Epigenetic Silencing of Mir-203 Contributes to IL2Rb Overexpression and Malignant Transformation in Cutaneous T-Cell Lymphoma

Introduction

Cutaneous T-cell lymphomas (CTCL) are rare malignancies, which present in the skin and presumably arise from malignant transformation of T-cells normally destined to home to the cutaneous environment. MicroRNAs (miRs) regulate gene expression at the post transcriptional level. Many studies have shown that altered miR expression is a central event in lymphomagenesis, and that miRs have potential as both diagnostic and predictive tumor markers.

In CTCL we have previously identified and validated a 3 miR classifier that distinguishes CTCL from BID with > 95% accuracy, based upon the up-regulation of miR-155 combined with the down-regulation of miR-203 and miR-205. In normal adult tissues, miR-203 is mainly associated with keratinocyte differentiation, acting to repress stemness, and to induce cell cycle arrest and differentiation. In cancer, miR-203 has been shown to hold tumor suppressor properties, and may be down-regulated by promoter hyper-methylation. The function and implications of miR-203 for CTCL has not previously been described. In this study we have investigated the regulation and function of miR-203 in primary CTCL biopsies and cell lines.

Materials and Methods

Twenty-one fresh frozen primary CTCL biopsies, IL-2 independent CTCL cell lines (MyLa2059 and PDB2B), and the IL-2 dependent CTCL cell lines (SeAx and SeZ4) were analyzed in this study. Promoter methylation was analyzed by methylation specific melting curve analysis. Cell lines were transfected by electroporation of miR-203 mimic or non-template-control (mirVana, Ambion). Proliferation was measured by 3H-Thymidine and apoptosis by MMT assays. MiR-203 mimic and mock transfected cells were examined by Affymetrix RNA expression arrays (GeneChip Human Gene 2.0 ST). IL2Rβ mRNA expression was confirmed by qPCR and IL2Rβ protein levels by flow cytometry as measured by CD122 (IL2Rβ-chain), compared to CD25 (IL2Rα-chain) and CD132 (IL2Rγ-chain). Cloning was done according to the manufacturers’ recommendation (In-Fusion, Clontech) and luciferase reporter assays were performed using the Dual-Glo system (Promega).

Results

We show that miR-203 is epigenetically silenced by DNA methylation in both CTCL cell lines and in 9 of 21 (43%) of primary CTCL samples, and that miR-203 can be up-regulated by the hypo-methylating agents 5-azacytidine and 5-aza-2-deoxycytidine in vitro. We also show, that forced miR-203 expression in CTCL cells targets known oncogenes such as p63, Survivin and CREB. Furthermore, it is shown that induction of miR-203 reduces cell viability and decreases proliferation.

mRNA array analysis of miR-203 mimic and mock transfected cells lead to the identification of 19 significantly de-regulated genes (P<0.5/log fold change>2), including the as yet unrecognized miR-203 target molecule IL2Rb, which is essential for IL-2 induced JAK/STAT signaling. qPCR and FACS analysis confirmed this up-regulation both at the mRNA and protein level. The IL-2 dependent cell line SeAx showed significantly more profound down-regulation of IL2Rβ upon in miR203 transfected cell lines. Preliminary luciferase reporter assays confirm that IL2Rβ expression is regulated by miR-203, providing novel evidence that miR-203 may act in concert with IL-2/STAT in CTCL pathogenesis. These experiments are currently being validated.

Conclusion

We provide the first evidence that miR-203 acts as a tumor suppressor in CTCL. Furthermore we show that down-regulation of miR-203 leads to increased expression of an as yet unidentified target gene, IL2Rβ, which is directly involved in JAK/STAT signaling, that plays an essential role in the regulation of T-cell proliferation. Thus, we suggest that epigenetic miR-203 down-regulation and IL2Rβ up-regulation are important early and driving events in CTCL pathogenesis.