ER stress and cancer: The FOXO forkhead transcription factor link

Accepted Manuscript ER stress and cancer: The FOXO forkhead transcription factor link Glowi Alasiri, Lavender Yuen-Nam Fan, Stefania Zona, Isabella Galeno Goldsbrough, Hui-Ling Ke, Holger Werner Auner, Eric Wing-Fai Lam PII:

S0303-7207(17)30296-4

DOI:

10.1016/j.mce.2017.05.027

Reference:

MCE 9958

To appear in:

Molecular and Cellular Endocrinology

Received Date: 24 March 2017 Revised Date:

17 May 2017

Accepted Date: 24 May 2017

Please cite this article as: Alasiri, G., Fan, L.Y.-N., Zona, S., Goldsbrough, I.G., Ke, H.-L., Auner, H.W., Lam, E.W.-F., ER stress and cancer: The FOXO forkhead transcription factor link, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.05.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

ER stress and cancer: the FOXO forkhead transcription factor link

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Glowi Alasiri1,3, Lavender Yuen-Nam Fan1,3, Stefania Zona1, Isabella Galeno Goldsbrough1, Hui-Ling Ke1, Holger Werner Auner2*, Eric Wing-Fai Lam1* 1

Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, UK.

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College London, Hammersmith Hospital Campus, Du Cane Road, London W12

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0NN,

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[email protected];

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Holger W. Auner, Department of Medicine, Imperial College London, Hammersmith

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Hospital Campus, Du Cane Road, London W12 0NN, UK Phone: +44-20-3313-4017;

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E-mail: [email protected];

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Abstract

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The endoplasmic reticulum (ER) is a cellular organelle with central roles in maintaining proteostasis due to its involvement in protein synthesis, folding, quality control, distribution and degradation. The accumulation of misfolded proteins in the ER lumen causes ‘ER stress’ and threatens overall cellular proteostasis. To restore ER homeostasis, cells evoke an evolutionarily conserved adaptive signalling and gene expression network collectively called the ‘unfolded protein response (UPR)’, a complex biological process which aims to restore proteostasis. When ER stress is overwhelming and beyond rectification, the normally pro-survival UPR can shift to induce cell termination. Emerging evidence from mammalian, fly and nematode worm systems reveals that the FOXO Forkhead proteins integrate upstream ER stress and UPR signals with the transcriptional machinery to decrease translation, promote cell survival/termination and increase the levels of ER-resident chaperones and of ER-associated degradation (ERAD) components to restore ER homeostasis. The high rates of protein synthesis/translation associated with cancer cell proliferation and metabolism, as well as mutations resulting in aberrant proteins, also induce ER stress and the UPR. While the pro-survival side of the UPR underlies its ability to sustain and promote cancers, its apoptotic functions can be exploited for cancer therapies by offering the chance to ‘flick the proteostatic switch’. To this end, further studies are required to fully reevaluate the roles and regulation of these UPR signalling molecules, including FOXO proteins and their targets, in cancer initiation and progression as well as the effects on inhibiting their functions in cancer cells. This information will help to establish these UPR signalling molecules as possible therapeutic targets and putative biomarkers in cancers.

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Department of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Contributed equally and are joint first authors

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+44-20-7594-2810;

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*Correspondence: Eric W.-F. Lam, Department of Surgery and Cancer, Imperial

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Fax:

+44-20-8383-5830;

E-mail:

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AKT, Protein Kinase B; AKT1, Protein Kinase B Alpha; AMPK, AMP-activated protein kinase; ASK1, Apoptosis Signal Regulated Kinase 1; ATF4, Activating transcription factor 4; ATF6, Activating Transcription Factor 6α; ATG5,Autophagy protein 5; ATG7,Autophagy protein 7 ; ATG10, Autophagy protein 10; Bak, BCL2 Antagonist/Killer 1; BAX,BCL2 Associated X; BBC3, p53-upregulated modulator of apoptosis; BCL-2, B-cell lymphoma 2; BECN1, Beclin 1; Bim, BCL2-Like 11; BiP, immunoglobulin heavy-chain-binding protein; B-Raf, B-Raf Proto-Oncogene, Serine/Threonine Kinase; bZIP, basic leucine zipper ;CHOP, CCAAT/enhancer-binding (C/EBP) homologous protein; CNX, calinexin; CRT, calreticulin; DR5, death receptor 5; EGFR, Epidermal Growth Factor Receptor; EIF2AK3, eukaryotic translation initiation factor 2α kinase 3; eIF2, eukaryotic initiation factor 2; EMT, epithelia-tomesenchymal transition; ER, Endoplasmic Reticulum; ERAD, ER-associated degradation; Ero1, ER oxidoreductin 1 Ero1; FOX, Forkhead box; FKBP,FK506‐binding protein; GADD34, Growth arrest and DNA-damageinducible protein 34; GRP58, glucose-regulated protein 58-kD; GRP, glucose regulated protein; GRP78, glucose-regulated protein of 78 kDa; GRP94, glucose regulated protein 94 kDa; GRP170, glucose regulated protein 170; Her2,human epidermal growth factor receptor 2; H-Ras, Harvey Rat Sarcoma Viral Oncogene Homolog; HSPA5, Heat Shock Protein Family A Hsp70 Member 5; IRE1α, Inositol Requiring Enzyme 1α; JNK, Jun N-terminal kinase; LAMP3, lysosomal-associated membrane protein 3; LCN2, lipocalin 2; MAP1LC3B, microtubule-associated protein 1 light chain 3β; MAPK8, Mitogen-Activated Protein Kinase 8; MEF, mouse embryo fibroblast; mTOR, Mechanistic Target Of Rapamycin; Myc, MYC proto-oncogene; PDIs,proteindisulphide isomerases; PERK, protein kinase R (PKR)-like ER kinase; PI3K, Phosphoinositide-3-kinase; p58IPK , Protein Kinase Inhibitor Of 58 KDa; PPI, peptidyl-prolyl isomerase; PUMA, P53 Up-Regulated Modulator Of Apoptosis; RIDD, IRE1α-dependent decay; RIP, regulated intramembrane proteolysis; S1P, site-1 proteases; S2P, site-2 proteases; Sequestosome-1 (SQSTM1); TNF, Tumour necrosis factor; TRAF2, TNF-receptor-associated factor 2; TRIB3, tribbles homologue 3; UGGT, UDP-glucose/glycoprotein glucosyl transferase; uORF , upstream short open reading frame; UPR, unfolded protein response; VCP/p97, Valosin-containing protein; VEGF, Vascular endothelial growth factor; XBP-1,X-box binding protein 1, YY1, Yin Yang 1
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Abbreviations:

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ER stress and unfolded protein response

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Cells orchestrate a finely-tuned balance between protein synthesis and degradation to

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maintain protein homeostasis (proteostasis). The endoplasmic reticulum (ER) is a cellular

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organelle dedicated to the folding and assembly of secretory and membrane-bound proteins

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and is enriched for enzymes that facilitate the folding process (Anelli and Sitia, 2010). The

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accumulation of misfolded/unfolded proteins in the ER lumen which causes a condition

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known as ‘ER stress’ and triggers the unfolded protein response (UPR) to maintain cellular

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proteostasis by adapting protein synthesis, degradation, trafficking, and folding (Dufey et al.,

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ACCEPTED MANUSCRIPT 2014). Upon ER stress, molecular sensors in the ER recognize unfolded proteins and relay

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information across the ER membrane to activate downstream signalling molecules and

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transcription factors in order to initiate a comprehensive adaptive gene expression program

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to alleviate ER stress. This gene expression program increases the ER folding capacity by

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expanding the size of the ER and by increasing the amounts of folding chaperones and

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modifying enzymes (Ron and Walter, 2007). UPR induction can also enhance the removal

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of irreparable misfolded proteins from the lumen via ER-associated degradation (ERAD) and

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autophagy (Rashid et al., 2015). Although UPR induction can promote cell survival in the

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short term, prolonged UPR activation without the reestablishment of proteostasis leads to

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cell death primarily by apoptosis.

