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Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

Gillissen B, Richter A, Richter A, Overkamp T, Essmann F, Hemmati PG, Preissner R, Belka C, Daniel PT - Cell Death Dis (2013)

Bottom Line: Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes.Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype.Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany.

ABSTRACT
TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

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TRAIL-induces pro-caspase-3 processing in Bax/Bak double-deficient cells but fails to overcome inhibition by XIAP and proteasomal caspase-3 degradation. (a) HCT116 wt and Bax−/Bak− cells were treated with 50 ng/ml TRAIL and pro-caspase-8 and -3 processing were analyzed by immunoblotting. TRAIL-treatment induced cleavage of pro-caspase-8 and -3 in both cell lines. Cleavage of pro-caspase-8 is accompanied by generation of its active subunits. In contrast, processing of pro-caspase-3 to its active subunits is detectable only in HCT116 wt but not in HCT116 Bax−/Bak− cells (left). Additional downregulation of XIAP in combination with TRAIL caused full processing of pro-caspase-3 to its active subunits in HCT116 Bax−/Bak− cells (right). (b) HCT116 wt and Bax−/Bak− cells were incubated with the proteasome inhibitor MG132 prior to TRAIL-treatment. Upon inhibition of the proteasome, TRAIL-treatment resulted in full processing of pro-caspase-3 to its active subunits in both cell lines. (c, d) In addition to TRAIL, HCT116 wt and Bax−/Bak− cells were treated with 1 μM of MG132 (c) or with 1 μM of bortezomib (BZM) (d). Cells were cultured for 24 h, harvested and apoptotic cells were determined by flow cytometric measurement of cellular DNA content. HCT116 wt cells were sensitized for TRAIL-induced apoptosis by MG132 or BZM. Furthermore, inhibition of the proteasome enables TRAIL to kill Bax/Bak-deficient HCT116 cells
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fig2: TRAIL-induces pro-caspase-3 processing in Bax/Bak double-deficient cells but fails to overcome inhibition by XIAP and proteasomal caspase-3 degradation. (a) HCT116 wt and Bax−/Bak− cells were treated with 50 ng/ml TRAIL and pro-caspase-8 and -3 processing were analyzed by immunoblotting. TRAIL-treatment induced cleavage of pro-caspase-8 and -3 in both cell lines. Cleavage of pro-caspase-8 is accompanied by generation of its active subunits. In contrast, processing of pro-caspase-3 to its active subunits is detectable only in HCT116 wt but not in HCT116 Bax−/Bak− cells (left). Additional downregulation of XIAP in combination with TRAIL caused full processing of pro-caspase-3 to its active subunits in HCT116 Bax−/Bak− cells (right). (b) HCT116 wt and Bax−/Bak− cells were incubated with the proteasome inhibitor MG132 prior to TRAIL-treatment. Upon inhibition of the proteasome, TRAIL-treatment resulted in full processing of pro-caspase-3 to its active subunits in both cell lines. (c, d) In addition to TRAIL, HCT116 wt and Bax−/Bak− cells were treated with 1 μM of MG132 (c) or with 1 μM of bortezomib (BZM) (d). Cells were cultured for 24 h, harvested and apoptotic cells were determined by flow cytometric measurement of cellular DNA content. HCT116 wt cells were sensitized for TRAIL-induced apoptosis by MG132 or BZM. Furthermore, inhibition of the proteasome enables TRAIL to kill Bax/Bak-deficient HCT116 cells

Mentions: As caspase-8 and -3 are crucial for TRAIL-induced apoptosis, we next examined to which extent Bax/Bak deficiency impacts on processing of caspase-8 and -3. We treated HCT116 wt and Bax−/Bak− cells with TRAIL and analyzed the cleavage pattern of these caspases. Pro-caspase-8 and -3 levels are comparable in both cell lines, respectively (Figure 2a, left). Following TRAIL-treatment, pro-caspase-8 is cleaved in both cell lines and processed to its active subunits to a similar extent. Interestingly, in both cell lines, caspase-8 activation goes along with cleavage of the pro-caspase-3 zymogen, indicating that caspase-8 activation upon TRAIL-treatment is sufficient to cleave pro-caspase-3. Nevertheless, cleavage of pro-caspase-3 is accompanied by processing to its active p18/p16 subunits only in TRAIL-sensitive HCT116 wt cells. In contrast, TRAIL-resistant HCT116 Bax−/Bak− cells showed no processing of caspase-3 to its active subunits (Figure 2a, left). Thus, in contrast to cleavage of the pro-caspase-3 zymogen, which is independent of Bax and Bak, processing of pro-caspase-3 to its active subunits relies on an intact intrinsic mitochondrial pathway.


Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

Gillissen B, Richter A, Richter A, Overkamp T, Essmann F, Hemmati PG, Preissner R, Belka C, Daniel PT - Cell Death Dis (2013)

TRAIL-induces pro-caspase-3 processing in Bax/Bak double-deficient cells but fails to overcome inhibition by XIAP and proteasomal caspase-3 degradation. (a) HCT116 wt and Bax−/Bak− cells were treated with 50 ng/ml TRAIL and pro-caspase-8 and -3 processing were analyzed by immunoblotting. TRAIL-treatment induced cleavage of pro-caspase-8 and -3 in both cell lines. Cleavage of pro-caspase-8 is accompanied by generation of its active subunits. In contrast, processing of pro-caspase-3 to its active subunits is detectable only in HCT116 wt but not in HCT116 Bax−/Bak− cells (left). Additional downregulation of XIAP in combination with TRAIL caused full processing of pro-caspase-3 to its active subunits in HCT116 Bax−/Bak− cells (right). (b) HCT116 wt and Bax−/Bak− cells were incubated with the proteasome inhibitor MG132 prior to TRAIL-treatment. Upon inhibition of the proteasome, TRAIL-treatment resulted in full processing of pro-caspase-3 to its active subunits in both cell lines. (c, d) In addition to TRAIL, HCT116 wt and Bax−/Bak− cells were treated with 1 μM of MG132 (c) or with 1 μM of bortezomib (BZM) (d). Cells were cultured for 24 h, harvested and apoptotic cells were determined by flow cytometric measurement of cellular DNA content. HCT116 wt cells were sensitized for TRAIL-induced apoptosis by MG132 or BZM. Furthermore, inhibition of the proteasome enables TRAIL to kill Bax/Bak-deficient HCT116 cells
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3674381&req=5

fig2: TRAIL-induces pro-caspase-3 processing in Bax/Bak double-deficient cells but fails to overcome inhibition by XIAP and proteasomal caspase-3 degradation. (a) HCT116 wt and Bax−/Bak− cells were treated with 50 ng/ml TRAIL and pro-caspase-8 and -3 processing were analyzed by immunoblotting. TRAIL-treatment induced cleavage of pro-caspase-8 and -3 in both cell lines. Cleavage of pro-caspase-8 is accompanied by generation of its active subunits. In contrast, processing of pro-caspase-3 to its active subunits is detectable only in HCT116 wt but not in HCT116 Bax−/Bak− cells (left). Additional downregulation of XIAP in combination with TRAIL caused full processing of pro-caspase-3 to its active subunits in HCT116 Bax−/Bak− cells (right). (b) HCT116 wt and Bax−/Bak− cells were incubated with the proteasome inhibitor MG132 prior to TRAIL-treatment. Upon inhibition of the proteasome, TRAIL-treatment resulted in full processing of pro-caspase-3 to its active subunits in both cell lines. (c, d) In addition to TRAIL, HCT116 wt and Bax−/Bak− cells were treated with 1 μM of MG132 (c) or with 1 μM of bortezomib (BZM) (d). Cells were cultured for 24 h, harvested and apoptotic cells were determined by flow cytometric measurement of cellular DNA content. HCT116 wt cells were sensitized for TRAIL-induced apoptosis by MG132 or BZM. Furthermore, inhibition of the proteasome enables TRAIL to kill Bax/Bak-deficient HCT116 cells
Mentions: As caspase-8 and -3 are crucial for TRAIL-induced apoptosis, we next examined to which extent Bax/Bak deficiency impacts on processing of caspase-8 and -3. We treated HCT116 wt and Bax−/Bak− cells with TRAIL and analyzed the cleavage pattern of these caspases. Pro-caspase-8 and -3 levels are comparable in both cell lines, respectively (Figure 2a, left). Following TRAIL-treatment, pro-caspase-8 is cleaved in both cell lines and processed to its active subunits to a similar extent. Interestingly, in both cell lines, caspase-8 activation goes along with cleavage of the pro-caspase-3 zymogen, indicating that caspase-8 activation upon TRAIL-treatment is sufficient to cleave pro-caspase-3. Nevertheless, cleavage of pro-caspase-3 is accompanied by processing to its active p18/p16 subunits only in TRAIL-sensitive HCT116 wt cells. In contrast, TRAIL-resistant HCT116 Bax−/Bak− cells showed no processing of caspase-3 to its active subunits (Figure 2a, left). Thus, in contrast to cleavage of the pro-caspase-3 zymogen, which is independent of Bax and Bak, processing of pro-caspase-3 to its active subunits relies on an intact intrinsic mitochondrial pathway.

Bottom Line: Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes.Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype.Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany.

ABSTRACT
TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

Show MeSH
Related in: MedlinePlus