Oncomedicine 2016; 1:14-17. doi:10.7150/oncm.16802

Short Research Paper

Cathelicidin LL-37 Promotes or Inhibits Cancer Cell Stemness Depending on the Tumor Origin

Guilherme Tude Coelho Neto1, Thais Martins de Lima1, Hermes Vieira Barbeiro1, Roger Chammas2, Marcel Cerqueira César Machado1, Fabiano Pinheiro da Silva1 Corresponding address

1. Emergency Medicine Department, University of Sao Paulo, Sao Paulo, Brazil
2. Oncology Department, University of Sao Paulo, Sao Paulo, Brazil

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How to cite this article:
Neto GTC, de Lima TM, Barbeiro HV, Chammas R, Machado MCC, Pinheiro da Silva F. Cathelicidin LL-37 Promotes or Inhibits Cancer Cell Stemness Depending on the Tumor Origin. Oncomedicine 2016; 1:14-17. doi:10.7150/oncm.16802. Available from http://www.oncm.org/v01p0014.htm

Abstract

Antimicrobial peptides play critical protective roles in a range of human diseases, including cancer. Multiple studies have demonstrated functions—such as proliferation, angiogenesis, apoptosis and immunomodulation—of these peptides in crucial cancer pathways. We investigated the role of the antimicrobial peptide LL-37 on stemness in breast cancer (SKBR3) and melanoma cells (A375). PCR array analysis of differential gene expression in SKBR3 and A375 cancer cell lines downregulated for LL-37 expression by siRNA revealed downregulation of genes related to stemness, including telomerase reverse transcriptase, forkhead box D3 and undifferentiated embryonic cell transcription factor 1, remarkably in breast cancer cells. Furthermore, SKBR3 cells knocked down for LL-37 expression showed a decreased production of oncospheres in comparison with negative controls, while A375 cells exhibited increased production. Taken collectively, our findings indicate a role for LL-37 in cancer cell stemness depending on the cell type.

Keywords: LL-37, cancer, stemness, pluripotency, self-renewal

Introduction

Antimicrobial peptides play crucial roles in critical molecular pathways in cancer, such as cell proliferation, epithelial cell migration, angiogenesis promotion, induction of apoptosis and immunomodulation (1).

The effects of the antimicrobial peptide LL-37 in cancer remain unclear. While LL-37 acts as a positive regulator of ovarian, breast, melanoma and lung cancer progression, it also suppresses colorectal and gastric cancer cell growth (2), indicating that its effects are tumor-specific.

Recently, the concept of clonal tumor evolution has been challenged by the observation of cancer stem cells (CSCs) in a variety of tumors. CSCs possess increased invasive and metastatic capabilities and render tumors more resistant to several microenvironmental stresses, including the action of several anti-cancer drugs (3).

Here we investigated the effects of LL-37 on stemness in both breast cancer and melanoma cells. We performed array analysis to examine the expression of 84 genes related to DNA damage in wild-type and LL-37-knockdown cancer cells.

Material and Methods

The study protocol was approved by the Hospital das Clinicas Ethical Committee, protocol 034/14.

Cell culture

Immortalized human breast cancer cells (SKBR-3) and skin malignant melanoma cells (A375) were used in this study. Cells were maintained according to the guidelines of the ATCC (American Type Culture Collection).

Real-time PCR

RNA was extracted from cultured cells using TRIZOL® protocol. RNA was quantified using NanoVeu (GE Heathcare) systems and RT-PCR was performed using the StepOne SuperScript® III (Applied Biosystems) protocol as provided by the manufacturer. Beta-2 microglobulin (B2M) gene was used as an internal control; primers were as follows: GAT GAG TAT GCC TGC CGT TGC, and CAA TCC AAA TGC GGC ATC T. The reactions were performed in a StepOne™ system (Applied Biosystems) at 50°C for 10 min, 95°C for 5 min and then 95°C for 15 s followed by 60°C for 30 s, and 72°C for 30 s for 40 cycles. Quantification was performed by 2-ΔΔCT method.

LL-37 gene silencing

Cells were plated at 2.5×105 cells per well in a 6-well plate overnight. LL-37 Silencer Selected Pre-designed short interfering RNA (siRNA) or negative scramble siRNA (Ambion®) (10 nM each) was combined with 5 µL of Lipofectamine™ RNAiMAX reagent for 20 min. Opti-MEM® I Reduced Serum Medium (Invitrogen) was added to a final volume of 2.5 mL per well after cells were rinsed with PBS. After 24 h (SKBR3) or 48 h (A375), experimental assays were performed.

