Down-modulation of expression, or dephosphorylation, of IG20/MADD in tumor necrosis factor-related apoptosis-inducing ligand-resistant thyroid cancer cells makes them susceptible to treatment with this ligand

Abstract

Background: The IG20/MADD gene is overexpressed in thyroid cancer tissues and cell lines, and can contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. The ability of the MADD protein to resist TRAIL-induced apoptosis is dependent upon its phosphorylation by Akt. Interestingly, while TRAIL induces a significant reduction in the levels of phospho-Akt (pAkt) and phospho-MADD (pMADD) in TRAIL-sensitive cells, it fails to do so in TRAIL-resistant cells. In this study, we investigated if MADD phosphorylation by Akt was contributing to TRAIL resistance in thyroid cancer cells.

Methods: We determined the susceptibility of different thyroid cancer cell lines to TRAIL-induced apoptosis by fluorescence-activated cell sorting (FACS) analysis. We tested for various TRAIL resistance factors by FACS analyses or for IG20/MADD expression by quantitative reverse transcription-polymerase chain reaction. We determined the levels of pAkt and pMADD upon TRAIL treatment in thyroid cancer cells by Western blotting. We tested if down-modulation of IG20/MADD gene expression using shRNA or phosphorylation using a dominant negative Akt (DN-Akt) or pretreatment with LY294002, a PI3 kinase inhibitor, could help overcome TRAIL resistance.

Result: BCPAP and TPC1 cells were susceptible, while KTC1 and FTC133 cells were resistant, to TRAIL-induced apoptosis. The differential susceptibility to TRAIL was not related to the levels of expression of death receptors, decoy receptors, or TRAIL. KTC1 and FTC133 cells showed higher levels of IG20/MADD expression relative to BCPAP and TPC1, and were rendered susceptible to TRAIL treatment upon IG20/MADD knockdown. Interestingly, upon TRAIL treatment, the pAkt and pMADD levels were reduced in TRAIL-sensitive BCPAP and TPC1 cells, while they remained unchanged in the resistant KTC1 and FTC133 cells. While expression of a constitutively active Akt in BCPAP and TPC1 cells rendered them resistant to TRAIL, pretreating KTC1 and FTC133 cells with LY294002 rendered them TRAIL-sensitive. Moreover, expression of a DN-Akt in KTC1 and FTC133 cells reduced the levels of pAkt and pMADD and sensitized them to TRAIL-induced apoptosis.

Conclusion: Our results show that pMADD is an important TRAIL resistance factor in certain thyroid cancer cells and suggest that down-modulation of either IG20/MADD expression or phosphorylation can render TRAIL-resistant thyroid cancer cells sensitive to TRAIL.

FIG. 1.

Susceptibility of different thyroid cancer cell lines to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis. Approximately 3×10⁵ of BCPAP, TPC1, KTC1, and FTC133 thyroid cancer cells were cultured in 6-well plates, and treated with different doses of TRAIL. Subsequently, cells were stained with tetramethyl-rhodamine methyl ester (TMRM) and subjected to fluorescence-activated cell sorting (FACS) analysis. Loss of TMRM staining, because of mitochondrial depolarization, was used as a marker of apoptosis. Summarized data are shown from three independent experiments.

FIG. 2.

Expression of TRAIL resistance factors in thyroid cancer cell lines. (A) Expression levels of death receptors (DRs), decoy receptors (DcRs), and TRAIL in different thyroid cancer cell lines; (B) expression of endogenous IG20/MADD in thyroid tissues and thyroid cancer cell lines detected by quantitative reverse transcription–polymerase chain reaction (RT-PCR). Sequences of primers and the probe have been published earlier (8), and 18s rRNA primer was used as internal control. The experiment was carried out in triplicate, the dash line indicate the average normal expression level of IG20/MADD gene.

FIG. 3.

IG20/MADD knockdown sensitizes resistant thyroid cancer cells to TRAIL-induced apoptosis. (A, B) Efficiency of IG20 knockdown using MID-shRNA in thyroid cancer cell lines: total of 6×10⁵ of KTC1 and FTC133 cells were cultured in 60-mm dishes and transduced with SCR or MID-shRNA carrying lentivirus for 3 days. (A) One microgram of total RNA was used for RT-PCR. The amplified products were separated using a 2% Tris-Borate-EDTA (TBE) agarose gel. The GAPDH served as a loading control. Western blot (B): fifty micrograms of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to nitrocellulose membranes. Blots were stained using anti-13L antibody. β-actin was used as a loading control. The protein bands were visualized by enhanced chemiluminescence. (C, D) Effect of IG20/MADD knockdown on TRAIL-induced apoptosis in TRAIL-resistant thyroid cancer cell lines: total of 3×10⁵ of KTC1 and FTC133 cells were cultured in six-well plates overnight, and then transduced with SCR or MID-shRNA carrying lentivirus for 72 hours. These cells were incubated with 100 ng/mL of TRAIL for an additional 6 hours. The cells were stained with TMRM and subjected to FACS analysis by gating for green fluorescent protein-positive cells. Summarized data are shown from three independent experiments. *p<0.05; **p<0.01.

FIG. 4.

Expression levels of phospho-MADD (pMADD) and phospho-Akt (pAkt) upon TRAIL treatment in thyroid cancer cell lines: total of 6×10⁵ of (A) BCPAP, (B) TPC1, (C) KTC1, and (D) FTC133 thyroid cancer cells were cultured in 60-mm dishes overnight and treated with TRAIL at the concentration and for the duration indicated. Cell lysates containing 50 μg of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Blots were stained using specific antibodies against Akt, pAkt, MADD, and pMADD. The protein bands were visualized by enhanced chemiluminescence.

FIG. 5.

Pretreatment with the PI3K inhibitor LY 294002 (LY) sensitizes KTC1 and FTC133 cells to TRAIL-induced apoptosis. Total of 1.6×10⁵ of KTC1 and FTC133 cells were cultured in 6-well plates. The cells were serum starved overnight, and treated with 10 μM of LY for 1 hour followed by treatment with TRAIL (100 μg/mL) for 6 hours. The cells were harvested and stained with PE-labeled anti-caspase-3 antibody and subjected to FACS analysis. Data are summarized from three experiments. **p<0.01.

FIG. 6.

Dominant-negative Akt (DN-Akt) sensitizes thyroid cancer cells to TRAIL-induced apoptosis. (A, C) Expression of DN-Akt reduces the levels of pAkt and pMADD in KTC1 and FTC133 cells. Total of 8×10⁴ of KTC1 and FTC133 cells were cultured in six-well plates and transfected with either an empty vector (Vec) or a DN-Akt and cultured for 42 hours. The cells were treated with 100 ng/mL of TRAIL for an additional 6 hours, washed in cold PBS, and then proteins were subjected to Western blotting to detect pAkt and pMADD; β-actin was used as a loading control. (B, D) Overexpression of DN-Akt leads to spontaneous as well as TRAIL-induced apoptosis in KTC1 and FTC133 cells. The same set of KTC1 and FTC133 cells as shown in (A) and (C) were stained with TMRM and subjected to FACS analysis. Summarized data are shown from three independent experiments, *p<0.05; **p<0.01.