Caspase-8 loss radiosensitizes head and neck squamous cell carcinoma to SMAC mimetic–induced necroptosis
Burak Uzunparmak, Meng Gao, Antje Lindemann, Kelly Erikson, Li Wang, Eric Lin, Steven J. Frank, Frederico O. Gleber-Netto, Mei Zhao, Heath D. Skinner, Jared Newton, Andrew G. Sikora, Jeffrey N. Myers, Curtis R. Pickering
Burak Uzunparmak, Meng Gao, Antje Lindemann, Kelly Erikson, Li Wang, Eric Lin, Steven J. Frank, Frederico O. Gleber-Netto, Mei Zhao, Heath D. Skinner, Jared Newton, Andrew G. Sikora, Jeffrey N. Myers, Curtis R. Pickering
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Research Article Cell biology Oncology

Caspase-8 loss radiosensitizes head and neck squamous cell carcinoma to SMAC mimetic–induced necroptosis

Abstract

Caspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCCs), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCCs suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, birinapant, in combination with pan-caspase inhibitors Z-VAD-FMK or emricasan and radiation. In a syngeneic mouse model of oral cancer, birinapant, particularly when combined with radiation, delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels rendered many HNSCC cell lines resistant to necroptosis, patient tumors maintained RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.

Authors

Burak Uzunparmak, Meng Gao, Antje Lindemann, Kelly Erikson, Li Wang, Eric Lin, Steven J. Frank, Frederico O. Gleber-Netto, Mei Zhao, Heath D. Skinner, Jared Newton, Andrew G. Sikora, Jeffrey N. Myers, Curtis R. Pickering

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Figure 4

Susceptibility to necroptosis is determined by levels of RIP3 in HNSCCs.

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Susceptibility to necroptosis is determined by levels of RIP3 in HNSCCs....
(A) CRISPR/Cas9 was used to knock out CASP8 in the mouse-derived MOC1 cell line. MOC1 parental cells were transiently transfected with 2 sgRNAs designed against mouse Casp8 (sgRNA-mCASP8 #1 and sgRNA-mCASP8 #2) or a nontargeting sgRNA, after which clonal selection/expansion was performed. Engineered clones were subjected to a Western blot screen to identify Casp8WT and Casp8KO MOC1 clones. (B) Indicated Casp8WT and Casp8KO MOC1 clones were treated with birinapant (B [1 μmol/L]), Z-VAD-FMK (Z [5 μmol/L]), necrostatin-1s (N [10 μmol/L]), or the combinations for 24 hours. Cell viability was assessed using CellTiter-Glo. Values normalized to nontreated cells from the same experiment to calculate percentage of cell density. All treatments were carried out in replicates of 4. (C) Indicated Casp8WT and Casp8KO MOC1 clones were subjected to Western blot analysis for necroptosis markers RIP1, RIP3, and MLKL. (D) RIP3 was knocked down using shRNA in 2 necroptosis-sensitive MOC1 clones: the Casp8WT C2 and Casp8KO g2-1 clones. Scrambled shRNA control and shRip3 cells were subjected to Western blot to validate knockdown of RIP3. (E) Control and shRip3 C2 (Casp8WT) and g2-1 (Casp8KO) MOC1 clones were treated with birinapant (B [1 μmol/L]), Z-VAD-FMK (Z [5 μmol/L]), necrostatin-1s (N [10 μmol/L]), or the combinations for 24 hours. Cell viability was assessed by CellTiter-Glo. All treatments were carried out in replicates of 4. (F) Necroptosis-resistant C4 (Casp8WT) and g2-2 (Casp8KO) MOC1 clones were transduced with control, HA-tagged WT Rip3, or HA-tagged D143N Rip3 (a kinase domain dead RIP3) inducible expression constructs. RIP3 expression was induced with doxycycline (50 ng/mL). Western blot analysis was performed to validate expression of WT or D143N Rip3 in the indicated MOC1 clones. Relative RIP3 expression was quantified using parental cells in C and F and C2 control cells in D as reference control, and β-actin was used as loading control. (G) Cells engineered in F were treated with birinapant (B [1 μmol/L]), Z-VAD-FMK (Z [5 μmol/L]), necrostatin-1s (N [10 μmol/L]), or the combinations for 24 hours. Cell viability was assessed by CellTiter-Glo. One-way ANOVA with post hoc Bonferroni-corrected t test was used for statistics. *P < 0.05; **P < 0.001 for the indicated pairwise comparisons. All experiments detailed above were repeated 3 times with similar results.