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. 2010 Jun 29;107(26):11936-41.
doi: 10.1073/pnas.1005667107. Epub 2010 Jun 14.

Overcoming cancer cell resistance to Smac mimetic induced apoptosis by modulating cIAP-2 expression

Sean L Petersen  1 , Michael Peyton, John D Minna, Xiaodong Wang
Affiliations

Overcoming cancer cell resistance to Smac mimetic induced apoptosis by modulating cIAP-2 expression

Sean L Petersen et al. Proc Natl Acad Sci U S A. .

Abstract

Smac mimetics target cancer cells in a TNFalpha-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNFalpha-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of cIAP2, which although initially degraded, rebounds and is refractory to subsequent degradation. cIAP2 is induced by TNFalpha via NF-kappaB and modulation of the NF-kappaB signal renders otherwise resistant cells sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate cIAP2. Using the PI3K inhibitor, LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
cIAP2 respond differently to Smac mimetic and TNF in resistant cell H1299 as compared with a sensitive cell line H2009. (A) Dose–response of H2009 cells to increasing concentrations of Smac mimetic with and without 50 ng/mL TNF. (B) Western blot analysis of cIAP1, cIAP2, and XIAP during a time course of response of H2009 to 100 nM of Smac mimetic (SM), 50 ng/mL of TNFα, and Smac mimetic/TNFα from 0 to 300 min. The same amounts of Smac mimetic and TNFα were used in all of the following experiments. (C) TNFR1 coimmunoprecipitation of H2009 cells treated with Smac mimetic, TNFα, or both as in B, and the pellet were analyzed by Western blotting using antibodies against RIPK1, cIAP1, and cIAP2 as indicated. (D) Caspase-8 coimmunoprecipitation of H2009 treated with Smac mimetic, TNFα, and Smac mimetic/TNFα as in B and the pellet were analyzed by Western blotting using antibodies against RIPK1, caspase-8, and FADD as indicated. (E) Dose–response of H1299 cells to increasing concentrations of Smac mimetic with and without 50 ng/mL TNFα. (F) Western blot analysis of cIAP1, cIAP2, and XIAP during a time course of response of H1299 to Smac mimetic, TNFα, and Smac mimetic/TNF from 0 to 300 min. Percentage of cell death is based on cell titer glo (Promega) cell viability assay in 96-well plates. Graphical representations indicate the mean ± SD of at least three independent experiments.
Fig. 2.
Fig. 2.
cIAP2 siRNA knockdown sensitizes resistant cells by preventing cIAP2 up-regulation during Smac mimetic/TNFα treatment. (A) Comparison of cIAP2 degradation in response to Smac mimetic/TNFα (SM/TNF) between mock-transfected versus cIAP2 siRNA-transfected H1299 cells. H1299 cells were treated as indicated and equal amounts of cell lysates were analyzed by Western blotting using antibodies against cIAP1, cIAP2, XIAP, and actin. (B) Time course in cIAP1 and XIAP siRNA knockdown H1299 cells treated as indicated and cell lysates were analyzed by Western blotting as in A. (C) Cell death of cIAP1, cIAP2, and XIAP siRNA treated H1299 cells in response to Smac mimetic, TNFα, or both. (D) Formation of the RIPK1–caspase-8–FADD complex as shown by caspase-8 coimmunoprecipitation in cIAP2 siRNA-treated cells in response to Smac mimetic/TNFα as compared with mock-transfected cells. Pellets from immunoprecipitation using an anti-caspase-8 antibody were analyzed by Western blotting with anti-RIPK1 and FADD antibodies as indicated. Percentage of cell death is based on cell titer glo (Promega) cell viability assay in 96-well plates. Graphical representations indicate the mean ± SD of at least three independent experiments.
Fig. 3.
Fig. 3.
Blocking NF-κB with a NEMO siRNA sensitizes resistant cell line H1299 to Smac mimetic and TNFα treatment. (A) Testing of four individual NEMO siRNA oligos for the ability to sensitize H1299 cells to Smac mimetic and TNFα. (B) Western blot analysis of efficiency of NEMO siRNA knockdown for each individual siRNA. (C) Determination of RIPK1 dependence in NEMO knockdown induced sensitivity. Cells were cotransfected with both NEMO and RIPK1 at both a 1:1 and 2:1 ratio and cell death was determined after the indicated treatments. (D) Western blot analysis of efficiency of double knockdown of RIPK1 and NEMO. (E) Time course comparison of indicated protein levels measured by Western blotting in mock-transfected and NEMO siRNA-transfected cells in response to Smac mimetic and TNFα. (F) Western blot analysis of pellets from caspases-8 immunoprecipitation using anti-RIPK1, caspase-8, and FADD from mock-transfected and NEMO siRNA-transfected cells treated for 300 min with Smac mimetic, TNFα, or both. Percentage of cell death is based on cell titer glo (Promega) cell viability assay in 96-well plates. Graphical representations indicate the mean ± SD of at least three independent experiments.
Fig. 4.
Fig. 4.
Low-dose chemical inhibition of IKK2 BMS-345541 sensitizes H1299 cells to Smac mimetic and TNFα treatment by preventing up-regulation of cIAP2. (A) Time course response of H1299 cells pretreated with 5 μM BMS-345541. These cells were then treated with Smac mimetic, TNFα, or both and the indicated protein levels were analyzed by Western blotting. (B) Dose–response of H1299 cells pretreated with increasing concentrations of BMS-345541 as indicated for 1 h. Cells were then treated with Smac mimetic, TNFα, or both overnight and cell death was measured and plotted. (C) H1299 cells pretreated either with DMSO or BMS-345541 as indicated. These cells were then treated with Smac mimetic, TNFα, or both for the times indicated. TNFR1 was then immunoprecipitated and the elutes were analyzed by Western blotting for recruitment of RIPK1, cIAP1, and cIAP2 to the receptor. (D) Caspase-8 coimmunoprecipitation comparing DMSO and BMS-345541 pretreated cells that were subsequently treated with Smac mimetic, TNFα, or both for the times indicated. Elutes were analyzed by Western blot for formation of the RIPK1–caspase-8–FADD complex. Percentage of cell death is based on cell titer glo (Promega) cell viability assay in 96-well plates. Graphical representations indicate the mean ± SD of at least three independent experiments.
Fig. 5.
Fig. 5.
Inhibition of PI-3K renders H1299 cells sensitive to Smac mimetic. (A) Effects of inhibition of AKT. H1299 cells were pretreated with 50 μM LY294002 or DMSO for 1 h and then treated with Smac mimetic, TNFα, or both for the indicated times and then analyzed by Western blot for AKT phosphorylation and levels of cIAP1, cIAP2, and XIAP as indicated. (B) Dose-dependent response of H1299 cells to pretreatment with increasing concentrations of LY294002 followed by overnight treatment with Smac mimetic, TNFα, or both. (C) Caspase-8 coimmunoprecipitation comparing DMSO and LY294002 pretreated cells followed by treatment with Smac mimetic, TNFα, or both for 300 min. Elutes were analyzed by Western blot for formation of the RIPK1–caspase-8–FADD complex. (D) Evaluation of RIPK1 dependence on LY294002 induced sensitivity. RIPK1 knockdown cells were pretreated with LY294002 followed by treatment with Smac mimetic, TNFα, or both. Percentage of cell death is based on cell titer glo (Promega) cell viability assay in 96-well plates. Graphical representations indicate the mean ± SD of at least three independent experiments.
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