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ER stress and UPR activation are also involved in the pathogenesis and development of

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many pathological conditions, including diabetes, obesity, neurodegenerative diseases and

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cancer (Kaushik and Cuervo, 2015). More than 30% of cellular proteins are folded in the ER,

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which provides a highly specialized microenvironment for protein processing(Anelli and

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Sitia, 2010). These ER-specific mechanisms are executed by ER-associated chaperones

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and polypeptide-modifying enzymes, such as glucose regulated proteins (GPRs), calinexin

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(CNX), calreticulin (CRT), peptidyl-prolyl isomerases (PPIs) and protein-disulphide

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isomerases (PDIs), Moreover, the ER and UPR components also play key roles in the

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degradation of irreparably misfolded proteins that need to be disposed of to ensure that only

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properly folded proteins are exported from the ER (Nakatsukasa and Brodsky, 2008).

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Protein folding: ER chaperones and Foldases

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In ER, chaperones and folding enzymes operate on the newly synthesized unfolded

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polypeptides to facilitate their folding into their native conformations and quality control

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(Braakman and Hebert, 2013). These ER chaperones bind to newly synthesized unfolded

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proteins, recruit folding enzymes and prepare the unfolded proteins for subsequent folding

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and assembly. The folding process is also enabled by many folding-enzymes (foldases)

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which include the PDI family of thiol oxidoreductases and PPIs.

120 121

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General chaperones

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The general chaperone GRP78/BiP(glucose-regulated protein of 78 kDa/immunoglobulin

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heavy-chain-binding protein), is one of the most important and abundant ER chaperones,

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and it recognizes polypeptides that have hydrophobic residues . GRP78/BiP expression is

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induced by the presence of misfolded or unfolded proteins in the ER (Dufey et al., 2014;

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ACCEPTED MANUSCRIPT Kozutsumi et al., 1988), and it identifies misfolded proteins and facilitate their folding into

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their native conformations. Moreover, GRP78/BiP also binds to nascent hydrophobic

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proteins and protects them from aggregation (Morris et al., 1997). However, if misfolded

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proteins are persistently resistant to the folding process, they will be targeted to the ERAD

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machinery for degradation (Chambers et al., 2012; Travers et al., 2000). Recent studies

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have shown that GRP78/BiP is also important for retro-translocating misfolded proteins to

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the cytosol for ERAD (Araki and Nagata, 2012; Chillaron and Haas, 2000; Hagiwara et al.,

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2011). Furthermore, GRP78/BiP also maintains the permeability barrier between the cytosol

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and the ER lumen by gating the heteromeric Sec61 translocon complex (Plemper et al.,

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1997). Like GRP78/BiP, the other common general chaperones GRP94 (also as gp96 and

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HSP90B1), and GRP170 (also as HSP110) also play similar critical roles in facilitating

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protein folding, assembly and transport as well as the export of misfolded proteins to the

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cytosol for degradation via the ERAD pathway; however, they have much narrower

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substrate specificities (Braakman and Hebert, 2013). After the proteins leave the translocon,

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their folding and post-translational modifications are mediated further by additional

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collections of chaperones and folding enzymes.

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143 Lectin Chaperones

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The membrane-bound CNX and the soluble CRT are two lectin chaperones that recognize

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glycoproteins in the ER lumen. These lectins specifically bind mono-glycosylated

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glycoproteins and are involved in their folding and quality control. When nascent proteins

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enter the ER lumen, this will activate the ER enzymes glucosidase I and II, which will cleave

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off excess oligosaccharides to generate the mono-glycosylated proteins so that they can be

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recognized by CNX and CRT(David et al., 1993; Wada et al., 1995). However, if the protein

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is still not folded properly, UDP-glucose/glycoprotein glucosyl transferase (UGGT) will act on

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the unfolded glycoproteins, allowing the proteins to rebind CNX and CRT, and channel them

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back into the CNX/CRT cycle to undergo more cycles of oxidative folding (Totani et al.,

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2009). This process will be repeated at least several times to help with protein folding, but

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persistently misfolded proteins are identified and destroyed via the ERAD pathway.

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Other Folding Chaperones and Enzymes

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A key step in the protein folding process is the donation of disulfide bonds to the newly

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synthesized client proteins and this is mediated by ER resident oxidoreductases, which

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include ER oxidoreductin 1 (Ero1). Ero1 and the thioredoxin-like PDIs are the major folding

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enzymes in the ER lumen, which catalyze the formation and breakage of disulfide bonds

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between cysteine residues within proteins (Frand and Kaiser, 1999). ERp57/GRP58 is

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another key member of PDI family, which functions to ensure correct folding and the quality

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ACCEPTED MANUSCRIPT control of newly-synthesized glycoproteins (Oliver et al., 1999) . This role of ERp57/GRP58

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requires its interaction with the CRT or CNX, which are responsible for recognizing and

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binding monoglucosylated proteins (Molinari and Helenius, 1999). PPI family of folding

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enzymes are comprised of Cyclosporin A (CsA)‐binding cyclophilins, the FK506‐binding

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proteins (FKBPs), and the Parvulin‐like PPIs; these catalyze the conversion of the cis and

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trans isomers of peptide bonds to proline, and have an essential role in the folding of newly

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synthesized proteins. In summary, PPIs and PDIs together with other foldases assemble the

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protein folding process (Jansen et al., 2012).

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ER stress sensors

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UPR signalling is activated upon the detection of misfolded proteins in the ER lumen

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primarily by the chaperone GRP78/BiP and is mediated through three signalling axes driven

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by three ER-transmembrane sensor proteins, PERK [protein kinase R (PKR)-like ER

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kinase], IRE1α (Inositol Requiring Enzyme 1α) and ATF6α (Activating Transcription Factor

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6α), respectively (Hetz, 2012). Under normal unstressed conditions, these ER stress sensor

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proteins bind GRP78/BiP and remain inactive (Bertolotti et al., 2000; Shen et al., 2002). In

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response to ER stress, GRP78/BiP binds to the unfolded or misfolded proteins in the ER

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lumen, and its dissociation from PERK, IRE1α and ATF6 results in their activation and the

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transduction of downstream UPR signals across the ER membrane to the cytosol and

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ultimately to the nucleus to trigger an adaptive gene expression program (Bertolotti et al.,

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2000; Shen et al., 2002)(Figure 1).

