Epigenetic deregulation of Ellis Van Creveld confers robust Hedgehog signaling in adult T-cell leukemia

Abstract

One of the hallmarks of cancer, global gene expression alteration, is closely associated with the development and malignant characteristics associated with adult T-cell leukemia (ATL) as well as other cancers. Here, we show that aberrant overexpression of the Ellis Van Creveld (EVC) family is responsible for cellular Hedgehog (HH) activation, which provides the pro-survival ability of ATL cells. Using microarray, quantitative RT-PCR and immunohistochemistry we have demonstrated that EVC is significantly upregulated in ATL and human T-cell leukemia virus type I (HTLV-1)-infected cells. Epigenetic marks, including histone H3 acetylation and Lys4 trimethylation, are specifically accumulated at the EVC locus in ATL samples. The HTLV-1 Tax participates in the coordination of EVC expression in an epigenetic fashion. The treatment of shRNA targeting EVC, as well as the transcription factors for HH signaling, diminishes the HH activation and leads to apoptotic death in ATL cell lines. We also showed that a HH signaling inhibitor, GANT61, induces strong apoptosis in the established ATL cell lines and patient-derived primary ATL cells. Therefore, our data indicate that HH activation is involved in the regulation of leukemic cell survival. The epigenetically deregulated EVC appears to play an important role for HH activation. The possible use of EVC as a specific cell marker and a novel drug target for HTLV-1-infected T-cells is implicated by these findings. The HH inhibitors are suggested as drug candidates for ATL therapy. Our findings also suggest chromatin rearrangement associated with active histone markers in ATL.

Keywords: ATL; EVC; HTLV-1; Hedgehog; epigenetics.

Fig 1

EVC overexpression in ATL. (a, b) Microarray heatmap (a) and box plot (b) of EVC. **P < 0.01. ***P < 0.001. (c) Schematic illustration of locus encoding EVC1/2. (d) Individual expression values (n = 52) between EVC1 and EVC2. (e) EVC1 mRNA level in ATL patient PBMC (total, n = 11; acute, n = 7; chronic, n = 4) and in CD4+ T cells from healthy donors (n = 6) evaluated using quantitative RT-PCR (qRT-PCR). **P < 0.01. (f) EVC1 and EVC2 levels in CADM1 versus CD7 plot subpopulations. Normal P, CD4+/CADM1−/CD7+ T cells from healthy donors; Indolent P, CD4+/CADM1−/CD7+ from indolent ATL patients; Indolent N, CD4+/CADM1+/CD7− from indolent ATL patients; Acute N, CD4+/CADM1+/CD7− from acute ATL patients. The gene expression microarray dataset is available in Kobayashi et al. (g) EVC1 levels in various cell lines examined using qRT-PCR (n = 3, mean ± SD).

Fig 2

Epigenetic reprogramming in the EVC locus. (a) Schematic of CpG islands and chromatin immunoprecipitation (ChIP) loci. (b) Results of bisulfite sequencing (+46 to +466 from EVC1 transcription start site [TSS]; +905 to +1206 from EVC2 TSS). The black and empty boxes represent methylated and unmethylated CpG, respectively. (c) EVC RNA levels in Jurkat cells in the presence or absence of epigenetic drugs (n = 3, mean ± SD). *P < 0.05. (d) Histone covalent modifications at EVC and GAPDH loci in three cell lines were analyzed using PCR-based ChIP assay with specific antibodies. Positions of primer sets for the real-time PCR are indicated in (a). Enrichment values relative to input samples are plotted. (e) TL-Om1 and MT-1 cells were treated with 1 or 5 μM of anacardic acid for 48 h and the EVC mRNA level was then analyzed (n = 3, mean ± SD). *P < 0.05. (f) Epigenetic changes in primary ATL samples. Three independent clinical samples were compared with normal PBMC (n = 3, mean ± SD).

Fig 3

Role of Tax in EVC regulation. (a) EVC1 RNA levels are affected by Tax. The 293T cells in different FBS condition were transfected with the indicated plasmids. Relative EVC1 levels were evaluated using quantitative RT-PCR (qRT-PCR) (top panel, n = 3, mean ± SD). **P < 0.01. Tax expression was confir-med using western blotting with an anti-Tax antibody (Lt-4) (bottom panel). (b) Levels of SHH and PTHrP in the presence or absence of Tax (n = 3, mean ± SD). *P < 0.05. **P < 0.01. (c) EVC and PTCH1 levels in Jurkat cells expressing Tax (n = 3, mean ± SD). *P < 0.05. (d) Tax-mediated epigenetic changes. Histone modifications at EVC and GAPDH loci in Tax-expressing Jurkat cells were analyzed using a ChIP assay. *P < 0.05 (Tax WT vs Empty). Primer positions are shown in Figure2(a).

Fig 4

EVC1 expression in ATL cells. (a–e) Immunohistochemistry-based EVC1 protein detection in paraffin-embedded samples: 293T transfected with the indicated plasmids (a), primary ATL lymph node (b, c, representative data are shown) and spleen from mice engrafted with primary ATL cells (d, tumor invasive, n = 3; e, non-invasive). These samples were stained with anti-EVC1 antibody and hematoxylin.

Fig 5

EVC supports Hedgehog (HH) activity. (a) Relative RNA levels in shRNA-expressing TL-Om1 cells (n = 3, mean ± SD). *P <0.05. (b) Luciferase reporter plasmid containing 7 × sequential GLI-binding sites. (c) GLI2ΔN activa-ted HH activity (n = 3, mean ± SD). **P < 0.01. (d–f) Hedgehog activity in various shRNA-expressing TL-Om1 (d), MT-2 (e) and Tax and shEVC1-expressing Jurkat (f) (n = 3∼4, mean ± SD). *P < 0.05. **P < 0.01.

Fig 6

Hedgehog (HH)-dependent ATL cell survival. (a) Time course of the abundance of Venus+ TL-Om1 (left) and MT-2 (right) infected with lentiviral vector expressing control shRNA (shCtrl), either of two shRNA targeting EVC1 and EVC2, or shRNA targeting GLI1 and GLI2, then cultured for 27 days together with uninfected cells. Data are representative of three independent experiments. Results are presented relative to those of cells at 4 days post-infection. (b) shRNA-mediated apoptosis induction. shRNA-expressing cells were cultured in 1% FBS for 72 h. The apoptotic pattern was defined by gating with Venus+ and Annexin V/7-AAD (n = 3, representative data). (c) GANT61 inhibited HH activity in ATL cells (n = 3, mean ± SD). *P < 0.05. (d–e) GANT61 reduced ATL cell viability (n = 3, mean ± SD). The cells were treated with the indicated concentrations of GANT61 for 96 h (d) or with 5 μM of GANT61 for the indicated time periods (e). Cells were maintained in 1% FCS. (f) GANT61-dependent apoptosis analyzed using Annexin V/7-AAD staining (n = 3, mean ± SD). **P < 0.01.

Fig 7

GANT61 treatment reduced cell viabilities of primary ATL samples. (a) Effect of GANT61 in primary PBMC samples. The PBMC from healthy donors (n = 7), asymptomatic carriers (n = 3) and ATL patients (n = 5) were exposed in 5 μM of GANT61 for 72 h. Cells were maintained in media with 1% self-serum. **P < 0.01. (b) GANT61-dependent apoptosis in ATL samples. The PBMC from healthy donors (n = 4) and ATL patients (n = 4) were treated with 5 μM of GANT61 for 72 h. Graphs show percentiles of apoptotic popula-tion in CD4+ cells **P < 0.01.