the Immunomodulatory IL6 Pathway to Induce Apoptosis in Cancer Cells

Abstract

While the immunomodulatory pathways initiated in immune cells contribute to therapeutic response, their activation in cancer cells play a role in cancer progression. Also, many of the aberrantly expressed immunomodulators on cancer cells are considered as therapeutic targets. Here, we introduce host defense peptide (HDP), a known immuomodulator, as a therapeutic agent to target them. The cationic host defense peptides (HDPs), an integral part of the innate immune system, possess membranolytic activity, which imparts antimicrobial and antitumor efficacy to it. They act as immunomodulators by activating the immune cells. Though their antimicrobial function has been recently reassigned to immunoregulation, their antitumor activity is still attributed to its membranolytic activity. This membrane pore formation ability, which is proportional to the concentration of the peptide, also leads to side effects like hemolysis, limiting their therapeutic application. So, despite the identification of a variety of anticancer HDPs, their clinical utility is limited. Though HDPs are shown to exert the immunomodulatory activity through specific membrane targets on immune cells, their targets on cancer cells are unknown. We show that SSTP1, a novel HDP identified by shotgun cloning, binds to the active IL6/IL6Rα/gp130 complex on cancer cells, rearranging the active site residues. In contrast to the IL6 blockers inhibiting JAK/STAT activity, SSTP1 shifts the proliferative IL6/JAK/STAT signaling to the apoptotic IL6/JNK/AP1 pathway. In IL6Rα-overexpressing cancer cells, SSTP1 induces apoptosis at low concentration through JNK pathway, without causing significant membrane disruption. We highlight the importance of immunomodulatory pathways in cancer apoptosis, apart from its established role in immune cell regulation and cancer cell proliferation. Our study suggests that identification of the membrane targets for the promising anticancer HDPs might lead to the identification of new drugs for targeted therapy.

Keywords: IL6 (IL-6) pathway; JNK/AP1 pathway; apoptosis; cancer; host defense peptides; immunoregulation; temporins.

Figure 1

SSTP1 induces apoptosis in oral cancer cells (A) HSC-4 cells were treated with peptides SSTP1 and SSTP2 at different concentrations in 0.5% DMEM for 48 h, and MTT assay was performed to calculate % growth inhibition. The graph was plotted using GraphPad Prism8.2.1using non-linear four parameter curve fit. The error bars indicate SEM of three biological replicates (B) HSC-4 cells were treated with 10 μM SSTP1 and SSTP2 for 22h and FACS analysis was performed after annexin/PI staining. (C)The quantified populations are represented in the graph. The error bars indicate standard deviation of triplicates. (D) The HSC-4 cells treated with 10 μM SSTP1 or SSTP2 for indicated time intervals were used for western blot analyses (E) Densitometric analysis was done for the bands, and fold change based on 0min was calculated. The error bars indicate standard deviation of biological duplicates. *p < .05, ***p < .001.

Figure 2

SSTP1 is internalized by active uptake. (A) Live HSC-4 cells were incubated with MitoTracker Deep Red and incubated for 10 min. Excess dye was washed off, and then the respective peptides were added, which were labeled with Alexa Fluor-488, and incubated either on ice or at 37°C. The unbound peptide was washed off and the images were acquired at the indicated time intervals. The arrowmarks indicate the internalized peptide. Magnified images along with DIC are provided in Supplementary Figure 3 . (B) The cells were treated with Alexa Fluor-488-labelled SSTP1 for indicated time and Alexa Fluor-568-labelled transferrin was added and incubated for one minute. The cells were washed, fixed and imaged. (C) The extent of co-occurrence for panel B was measured and plotted as Manderson’s overlap. The median value is shown in the graph. (D) The high magnification representative images of (B) The scale bars represent 10 μm.

Figure 3

RNA-Seq analysis suggests the activation of cytokine signaling pathway upon SSTP1 treatment. (A) HSC-4 cells were treated with 10 μM SSTP1 and SSTP2 for 3h, and total RNA was isolated to perform RNA-Seq analysis. The Venn diagram represents the differential expression of transcripts upon SSTP1 treatment compared to SSTP2-treatment. (B) Volcano plot showing differential expression profile of genes showing significant up regulation and down regulations on treatment with SSTP1 with respect to control peptide SSTP2. Red dots indicate absolute log2 fold change≥2 and FDR- adjusted p value ≤ 0.05 and blue dots indicate log2 fold change≥2 and p value ≤ 0.05. (C, D) The results were confirmed by RT-PCR and qPCR for the indicated molecules. (E) A pathway enrichment analysis was performed based on the differential gene expression analysis, and top 3 pathways that were regulated are tabulated.

Figure 4

Cytokine signaling pathway involved in SSTP1 response (A) STAT/.JAK, JNK/AP1 MAPK1/2, and AKT pathways are reported to be downstream of Cytokine signaling. Heat map showing differential expression of molecules involved in cytokine signaling are shown. (B) Schematic diagram of cytokine signaling pathway in SSTP1 response.

