PERK-dependent molecular mechanisms in breast cancer progression and treatment
Sultana, Afrin
Sultana, Afrin
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Identifiers
http://hdl.handle.net/10379/17813
https://doi.org/10.13025/17313
https://doi.org/10.13025/17313
Repository DOI
Publication Date
2023-06-16
Type
Thesis
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Abstract
Stressful conditions of the tumour microenvironment, which overwhelm the folding capacity of the endoplasmic reticulum (EnR), activate an evolutionarily conserved signalling pathway known as the unfolded protein response (UPR). Three central signalling nodes that comprise the UPR are inositol-requiring enzyme 1α (IRE1), activating transcription factor 6α (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). During resting conditions activity of PERK, IRE1 and ATF6 are kept in check by the interaction of their luminal domain with the EnR chaperone, glucose-regulated protein 78 (GRP78); however, upon the build-up of misfolded proteins in EnR, GRP78 dissociates from these sensor molecules, leading to their activation. Upon activation, IRE1 undergoes autophosphorylation and oligomerization, which leads to the activation of its endoribonuclease activity. The primary function of IRE1's endoribonuclease activity is the unconventional splicing of the XBP1 mRNA and regulated IRE1-dependent decay of transcripts (RIDD). ATF6 exists as a 90-kDa type-II transmembrane protein located in the EnR membrane. During conditions of UPR, ATF6 is first transported from the endoplasmic reticulum to the Golgi apparatus, where it is proteolytically processed by site-1 and site-2 proteases, thereby translocating released 50-kDa ATF6 fragment to the nucleus to activate transcription of chaperones via a cis-acting EnR stress response element (ERSE). During UPR, active PERK phosphorylates its downstream targets, such as the eukaryotic translation initiation factor 2α (eIF2α) and nuclear factor erythroid 2-related factor 2 (NRF2). Phosphorylation of eIF2 leads to a general translational block and preferential translation of a subset of mRNAs, such as activating transcription factor 4 (ATF4). Phosphorylation of NRF2 by PERK attenuates KEAP1-mediated degradation of NRF2 and promotes the expression of antioxidant enzymes via cis-acting antioxidant response elements (ARE). The degradation of misfolded proteins and enhanced protein folding capacity, mediated by coordinated efforts of UPR, attempts to restore protein homeostasis and increase cell survival during EnR stress. The UPR can exert both pro-survival and deleterious effects on the survival of cancer cells. Through several mechanisms, PERK-mediated eIF2α phosphorylation directly regulates the cellular expression of fate-determining genes. ATF4 is translationally induced by PERK and regulates the transcription of a complex network of genes that ultimately determine cell fate. The expression of multiple EnR-resident chaperone proteins (such as BIP/GRP78) is induced by ATF4, which results in increased folding capacity, amino acid metabolism and glutathione synthesis, and increased resistance to oxidative stress. Furthermore, ATF4 transcriptionally induces the expression of autophagy genes, which is important in autophagosome formation and function. Inhibitors of apoptosis, such as cIAP1 and 2, are also induced during EnR stress in a PERK-dependent but ATF4-independent manner. A well-known ATF4 pro-death target gene named the transcription factor C/EBP homologous protein (CHOP) further promotes the transcription of pro-death genes. ATF4/CHOP induces GADD34, which encodes a protein that directs protein phosphatase 1 (PP1) to eIF2α. The induction of GAAD34 creates a negative feedback loop to re-establish global protein translation and can increase protein load during EnR stress, exacerbating stress and causing cell death. CHOP is directly responsible for inducing the expression of two BH3-only pro-apoptotic BCL-2 family members: Bim and Puma. CHOP indirectly induces the expression of another BH3-only pro-apoptotic BCL-2 family member, NOXA, through the induction of the transcription factor ATF5. Furthermore, CHOP induces expression of death receptor 5 (DR5), binding the Fas-associated death domain (FADD) independent of the DR5 ligand, Apo2/TRAIL. FADD transduces pro-death signals by activating caspase 8. The EnR and mitochondrial membranes are connected via mitochondria associated EnR membranes (MAMs). PERK has been localized to MAMs, suggesting that PERK's activation and downstream signalling pathways can influence mitochondrial-mediated cell survival. Small non-coding RNAs, microRNA (miRNA), are regulated by the UPR and play significant roles in cell survival and cell death signalling. Downstream of PERK, ATF4- dependent expression of miR-211 and NRF2-dependent repression of miR-214 promotes cell survival. Cell survival is also promoted by EnR stress-induced expression of miR-7a and subsequent indirect repression of pro-apoptotic transcription factor CHOP. CHOP induces expression of miR-216b, which mediates translational repression of c-Jun and sensitizes cells to EnR stress-induced apoptosis. PERK-mediated induction of ATF4 and NRF2 downregulates expression of the miR-106b-25 cluster and increases cell death. PERK-mediated signalling pathways are complex and mediate pro-death and pro-survival outcomes; as such, PERK signalling plays a clear role in cell fate and tumorigenesis. Activation of PERK in cancers has been reported by several publications and was the motivation for multiple pharmaceutical companies to invest continuous efforts to generate PERK inhibitors. Despite significant progress in elucidating the signals that drive these PERK-mediated responses, substantial gaps in our knowledge will compromise our ability to successfully translate inhibitors into the clinic. Furthermore, the PERK effectors' role and molecular mechanisms are not fully understood. The work presented in this thesis describes identifying and characterising novel downstream effectors of PERK in breast cancer.
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Publisher
NUI Galway