Abstract
IntroductionHuman cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 is a multifunctional protein. In addition to being important in antimicrobial defense, it induces chemotaxis, stimulates angiogenesis and promotes tissue repair. We previously showed that human breast cancer cells express high amounts of hCAP18, and hypothesised that hCAP18/LL-37 may be involved in tumour progression.MethodshCAP18 mRNA was quantified in 109 primary breast cancers and compared with clinical findings and ERBB2 mRNA expression. Effects of exogenous LL-37 and transgenic overexpression of hCAP18 on ErbB2 signalling were investigated by immunoblotting using extracts from breast cancer cell lines ZR75-1 and derivatives of MCF7. We further analysed the impact of hCAP18/LL-37 on the morphology of breast cancer cells grown in soft agar, on cell migration and on tumour development in severe combined immunodeficiency (SCID) mice.ResultsThe expression of hCAP18 correlated closely with that of ERBB2 and with the presence of lymph node metastases in oestrogen receptor-positive tumours. hCAP18/LL-37 amplified Heregulin-induced mitogen-activated protein kinase (MAPK) signalling through ErbB2, identifying a functional association between hCAP18/LL-37 and ErbB2 in breast cancer. Treatment with LL-37 peptide significantly stimulated the migration of breast cancer cells and their colonies acquired a dispersed morphology indicative of increased metastatic potential. A truncated version of LL-37 competitively inhibited LL-37 induced MAPK phosphorylation and significantly reduced the number of altered cancer cell colonies induced by LL-37 as well as suppressed their migration. Transgenic overexpression of hCAP18 in a low malignant breast cancer cell line promoted the development of metastases in SCID mice, and analysis of hCAP18 transgenic tumours showed enhanced activation of MAPK signalling.ConclusionsOur results provide evidence that hCAP18/LL-37 contributes to breast cancer metastasis.
Highlights
Human cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 is a multifunctional protein
Results hCAP18 expression correlates with the expression of ERBB2 and is associated with lymph node metastasis in estrogen receptor positive human breast cancer Previously we have shown that hCAP18 mRNA and protein are overexpressed in human breast cancer samples [17]
Stratifying the patient material based on ER expression, and the presence of lymph node metastasis we found that hCAP18 expression was significantly higher (p < 0.001) in ER-positive tumours (n = 38) with lymph node metastases than in tumours without lymph node metastasis (n = 42) associating the expression of hCAP18 with metastasis formation in breast cancer (Figure 1a)
Summary
HCAP18, and its C-terminal peptide LL-37 is a multifunctional protein. ANOVA: analysis of variance; BSA: bovine serum albumin; DMEM: Dulbecco's modified eagle's media; eGFP: enhanced green fluorescent protein; EGFR: epidermal growth factor receptor; ER: oestrogen receptor; FACS: fluorescence-activated cell sorting; FCS: fetal calf serum; FPRL-1: formyl peptide receptor like-1; hCAP: human cathelicidin antimicrobial protein; HLA: human leucocyte antigen; HPLC: high-performance liquid chromatography; HRG: heregulin β3; MAPK: mitogen-activated protein kinase; PBS: phosphate buffered saline; RT-PCR: reverse-transcription polymerase chain reaction; SCID: severe combined immunodeficiency. Cytokine release and cell migration [7,8] and stimulates chemotaxis and angiogenesis through the G-protein coupled receptor, the formyl peptide receptor like-1 (FPRL-1) [9,10,11] In line with these findings, current research indicates that hCAP18/ LL-37 is actively involved in tissue repair and wound healing [12,13] processes that share fundamental biological features with tumour growth and progression [14]
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