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PERK Pathway

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PERK (eukaryotic translation initiation factor 2α kinase 3, EIF2AK3), is a type I

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transmembrane kinase with a luminal and a cytoplasmic domain. When active, its key

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function is to phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2) on serine 51.

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This reduces the levels of eIF2-GTP available for translation initiation, resulting in the

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attenuation of global protein synthesis and thus alleviating the demands for the ER protein

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folding machinery. (Figure 1).

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Although general translation is decreased in response to ER stress, specific mRNAs are

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preferentially translated. Activating transcription factor 4 (ATF4) is central to PERK-governed

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signaling and regulates multiple functions to aid cells recovering from ER stress. For

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example, under normal unstressed conditions, ATF4 mRNA translation is repressed

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because its upstream short open reading frame (uORF) is within the 5´- untranslated region

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of its mRNA, an inhibitory element that blocks ATF4 expression (Harding et al., 2000;

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Vattem and Wek, 2004). However, the phosphorylation of eIF2α overcomes this inhibition

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and increases ATF4 protein synthesis. The upregulated ATF4 transcription factor triggers

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the expression of pro-survival gene products, which include antioxidant response molecules

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and those involved in protein folding and differentiation (Wang and Kaufman, 2014).

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Moreover, ATF4 also induces the transcription of CHOP (CEBPz), another key ER stress

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transcription factor which has both pro-apoptotic and survival functions. In addition, ATF4

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and CHOP heterodimers also activate the transcription of genes that control autophagy,

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such as p62/SQSTM1, Atg5 Atg7, and Atg10, to promote cell survival (Huggins et al., 2015;

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Rouschop et al., 2010). PERK activation also induces the expression of miR-211, which

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represses CHOP expression and restricts it from activating pro-apoptotic genes (Chitnis et

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al., 2012). Conversely, ATF4 and CHOP also co-operate to promote the transcription of the

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GADD34, the regulatory subunit of protein phosphatase 1, which functions as a negative

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regulator in a feedback loop in PERK signalling to restore protein synthesis. GADD34

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dephosphorylates

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eIF2α,

resulting

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eventual

restoration

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global

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ACCEPTED MANUSCRIPT translation(Han et al., 2013; Novoa et al., 2003). ATF4 and CHOP also upregulate other

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gene families that promote protein synthesis, and while this function is essential for the

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restoration of steady-state protein synthesis in cells with resolved ER stress, it contributes to

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cell death in cells that cannot rectify the underlying stress (Han et al., 2013). In addition,

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another ATF4/CHOP target, p58(IPK) can also bind to the PERK kinase domain and inhibit

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its auto-phosphorylation activity, resulting in a reduction in eIF2α phosphorylation and the

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synthesis of GRP78/BiP(Yan et al., 2002). Under chronic stress conditions, persistent CHOP

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induction will also drive cells to undergo apoptosis through promoting the transcription of

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pro-apoptotic genes, such as the p53-upregulated modulator of apoptosis (also called

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BBC3), tribbles homologue 3 (TRIB3), the B-cell lymphoma 2 (BCL-2) family of apoptotic

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regulators, lipocalin 2 (LCN2) and death receptor 5 (DR5 or TNFRSF10B(Hsin et al., 2012;

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Li et al., 2006; Yamaguchi and Wang, 2004))However, the role of DR5 in ER stress-induced

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apoptosis has recently been called into question(Glab et al., 2017).

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IRE1α Pathway

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Upon ER stress, the IRE1α released by the chaperone GRP78/BiP undergoes dimerization,

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autophosphorylation, and activation, although some argue that misfolded proteins binds to

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Ire-1 ligands

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endonuclease activity of IRE1α to splice off a 26-base pair intron from the X-box binding

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protein 1 (XBP1) mRNA (Yoshida et al., 2001) to generate a specifically spliced XBP1s

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mRNA species that encodes for the transcriptionally active form of XBP1 (XBP1s). XBP1s

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drives the expression of genes. chaperones and foldases) that are involved in protein folding

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as well as ERAD }(Calfon et al., 2002; Lee et al., 2003; Travers et al., 2000). Activated

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IRE1α also cleaves a range of mRNA species to reduce the protein load in ER, a process

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named regulated IRE1α-dependent decay (RIDD)(Hollien et al., 2009) (Wang and Kaufman,

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2016). It has also been demonstrated that activated IRE1α is able to bind to the TNF-

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receptor-associated factor 2 (TRAF2) and recruit Apoptosis Signal Regulated Kinase 1

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(ASK1) to an IRE1α-TRAF2-ASK1 complex, which promotes ER stress-induced c-Jun N-

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terminal kinase (JNK) activation(Urano et al., 2000). Furthermore, the IRE1α-TRAF2

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complex is also required for the activation of the ER-resident Caspase-12 to facilitate ER

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stress-induced apoptosis(Yoneda et al., 2001). Another link between the UPR and the

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apoptotic pathway is the regulation of IRE1α-XBP1 by some members of the Bcl-2 family.

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For example, the pro-apoptotic Bcl-2-like proteins Bax and Bak are able to bind to the

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cytosolic domain of IRE1α directly and trigger the downstream signalling (Hetz et al., 2006).

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Another example is that the BH3-only proapoptotic Bcl-2 family members Bim and PUMA

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are involved in the regulation of XBP1 splicing via interaction with IRE1α (Rodriguez et al.,

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in yeast to regulate UPR(Gardner and Walter, 2011) . This triggers the

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ACCEPTED MANUSCRIPT 2012). Furthermore, it has been shown that RIDD is up-regulated upon ER stress and

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exhibits pro-apoptotic effects (Maurel et al., 2014). (Figure 2)

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ATF6 Pathway

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ATF6 is an ER stress transmembrane protein sensor that functions as a transcription factor

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upon cleavage. In response to ER stress, the loss of BiP binding releases ATF6 allowing it

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to translocate from the ER to the Golgi. In the Golgi, ATF6 is cleaved by site-1 (S1P) and

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site-2 proteases (S2P) through regulated intramembrane proteolysis (RIP) to release a

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cytosolic amino terminal fragment that functions as an active basic leucine zipper (bZIP)

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transcription factor, named ATF6f. The cleaved ATF6 fragment migrates to the nucleus to

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transcriptionally up-regulate protein-folding enzymes and chaperones. GRP78, GRP94,

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ERp72, and PDI) (Teske et al., 2011) as well as components of ERAD (Shen et al., 2002).

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For example, upon ER stress, the activated ATF6 relocates to the nucleus to cooperate with

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the transcription factor Yin Yang (YY1) to transactivate the promoter of the gene (HSPA5)

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encoding GRP78/BiP, a prosurvival ER chaperone (Baumeister et al., 2005).

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ATF6 also plays a pivotal role in transcriptional induction of other critical ER-stress

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transcription factors, such as XBP1 (Lee et al., 2002; Yoshida et al., 2001; Yoshida et al.,

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2000) and CHOP (Nakanishi et al., 2005; Yoshida et al., 2000). Accordingly, XBP1 mRNA

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expression is induced by ATF6 and spliced by IRE1 to be activated in response to ER

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stress. In addition, ATF6 also cooperates with XBP1, to upregulate genes, encoding ER

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protein chaperones, ERAD signalling complexes, and lipid biosynthetic enzymes.

