FAS system deregulation in T-cell lymphoblastic lymphoma

Abstract

The acquisition of resistance towards FAS-mediated apoptosis may be required for tumor formation. Tumors from various histological origins exhibit FAS mutations, the most frequent being hematological malignancies. However, data regarding FAS mutations or FAS signaling alterations are still lacking in precursor T-cell lymphoblastic lymphomas (T-LBLs). The available data on acute lymphoblastic leukemia, of precursor origin as well, indicate a low frequency of FAS mutations but often report a serious reduction in FAS-mediated apoptosis as well as chemoresistance, thus suggesting the occurrence of mechanisms able to deregulate the FAS signaling pathway, different from FAS mutation. Our aim at this study was to determine whether FAS-mediated apoptotic signaling is compromised in human T-LBL samples and the mechanisms involved. This study on 26 T-LBL samples confirms that the FAS system is impaired to a wide extent in these tumors, with 57.7% of the cases presenting any alteration of the pathway. A variety of mechanisms seems to be involved in such alteration, in order of frequency the downregulation of FAS, the deregulation of other members of the pathway and the occurrence of mutations at FAS. Considering these results together, it seems plausible to think of a cumulative effect of several alterations in each T-LBL, which in turn may result in FAS/FASLG system deregulation. Since defective FAS signaling may render the T-LBL tumor cells resistant to apoptotic cell death, the correct prognosis, diagnosis and thus the success of anticancer therapy may require such an in-depth knowledge of the complete scenario of FAS-signaling alterations.

Figure 1

FAS mutations in T-LBL samples. Schematic representation of mutations found in FAS in human T-LBL samples, indicating FAS exons and FAS protein domains affected. An abbreviated form to indicate the mutations is included and explained in the legend

Figure 2

In vitro expression levels of mutant FAS variants. (a) JK cells transfected with wild-type or mutant FAS variant pKEX constructs. Flow cytometry showing endogenous (shaded) and exogenous (solid) surface FAS expression levels 48 h after transfection. Figure shows a representative of three independent experiments. (b) HEK-293T cells transfected with wild-type or mutant FAS variant pKEX constructs. Western blot of (panel 1) cellular protein extracts and (panel 2) conditioned medium protein extracts. NTC, non-transfected control

Figure 3

Functionality of mutant FAS variants. (a) Set-up of the effective dose for CH11-mediated cell death induction, showing the normalized cell death of JK cells treated with increasing doses of CH11. Normalization was made as follows: first, all data were divided by the result for PI staining of non-stimulated cells (0 ng/ml CH11), thus setting the latter as 1. Second, the results for PI staining of irrelevant IgM-treated cells were subtracted from their corresponding CH11-treated cells. (b) FAS-mediated cell death induction in JK cells transfected with wild-type or mutant FAS variant pKEX constructs. Histogram represents the mean and S.D. from at least three independent experiments of 50 and 100 ng/ml of irrelevant IgM and CH11 induction. The percentage of PI-positive cells is showed, previously subtracting from all data the result for non-stimulated cells. Dotted lines represent the result for % PI-positive cells treated with 50 ng/ml (black line) or 100 ng/ml (gray line) CH11 in JK cells not transfected. Significance is indicated by the corresponding P-value. n.s., not significant. (c) Representative experiment of flow cytometry showing PI positivity 24 h after 100 ng/ml CH11-triggering by endogenous FAS (dashed line), or exogenous wild-type (shaded) or mutant FAS variants (solid line). (d) FAS-mediated caspase 8 activation in JK cells transfected with wild-type or mutant FAS variants pKEX constructs, 8 h after 100 ng/ml CH11-triggering. Numbers indicate fluorescence intensity corresponding to the cleaved caspase 8 staining

Figure 4

FAS mRNA expression level in RNA samples. qRT-PCR determining FAS mRNA expression levels in human T-LBL samples where only RNA was available. The mean values were normalized to that of JK. Error bars represent S.D. Data represent two independent experiments. *Denotes statistical significance compared with the JK control, with P<0.05

Figure 5

Fas pathway expression levels in human T-LBL samples. Western blot showing FAS, FASLG, FADD, CASP8 and CASP3 protein levels in human T-LBL protein extracts. kDa, kiloDaltons. cl, cleaved