The soy-derived peptide Lunasin inhibits invasive potential of melanoma initiating cells

Abstract

Lunasin is a 44 amino acid peptide with multiple functional domains including an aspartic acid tail, an RGD domain, and a chromatin-binding helical domain. We recently showed that Lunasin induced a phenotype switch of cancer initiating cells (CIC) out of the stem compartment by inducing melanocyte-associated differentiation markers while simultaneously reducing stem-cell-associated transcription factors. In the present study, we advance the hypothesis that Lunasin can reduce pools of melanoma cells with stem cell-like properties, and demonstrate that Lunasin treatment effectively inhibits the invasive potential of CICs in vitro as well as in vivo in a mouse experimental metastasis model. Mice receiving Lunasin treatment had significantly reduced pulmonary colonization after injection of highly metastatic B16-F10 melanoma cells compared to mice in the control group. Mechanistic studies demonstrate that Lunasin reduced activating phosphorylations of the intracellular kinases FAK and AKT as well as reduced histone acetylation of lysine residues in H3 and H4 histones. Using peptides with mutated activity domains, we functionally demonstrated that the RGD domain is necessary for Lunasin uptake and its ability to inhibit oncosphere formation by CICs, thus confirming that Lunasin's ability to affect CICs is at least in part due to the suppression of integrin signaling. Our studies suggest that Lunasin represents a unique anticancer agent that could be developed to help prevent metastasis and patient relapse by reducing the activity of CICs which are known to be resistant to current chemotherapies.

Keywords: Lunasin; cancer stem cells; histone acetylation; integrin signaling; melanoma.

Figure 1. Amino acid sequence of the Lunasin peptide

Lunasin is a 44 amino acid peptide with 3 functional domains attributed with its anticancer activity: 1) a helical regional conserved in chromatin-binding proteins (blue), 2) a RGD motif recognized by integrins (red), and 3) a poly-aspartic acid tail involved in histone-tail binding (green).

Figure 2. Lunasin is readily internalized by A375 melanoma cells

A375 cells treated with Lunasin for up to 24 h internalized Lunasin, which was found to colocalize with integrin α_V subunits. Additionally, we observed nuclear localization of Lunasin that persisted after 24 h of treatment. We also observed the clustering of integrin subunits around the nucleus when A375 cells were treated with Lunasin suggesting the endocytic mechanism for Lunasin internalization involved integrins. Representative images from three independent experiments were used, and were taken at 40x magnification. (Blue = dapi, green = Lunasin, red = integrin α_V).

Figure 3. Lunasin disrupted oncosphere formation and reduced ALDH^high populations

B16-F10 ALDH^high cells were plated in low adherent culture in stem cell media and allowed to form floating oncospheres. When media was amended with 100 μM Lunasin, we observed a significant decrease in oncosphere formation compared to control samples (A, B). V = vehicle, L = Lunasin. ALDH activity was measured as previously described. When B16-F10 cells were treated with Lunasin, we observed a significant reduction in cells displaying the ALDH^high phenotype (C, D). Figures represent data obtained from three independent experiments and are shown as mean ± s.d. Statistical significance (p < 0.05) was determined by student's t-test and denoted by an asterisk (*).

Figure 4. CIC invasion was suppressed in Lunasin-treated cells

In vitro invasion assays demonstrate that Lunasin-treated A375 and B16-F10 ALDH^high cells had less invasive capacity than cells treated with vehicle (A). Invading cells were stained with toluidine blue and representative images are shown at 20x magnification (B). Data from three independent experiments are shown as mean ± s.d. Statistical significance was determined by student's t-test and denoted by an asterisk.

Figure 5. Lunasin reduced pulmonary metastases in vivo

B16-F10 melanoma cells were injected IV into female C57BL/6 mice. Lunasin-treated mice had less incidence of macrometastases (A) as well as significantly reduced average lesion area (B). Representative images of pulmonary tissues resected from control (C) and Lunasin (D) treated mice are shown. H&E stained sections demonstrate the significant difference between average lesion area in vehicle (E) and Lunasin (F) treated mice. Stained sections were imaged at 10x (left) and 40x (right), scale bar = 1 mm. Graphs represent data plotted as mean ± s.e.m. Means were determined to be statistical significant by student's t-test and significance is denoted by an asterisk.

Figure 6. Lunasin suppressed integrin signal transduction

ALDH^high and ALDH^low cells derived from the A375 melanoma cell line were treated with 100 μM Lunasin in low adherent culture for 24 h, and the resulting lysates were probed for integrin-associated signaling proteins (A). We observed a significant difference in phosphorylation patterns of FAK, AKT, and ERK in ALDH^high cells, while only a modest effect was observed in ALDH^low cells (A). Actin was used as a reference protein. Additionally, we used PLA assays to validate that Lunasin was targeting integrin signal transduction. Our results suggest Lunasin decreased the interactions between integrin β subunits and the intracellular kinases FAK and ILK (B). Furthermore, we show that Lunasin specifically interacts with the RGD-recognizing α_V integrin subunit (B).

Figure 7. Lunasin inhibited phosphorylation of FAK, AKT, and ERK as well as histone acetylation

ALDH^high cells derived from human A375 and murine B16-F10 melanomas were treated with Lunasin for 24 h, and the resulting cell lysates were subjected to SDS-PAGE and probed for integrin-associated signaling pathways. We observed a decrease in histone acetylation at H3K9 and H4K12 (A), which suggests epigenetic modification may play a role in Lunasin's effects on melanoma CICs. As previously shown, these changes are concomitant with inhibition of activating phosphorylations of AKT (B), ERK (B), and FAK (C). Actin was used as a reference protein for all immunoblot analysis.

Figure 8. Lunasin's RGD motif is essential for disrupting oncosphere formation

Mutated peptides with alterations in the RGD domain and the poly-aspartic-acid tail were used to treat A375 ALDH^high cells in low adherent culture. Vehicle-treated cells readily formed oncospheres, but native Lunasin disrupted oncosphere formation (A). When the RGD sequence was mutated to RAD, Lunasin lost its ability to inhibit oncosphere formation, while a peptide containing a scrambled tail retained the ability to inhibit oncosphere generation (A). Representative images taken at 10x (left) and 20x (right) demonstrate the ability of the peptide to inhibit oncosphere formation (B). Averages from three independent experiments were plotted as mean ± s.d. Statistical significance was determined by student's t-test and denoted by an asterisk.

Figure 9. Lunasin uptake is an integrin-dependent process

RGD (A) and RAD (B) Lunasin peptides were incubated with A375 cells for various time points up to 1 h. While some RAD-Lunasin was detected, cells treated with RGD-Lunasin (native) showed significantly more abundant intracellular localization as illustrated by a significantly increased fluorescent signal. Additionally, we observed that native Lunasin (i.e. RGD) was localized in the nucleus of A375 cells after just 10 minutes, while RAD-Lunasin was detected mainly in the cytoplasm. Images are representative of data obtained from two independent experiments, and were analyzed using ImageJ software.