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ER stress and Cancer

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ER Chaperones and Cancer regulation:

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The high rates of translation associated with cancer cell proliferation and metabolism can

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induce ER stress and consequently UPR (Ozcan et al., 2008). In addition, large-scale gains

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and losses of genetic material as well as point mutations generate imbalanced protein levels

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and proteins that are difficult to fold, thereby challenging proteostasis-maintaining

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mechanisms in cancer cells (Deshaies, 2014). In spite of the precarious proteostatic balance

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in cancer cells there is considerable evidence to support the notion that the UPR has a

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central role in cancer initiation, progression and chemotherapeutic resistance (Wang and

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Kaufman, 2014). For example, GRP78/BiP overexpression is commonly observed in

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cancers and is associated with aggressive cancer growth and metastasis (Miao et al., 2013).

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Similar observations have been made for other proteostasis-promoting molecules, including

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the ubiquitously expressed protein degradation mediator VCP/p97 (Yamamoto et al., 2003).

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ER sensors and Cancer:

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Oncogenic stress is increasingly recognized as a cause of ER stress and a UPR trigger.

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Oncogene activation, such as B-Raf proto-oncogene mutations, H-Ras proto-oncogene

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mutations, and c-Myc amplification, as well as chemotherapeutic drug treatments, can

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induce ER stress in cancer cells (Bu and Diehl, 2016). For example, expression of the c-Myc

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oncogene can increase general transcription and translation, which causes the production of

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high levels of misfolded and unfolded proteins in the ER lumen. As a result, PERK will be

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induced to activate survival mechanisms, such as autophagy, which can promote tumor

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initiation and progression(Hart et al., 2012). This has been observed in both human

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lymphoma cells and mouse models carrying c-Myc translocations (Hart et al., 2012).

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Another oncogenic molecule involved in inducing ER stress is B-RAF, which plays an

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important role in the development of melanoma. Mutant B-RAF can induce chronic ER

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stress via the binding to BiP in the ER lumen, and this

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GRP78/BiP from the three ER sensor arms. As a result, PERK and IRE1α are activated to

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leads to the dissociation of

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promote autophagy to promote cell survival(Corazzari et al., 2015)

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adenocarcinoma, another oncogene HER2/Neu, has also been found to activate PERK

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signalling and thereby, cancer progression(Bobrovnikova-Marjon et al., 2010). Furthermore,

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PERK activation also helps cancer cells to overcome environmental stress, such as hypoxia

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and a lack of nutrition. For instance, at low glucose levels, PERK activation induces cancer

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cell survival via Akt activation and hexokinase II transportation to the mitochondria(Hou et

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al., 2015). The UPR is also intimately involved in angiogenesis, a tumor progression process

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that facilitates the enrichment of cancer cells with oxygen and nutrition’s via the expansion

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of the vascular network. For example, VEGF, a proangiogenic factor, has been found to

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induce cell survival and angiogenesis in endothelial cells through activating PERK signalling

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and ATF6. Furthermore, PERK-ATF4 arm has also been shown to repress anti-

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angiogenesis factors and enhance Vascular endothelial growth factor VEGF expression

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(Blais et al., 2006). In addition, the PERK/elF2α arm and its downstream molecules also

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have a broad role in the regulation of autophagy to promote cancer cell survival. In human

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cancer cells, hypoxia can upregulate PERK to induce the expression of autophagy

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molecules ATG5 and microtubule-associated protein 1 light chain 3β (MAP1LC3B), which

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are responsible for the expansion of phagophores. Conversely, PERK inhibition causes the

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downregulation of both MAP1LC3B and ATG5, further suggesting a role of PERK in

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promoting autophagy and thus cancer development (Rouschop et al., 2010). In addition, the

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oncogene c-Myc can also regulate PERK to activate the cytoprotective autophagy

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mechanism in cancer cells (Yang et al., 2013). The PERK/elF2-α/ATF4 signalling axis has

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been shown to be involved in autophagy activation in neural cells after ionic irradiation by

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increasing the expression of autophagic regulator LC3. In agreement, PERK-knockdown

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neural cells showed enhanced sensitivity to radiation (Yang et al., 2013).

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Moreover, in

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The transcription factors ATF4 and CHOP are downstream targets of PERK and have been

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found to be involved in autophagy regulation and autophagosome formation. The PERK-

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elF2α-ATF4 signalling axis regulates the expression of p62/SQSTM1, a regulator and

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established genetic marker of autophagy, in response to leucine starvation in mouse embryo

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fibroblast (MEFs), p62/SQSTM1 also enhances stem-like properties by stabilizing MYC

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mRNA in breast cancer. Moreover, CHOP is one of the transcription factors that induces

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p62/SQSTM1 expression, and appropriately, CHOP knockdown restricts p62/SQSTM1

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expression and autophagy (B'Chir et al., 2013). Furthermore, apart from PERK activation,

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ATF4 also controls LC3B expression in response to the bortezomib (proteasome inhibitor)

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treatment, inducing autophagy and drug resistance in the breast carcinoma cells (Milani et

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al., 2009).

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ACCEPTED MANUSCRIPT 342 PERK signalling also plays an important role in cancer metastasis and migration in hypoxic

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microenvironments. ATF4 stimulates the expression of the lysosomal-associated membrane

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protein 3 (LAMP3) to promote epithelia-to-mesenchymal transition (EMT) and metastatic

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distribution in several cancer types (Pytel et al., 2016). In conclusion, PERK signalling plays

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a crucial role in regulating cell fate in response to internal and external stresses; as such,

348

PERK might help cancer cells to adapt to stress conditions and activate the survival

349

pathway,

350

chemotherapeutic drug resistance.

such

as

autophagy,

angiogenesis,

cell

cycle

351

RI PT

343

arrest,

metastasis

and

In human prostate cancer cells, IRE1α regulates cyclin A1 activity and increases cell

353

proliferation via the control of XBP1 splicing (Thorpe and Schwarze, 2009). XBP1, a key

354

transcription factor of the UPR, is essential for the survival of cancer cells under hypoxia and

355

for tumor growth (Romero-Ramirez et al., 2004). For instance, human lung carcinoma and

356

fibrosarcoma cells increase XBP1 splicing and GRP78/BiP expression in response to

357

hypoxic stress (Vandewynckel et al., 2013). In addition, XBP1 expression and splicing were

358

also up-regulated in breast cancer as well as hepatocellular carcinoma (HCC), which may

359

promote cell survival via the expression of GRP78/BiP (Fujimoto et al., 2003; Shuda et al.,

360

2003). Furthermore, XBP1 overexpression is necessary for plasma cell differentiation and

361

expansion, which may be important for the initiation of multiple myeloma (Carrasco et al.,

362

2007). Nutrient deprivation is also one of the tumor microenvironment factors that trigger ER

363

stress and thereby UPR. XBP1 activity is also associated with the stress response towards

364

glucose deprivation, where glucose deprivation enhances XBP1 splicing (Spiotto et al.,

365

2009). VEGF is an essential regulator of tumor angiogenesis and therefore expansion. UPR

366

promotes

367

accordingly, depletion of either IRE1α or ATF6 causes downregulation of VEGF expression

368

and in vivo angiogenesis in tumor neovascularization mouse models (Ghosh et al., 2010; Liu

369

et al., 2013).

