Efficient recruitment of c-FLIPL to the death-inducing signaling complex leads to Fas resistance in natural killer-cell lymphoma

Abstract

Activation-induced cell death (AICD) mediated by the Fas/Fas ligand (FasL) system plays a key role in regulating immune response. Although normal natural killer (NK) cells use this system for their homeostasis, malignant NK cells seem to disrupt the process. Extranodal NK/T-cell lymphoma, nasal type (ENKL) is a rare but fatal disease, for which novel therapeutic targets need to be identified. We confirmed that ENKL-derived NK cell lines NK-YS and Hank1, and primary lymphoma cells expressed procaspase-8/FADD-like interleukin-1β-converting enzyme (FLICE) modulator and cellular FLICE-inhibitory protein (c-FLIP), along with Fas and FasL. Compared with Fas-sensitive Jurkat cells, NK-YS and Hank1 showed resistance to Fas-mediated apoptosis in spite of the same expression levels of c-FLIP and the death-inducing signaling complex (DISC) formation. Unexpectedly, the long isoform of c-FLIP (c-FLIP_L ) was coimmunoprecipitated with Fas predominantly in both ENKL-derived NK cell lines after Fas ligation. Indeed, c-FLIP_L was more sufficiently recruited to the DISC in both ENKL-derived NK cell lines than in Jurkat cells after Fas ligation. Knockdown of c-FLIP_L per se enhanced autonomous cell death and restored the sensitivity to Fas in both NK-YS and Hank1 cells. Although ENKL cells are primed for AICD, they constitutively express and efficiently utilize c-FLIP_L , which prevents their Fas-mediated apoptosis. Our results show that c-FLIP_L could be a promising therapeutic target against ENKL.

Keywords: Fas; FasL; activation-induced cell death; c-FLIPL; extranodal NK/T-cell lymphoma, nasal type.

Figure 1

Extranodal natural killer (NK)/T‐cell lymphoma, nasal type (ENKL) expresses cellular Fas‐associated death domain‐containing protein (FADD)‐like interleukin‐1β‐converting enzyme (FLICE)‐inhibitory protein (c‐FLIP) along with Fas and Fas ligand (FasL). A, Flow cytometry showing that ENKL‐derived NK cell lines, NK‐YS and Hank1, clearly expressed cell surface Fas and intracytoplasmic FasL. B, FasL, tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL), and TNF‐α levels in culture supernatants of Hank1 and Jurkat. Each cytokine concentration was measured three times and the mean value was represented in the time‐course graph. Hank1 secretes FasL and abundant TNF‐α. C, Western blot analysis detected Fas, FADD, procaspase‐8/FLICE, and long and short forms of c‐FLIP (c‐FLIP_L and c‐FLIP_S, respectively) at approximately the same levels in NK‐YS, Hank1, and Jurkat cells. D, Immunohistochemistry for Fas, FasL, and c‐FLIP was carried out using diagnostic specimens of 9 cases of ENKL. Simultaneous expression of Fas, FasL, and c‐FLIP was observed in 7 of 9 examined cases (78%). Two representative cases (UPN1 and UPN2) are presented

Figure 2

Extranodal natural killer (NK)/T‐cell lymphoma, nasal type cells show resistance to Fas‐mediated apoptosis. A, MTT assay showed that the stimulation of Fas with agonistic 7C11 but not with control mouse (Ms) IgM or antagonistic ZB4 decreased cell viability, particularly in Fas‐sensitive Jurkat cells. Although the viability of Jurkat cells was decreased to 10% 1 h after Fas ligation with 7C11, those of NK‐YS and Hank1 stayed at approximately 50% and 70%, compared with each control, respectively. The effects were statistically significant (Jurkat, *P < .001; NK‐YS, **P < .001; Hank1, ***P < .001; Student’s t test). B, Flow cytometry detected that only Fas‐sensitive Jurkat cells increased annexin V‐positive cell fractions 1 h after Fas ligation with 7C11. C, More than 40% of Jurkat cells showed apoptotic changes, whereas most NK‐YS and Hank1 cells failed to increase apoptotic changes. Only events in Jurkat cells reached significance (*P < .001, Student's t test)

