Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells

Abstract

Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

FIGURE 1

FPRL1 is expressed on ovarian cancer cells. Ovarian cancer cell lines, representing different histologic subtypes, were analyzed for FPRL1 expression by flow cytometry. Primary antibodies were detected with Alexa-488–conjugated goat anti-rabbit antibodies. Black line, FPRL1 expression; gray line, isotype control (n = 3).

FIGURE 2

LL-37 does not signal through a GPCR, such as FPRL1, to stimulate ovarian cancer cell proliferation. Graphic representation of ovarian cancer cell growth after exposure to LL-37 or EGF. Serum-starved cells were pretreated with or without 10 ng/mL of Ptx for 1 h, followed by LL-37 or EGF treatment. After 48 h, cellular DNA was measured using fluorescent probes. MFI, mean fluorescence intensity. Columns, mean of three or more independent experiments; bars, SE.

FIGURE 3

LL-37 mediates ovarian cancer cell migration and invasion through FPRL1. A. Graphic representation of ovarian cancer cell invasion. Serum-starved cells were seeded onto Matrigel-coated inserts in medium containing 10 μg/mL of LL-37 or 10 ng/mL of EGF. Columns, mean fold change of the mean fluorescence intensity of invaded cells compared with unstimulated controls; bars, SE (n = 3). B. Graph depicting fprl1 gene expression in knockdown (KD) cells. SK-OV-3 cells stably transduced with lentiviruses containing FPRL1-specific shRNA (KD-1 to KD-5) or nontarget (NT) sequences were analyzed by qPCR. Columns, mean of three independent experiments compared with nontarget cells; bars, SE. C. Graphic representation of SK-OV-3 ovarian cancer cell invasion through Matrigel-coated inserts. Untreated and Ptx-treated SK-OV-3 cells were stimulated with LL-37 or EGF as described above. P values for LL-37 or EGF groups were determined from their respective untreated or Ptx-treated alone controls. D. Graphic representation of SK-OV-3 nontarget cells and FPRL1 KD-2 cell invasion stimulated with LL-37 or EGF as above (*, P < 0.05; **, P < 0.01).

FIGURE 4

Activation of MAPK signaling pathways by LL-37 does not occur through FPRL1. A. The influence of recombinant LL-37 (5 μg/mL) on phosphorylation of AKT, ERK, and STAT3 in SK-OV-3 cells. Images are representative of three or more independent experiments. B. Cellular protein was isolated from Ptx-treated SK-OV-3 cells, SK-OV-3 nontarget cells, and SK-OV-3/FPRL1 KD-2 cells that were treated with LL-37 for 30 min. Phosphorylation and expression of ERK was measured by Western blot analysis. β-actin levels were assessed to ensure equal loading.

FIGURE 5

Inhibition of FPRL1 negatively affects LL-37–induced nuclear accumulation and activity of multiple transcription factors. A. Graphic representation of transcription factor-DNA binding. Nuclear extracts from serum-starved ovarian cancer cells, treated or untreated with 5 μg/mL of LL-37 for 1 h, were analyzed for transcription factors bound to specific fluorescently labeled oligonucleotide probes. MFI, mean fluorescence intensity. Columns, mean; bars, SE. B. Analysis of transcription factor activity in SK-OV-3/FPRL1 KD and Ptx-treated cells after LL-37 treatment as described above. Columns, mean of four independent experiments; bars, SE (*, P < 0.05; **, P < 0.01).

FIGURE 6

LL-37 modulates target gene expression through FPRL1 signaling. A. Graphic representation of genes regulated by LL-37. Serum-starved SK-OV-3 and OVCAR-3 cells were treated with 5 μg/mL of LL-37 for 6 h. RNA was isolated and analyzed by qPCR using the δCt method. B. Analysis of target gene expression in SK-OV-3/FPRL1 KD and Ptx-treated cells after LL-37 treatment. Columns, mean of three or more independent experiments; bars, SE (*, P < 0.05; **, P < 0.01).

FIGURE 7

LL-37 increases the release of protumorigenic molecules from ovarian cancer cells. A. Serum-starved ovarian cancer cells were treated with 5 μg/mL of LL-37 for 48 h, then conditioned medium was analyzed by Luminex-based cytokine arrays. The amount of cytokines and growth factors in conditioned medium is represented graphically. B. Ptx-treated SK-OV-3 cells, SK-OV-3 nontarget cells, and SK-OV-3/FPRL1 KD-2 cells were treated with LL-37 as above. The levels of EGF, CXCL10, and PDGF-BB were measured in conditioned medium after 48 h. Columns, mean of three or more independent experiments; bars, SE (*, P < 0.05; **, P < 0.01; ***, P < 0.001).