Upregulation of c-FLIP-short in response to TRAIL promotes survival of NSCLC cells, which could be suppressed by inhibition of Ca2+/calmodulin signaling

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for killing tumor cells. However, a number of studies have demonstrated that different cancer cells resist TRAIL treatment and, moreover, TRAIL can promote invasion and metastasis in resistant cells. Here we report that TRAIL rapidly activates caspase-8 in a panel of non-small-cell lung carcinomas (NSCLCs). Adenocarcinomas derived from the lung in addition to high caspase-8 expression are characterized by increased expression of DR4 compared with adjacent non-neoplastic tissues. Blocking DR4 or lowering caspase-8 expression significantly reduced apoptosis in NSCLC cell lines, indicating the importance of DR4 and signifying that higher levels of caspase-8 in lung adenocarcinomas make them more susceptible to TRAIL treatment. Despite rapid and robust initial responsiveness to TRAIL, surviving cells quickly acquired resistance to the additional TRAIL treatment. The expression of cellular-FLIP-short (c-FLIPS) was significantly increased in surviving cells. Such upregulation of c-FLIPS was rapidly reduced and TRAIL sensitivity was restored by treatment with cycloheximide. Silencing of c-FLIPS, but not c-FLIP-long (c-FLIPL), resulted in a remarkable increase in apoptosis and significant reduction of clonogenic survival. Furthermore, chelation of intracellular Ca(2+) or inhibition of calmodulin caused a rapid proteasomal degradation of c-FLIPS, a significant increase of the two-step processing of procaspase-8, and reduced clonogenicity in response to TRAIL. Thus, our results revealed that the upregulation of DR4 and caspase-8 expression in NSCLC cells make them more susceptible to TRAIL. However, these cells could survive TRAIL treatment via upregulation of c-FLIPS, and it is suggested that blocking c-FLIPS expression by inhibition of Ca(2+)/calmodulin signaling significantly overcomes the acquired resistance of NSCLC cells to TRAIL.

Figure 1

Expression of DISC components and apoptotic response in NSCLC cells upon treatment with TRAIL. (a) Expression of c-FLIP_S, procaspase-8, DR4 and DR5, and FADD in a panel of NSCLC cells. (b) TRAIL-mediated activation of caspase cascade in NSCLC cells. NSCLC cells were treated with TRAIL (3 h, 200 ng/ml) and processing of procaspase-8 and formation of active forms of caspase-9 and -3 and specific cleavage (Cl) of PARP-1 were analyzed by immunoblot. (c and d) NSCLC cells were treated with TRAIL (24 h, 100 ng/ml) and MMP was assessed using TMRE staining. Apoptotic cell death was measured by Annexin V staining. Error bars represent S.E. *P<0.05

Figure 2

DR4 mediates apoptosis of NSCLC cells in response to TRAIL treatment. (a) DR4 and DR5 in A549 cells were blocked using specific antagonizing antibodies for 30 min before TRAIL treatment (3 h, 200 ng/ml). Processing of procaspase-8 and cleavage (Cl) of PARP were analyzed by immunoblot. (b) DR5 was silenced in A549 cells using specific small interfering RNA (siRNA), and 48 h post-siRNA transfection, cells were treated with TRAIL (3 h, 200 ng/ml). (c) Expression of surface DR4 assessed by flow cytometry as described in Materials and Methods section. Filled histograms represent antibody control and green histograms represent staining with DR4 antibody. (d) Surface DR4 in a panel of NSCLC cells was blocked using a neutralizing antibody for 30 min prior TRAIL treatment (3 h, 200 ng/ml). Cell death was analyzed by Annexin V staining using flow cytometry. (e) Expression of procaspase-8 and DR4 in lung adenocarcinomas and adjacent non-neoplastic tissues was analyzed by immunoblot. Error bars represent S.E. *P<0.05. Ctr, control; a-DR, antagonizing DR4, or DR5 antibody; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; N, normal tissue; T, tumor tissue; scr, scrambled; Untr, untreated

Figure 3

Survival of NSCLC cells in response to TRAIL treatment. (a and b) Cells were seeded in 6-well plates, and 24 h after the seeding, cells were treated with TRAIL (200 ng/ml). At 14 days after treatment, cells were fixed in paraformaldehyde and colonies were counted. (c) A549 cells were repeatedly treated for 3 h with TRAIL (200 ng/ml). At 48 and 96 h after the initial treatment, fresh TRAIL (200 ng/ml) was added for 3 h and cells were collected. Procaspase-8 processing and PARP-1 cleavage was analyzed by immunoblot. Error bars represent S.E. *P<0.05. Cl, cleaved; Unt, untreated