Figure 3

Death‐inducing signaling complex was generated equally among both extranodal natural killer (NK)/T‐cell lymphoma, nasal type cells and Fas‐sensitive Jurkat cells after Fas ligation. A, Immunoprecipitation (IP) and western blot (WB) analyses similarly detected Fas‐associated death domain‐containing protein (FADD) and the cleaved forms of caspase‐8 (Casp‐8), which were coimmunoprecipitated with Fas after treatment with 7C11 but not with control mouse (Ms) IgM in NK‐YS, Hank1, and Fas‐sensitive Jurkat cells. B, Confocal microscopy showed the fusion yellow signals of Fas (red) and procaspase‐8/FLICE (Casp‐8, green) at the cell membrane after treatment with 7C11, but not with control Ms IgM in the 3 cell lines

Figure 4

Long isoform of cellular FLICE‐inhibitory protein (c‐FLIP_L) is efficiently recruited to the death‐inducing signaling complex in extranodal natural killer (NK)/T‐cell lymphoma, nasal type (ENKL) cells after Fas ligation. A, A cleaved form of c‐FLIP_L (indicated by red arrowhead) was coimmunoprecipitated with Fas predominantly in both ENKL cells after Fas ligation. Cleaved c‐FLIP_L was also bound to caspase‐8 after Fas ligation predominantly in both ENKL cells. B, Confocal microscopy also confirmed the colocalization of Fas (red) and c‐FLIP_L (green) predominantly at the cell membrane in both NK‐YS and Hank1, compared with Jurkat cells. C, Yellow fusion signals between Fas and c‐FLIP_L were counted in each 100 cells, in triplicate. Compared with Jurkat cells, the mean values were significantly high in NK‐YS and Hank1 (Jurkat vs NK‐YS, *P < .001; Jurkat vs Hank1, **P < .001; Student’s t test). IP, immunoprecipitation; WB, western blot

Figure 5

Long isoform of cellular FLICE‐inhibitory protein (c‐FLIP_L) plays a critical role in resistance to Fas‐mediated apoptosis in extranodal natural killer (NK)/T‐cell lymphoma, nasal type (ENKL) cells. A, RNA interference for c‐FLIP_L showed effective knockdown of c‐FLIP_L in NK‐YS and Hank1 cells, while the expression levels of the short isoform (c‐FLIP_S), receptor‐interacting protein (RIP)1, and antiapoptotic molecules including BCL2, BCL‐xL, and MCL1 were unchanged. B, Although the cells transfected control (CNT) siRNA hardly showed apoptotic changes even after Fas ligation, knockdown of c‐FLIP_L per se the increased annexin V‐positive fraction even after treatment with mouse (Ms) IgM in both ENKL cell lines. Fas ligation obviously enhanced the effect. Flow cytometry detected that more than 50% of both ENKL‐derived NK cell lines were positive for annexin V. C, These events were statistically significant (CNT siRNA‐7C11 vs c‐FLIP_L siRNA‐Ms IgM, *P = .005 in NK‐YS and ^# P = .002 in Hank1; c‐FLIP_L siRNA‐Ms IgM vs c‐FLIP_L siRNA‐7C11, **P < .001 in NK‐YS and ^## P < .001 in Hank1; Student’s t test). D, Although Fas ligation with CH‐11 promoted the processing of procaspase‐3, knockdown of c‐FLIP_L clearly accelerated this process and increased the cleavage of poly (ADP‐ribose) polymerase (PARP)1 in NK‐YS and Hank1 cells. Only this experiment was carried out using CH‐11 for Fas ligation

Figure 6

Predicted mechanisms of resistance to activation‐induced cell death (AICD) in extranodal natural killer (NK)/T‐cell lymphoma, nasal type (ENKL) cells. Although ENKL cells are similar to activated normal NK cells in being primed for AICD, they efficiently utilize the long isoform of cellular Fas‐associated death domain‐containing protein (FADD)‐like interleukin‐1β‐converting enzyme (FLICE)‐inhibitory protein (c‐FLIP_L), which prevents their Fas‐mediated apoptosis, and can survive in spite of the coexpression of Fas and Fas ligand (FasL)