Delineating the Molecular Mechanism Behind the Role of HuR in Cell Death and Drug Resistance

Abstract

Apoptosis plays an essential role in the development and maintenance of tissue hemostasis. However, failure to undergo apoptosis is thought to represent the key to the development of several human diseases including cancers. The RNA-binding protein HuR (human antigen R) plays an important role in apoptosis and in carcinogenesis, as well as other cellular processes, including proliferation, and cell differentiation. We previously showed that HuR is required for both pro-survival and pro-apoptotic pathways, where the caspase-mediated cleavage of HuR determines the fate of the cell that is favored. Other posttranslational modifications such as phosphorylation and methylation have been shown to regulate the function of HuR. Recently, PARylation of HuR by PAR polymerase enzyme-1 (PARP1) was also shown to regulate the function of HuR during inflammation. However, the regulatory mechanism(s) of the pro-apoptotic function of HuR and the involvement of posttranslational modifications such as PARylation in this process is still elusive. In the first part of this thesis, I have identified PARylation as a regulatory mechanism that modulates the function of HuR in determining cell fate. My results showed that PARP1/2-mediated PARylation prevents the accumulation of HuR in the cytoplasm, subsequently resulting in a decrease in its cleavage, thereby inhibiting the pro-apoptotic function of HuR. I demonstrated that the combined depletion of PARP1 and PARP2 increases the cytoplasmic accumulation of HuR and thus increases its cleavage. HuR cleavage, consequently, increases its pro-apoptotic function as evidenced by the significant increase in the level of caspase-3 cleavage and in the number of apoptotic cells. Furthermore, I showed that the polymers of ADP-ribose (PAR), which is the product from PARPs’ catalytic activity, binds HuR non-covalently through a consensus motif and that this binding is required for the nuclear localization of HuR as well as its association with the import factor Transpotin-2 (TRN2). Indeed, mutating the HuR PAR-binding site (HuR-PBS) prevented PAR from binding to HuR, resulting in the cytoplasmic accumulation of HuR, and therefore advancing apoptosis. Thus, this work provides evidence for the importance of the PARP-mediated PARylation and the resulting PAR binding to HuR in regulating the function of HuR during apoptosis. For decades, chemotherapeutic drugs have been shown to induce apoptosis in several cancer cells and tumors, yet many cells conferred multidrug resistance (MDR) which represents a major obstacle in cancer treatment, usually associated with resistance to apoptosis. Several studies associated HuR with the development of chemotherapeutic resistance in a variety of tumors. However, the mechanism in which HuR affects drug resistance in cancer cells and mediates MDR1 mRNA expression, in particular, is not fully understood. In the second part of this thesis, using KB human cervical adenocarcinoma cells, I established the importance of HuR in the regulation of MDR1 mRNA expression. I showed that HuR knockdown decreased the expression of MDR1 mRNA and protein in the drug resistant KB-V1 cells. This effect, interestingly, is not due to a change in HuR expression nor a change in HuR cellular localization. Additionally, I showed that HuR binds an ARE in MDR1 mRNA in drug resistant KB-V1 cells. Together, this work reveals a new role for PARylation in cell fate determination and implicates the non-covalent interaction of PAR and HuR as an important regulatory process required for the regulation of the pro-apoptotic function of HuR. Additionally, this work provides further insight on the HuR-mediated regulation of the MDR1 mRNA thus linking HuR to the resistance of cells to drug treatment.