Project 3: PHLDA2-mediated Phospholipid Oxidation in Ferroptosis and Tumor Suppression

Project Summary Cells employ several ferroptosis surveillance pathways to protect themselves from inappropriate ferroptosis including GPX4-mediated reduction of phospholipid peroxides, the antioxidant activity by free radical trappers such as CoQ10 and MUFA-mediated phospholipid remodeling. Interestingly, although the canonical ferroptosis response is suppressed by both GPX4-dependent and GPX4-independent manners, initiation of ferroptosis in cells often requires the treatment of GPX4 inhibitors. Moreover, only oxidized forms of phosphatidylethanolamines phospholipids (PL- PUFA (PE)-OOH) are associated with execution of ferroptosis induced by GPX4 inhibitors. Thus, it remains unclear whether oxidation of other classes of phospholipids is directly involved in triggering ferroptosis and how non-canonical ferroptosis responses are regulated in vivo. In our preliminary studies, we have discovered a new non-canonical ferroptosis pathway controlled by pleckstrin homology-like domain family A member 2 (PHLDA2), a membrane- associated phospholipid binding protein through the Crispr-Cas9 screening. In contrast to the canonical ferroptosis induced by GPX4 inhibitors, PHLDA2-mediated ferroptosis is neither ACSL4-dependent nor acts through peroxidation of phosphatidylethanolamine (PE); instead, PHLDA2 has a strong affinity for phosphatidic acid (PA) and, by recruiting ALOX12 lipoxygenase, PHLDA2 catalyzes peroxidation of PA (PL-PUFA (PA)-OOH) to induce ferroptosis upon high levels of ROS. To fuel their enhanced growth and proliferation, cancer cells undergo metabolic reprogramming, generally accompanied by increased ROS production. Indeed, ROS-induced ferroptosis occurs in vivo in tumor cells without any treatment of GPX4 inhibitors and strikingly, loss of PHLDA2 abrogates ROS-induced ferroptosis and promotes tumor growth in mouse xenograft tumor models. More importantly, by using PHLDA2-null mice, we found that PHLDA2-mediated ferroptosis is crucial for tumor development in well-established tumor mouse models. These data demonstrate that PHLDA2-mediated phospholipid peroxidation triggers a non-canonical ferroptosis response critical for tumor suppression in both immunodeficient and immunocompetent environments. The central hypothesis to be tested here is whether PHLDA2-mediated ferroptosis plays a key role in tumor growth suppression without the treatment of GPX4 inhibitors and how this new pathway is modulated by the ferroptosis surveillance pathways involving MBOAT1/2 and ADCK3 in vivo. In Aim 1, we will elucidate the molecular mechanism of PHLDA2-mediated ferroptosis through lipid peroxidation and its regulation by ferroptosis surveillance pathways involving MBOAT1/2 and ADCK3. In Aim 2, we will dissect the physiological significance of PHLDA2-dependent ferroptosis in regulating tumor growth of breast and liver tumors by using different mouse models.