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angiogenesis

through

IRE1α and

ATF6-mediated

VEGF

induction,

and

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352

371

The cross talk between ER stress and Autophagy in Cancer:

372

Autophagy is an important means by which tumor cells defend against microenvironmental

373

stress and chemotherapeutic drugs. ER stress is intimately involved in macro-autophagy

374

activation and autophagosomal membrane formation (Hart et al., 2012). UPR regulates

375

autophagy through the IRE1α, the PERK/elF2α and the intracellular calcium-dependent

376

signalling axes (Hart et al., 2012; Høyer-Hansen and Jäättelä, 2007) via three key cellular

377

signalling molecules: Jun N-terminal kinase (JNK), AKT1 and mTOR. IRE1α can activate

11

ACCEPTED MANUSCRIPT JNK (also called MAPK8; Mitogen-activated protein kinase 8), which has the ability to

379

phosphorylate anti-apoptotic protein BCL-2 and inhibit its interaction with BECN1, a

380

molecule responsible for the activation of PtdIns3K and phagophore formation in autophagy

381

(Clarke et al., 2012; Strappazzon et al., 2011). Moreover, the post-transcriptional splicing of

382

XBP1 mRNA by IRE1α gives rise to the transcription factor XBP1s, which induces the

383

expression of BCL-2. Furthermore, XBP1s can also transcriptionally activate the expression

384

of BECN1 directly (Rashid et al., 2015).

385

The PERK/elF2α arm and its downstream signalling molecules play a crucial role in the

386

regulation of autophagy. In human cancer cells, PERK induces the expression of the

387

autophagy molecules, ATG5 and MAP1LC3B, which are responsible for the expansion of

388

phagophore (Rouschop et al., 2010). The PERK/elF2α/ATF4 arm is also involved in

389

autophagy activation by increasing the expression of LC3 in nerve cells in response to

390

radiotherapy (Yang et al., 2013).

391

ATF4 and CHOP are downstream target molecules of PERK, which has been found to be

392

involved in autophagy regulation and autophagosomal formation. elF2α/ATF4 regulate p62,

393

a marker gene and regulator of autophagy, after inducing leucine starvation in mouse

394

embryo fibroblasts (MEFs). Moreover, CHOP is one of the transcription factors that induces

395

p62 expression, and accordingly, knockdown of CHOP causes the depletion of p62 and the

396

repression of autophagy (B'Chir et al., 2013). Furthermore, apart from PERK activation,

397

ATF4 also regulates LC3B in response to the Bortezomib treatment in the breast cancer

398

MCF-7 cells to induce autophagy and resist the cytotoxic effects of bortezomib (Milani et al.,

399

2009). The ATF6 signalling arm plays a crucial role in mediating autophagy. In collaboration

400

with CHOP, ATF6 inhibits the expression of death-associated protein kinase 1 (DAPK1), an

401

activator of IFNγ-induced cell death, to promote autophagy (Kalvakolanu and Gade, 2012).

402

Moreover, it is well established that ATF6 transcriptionally activates BiP/GRP78, which

403

indirectly represses mTOR through AKT1 downregulation (Yung et al., 2011). However,

404

GPR78 knockout in mice caused the activation of AKT, which suggests that GRP78 has two

405

different regulatory roles in the regulation of autophagy (Chang et al., 2012).

406

UPR can regulate autophagy indirectly through molecules that stimulate or inhibit mTOR.

407

For example, TSC1/2 (tuberous sclerosis) is a negative regulator of mTOR. Indeed,

408

research showed that the induction of ER stress in TSC-deficient MEF cells did not

409

downregulate mTOR activity (Qin et al., 2010). Another ER stress modulator and

410

transcription factor regulated by CHOP and ATF4 is TRIB3, which is a negative regulator for

411

AKT-mTOR, which causes autophagy activation (Verfaillie et al., 2010).

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378

412 413

The relationship between FOXO, ER stress and cancer

12

ACCEPTED MANUSCRIPT The FOXO subgroup of Forkhead box (FOX) transcription factors were first identified in the

415

nematode Caenorhabditis elegans (C. elegans) as Daf-16 for its role in metabolic signalling

416

and longevity (van der Horst et al., 2006). In mammals, there are four FOXO proteins:

417

FOXO1, FOXO3, FOXO4, and FOXO6. Structurally, they are all composed of a highly

418

conserved DNA binding domain, located upstream of a nuclear localization signal, a nuclear

419

export sequence and a C-terminal transactivation domain (Anderson et al., 1998). Amongst

420

all FOXO proteins, FOXO3 is the most ubiquitously expressed within both embryonic and

421

adult tissues (Furuyama et al., 2000; Greer and Brunet, 2005). FOXO3 knock-down results

422

in premature ovarian failure, spontaneous lympho-proliferation, organ inflammation, and

423

helper T cell hyperactivation (Lin et al., 2004; Paik et al., 2007; Tothova et al., 2007). The

424

relatively mild and tissue-restricted phenotype observed in FOXO3-deficient animals

425

indicates functional and expression compensation exists between these different FOXO

426

proteins. FOXO activity is tightly regulated but is also fine-tuned by a range of post-

427

translational modifications, including phosphorylation, ubiquitination, acetylation and

428

methylation (Calnan and Brunet, 2008; Lam et al., 2013). The most extensively researched

429

post-translational modification is Akt(PKB)-mediated phosphorylation, which promotes

430

FOXO3 cytoplasmic translocation and thereby inhibition in response to growth factor

431

signalling (Lam et al., 2013; Myatt and Lam, 2007). By contrast, phosphorylation by AMP-

432

activated protein kinase (AMPK)(Greer et al., 2007), p38-MAPK (Consolaro et al., 2015; Ho

433

et al., 2012), JNK (Sunters et al., 2006) promotes nuclear localization and the activation of

434

FOXO protein activation. For example, JNK recognizes FOXO3 through at least 4

435

acetylation sites (K242, K259, K290 and K569) to mediate its phosphorylation. The JNK-

436

dependent S574 phosphorylated form of FOXO3 has been shown to be involved in the

437

induction of apoptosis (Li et al., 2016). FOXO proteins function by binding to the promoter

438

consensus sequences 5’-GTAAA(T/C)A-3’, and modulating the transcription of target genes

439

through their transactivation domain and interaction with a myriad of co-factors (Greer and

440

Brunet, 2005; Lam et al., 2013). Amongst their many functions, FOXO proteins can

441

negatively regulate cell cycle progression through activating negative cell cycle regulators,

442

including p21Cip1, p27Kip1, and p130(RB2). In addition, FOXO is also involved in the

443

regulation of programmed cell death through the transcriptional control of proapoptotic

444

genes, such as tumor necrosis factor (TNF), and the Bcl-2 related Bim and PUMA (Lam et

445

al., 2013). Moreover, the cytostatic and cytotoxic activity of common cancer therapeutic

446

agents, such as anthracyclines, taxanes and platinum compounds, also relies on FOXO3

447

activity (Koo et al., 2012). One of the downstream target genes repressed by FOXOs is the

448

potent oncogene FOXM1. FOXM1 is another member of the FOX family of transcription

449

factors that is widely expressed in actively proliferating tissues and plays a key role in

