Abstract 3550: Activation of a novel form of regulated necrosis to eliminate extracellular matrix detached epithelial cells

The overwhelming majority of breast cancer deaths are caused by the metastasis of cancer cells from the primary tumor to distant sites in the body. For cancer cells to successfully metastasize, they must: detach from the primary tumor, move out of the original tissue into the circulatory or the lymphatic system, travel to a new site followed by arresting their movement, and then extravasate to begin colonizing a secondary site. When normal epithelial cells detach from the extracellular matrix (ECM), they induce caspase-dependent cell death which is known as anoikis. Anoikis can therefore serve as a critical barrier to metastasis as cancer cells are exposed to limited and variable matrix conditions during each step of the metastatic cascade. However, our previous studies suggest that anoikis evasion is not sufficient to protect ECM-detached cells from cell death. Using MCF10A mammary epithelial cells, we have previously shown that ECM-detachment induced metabolic changes can compromise the survival of detached cells, although the precise mechanism controlling cell death remains unclear. Here, we present data suggesting that ECM-detached cells can also be eliminated by regulated necrosis (RN), a genetically programmed, caspase-independent form of necrosis that is morphologically indistinguishable from classical necrosis. In general, the molecular mechanisms involved in RN are poorly understood. With this in mind, the current most appreciated subtype of RN, termed necroptosis, is dependent upon TNFα, RIP1K, RIP3K, MLKL, and PGAM5 to execute cell death. Our data in ECM-detached cells suggest that cells are being eliminated by a mechanism that is dependent on the kinase activity of RIP1K. Further studies have shown an increase in the expression of CYLD, a deubiquitinating enzyme that specifically removes ubiquitin chains from RIP1K, in turn stabilizing both RIP1K expression and activation. Upon analyzing other dependent executioners of necroptosis, we have strikingly found that TNFα, RIP3K, and MLKL are all dispensable for RN to occur in ECM-detachment. Furthermore, using a variety of molecular tools, such as 3D cell culture, we have found that PGAM5 is necessary for the execution of ECM-detachment induced RN. These findings uncover a novel and distinct RN pathway and highlight the need to more thoroughly understand RN as well as the mechanisms employed by ECM-detached cells to antagonize RN and promote their survival in detachment. Citation Format: Mark A. Hawk, Cassandra L. Buchheit, Luqun Shen, Patrick Fagan, Zachary T. Schafer. Activation of a novel form of regulated necrosis to eliminate extracellular matrix detached epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3550.