PCR array

Total RNA was converted into cDNA using the RT2 First Strand Kit (SABiosciences, Frederick, USA) and cDNA was then combined with the RT2 SYBR Green qPCR Master Mix (SABiosciences). Each sample was added to 24 Human DNA Damage PCR arrays (Qiagen, USA) according to the StepOne equipment protocol (Applied Biosystems). PCR-array data analysis was performed at the manufacturer's website (http://www.sabiosciences.com/pcrarraydataanalysis.php).

Sphere-forming assays

The assay was performed as previously reported (4, 5). Briefly, after siRNA treatment, cells were detached and a single cell suspension was obtained after passing cells through 25 G needles. Cells (1.0×105) were plated in their respective cell culture media containing B27 supplement and rEGF (100 ng/ml; Sigma Aldrich, Poole, UK; E-9644). After 5 days, the number of spheres that were greater than 50 µm in diameter were counted and sphere forming efficiency (%) was determined.

Statistical analysis

Results were analyzed suing Mann-Whitney test and are shown as mean ± standard deviation. A p-value < 0.05 was considered significant.

Results

LL-37 production upregulates several pathways related to stemness

We next analyzed differential gene expression in cancer cell lines downregulated for LL-37 expression by siRNA. Knockdown efficiency of LL-37 expression was more than 90%, as evaluated by qPCR (data not shown). PCR arrays showed a downregulation of several genes related to stemness, especially in SKBR3, for LL-37 compared with control cells (Tables 1 and 2).

LL-37 knockdown cells show decreased production of oncospheres in breast cancer cells and increased production in melanoma cells

We next analyzed the production of cancer-derived extracellular vesicles, oncospheres, as a hallmark of stemness in cancer cell lines depleted for LL-37 expression. Our results showed that SKBR3 cells knocked down for LL-37 expression produced a decreased number of oncospheres compared with negative controls (Figure 1A), while A375 produced an increased number of oncospheres compared with negative controls (Figure 1B).

 Figure 1 

Oncosphere-forming efficiency in SKBR3 (A) and A375 (B) cell lines depleted for LL-37 expression by siRNA.

Oncomedicine Image (Click on the image to enlarge.)
 Table 1 

Genes upregulated in SKBR3 breast cancer cells compared with SKBR3 cells transfected with LL-37 siRNA treatment. Genes related to stemness are in bold.

SymbolGene NameFold Changep-value
KAT2AK(lysine) acetyltransferase 2A1.63370.008484
COL2A1Collagen, type II, alpha 11.89030.012853
GDF3Growth differentiation factor 31.84020.014975
HNF4AHepatocyte nuclear factor 4, alpha1.90640.016682
TERTTelomerase reverse transcriptase2.24680.018056
HAND1Heart and neural crest derivatives expressed 12.10760.02039
HSPA9Heat shock 70kDa protein 9 (mortalin)3.79970.020459
SOX15SRY (sex determining region Y)-box 152.10140.02309
NAT1N-acetyltransferase 1 (arylamine N-acetyltransferase)26.9250.026202
ALPLAlkaline phosphatase, liver/bone/kidney1.8380.026346
PARD6APar-6 partitioning defective 6 homolog alpha (C. elegans)14.23260.026805
OLIG2Oligodendrocyte lineage transcription factor 21.75980.031648
FOXD3Forkhead box D32.65020.03305
MYBL2V-myb myeloblastosis viral oncogene homolog (avian)-like 21.68630.035958
LIN28ALin-28 homolog A (C. elegans)1.81080.036787
TP53Tumor protein p531.97570.040021
FGF2Fibroblast growth factor 2 (basic)2.03550.044985
NESNestin1.84980.047692
UTF1Undifferentiated embryonic cell transcription factor 15.21390.048209

Discussion

Self-renewal and pluripotency are the key characteristics of stem cells. Here, our findings suggest that LL-37 regulates stemness. Cancer stem cells (CSCs) are a considerable clinical problem, since they are highly resistant to radiation and chemotherapy (6). The mechanism of resistance is not fully understood, but enhanced DNA repair capacities and low intracellular reactive oxidative species concentrations are implicated (7). CSCs also proliferate more slowly than non-stem carcinoma cells, circulate in the bloodstream (8) and can lead to tumor metastasis and relapse. Definitive markers of CSCs do not exist, but our results demonstrated many interesting results in SKBR3 and A375 wild-type cells, when compared with LL-37 knockdown cells. Nestin, for example, is an important marker of CSCs, regulates proliferation, migration and invasion of cancer cells (9) correlating to a worse prognosis (10).

 Table 2 

Genes upregulated in A375 melanoma cells compared with A375 cells transfected with LL-37 siRNA treatment. Genes related to stemness are in bold.