450

tumorigenesis and cancer progression (Bella et al., 2014; Koo et al., 2012; Lam et al., 2013;

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SC

RI PT

414

13

ACCEPTED MANUSCRIPT Zona et al., 2014). FOXO3 and FOXM1 transcription factors regulate the expression of a

452

common group of genes, but have antagonistic functions in regulating their targets. FOXO3

453

and FOXM1 have also been shown to negatively regulate one another’s expression and

454

activity, and appropriately, FOXM1 is described as an antagonist of FOXO proteins (Fu and

455

Tindall, 2008). Recent evidence also suggests FOXM1 can protect cells from

456

chemotherapeutic drug-induced senescence and cell death, and appropriately, FOXM1 is

457

overexpressed in genotoxic and cytotoxic agent-resistant cancer cells (Karunarathna et al.,

458

2016; Khongkow et al., 2015; Khongkow et al., 2014; Kwok et al., 2010; Monteiro et al.,

459

2013; Myatt et al., 2014). Collectively, these findings illustrate that FOXO proteins, and

460

particularly FOXO3, have a crucial tumor suppressive role (Lam et al., 2013).

461

PERK pathway and FOXO3 story:

462

FOXO3 is an integral molecule at the heart of ER-stress and UPR signalling. PERK, one of

463

the three most important ER stress sensors, has been shown to be able to modulate

464

FOXO3 activity both directly and indirectly. PERK, one of the main signalling arms of UPR,

465

has also been found to induce FOXO transcription factor activity to promote insulin

466

resistance (Zhang et al., 2013). (Figure 3).

467 468

The PERK pathway plays a key role in insulin resistance, obesity and adipocytic

469

differentiation. In these conditions, PERK can cause mTOR induction as well as AKT

470

phosphorylation on Ser473 and activation (Bobrovnikova-Marjon et al., 2012). Thus, upon

471

ER stress and PERK activation, Akt can phosphorylate FOXO proteins and cause them to

472

relocate from the nucleus to the cytoplasm where FOXOs are sequester by head shock

AC C

EP

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SC

RI PT

451

14

ACCEPTED MANUSCRIPT proteins and become inactivated (Zhang et al., 2015). In consequence, constitutive PERK

474

activation can negatively regulate FOXO activity indirectly via Akt to restrict apoptotic

475

signalling and thus, promote cancer progression. Paradoxically, PERK can also mediate

476

FOXO3 phosphorylation directly, in an Akt-independent manner, at specific serine residues,

477

such as S261, S298, S301, S303 and S311 to promote FOXO3 its nuclear relocation and

478

activation(Zhang et al., 2013). In addition, downstream of PERK, CHOP-induced

479

enhancement of ERO1α expression can also trigger ROS production and thereby, the

480

induction of the JNK. This can, in turn, phosphorylate and activate FOXO proteins.

481

Furthermore, in cells treated with the ER-stress inducing agent tunicamycin, CHOP

482

cooperates with FOXO3 to induce the expression of proapoptotic genes, including Bim and

483

PUMA in response to ER stress (Ghosh et al., 2012). Nevertheless, the mechanism that

484

dictates whether PERK signalling activates or restrains FOXO3 activity remains enigmatic.

485

Interestingly, recent work on B-Raf-induced melanoma has demonstrated that PERK is a

486

gene dose-dependent tumor suppressor, where the level of its activity determines whether it

487

functions as a tumor suppressor or an oncogene (Pytel et al., 2016). The finding that the

488

nature of PERK function is determined by gene dose and possibly the duration of its activity

489

might help to explain the dual contradictory roles of PERK signalling on FOXO3 regulation.

M AN U

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473

490 IRE-1 and FOXO regulation:

492

Another possible means by which ER stress may impact on FOXO activity is through the

493

other key ER stress sensor IRE1α. A recent study has shown that the C. elegans FOXO

494

transcription factor Daf-16 and its human homologue FOXO3 can restore proteostasis in Ire-

495

1 mutant and cause ER stress resistance in the nematode C. elegans (Safra et al., 2014).

496

This suggest that FOXO transcription factors can maintain protein homeostasis and cause

497

drug resistance even if the IRE1α-mediated ERAD system is defective and that FOXO

498

proteins function downstream of IRE1α in ER stress signalling. Moreover, XBP1u, which is

499

targeted by IRE1α upon ER stress, can interact with FOXO1 to promote its degradation

500

through 20S proteasome pathway in cancer cells (Zhao et al., 2013). Furthermore, XBP1s

501

also interacts with FOXO1 to direct it toward proteasome-mediated degradation to prevent

502

insulin resistance in type 2 diabetes (Zhou et al., 2011). Moreover, activation of IRE1α can

503

also stimulate JNK (Nishitoh et al., 2002), which has been shown to augment FOXO3

504

activity (Sunters et al., 2003; Sunters et al., 2006). Consistently, recent research also

505

demonstrates that in response to elevated ROS (reactive oxygen species) levels, JNK-

506

mediated

507

cells. Accordingly, treatment of HMSCs with ROS-inducing agent H2O2 leads to JNK-

508

mediated FOXO3 phosphorylation at Ser294 and nuclear translocation to transactivate

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491

FOXO3 induces

autophagy

in

15

human

mesenchymal

stem

(HMSCs)

ACCEPTED MANUSCRIPT 509

autophagy genes (Gomez-Puerto et al., 2016). Collectively, these findings propose a key

510

role of FOXO proteins in linking ER stress to cancer development as well as cancer

511

microenvironmental and cytotoxic stress resistance.

512 513 Chaperones and FOX regulation:

515

Furthermore, in colorectal cancer, FOXM1 can bind to promoter of GRP78/BiP and

516

regulated its expression at the transcriptional level to increase the levels of chaperone

517

protein and cope with ER stress induced by the accumulation of unfolded proteins

518

(Baumann et al., 2016). Conversely, the ability of FOXM1 to promote invasion and migration

519

of colorectal cancer cells depends on GRP78/BiP (Luo et al., 2016).

520

downregulation of GRP78/BiP reduces the ability of FOXM1 to induce colorectal cancer cell

521

migration and invasion (Luo et al., 2016). In agreement, Heat shock proteins Hsp70 has also

522

been shown to be able to regulate the

523

indirectly by promoting their maturation(Colvin et al., 2014). This indicates the existence of a

524

feed forward signalling loop involving FOXM1 and GRP78/BiP in the regulation of cancer

525

progression.

526

ER stress and FOX regulation in Worms:

527

The nematode worm Caenorhabditis elegans and fruit fly Drosophila melanogaster share

528

numerous diverse biological processes with mammals, which include the highly conserved

529

ER stress and UPR signalling cascades. The C. elegans abnormal dauer formation-16 (Daf-

530

16) and Drosophila dFoxO are sole orthologues of mammalian FOXO transcription factors,

531

including FOXO1, FOXO3 FOXO4 and FOXO6 (Greer and Brunet, 2005). Our

532

understanding of the contribution of the FOXO family of transcription factors in the ER stress

533

response has been accelerated by studies in worm and fly models. Various C. elegans and

534

D. melanogaster ER stress and UPR-related genes have structural orthologues in humans,

535

and these nematode worms and fly orthologues also share their functional relationships with

536

FOXO proteins as their mammalian counterparts (summarized in Table 1 and Table 2,

537 538

respectively).

