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 2

Knockdown of CASP8 sensitizes HNSCCs to necroptotic death by birinapant and Z-VAD-FMK.

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Knockdown of CASP8 sensitizes HNSCCs to necroptotic death by birinapant ...
UMSCC 17A and MOC1 control and shRNA knockdown (shCASP8) cells were treated with birinapant (B [200 nmol/L for the UMSCC 17A cells; 1 μmol/L for the MOC1 cells]), Z-VAD-FMK (Z [5 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. (A) 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. More detailed analysis can be found in Supplemental Figure 2, A and B, and Supplemental Figure 3. (B) Representative images of clonogenic survival assays. Twenty-four hours after treatments (birinapant doses reduced to 50 nmol/L and 250 nmol/L for the UMSCC 17A and MOC1 cells respectively, for this assay, and other drugs used at the same concentrations as stated above), drug dilutions were washed out, and colonies were allowed to form for 5–12 days, after which they were stained and counted. Surviving colonies were normalized to nontreated cells from the same experiment to calculate percentage of colony count. All treatments were carried out in triplicates. Please refer to Supplemental Figure 4 for more detailed version. (C) Annexin V-APC/SytoxBlue staining was performed 24 hours after treatments. Percentage of annexin V positivity was used as a measure to assess cell death. All treatments were carried out in triplicates. More detailed analysis can be found in Supplemental Figure 5. (D) Whole-cell lysates were collected 24 hours after treatments and subjected to Western blot analysis for the indicated key cell death markers; β-actin was used as loading control. 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. B, birinapant; NT, no treatment; X, radiation; Z, Z-VAD-FMK.