SymbolGene NameFold Changep-value
LIN28ALin-28 homolog A (C. elegans)1.52520
TCF3Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47)2.98680
RUNX2Runt-related transcription factor 21.79980.000007
CCNA2Cyclin A21.37860.000011
FOXA2Forkhead box A21.59970.000018
CD34CD34 molecule1.33550.000025
BGLAPBone gamma-carboxyglutamate (gla) protein1.51470.000044
CDH2Cadherin 2, type 1, N-cadherin (neuronal)1.68440.000044
FABP7Fatty acid binding protein 7, brain1.61570.000056
CCNE1Cyclin E11.4720.000063
OLIG2Oligodendrocyte lineage transcription factor 21.57130.000086
NCAM1Neural cell adhesion molecule 11.21640.00013
RESTRE1-silencing transcription factor1.39960.000231
TUBB3Tubulin, beta 31.49530.000248
CDC42Cell division cycle 42 (GTP binding protein, 25kDa)1.58890.00025
KRT15Keratin 151.4690.000278
GATA2GATA binding protein 21.55470.000372
ALPLAlkaline phosphatase, liver/bone/kidney1.48160.000379
GDF3Growth differentiation factor 31.71020.000422
TBX3T-box 31.57750.000669
AICDAActivation-induced cytidine deaminase1.38630.001057
RUNX1Runt-related transcription factor 11.22380.001949
KLF4Kruppel-like factor 4 (gut)1.11390.00216
FGF2Fibroblast growth factor 2 (basic)1.42190.002267
NAT1N-acetyltransferase 1 (arylamine N-acetyltransferase)1.38880.002528
LEFTY2Left-right determination factor 21.34890.003161
MYCNV-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)1.16720.003271
HAND1Heart and neural crest derivatives expressed 11.21170.003824
ZFP42Zinc finger protein 42 homolog (mouse)1.24210.004222
HSPA9Heat shock 70kDa protein 9 (mortalin)1.40290.005582
FGFR1Fibroblast growth factor receptor 11.36260.005687
FGF4Fibroblast growth factor 41.17890.006383
NODALNodal homolog (mouse)1.69440.0064
ALDH1A1Aldehyde dehydrogenase 1 family, member A11.25440.007374
KAT2AK(lysine) acetyltransferase 2A1.20060.007585
PAX6Paired box 61.15780.008689
DNMT3BDNA (cytosine-5-)-methyltransferase 3 beta1.20050.011056
HNF4AHepatocyte nuclear factor 4, alpha1.41460.011275
CDK1Cyclin-dependent kinase 11.14710.012381
COL1A1Collagen, type I, alpha 11.17610.015877
LEFTY1Left-right determination factor 11.2260.015976
DPPA3Developmental pluripotency associated 21.31380.024156
NR5A2Nuclear receptor subfamily 5, group A, member 21.14120.026799
EP300E1A binding protein p3001.38470.029084
KAT7K(lysine) acetyltransferase 71.9670.029885
PARD6APar-6 partitioning defective 6 homolog alpha (C. elegans)1.27030.033506
POU5F1POU class 5 homeobox 11.11310.042862
ESRRBEstrogen-related receptor beta1.20210.044154

Telomerase reverse transcriptase, forkhead box D3 (FOXD3) and undifferentiated embryonic cell transcription factor 1 (UTF1) are other genes related to stemness, so we hypothesized that LL-37 should be important to maintain stem cell identity in breast cancer cells (Table 1). Upregulation of telomerase is a prerequisite for cellular immortalization and has been associated with stemness in various human cancers (11). FOXD3 induces cancer progression by epithelial-mesenchymal transition (12) and UTF1 increases stem cells reprogramming to pluripotency (13).

The results were not so evident in A375 melanoma cells, but the presence of Runt-related transcription factors 1 and 2 (14-16), Cadherin 2 (17, 18), Nodal homolog (mouse) (19) and Aldehyde dehydrogenase 1 (family member A1) pointed to the same direction (20) (Table 2). However, the production of oncospheres in A375 cells put in evidence that LL-37 has opposing effects on cancer cell stemness depending on the cell type.

Conclusion

Emerging evidence highlights the role of antimicrobial peptides in non-infectious diseases, such as cancer. The mechanisms triggered by antimicrobial peptides are broad and lead to unexpected cell responses. Here, we show that the antimicrobial peptide LL-37 is implicated in cancer stemness. Further research needs to be directed to better clarify this phenomenon and the role of other antimicrobial peptides in this scenario.

Acknowledgements

This work was supported by FAPESP, the Sao Paulo Research Foundation (grant # 2015/00892-4).

Competing Interests

The authors have declared that no competing interest exists.

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Author contact

Corresponding address Corresponding author: Fabiano Pinheiro da Silva, M.D., Ph.D. Faculdade de Medicina da Universidade de São Paulo, Laboratório de Emergências Clínicas (LIM-51), Av. Dr. Arnaldo, 455 sala 3189, CEP 01246-000, São Paulo - SP, Brazil. Phone: +55 11 3061 8480; Fax: +55 11 3061 8480; E-mail: pinheirofabianocom