RI PT

514

SC

Accordingly,

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expression of oncoproteins, such as FOXM1,

C elegans orthologues pek-1

Human gene

Function

eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), also known as PERK kinase

pek-1 encodes a predicted ncodes a predicted transmembrane protein kinase Eif2ak3 which is required for the UPR. Pek-1 functions in the ER to phosphorylate eIF2α which inhibits assembly of 80S ribosomes and subsequent protein translation initiation, thus counteract endogenous ER stress by reducing the load of proteins to be processed in the ER (Safra et al., 2013; Shen et al., 2001; Shen et al., 2005).

16

Relationship with daf-16/FOXO in C. elegans Pek-1 regulates genes that are involved in inducible-UPR (i-UPR) (Shen et al., 2005). Pek-1 (PERK) modulates Daf-16/FOXO activity and phosphorylates the human and fly orthologues (Wang and Kaufman, 2014). It acts through Ire-1/Xbp-1, which cooperate with Daf-16/FOXO to maintain ER homeostasis (Shen et al., 2001).

ACCEPTED MANUSCRIPT endoplasmic 1 is a transmembrane serine/threonine protein kinase and sitereticulum-tospecific endoribonuclease which is required for UPR that nucleus signaling 1 counteracts cellular stress induced by accumulation of (ERN1), also unfolded proteins in the ER. Upon ER stress, activated IRE-1 known as removes an intron from X-box binding protein-1 (xbp-1) ribonuclease mRNA through unconventional splicing to produce inositol-requiring transcriptionally active Xbp-1(Calfon et al., 2002; Shen et al., protein-1 (IRE-1) 2001; Shen et al., 2005; Urano et al., 2002). The ire-1/xbp-1 pathway regulates the majority of genes in both i-UPR and cUPR, including those that are implicated in protein folding and ERAD. The ire-1/xbp-1 pathway has broader functions and plays a more important role in ER homeostasis than that of pek-1 and atf-6 when compared to mammalian cells (Henis-Korenblit et al., 2010; Shen et al., 2005). XBP-1 It is a bZIP transcription factor which plays an important role in maintaining ER homeostasis and promoting normal development via regulation by ire-1 as mentioned above (Kaufman, 1999; Shen et al., 2001; Shen et al., 2005; Urano et al., 2002). Cullin-1 (CUL-1) Encodes a cullin which is an core component of the SCF (Skp1p, Cullin, and F-box) ubiquitin-ligase complex (E3) that interact directly with various Skr proteins to facilitate ubiquitin-mediated protein degradation via autophagy (Ghazi et al., 2007; Safra et al., 2014). Cullin-2 (CUL-2) Encodes an E3 ubiquitin ligase that regulates diverse biological processes via ubiquitin-mediated proteolysis. CUL-2 functions as part of a Cul2-RING ubiquitin-ligase complex that regulates cell cycle progression, such as the initiation of meiotic anaphase II, cytokinesis and mitotic chromosome segregation (Feng et al., 1999; Sonneville and Gonczy, 2004). Targets of CUL-2-mediated degradation include CYB-1/cyclin B and the zinc-finger protein TRA-1(Liu et al., 2004). SKP1-CUL1-F-box All three genes encode proteins that are core components of (SCF) protein E3 the E3-ubiquitin ligase complex that facilitates ubiquitinubiquitin ligase mediated protein degradation via autophagy. In particular, these proteins are shown to interact with CUL1, C. elegans cullin homolog (Ghazi et al., 2007; Nayak et al., 2002; Yamanaka et al., 2002). SCF protein E3 Functions within the E3 complex to maintain ER ubiquitin ligase homeostasis(Liu et al., 2011; Safra et al., 2014)

xbp-1

skr-2, skr-8, skr-10

skr-5

TE D

cul-2

M AN U

SC

cul-1

RNF-121

hrd-1/sel-11

ERAD-associated E3 ubiquitin-protein ligase (HRD) -1

RNF-121 is a RING finger E3 ubiquitin ligase localized to membranes of the ER, the sarcoplasmic reticulum, and the Golgi. It exhibits E3 ubiquitin-protein ligase activity in vitro and in vivo and functions in ERAD pathway downstream of pek-1(Darom et al., 2010). Encodes an E3 ubiquitin ligase that has similar function as the E3 ubiquitin ligase HRD-1 in human. Hrd1/sel-11, together with its substrate receptor Hrd3/Sel-1, are core components of a large multi-subunit ER-embedded ubiquitin ligase complex that mediate the ubiquitylation of misfolded proteins from the ER lumen as they are retro-translocated out of the ER (Munoz-Lobato et al., 2014; Sasagawa et al., 2007). The gene encodes the Derlin-1 protein, also known as degradation in endoplasmic reticulum protein 1 (DERL1), is part of a complex that includes VIMP, SEL1, HRD1, and HERP. Derlin-1 localizes to endosomes and to the ER and mediates ERAD that detects misfolded proteins in the ER and targets them for degradation (Dang et al., 2011; Mehnert et al., 2014; Schaheen et al., 2009).

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EP

rnf-121

cup-2

Ire-1 acts upstream of Xbp-1, which cooperates with Daf-16/FOXO to modulate ER stress response (Lee et al., 2015; Shen et al., 2001).

RI PT

ire-1

Derlin-1 (DERL1)

cdc-48.1 and cdc48.2

ATP-driven chaperone valosincontaining protein (VCP)/p97

hsp-3 and hsp-4

glucose regulated protein 78/immunoglobulin

The two genes encode a type II AAA ATPase which is an essential component in ERAD. It functions as an ubiquitinselective chaperone which target unfolded proteins for dislocation across the ER membrane, and subsequent degradation by 26S proteasome in the cytosol (Janiesch et al., 2007; Meyer et al., 2012; Nishikori et al., 2008). Encodes a major ER chaperone protein which is involved in many cellular processes, including translocating newly synthesized polypeptides across the ER membrane,

17

Downstream of Ire-1, Xbp-1 collaborates with daf-16/FOXO to activate genes to promote ER stress resistance (Henis-Korenblit et al., 2010). The isoform of Cul-1, which acts upstream of Daf-16/FOXO to promote its nuclear accumulation and transcriptional activity including skr genes. The skr genes encode Skr proteins that are co-factors of Cul-1 (Ghazi et al., 2007).

As part of the SCF protein complex, these genes function upstream of Daf16/FOXO to promote transcriptional activity of Daf-16/FOXO by facilitating its nuclear accumulation (Ghazi et al., 2007). Transcriptional target of Daf-16/FOXO and its regulation is independent of the Ire-1/Xbp-1 pathway (Liu et al., 2011; Safra et al., 2014). Downstream of Pek-1 and Daf16/FOXO signalling (Darom et al., 2010).

Downstream targets of Ire-1/Xbp-1 and Daf-16/FOXO pathway (Safra et al., 2014).

Downstream of Ire-1/Xbp-1 pathway and likely a target of Daf-16/FOXO which is a cofactor of Xbp-1 in ER stress response (Safra et al., 2014).

