High expression of the evolutionarily conserved alpha/beta hydrolase domain containing 6 (ABHD6) in Ewing tumors

Abstract

Despite improvements in the treatment of patients with Ewing family tumors (EFT), the prognosis for patients with advanced disease is still unsatisfactory. Recently, we identified lipase I as an EFT-associated gene that might be interesting for the development of new immunological or pharmacological treatment strategies. Lipase I is a member of the large protein superfamilies of alpha/beta hydrolases and serine hydrolases. In the present paper we describe high expression of another member of these superfamilies in EFT. By DNA microarray data base mining we found exceptional high expression of alpha/beta hydrolase domain containing 6 (ABHD6) in EFT but not in other sarcomas. Expression of ABHD6 in EFT correlated with expression of another EFT-associated gene, aristaless. Analysis of ABHD6-associated GGAA microsatellites revealed shorter microsatellites in EFT with lack of ABHD6 expression. ABHD6 homologues were found in varying chordata but not in other animal species. Based on homology modeling we predicted the 3D-structure of ABHD6, which shows high similarity with bacterial homoserine transacetylases. High expression of ABHD6 in EFT in comparison to normal tissues and other tumors suggests that ABHD6 might be an interesting new diagnostic or therapeutic target for EFT. However, knock down of ABHD6 in EFT cells did not inhibit tumor cell growth.

Figure 1

High expression of lipase I (LIPI), carboxylesterase 1 (CES1), and α/β hydrolase domain containing 6 (ABHD6) in Ewing family tumors (EFT). (a) Consensus motif of human members of the α/β hydrolase superfamily. Sequence logos were generated on the basis of the following human α/β hydrolases: ABHD1, ABHD2, ABHD3, ABHD4, ABHD6, ABHD8, ABHD11, CES1, CES2, CES3, DAGLB, LYPLA1, LYPLA2, LIPA, LIPC, LIPD, LIPF, LIPG, LIPH, LIPI, PNLIP, PNLIPRP1, PNLIPRP2, PNLIPRP3, and PSPLA1. Approximately half of these enzymes encode the central serine by the codon AGY. The other enzymes encode this amino acid by the codon TCN. (b) Unsupervised hierarchical clustering of EFT samples and normal tissue samples (NBA). For cluster analysis only probe sets corresponding to lipases and other α/β hydrolases were used. EFT was clearly separated from NBA. The position of probe sets corresponding to CES1, ABHD6, and LIPI is indicated. See Materials and Methods for a description of NBA samples.

Figure 2

 High expression of lipase I (LIPI) and CES1 in Ewing family tumors (EFT) but not in other tumor samples. Presented are signal intensities (Affymetrix HG_U133Plus2.0 microarrays) from normal tissues (NBA), EFT, and other tumor samples (Non‐EFT) for CES1 and LIPI. Whereas LIPI is only detectable in EFT, CES1 is highly expressed in normal liver. See Materials and Methods for a description of NBA and tumor samples.

Figure 3

 High expression of α/β hydrolase domain containing 6 (ABHD6) in Ewing family tumors (EFT) but not in other tumor samples. (a) Presented are signal intensities (Affymetrix HG_U133Plus2.0 microarrays) from normal tissues (NBA), EFT, and other tumor samples (Non‐EFT) for ABHD6. (b) High expression of ABHD6 in an independent database based on Affymetrix HG_U133A microarray data. See Materials and Methods for a description of NBA and tumor samples.

Figure 4

 Validation of high expression of α/β hydrolase domain containing 6 (ABHD6) in Ewing family tumors (EFT) cell lines by RT‐PCR. Presented are results from RT‐PCR analysis with ABHD6‐specific primers. β‐Actin (ACTB) was used as control. NTC, no template control. (a) ABHD6 was only detected in EFT cell lines, not in neuroblastoma (NB) cell lines. The following cell lines were analyzed: (from left to right) A673, SK‐N‐MC, RD‐ES, SK‐ES, TC‐32, STA‐ET8, TC‐71, TTC‐466 (EFT), SiMa, SH‐SY5Y, IMR5, Kelly, and CHP‐134 (neuroblastoma). (b) ABHD6 was only detected in primary EFT samples, not in rhabdomyosarcoma (RMS) samples.

Figure 5

 Analysis of putative EWSR1–ETS binding sites upstream of α/β hydrolase domain containing 6 (ABHD6). (a) Analysis of the human ABHD6 locus. Exons are shown as filled boxes, introns as open boxes. The predicted CpG island is shown in gray, the predicted promoter region is shaded. The position of the start codon, stop codon, and probable ETS binding site are indicated. (b) Analysis of ABHD6‐associated GGAA repeats in Ewing family tumor (EFT) cell lines. Length of ABHD6‐associated microsatellites from five EFT cell lines (1, SK‐ES; 2, TC71; 3, A673; 4, SK‐N‐MC; 5, RD‐ES) was visualized by RT‐PCR as described in Materials and Methods (right panel). In parallel, expression of ABHD6 was quantified by quantitative RT‐PCR (qRT‐PCR) in the same cell lines (left panel). For comparative gene expression analysis the signal of one neuroblastoma cell line (not shown) was set as one. NTC, no template control.

Figure 6

 Correlation of expression of α/β hydrolase domain containing 6 (ABHD6) and aristaless (ARX) in Ewing family tumors (EFT). (a) Presented are signal intensities (Affymetrix HG_U133Plus2.0 microarrays) for ABHD6 and ARX in EFT cell lines. (b) Presented are results from conventional RT‐PCR analysis with ABHD6‐ and ARX‐specific primers. ABHD6 signals were only detectable in EFT cell lines, with the exception of the neuroblastoma cell line Kelly, ARX was also detectable only in EFT. β‐Actin (ACTB) was used as a control (not shown). The following cell lines were analyzed: (from left to right) A673, SK‐N‐MC, RD‐ES, SK‐ES, TC‐32, STA‐ET8, TC‐71, TTC‐466 (EFT), SiMa, SH‐SY5Y, IMR5, Kelly, and CHP‐134 (neuroblastoma). (c) Presented are results from quantitative RT‐PCR analysis with ABHD6‐ and ARX‐specific primers. EFT samples are presented as filled circles, neuroblatsoma samples as open circles (same samples as in [b]). For comparative gene expression analysis GAPDH was used as a calibrator. The signal of one neuroblastoma cell line was set as one. The position of the Kelly cell line is indicated.

Figure 7

 Knock down of α/β hydrolase domain containing 6 (ABHD6) in Ewing family tumor (EFT) cells did not influence cell growth. ABHD6 was knocked down in the EFT cell line SK‐N‐MC as described in Materials and Methods. Cells transfected with empty vector were used as a control. (a) After selection of stable blasticidin‐resistant cells, expression of ABDH6 was analyzed by RT‐PCR. β‐Actin (ACTB) and lipase I (LIPI) served as controls. (b) The same cells as in (a) were placed at a cell density of 1 × 10⁵/mL in 10 mL culture medium. After 4 days cells were harvested and counted. Bars represent means and standard deviations from three independent experiments. In each experiment cell numbers were determined in quadruplicate. miR ABHD6, cells transfected with ABHD6‐specific RNAi expression vector.