Tumor suppressor Par-4 activates autophagy-dependent ferroptosis.

Ferroptosis is a unique iron-dependent form of non-apoptotic cell death characterized by devastating lipid peroxidation. Whilst growing evidence suggests that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms regulating ferroptosis are largely unknown. In this study, through an unbiased RNA-sequencing screening, we demonstrate the activation of a multi-faceted tumor-suppressor protein Par-4/PAWR during ferroptosis. Functional studies reveal that genetic depletion of Par-4 effectively blocks ferroptosis, whereas Par-4 overexpression sensitizes cells to undergo ferroptosis. More importantly, we have determined that Par-4-triggered ferroptosis is mechanistically driven by the autophagic machinery. Upregulation of Par-4 promotes activation of ferritinophagy (autophagic degradation of ferritin) via the nuclear receptor co-activator 4 (NCOA4), resulting in excessive release of free labile iron and, hence, enhanced lipid peroxidation and ferroptosis. Inhibition of Par-4 dramatically suppresses the NCOA4-mediated ferritinophagy signaling axis. Our results also establish that Par-4 activation positively correlates with reactive oxygen species (ROS) production, which is critical for ferritinophagy-mediated ferroptosis. Furthermore, Par-4 knockdown effectively blocked ferroptosis-mediated tumor suppression in the mouse xenograft models. Collectively, these findings reveal that Par-4 has a crucial role in ferroptosis, which could be further exploited for cancer therapy.

Targeting oxeiptosis-mediated tumor suppression: a novel approach to treat colorectal cancers by sanguinarine.

Oxeiptosis is a recently identified reactive oxygen species (ROS)-sensitive, caspase independent, non-inflammatory regulated cell death pathway. The activation of Kelch-like ECH-associated protein 1-Phosphoglycerate mutase 5-Apoptosis inducing factor mitochondria associated 1 (KEAP1-PGAM5-AIFM1) pathway is the key signaling event in the execution of oxeiptosis. In the present study, we demonstrate that sanguinarine (SNG), a quaternary benzophenanthridine alkaloid, induces oxeiptosis in human colorectal cancer (CRC) cells via ROS, specifically hydrogen peroxide (H2O2)-dependent activation of KEAP1-PGAM5-AIFM1 signaling axis. Whilst, knockdown of KEAP1, PGAM5, and AIFM1 largely abolishes SNG-induced oxeiptosis, hence reinforcing the importance of the role of this pathway in the SNG-mediated cytotoxicity. Moreover, extracellular addition of H2O2 sensitizes SNG-induced oxeiptosis in CRC cells, while removal of intracellular ROS by ROS scavengers, not only alleviated the overproduction of ROS caused by SNG, but also reversed the biochemical events associated with oxeiptosis. Finally, in vivo study demonstrates that SNG effectively reduces the tumor growth in HT-29 xenograft mouse model through features associated with oxeiptosis. This study highlights oxeiptosis as a novel tumor suppressive mechanism and further investigation of the role of oxeiptosis in cancer treatment is warranted.

Sanguinarine Induces H2O2-Dependent Apoptosis and Ferroptosis in Human Cervical Cancer.

Sanguinarine (SNG) is a benzophenanthridine alkaloid isolated mainly from Sanguinaria canadensis, Chelidonium majus, and Macleaya cordata. SNG is considered an antineoplastic agent based on its cytotoxic activity against various tumors. However, the exact molecular mechanism through which SNG mediates this activity has not been elucidated. Here, we report that SNG induces death in human cervical cancer (HeLa) cells through activation of two interdependent cell death pathways-apoptosis and ferroptosis. SNG-induced apoptosis was characterized by caspase activation and PARP cleavage, while ferroptosis involved solute carrier family 7 member 11 (SLC7A11) down-regulation, glutathione (GSH) depletion, iron accumulation, and lipid peroxidation (LPO). Interestingly, incubation with caspase inhibitor z-VAD-fmk not only inhibited the features of apoptosis, but also negated markers of SNG-induced ferroptosis. Similarly, pretreatment with ferroptosis inhibitor ferrostatin-1 (Fer-1), apart from rescuing cells from SNG-induced ferroptosis, also curbed the features of SNG-induced apoptosis. Our study implies that, together, apoptosis and ferroptosis act as partners in the context of SNG mediated tumor suppression in HeLa cells. Importantly, SNG increased the generation of reactive oxygen species (ROS), and ROS inhibition blocks the induction of both apoptosis and ferroptosis. These findings highlight the value of continued investigation into the potential use of SNG as an antineoplastic agent.

Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis.

AMP-activated protein kinase (AMPK) coordinates energy homeostasis during metabolic and energy stress. We report that the catalytic subunit isoform AMPK-α1 (but not α2) is cleaved by caspase-3 at an early stage during induction of apoptosis. AMPK-α1 cleavage occurs following Asp529, generating an ∼58-kDa N-terminal fragment (cl-AMPK-α1) and leading to the precise excision of the nuclear export sequence (NES) from the C-terminal end. This cleavage does not affect (1) the stability of pre-formed heterotrimeric complexes, (2) the ability of cl-AMPK-α1 to become phosphorylated and activated by the upstream kinases LKB1 or CaMKK2, or (3) allosteric activation by AMP or A-769662. Importantly, cl-AMPK-α1 is only detectable in the nucleus, consistent with removal of the NES, and ectopic expression of cleavage-resistant D529A-mutant AMPK-α1 promotes cell death induced by cytotoxic agents. Thus, we have elucidated a non-canonical mechanism of AMPK activation within the nucleus, which protects cells against death induced by DNA damage.

Prostate apoptosis response-4 and tumor suppression: it's not just about apoptosis anymore.

The tumor suppressor prostate apoptosis response-4 (Par-4) has recently turned 'twenty-five'. Beyond its indisputable role as an apoptosis inducer, an increasing and sometimes bewildering, new roles for Par-4 are being reported. These roles include its ability to regulate autophagy, senescence, and metastasis. This growing range of responses to Par-4 is reflected by our increasing understanding of the various mechanisms through which Par-4 can function. In this review, we summarize the existing knowledge on Par-4 tumor suppressive mechanisms, and discuss how the interaction of Par-4 with different regulators influence cell fate. This review also highlights the new secretory pathway that has emerged and the likely discussion on its clinical implications.

Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis.

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.