Molecular Mechanisms of TAZ-regulating Ferroptosis in Cancer Cells and tRNA Fragment in Erythrocytes

Abstract

Here, I sought to determine the molecular mechanisms of the cellular response to stresses in two contexts. In the first part of my thesis, I focus on how ferroptosis, a lipid oxidative stress-induced cell death, can be regulated by cell density via an evolutionarily conserved pathway effector. In the second part, I focus on the transcriptional response of red blood cells (RBCs) during the refrigerated storage. Ferroptosis is a novel form of programmed cell death characterized by the accumulation of lipid peroxidation. It can be induced by the oxidative stress caused by starvation of cystine, inhibition of glutathione peroxidase 4, or activation of NADPH oxidase(s). The canonical ferroptosis inducer, erastin, is a small molecule which triggers oxidative stress by inhibiting the cystine-glutamate transporter (xCT) and thus reduces intracellular cysteine level and glutathione biosynthesis. Recent studies indicate ferroptosis may have therapeutic potential toward cancer. However, much remains unknown about the determinants of ferroptosis susceptibility. We observed that vulnerability to the ferroptosis of cancer cells is highly influenced by cell confluency. Since cell density can be sensed by the evolutionarily conserved Hippo pathway effectors, YAP/TAZ, we hypothesize if these Hippo pathway effectors are involved in erastin-induced ferroptosis response. My data show that TAZ, instead of YAP, is abundantly expressed in both renal and ovarian cancer cells and undergoes density-dependent nuclear/cytosolic translocation. TAZ removal confers ferroptosis resistance, while overexpression of constitutively active form of TAZ, TAZS89A, sensitizes cells to ferroptosis. Similarly, I found that a lower TAZ level in the recurrent ovarian cancer is responsible for reduced ferroptosis susceptibility of these cells. I further investigated the mechanisms by which TAZ regulates ferroptosis. and found that TAZ regulates ferroptosis through EMP1-NOX4 axis in renal cancers and ANGPTL4-NOX2 axis in ovarian cancers. The relevance of the Hippo pathway effector with ferroptosis suggests that ferroptosis-inducing agents may be used to target the TAZ-activated tumors.The second part of my dissertation investigated the molecular mechanisms of transcriptome changes inside RBCs during ex vivo storages. RBCs are the major component of blood transfusions, one of the most common procedures in the hospital. In addition, some athletes utilize blood transfusion of stored RBCs to increase athletic performance, a practice banned by the world anti-doping agency. Currently, RBCs can be stored for up to 42 days at ~4°C before transfusion. However, transfusion with RBCs after long storage duration may correlate with a poorer prognosis compared with fresh RBCs and results in increased morbidity and mortality. To recognize the undesirable effects of prolonged RBC storage on transfusion recipients, it is critical to understand storage-associated RBC changes. To this end, our lab has previously identified a variety of RNA species in mature RBCs and profiled the miRNA changes that occur in RBCs at different time intervals during in vitro storage. This profiling demonstrates that the abundance of most RBC miRNAs did not change significantly during the 42 days of refrigerated storage, indicating extremely long decay half-lives. Unexpectedly, miR-720, a cleavage product of tRNAThr, increased dramatically in the first two weeks and persisted during storage. Furthermore, I present evidence for a role of angiogenin in tRNA cleavage to generate miR-720 during RBC storage. The dramatic increase in miR-720 may be used to monitor transfused RBCs in clinical patients, athletes performing blood doping, and other settings. Additionally, the increase in miR-720 levels in the stored RBC may potentially contribute to the cellular and clinical phenotypes associated with storage lesions.Taken together, these studies on how human cells respond to stresses have the potentials as guidance for cancer patients toward ferroptosis-inducing chemotherapeutics or provide a novel way of detecting blood doping or understanding the RBC storage lesions.