Figure 4

Upregulation of c-FLIP_S is essential for clonogenic survival of NSCLC cells treated with TRAIL. (a and b) NSCLC cells were treated with TRAIL (3 days, 200 ng/ml) and DISC components were analyzed by immunoblot. (c) A549 cells that survived 3 days treatment with TRAIL were untreated (Untr) or treated with CHX (10 μg/ml) simultaneously with freshly added TRAIL (200 ng/ml). Processing of procaspase-8, expression of c-FLIP_S, and formation of cleaved (Cl) PARP-1 were detected by immunoblot. (d) The effect of silencing of c-FLIP_L and c-FLIP_S on TRAIL-mediated activation of procaspase-8 and -3, and cleavage of PARP-1. (e and f) Clonogenic survival of A549 cells transfected with small interfering (si)RNAs targeting c-FLIP_L, c-FLIP_S, and procaspase-8. At 24 h after siRNA transfection, cells were seeded in 6-well plates, and 24 h after the seeding, cells were treated with TRAIL (200 ng/ml). Cell colonies were fixed and counted 14 days after the treatment. Ratio of the number of colonies in TRAIL-treated divided by the number of colonies in TRAIL-untreated samples for each specific siRNA is presented as bars in (f). (g) Processing of procaspase-8 in A549 cells transfected with siRNAs targeting procaspase-8, c-FLIP_L, and c-FLIP_S, and treated with TRAIL (200 ng/ml, 2 h). Cells from the same transfection experiment were used for clonogenic assay. Error bars represent S.E. *P<0.05. FADD, Fas-associated protein with death domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; scr, scrambled

Figure 5

Difference in c-FLIP_S mRNA expression and regulation of its degradation in NSCLC cells. (a) The expression of c-FLIP_S in NSCLC cells was analyzed by quantitative real-time polymerase chain reaction (PCR). (b) NSCLC cells were untreated (Untr) or treated with either inhibitor of proteasomal proteolysis MG132 (5 μM, 3 h) or bafilomycin A1 (Baf) (200 nM, 3 h). The expression of c-FLIP_S was analyzed by immunoblot. (c) A549 cells were treated with MG132 (5 μM, 3 h) alone or in combination with Ca²⁺ chelator BAPTA-AM (10 μM, 3 h) or the inhibitors of calmodulin, CMZ (10 μM, 3 h) and FPZ (10 μM, 3 h). The expression of c-FLIP_S was analyzed by immunoblot. (d) A549 cells were treated with calmodulin inhibitors and c-FLIP_S and procaspase-8 expression were analyzed by immunoblot. Error bars represent S.E. GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Figure 6

Inhibition of Ca²⁺/calmodulin signaling reduces survival of NSCLC cells in response to TRAIL. (a) A549 cells were pretreated (1 h) with calmodulin inhibitor CMZ (10 μM) or FPZ (10 μM), followed by 2-h treatment with TRAIL (200 ng/ml). Processing of procaspase-8 was analyzed by immunoblot. (b) A549 cells were pretreated with calcium chelator BAPTA-AM (10 μM, 1 h), followed by 1-h treatment with biotinylated TRAIL (500 ng/ml). Protein complexes were precipitated using streptavidin agarose as described in Materials and Methods section. Biotinylated TRAIL was also added to untreated cell lysate at 500 ng/ml. DR4, FADD, c-FLIP_S, and procaspase-8 cleavage were detected by immunoblot using specific antibodies. (c) A549 cells were pretreated with calmodulin inhibitors FPZ and CMZ or calcium chelator BAPTA-AM (10 μM, 1 h). To avoid toxicity, cells were washed and treated for an additional 24 h with TRAIL (200 ng/ml). Cell death was analyzed by flow cytometry using Annexin V staining. (d) The effect of calcium chelator BAPTA-AM on clonogenic survival of A549 and H661 cells. Cells were either untreated (Untr) or pretreated with BAPTA-AM (10 μM, 1 h), followed by TRAIL treatment (200 ng/ml, 3 h). Then, cells were washed and incubated for an additional 14 days and a clonogenic assay was performed as described above. Error bars represent S.E. *P<0.05. Cl, cleaved; FADD, Fas-associated protein with death domain; IP, immunoprecipitation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase