Examining MLKL phosphorylation to detect necroptosis in murine mammary tumors.

Necroptosis occurs predominantly in the center of late-stage tumors and necroptotic cells are dispersed and difficult to be detected by Western blotting of key markers without enrichment by microdissection. To overcome these obstacles, this protocol provides a detailed immunohistochemistry-oriented approach including the steps of tumor isolation from mouse mammary tumor models, necrotic region identification by H&E staining, and necroptosis detection through examining mixed lineage kinase domain-like protein (MLKL) phosphorylation. This protocol could be applied to other types of solid tumors. For complete details on the use and execution of this protocol, please refer to Baik et al. (2021).

ZBP1 not RIPK1 mediates tumor necroptosis in breast cancer.

Tumor necrosis happens commonly in advanced solid tumors. We reported that necroptosis plays a major role in tumor necrosis. Although several key necroptosis regulators including receptor interacting protein kinase 1 (RIPK1) have been identified, the regulation of tumor necroptosis during tumor development remains elusive. Here, we report that Z-DNA-binding protein 1 (ZBP1), not RIPK1, mediates tumor necroptosis during tumor development in preclinical cancer models. We found that ZBP1 expression is dramatically elevated in necrotic tumors. Importantly, ZBP1, not RIPK1, deletion blocks tumor necroptosis during tumor development and inhibits metastasis. We showed that glucose deprivation triggers ZBP1-depedent necroptosis in tumor cells. Glucose deprivation causes mitochondrial DNA (mtDNA) release to the cytoplasm and the binding of mtDNA to ZBP1 to activate MLKL in a BCL-2 family protein, NOXA-dependent manner. Therefore, our study reveals ZBP1 as the key regulator of tumor necroptosis and provides a potential drug target for controlling tumor metastasis.