Human antimicrobial protein hCAP18/LL-37 promotes a metastatic phenotype in breast cancer

Abstract

Introduction: Human 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.

Methods: hCAP18 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.

Results: The 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.

Conclusions: Our results provide evidence that hCAP18/LL-37 contributes to breast cancer metastasis.

Figure 1

hCAP18 transcript levels in relation to ERBB2 levels, lymph node status and oestrogen receptor (ER) status. All transcription levels determined by real-time reverse transcriptase PCR are displayed relative to the mean of four control samples. (a) Significantly higher levels of hCAP18 transcript for lymph node positive compared with lymph node-negative ER-positive patients (p < 0.001). (b) No significant difference in hCAP18 levels with respect to lymph node status was observed for ER-negative patients (a, b) filled markers and error bars represent geometric means and 95% confidence intervals. The association between hCAP18 levels and lymph node status was significantly different for (a) ER-positive and (b) ER-negative patients (p = 0.01). (c, d) Significant correlation between levels of hCAP18 and ERBB2 was observed in all groups, whether (c) ER positive or (d) ER negative, and irrespective of lymph node status (squares versus circles).

Figure 2

LL-37 synergistically enhances Heregulin-induced mitogen-activated protein kinase (MAPK) phosphorylation through the ERBB2 receptor. (a) Phosphorylation of ERBB2 (upper panel) and MAPK (lower panel) by treatment of ZR75-1 cells with LL-37 and Heregulin β3 (HRG) on their own and in combination. The diagram shows the mean phosphorylation level as evaluated by Western blot analysis, normalised against Ponceau staining and relative to untreated samples. On the left side is shown one representative of the triplicates used for evaluation. The lower rows show Western blots against total ERBB2 and MAPK, demonstrating an unaltered protein level, and Ponceau staining of the corresponding sections of the blot. (b) Inhibition of MAPK activation in ZR75-1 by the ERBB inhibitor PD153035 but not by pertussis toxin or metalloprotease inhibitor GM6001. The arrow points to the histogram for PD153035 treatment. (c) LL-25 inhibits the LL-37 dependent activation of MAPK. Experiments were performed in the MJ1105 control cell line using HRG at 2 ng/ml, and by varying the concentrations of both LL-37 and LL-25 as displayed. The diagram shows the levels of MAPK phosphorylation relative to the conditions of 2 μM LL-37. (d) Synergistic effect between HRG and endogenous production of transgenic hCAP18 in the MCF-7 derivative MJ1105. For all experiments, the Ponceau staining used for normalisation is shown. All conditions were run in triplicates (n = 3), and in addition repeated at least on one independent occasion.

Figure 3

LL-37 induces morphological changes in MJ1105 soft agar clones. The upper row shows an example of a colony in absence and in presence of LL-37. For the quantitative evaluation displayed in the diagram, cell culture experiments for each condition (n = 8) were arranged in a factorial design and the resulting data analysed by analysis of variance (ANOVA), the significance level set to α = 0.05. The proportion of cell colonies with LL-37 induced morphological alterations was significantly lower in presence of LL-25 (p < 0.01). The experiment was repeated on three independent occasions.

Figure 4

LL-37 at 2 μM increases the migration of MCF-7 breast cancer cells in a Boyden Chamber assay. The stimulatory effect induced by LL-37 was abrogated when LL-25 at 1 μM was added together with LL-37 in the lower chamber. Control experiments were with medium only in the lower chamber, or with LL-25 in the medium, respectively. The results are given as mean ± standard error of the mean, n = 4. * p < 0.05, ** p < 0.01, ns = non significant.

Figure 5

Overexpression of hCAP18 in MJ1105 cells increases metastasis formation in severe combined immunodeficiency (SCID) mice. (a) Metastasis formation displayed in a mouse injected with MJ1105-hCAP18 cells compared with control mouse treated with MJ1105 lacking hCAP18. The right panel shows tumour cells from ascites fluid detected by fluorescent transgene-coupled enhanced green fluorescent protein (eGFP) expression. (b) Expression analysis of hCAP18/L-37 in mouse tumours. (Left) Immunohistochemistry with anti-LL-37 antibodies demonstrating strong expression in transgenic tumours, and minor local induction in control tumours. Right, the expression of hCAP18 mRNA determined by reverse transcriptase PCR in tumours and transgenic cell lines. All comparisons are made to the expression of MJ1105 control cell line, which is set to one. (c) Western blot analysis demonstrates increased mitogen-activated protein kinase (MAPK) phosphorylation and increased degradation of phosphorylated ERBB2, in hCAP18 transgenic primary tumours compared with control primary tumours. On the left side of the blot, the control cell line is shown for comparison. Diagrams to the right illustrate the expression levels for respective tumour group, their statistic significance (two-tailed t-test, equal variance) indicated by asterix.