Figure 5

SSTP1 interacts with IL6Rα and mediate the uptake of SSTP1 (A) HSC-4 cells were treated with Biotin labeled-SSTP1 or SSTP2 (10 μM) for 30 minutes and then fixed and probed without permeabilization using mouse IL6Rα (2B2.3). (B) The co-localization of SSTP1 or SSTP2 to IL6Rα was analyzed for 11 cells, and the Pearson’s correlation was plotted. (C) Cells were treated as in panel A and fixed and permeabilized to probe for IL6Rα, SSTP1 and Rab5C. The arrow mark indicates co-localization. The scale bars represent 4 μm (D) The Mander’s overlap in SSTP1 foci were quantified for 10 cells. The average value for each cell is plotted (E) Pull-down assay using SSTP1-biotin or SSTP2-biotin and probed for IL6Rα. (F) FRET analysis to show the SSTP1 binding to IL6Rα as described in Methods. The scale bars represent 10 μm for panel (A, F). ***p < .001.

Figure 6

Binding of SSTP1 and SSTP2 to IL6/IL6Rα/gp130 complex alters the active site interactions. (A) The predicted structure of SSTP1 and SSTP2 was docked to the crystal structure of trimeric complex 1p9M, and the stable structures identified were obtained after dynamic simulation. (B) The SSTP1 or SSTP2-bound active trimeric complex was superimposed to 1p9M. The white circle represents the site with unique changes. (C) High resolution images of the interacting residues of IL6 and IL6Rα. The residues of IL6 chain is marked using white font color and the green font represents the residues of IL6Rα. The circle with dotted line denotes the site with unique changes.

Figure 7

SSTP1 preferentially activates JNK/AP1 pathway (A) HSC-4 cells were treated with the peptides as indicated before for the indicated time points, and the lysates were used for western blot. (B) Densitometric analysis was performed and relative phosphorylation of each molecule was calculated. Fold change based on untreated from biological duplicates were plotted. (C) A luciferase reporter assay was performed using 3×AP1pGL3 reporter construct after treatment with the peptides (10 μM) for 48h. The beetle luciferase activity was normalized with renilla luciferase activity. *p < .05, **p < .01.

Figure 8

SSTP1-induced pcJUN activation depends on JNK and on gp130 and IL6Rα. (A) HSC-4 cells treated with SSTP1 (30 minute) and the indicated inhibitors were used for western blot analysis of the indicated molecules (B) Densitometric analysis was done for the bands of (a) and relative phosphorylation based on untreated is plotted. (C) The cells treated with SSTP1 (30 minutes) and IL6Rα antibody or SC144 were used for western blot analysis of the indicated molecules. (D) Densitometric analysis was done for the bands of (C) and relative phosphorylation based on untreated is plotted. (E) Growth inhibition analyzed by MTT assay after 4h treatment with SSTP1(10 μM) with or without the inhibitors. LMT-28 (60 μM). For all the experiments SSTP1 (10 μM) was used with or without LMT-28 (60 μM), Ruxolitinib (30 μM) or SP600125 (20 μM), Anti-IL6Rα (10µg/ml) or SC144 (40 μM). Cells were pre-treated with the inhibitors LMT-28 (1.5 h), Ruxolitinib (1.5h) or SP600125 (1h), Anti-IL6Rα (2h) or SC144 (2h) prior to the addition of SSTP1. *p < .05, **p < .01, ***p < .001.

Figure 9

Knock-down of IL6Rα abolishes the apoptotic activity of SSTP1. (A) HSC-4 cells stably expressing either control shRNA or IL6Rα shRNA were treated with SSTP1(10 μM) for 30 minutes and western blot was performed for the indicated molecules. The relative phosphorylation based on untreated control ShRNA is plotted. (B) The control and IL6Rα- knocked-down cells were treated with SSTP1 for 4h and MTT was performed. The percentage growth inhibition based on the untreated cells is plotted. The western blot showing the knock-down is also shown. *p < .05, **p < .01, ***p < .001.

Figure 10

Sensitivity of cancer cells to SSTP1 depends on the level of IL6Rα. (A) Western blot performed using cell lysates of different cancer cells. (B) The densitometric analysis of the bands of the blot using ImageJ. (C) Growth inhibition analyzed by MTT assay using 10 μM SSTP1 treated for 48h. (D) Growth inhibitory curve of SSTP1 for MDA-MB-231 cells and HEK-293 cells analyzed after 48h treatment with SSTP1 and Hemolysis induced by SSTP1 on human RBC. The error bars indicate SEM of three biological replicates (E) MDA-MB-231 cells were treated with 4.5 μM SSTP1 for the indicated time intervals and probed for the indicated molecules by western blot. (F) The blots were quantified using ImageJ, and the graphs were plotted. *p < .05, **p < .01, ***p < .001.