Elsevier

European Journal of Cancer

Volume 47, Issue 12, August 2011, Pages 1908-1918
European Journal of Cancer

IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF

https://doi.org/10.1016/j.ejca.2011.04.012Get rights and content

Abstract

Background

IL-20 is a pleiotrophic member of the IL-10 family and plays a role in skin biology and the development of haematopoietic cells. Recently, IL-20 has been demonstrated to have potential anti-angiogenic effects in non-small cell lung cancer (NSCLC) by down regulating COX-2.

Methods

The expression of IL-20 and its cognate receptors (IL-20RA/B and IL-22R1) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of this family was examined in normal bronchial epithelial and NSCLC cell lines. Furthermore, the effect of IL-20 on VEGF family members was examined.

Results

The expression of IL-20 and its receptors are frequently dysregulated in NSCLC. IL-20RB mRNA was significantly elevated in NSCLC tumours (p < 0.01). Protein levels of the receptors, IL-20RB and IL-22R1, were significantly increased (p < 0.01) in the tumours of NSCLC patients. IL-20 and its receptors were found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation. In addition, treatment with recombinant IL-20 resulted in decreased expression of the VEGF family members at the mRNA level.

Conclusions

This family of genes are dysregulated in NSCLC and are subject to epigenetic regulation. Whilst the anti-angiogenic properties of IL-20 require further clarification, targeting this family via epigenetic means may be a viable therapeutic option in lung cancer treatment.

Introduction

IL-20 was initially identified in a human keratinocyte library.1 It is a pleiotrophic member of the extended IL-10 cytokine family, including IL-19, 22, 24, 26, 28 and 29, all of which have well defined immune functions.2, 3 IL-20 is a pro-inflammatory cytokine, playing key roles in skin diseases, particularly psoriasis,4, 5 and has been implicated in arthritis, atherosclerosis and lupus nephritis,6, 7, 8, 9 and treatment with an anti-IL-20 antibody produced a therapeutic benefit in arthritis.10 Lipopolysaccharide (LPS) induced IL-20 expression in glial cells suggests that IL-20 may also play a role in inflammatory conditions in the brain.11

IL-20 is expressed in a wide variety of cell types, particularly in lung and skin,4, 7 as well as bronchial epithelial cells.7, 12 In addition, expression of IL-20 has been found in breast13 and occurs in cells of the immune system such as monocytes, T cells and maturing dendritic cells.14, 15

IL-20 signals through a hetero-dimeric receptor complex, which consists of IL-20RB, combined with either IL-20RA (Type 1 receptor complex), or IL-22R1 (Type 2 receptor complex).7, 16, 17 The expression pattern of the IL-20 receptors indicate that cells in the skin, lung and reproductive organs are targets of IL-20 and other members of the extended IL-10 family.1, 7 IL-20 signalling has been shown to induce cellular proliferation and activate STAT3 through both receptor complexes.1, 16, 17

IL-20 may also have either pro- or anti- angiogenic properties, but this would appear somewhat cell line and in vivo/in vitro dependant.18, 19 However, it has recently been shown to demonstrate anti-angiogenic properties in NSCLC (non-small cell lung cancer).18

Inflammation plays a role in lung carcinogenesis.20, 21 As IL-20 has such a significant role in inflammatory skin biology, this suggests that IL-20 may be important in lung disease. Evidence has emerged linking decreased expression of IL-20RA via DNA CpG hypermethylation22 with poorer disease free survival in NSCLC.23 This suggests that the IL-20 signalling pathway may be important to NSCLC pathogenesis and could potentially be amenable to epigenetic targeting therapies.

We examined the expression of IL-20 and its receptors in a series of primary NSCLC tumour samples and an additional panel of normal and NSCLC cell lines, and tested whether epigenetic mechanisms play a role in their regulation. Our results demonstrate that the expression of IL-20 and its receptors are; frequently dysregulated in NSCLC; regulated via epigenetic mechanisms (DNA CpG methylation and histone post-translational modifications); and may represent a candidate anti-angiogenic therapeutic approach in treating NSCLC as IL-20 treatment results in the down regulation of VEGF family members.

Section snippets

Cell lines

The A549 (adenocarcinoma), SK-MES-1 (squamous cell carcinoma), H460, H647 and H1299 (large cell carcinoma) and BEAS2B (transformed normal bronchoepithelial) cell lines were purchased from the ATCC (LGC Promochem, Teddington, United Kingdom). HBEC cell lines24 were a gift from Prof. John D Minna (Hamon Centre for Therapeutic Oncology Research, UTSoutwestern, Dallas, TX, United States of America). All cell culture reagents were purchased from Lonza (Walkersville, MD, USA) unless stated otherwise.

Gene expression analysis of IL-20, IL-20RA/B and IL-22R1 in primary lung cancer tumour specimens

RT-PCR was performed to assess the expression of IL-20 family members in a panel of normal/tumour matched chemo-naïve patient samples (Fig. 1A). Densitometric analysis of the gels revealed a significant increase in the expression of IL-20RB in NSCLC tumour samples compared with normal (p < 0.01) (Fig. 1B). Overall, increased gene expression was observed for IL-20 (10/18, 55.5%), IL-20RB (14/18, 77.7% – p < 0.01) and IL-22R1 (13/18, 72.2%), whilst the IL-20RA receptor had reduced expression in the

Discussion

The IL-20 cytokine is a member of the IL-10 family and has a number of key roles in pro-inflammatory joint and skin conditions but has yet to be studied to any great extent in the lung.

In a series of tumour/normal matched chemo-naïve patient samples, the mRNA expression of IL-20 and its receptors were examined. In general, increased mRNA was observed in the tumours compared to their matched normal counterparts (Fig. 1B). In particular, significantly elevated levels of IL-20RB were observed

Cell lines

The HBEC cell lines were a gift from Prof. John D. Minna (Hamon Centre for Therapeutic Oncology Research, UTSouthwestern, Dallas, TX, USA).

Conflict of interest statement

None declared.

Acknowledgement

Grant support: This work was supported by an unrestricted educational grant from Pfizer.

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