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 6

Loss of CASP8 increases sensitivity to single-agent birinapant and birinapant plus radiation in vivo.

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Loss of CASP8 increases sensitivity to single-agent birinapant and birin...
(A) 2 × 106 MOC1 cells transduced with an inducible shRNA against Casp8 were injected into the right flank of WT female C57BL/6 mice. Mice were randomized and placed on control or DOX diet 3 days after injection to induce knockdown of Casp8 in vivo (Supplemental Figure 9, WB images). Control and shCasp8 mice were randomized into 4 treatment groups (vehicle control [black lines], 15 mg/kg birinapant [red lines], 5 × 2 Gy radiation [blue lines] or combination [cerulean lines], n = 7–10/each) 27 days after inoculation when the average tumor volume reached ~150 mm3. Solid and dashed lines represent control and shCasp8 mice, respectively, for the indicated treatment groups (Supplemental Figure 10, schema). Error bars represent standard deviation. Two-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.001 for indicated pairwise comparisons (Table 1 and Supplemental Tables 7–9). (B) Kaplan-Meier survival curves representing each treatment group. Log-rank (Mantel-Cox) test was used for statistical analysis. *P < 0.05, **P < 0.001 for indicated pairwise comparisons. (C) UMSCC 17A and MOC1 parental cells were treated with radiation (X [2, 4, and 6 Gy]), birinapant (B [50 nmol/L for the UMSCC-17A cells; 250 nmol/L for the MOC1 cells]), emricasan (E [1 μmol/L for both the cell lines]), necrostatin-1s (N [10 μmol/L for both the cell lines]), or the combinations as indicated for 24 hours. Cell viability was assessed using CellTiter-Glo as per Methods. One-way ANOVA with post hoc Bonferroni-corrected t test was used for statistics. *P < 0.05, **P < 0.001 for indicated pairwise comparisons. X (6 Gy) data are shown for simplicity (Supplemental Figure 11). (D) Representative images of clonogenic survival assays for the X (6 Gy) conditions. Clonogenic survival assay was performed as detailed in the Methods (birinapant doses reduced to 25 nmol/L and 125 nmol/L for the UMSCC 17A and MOC1 cells, respectively, for this assay, and other drugs used at the same concentrations as stated above). One-way ANOVA with post hoc Bonferroni-corrected t test was used for statistics. *P < 0.05, when comparing X+B to X alone for indicated radiation dose. ǂP < 0.05 and ǂǂP < 0.001, when comparing X+B+E to X alone for the indicated radiation doses. ƞP < 0.05 and ƞƞP < 0.001, when comparing X+B+E to X+B+E+N for the indicated radiation doses (Supplemental Figure 12). Experiments detailed above were repeated 3 times with similar results.