Ether à go-go potassium channel expression in soft tissue sarcoma patients

Abstract

Background: The expression of the human Eag1 potassium channel (Kv10.1) is normally restricted to the adult brain, but it has been detected in both tumour cell lines and primary tumours. Our purpose was to determine the frequency of expression of Eag1 in soft tissue sarcoma and its potential clinical implications.

Results: We used specific monoclonal antibodies to determine the expression levels of Eag1 in soft tissue sarcomas from 210 patients by immunohistochemistry. Eag1 was expressed in 71% of all tumours, with frequencies ranging from 56% (liposarcoma) to 82% (rhabdomyosarcoma). We detected differences in expression levels depending on the histological type, but no association was seen between expression of this protein and sex, age, grade or tumour size. Four cell lines derived from relevant sarcoma histological types (fibrosarcoma and rhabdomyosarcoma) were tested for Eag1 expression by real-time RT-PCR. We found all four lines to be positive for Eag1. In these cell lines, blockage of Eag1 by RNA interference led to a decrease in proliferation.

Conclusion: Eag1 is aberrantly expressed in over 70% sarcomas. In sarcoma cell lines, inhibition of Eag1 expression and/or function leads to reduced proliferation. The high frequency of expression of Eag1 in primary tumours and the restriction of normal expression of the channel to the brain, suggests the application of this protein for diagnostic or therapeutic purposes.

Figure 1

Typical staining of tumour samples with a single-chain antibody against Eag1. Eag1 shows homogeneous cytoplasmic staining with perinuclear localization. Shown are representative examples for the different intensities of Eag1 staining leading to the scoring of 0 (A, morphologically non-malignant skeletal muscle from a rhabdomyosarcoma case), 1+ (B, malignant fibrous histiocytoma), 2+ (C, leiomyosarcoma) and 3+ (D, rhabdomyosarcoma) used for further analysis. Magnification: 20×.

Figure 2

Preservation of Eag1 antigen in paraffin-embedded tissue. The percentage of Eag1-expressing tumours decreases with the elapsed storage time of samples before the immunohistochemistry procedure was performed. Insert: The percentage of Eag1-expressing tumours increases over the years (1997–2004) indicating that the Eag1 antigen is not stable in paraffin-embedded tissue for long periods of time. The numbers indicate the number of cases tested for each data point. *p < 0.01, **p < 0.001

Figure 3

Representative images of normal and tumour tissue. Alkaline phosphatase staining using anti-Eag1 antibody. Examples of fibrosarcoma (A, G), leimyosarcoma (B, H), liposarcoma (C, I), malignant fibrous histiocytoma (D, J), rhabdomyosarcoma (E, K) and synovial sarcoma (F, L). In all cases staining of Eag1 was restricted to tumours cells in comparison with the surrounding normal tissue. Magnification: 10× (A-F), 40× (G-L)

Figure 4

Inhibition of proliferation of sarcoma cell lines by imipramine or siRNAs. Soft tissue sarcoma cells (rhabdomyosarcoma – TE-671, A-204 and firbosarcoma – HT-1080, Hs633t) were transfected with siRNAs against Eag1 or were exposed to 10 μM imipramine for 96 hours prior to performing the MTT assay. Data are relative values compared to proliferation in the presence of vehicle and presented as mean ± standard error. Statistical analysis compared imipramine with non-treated cells, and siRNA-Eag1 and -GAPDH against scrambled siRNA molecules, p values are shown.