Downstream of the Ire-1/Xbp-1 and Atf6 pathways which are also the downstream targets of daf-16/FOXO (Caruso et al., 2008; Safra et al., 2014). Downstream of the Ire-1/Xbp-1 pathway, as well as Daf-16/FOXO, and are both up-regulated in response to

ACCEPTED MANUSCRIPT

endoplasmic reticulum oxidoreductase-1, ERO-1

sir-2.1

Sirtuin 1 (SIRT-1)

Upstream activator of Pek-1(Harding et al., 2003).

Upstream activator of Daf-16/FOXO in the insulin-like signaling pathway (Tissenbaum and Guarente, 2001; Wolff and Dillin, 2006).

SC

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553 Human gene

PERK

Function

Relationship with dFOXO/FOXO in D. melanogaster

It encodes a kinase that phosphorylates dFOXO at S243, promoting its nuclear localization and activity, enhancing the ER stress response. Interestingly, this contrasts the effect of dFOXO phosphorylation by Akt, which results in its sequestration in the cytoplasm (Zhang et al., 2013). It regulates the expression of heat shock proteins (Hsps) at transcription level (Fernandes et al., 1995).

The kinase phosphorylates dFOXO and promotes transcription of downstream ER stress-related genes (Zhang et al., 2013).

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EP

D. melanogaster orthologues perk

554 555 556 557

ER stress (Kapulkin et al., 2005; Urano et al., 2002).

Table 1 Functional human orthologues of C. elegans ER stress-related genes and their functions and relationship with Daf-16/FOXO. A role in the regulations of ER stress response to promote ER stress resistance and longevity. In C. elegans, ER stress responses, particularly the unfolded protein response (UPR), are mainly dependent on the Ire-1/Xbp-1 pathway. In fact, the downstream gene targets of the ire-1/xbp-1 pathway accounts for the majority of UPR genes which are implicated in protein folding and endoplasmic reticulum-associated protein degradation (ERAD), such as cul-1, cul-2, skr, and cup-2. Interestingly, xbp-1 was found to collaborate with daf-16 to activate transcription of these downstream targets. Studies of C. elegans also showed that pek-1 acts synergistically with atf-6 to maintain ER homeostasis as an alternative mechanism to the ire1/xbp-1 pathway. Furthermore, pek-1 and atf-6 are both found to interact with daf-16 during ER stress response, suggesting a role of daf-16 in maintain ER homeostasis that is independent of the ire-1/xbp-1 signaling pathway.

TE D

539 540 541 542 543 544 545 546 547 548 549 550 551 552

ero-1

facilitating folding and assembly of proteins, targeting misfolded proteins for ERAD and acting as an ER stress sensor (Calfon et al., 2002; Lee, 2005). Both proteins have compensatory functions for each other (Kapulkin et al., 2005) It is an ER-localized enzyme that is required specifically for protein oxidation in the ER lumen (Harding et al., 2003). This enzyme mediates disulfide bond formation in the ER by re-oxidizing P4HB/PDI, the enzyme catalyzing protein disulfide formation, which results in the production of reactive oxygen species (ROS) in the cell. Therefore, it plays an important role in ER stress-induced, CHOPdependent apoptosis by activating the inositol 1,4,5trisphosphate receptor (IP3R1) (Sevier and Kaiser, 2008). A histone deacetylase (HDAC) that is known to negatively regulate ER stress response by deacetylating XBP-1, thus inhibiting its transcriptional activity to promote ER stressinduced apoptosis (Tissenbaum and Guarente, 2001; Wang et al., 2011). SIRT1 is also implicated in the regulation of eIF2α phosphorylation which is upstream of CHOP and GADD34, important mediators of stress-induced response(Ghosh et al., 2011).

RI PT

heavy chainbinding protein, (grp78/BiP)

Heat shock transcription factor (hsf)

HSF

hsp70

HSP70

It encodes a protein with ATP-dependent chaperone activity which directs the re-folding of misfolded proteins, thus acts against ER stress (Hartl and Hayer-Hartl, 2002).

Upon induction of ER stress, dFOXO binds to the promoter region of HSF to up-regulate transcription of target genes (Donovan and Marr, 2016). Direct downstream transcription target of HSF as well as dFOXO/FOXO as HSF transcription is regulated by dFOXO/FOXO (Donovan and Marr, 2016; Fernandes et al., 1995).

Table 2. Functional human orthologues of D. melanogaster ER stress-related genes and their functions and relationship with dFOXO/ FOXO. Similarly, in D. melanogaster, dFOXO is shown to regulate ER homeostasis at the transcriptional level by binding to promoter regions of ER stress related genes

18

ACCEPTED MANUSCRIPT Daf-16 and dFOXO and regulation of Ire-1 arm:

559

Appropriately, emerging evidence has revealed that the orthologues of human FOXO

560

proteins, Daf-16 and dFOXO are implicated in the regulation of ER homeostasis at the

561

transcriptional level in both nematodes and flies, respectively. In C. elegans, ER stress

562

responses, particularly UPR, are dependent largely on the Ire-1/Xbp-1 pathway. In fact, the

563

downstream gene targets of the Ire-1/Xbp-1 pathway account for the majority of UPR genes

564

which are implicated in protein folding and ERAD, including cul-1, cul-2, sel-1, sel-2 and

565

cup-2. Interestingly, Daf-16/FOXO also activate transcription of these ER stress downstream

566

targets upon ER stress (Ghazi et al., 2007), suggesting Daf-16/FOXO and Xbp-1 cooperate

567

to regulate genes important for resisting ER stress. Consistently, Xbp-1 has been found to

568

function synergistically with Daf-16 to enhance transcription of ER-stress resistance genes,

569

as well as specific longevity-promoting genes, such as dox-1 (Henis-Korenblit et al., 2010).

570

In C. elegans, activation of the IGFR homologue, Daf-2, stimulates the PI3K-Akt pathway,

571

which will, in turn, phosphorylate Daf-16, promoting its sequestration in the cytoplasm, and

572

thus, restraining its transcriptional activity (Murphy et al., 2003). Studies into long-lived daf-2

573

mutants, where Daf-16 is constitutively activated, have revealed the many diverse roles of

574

Daf-16/FOXO in regulating downstream target genes in the response to ER stress (Lin et

575

al., 2001; Ogg et al., 1997; Tullet et al., 2008). In these daf-2 mutants, the ER stress-

576

inducing agent tunicamycin can induce increased load of misfolded proteins in the ER and

577

activate an arm of the UPR, involving the endoribonuclease Ire-1, and its downstream target

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Xbp-1. Daf-16/FOXO has also been found to be able to act independently of the Ire-1/Xbp-1

579

pathway in ire-1 and/or xbp-1-deficient animals (Safra et al., 2014). Accordingly, activated

580

Daf-16 has been shown to employ an alternative pathway for the degradation of misfolded

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ER proteins in the situations of a deleted ire-1 or xbp-1, via transcriptional activation of skr

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genes, including skr-5 (Safra et al., 2014). These skr gene products form a core component

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of an E3-Ubiqiutin ligase complex, that mediates degradation of unfolded proteins under

584

conditions of ER stress (Safra et al., 2014). This suggests that upon ER stress the activated

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Daf-16/FOXO can enable the autophagy-mediated ERAD as well as the proteasome-

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mediated ERAD to resolve ER protein homeostasis, and that Daf-16 can circumvent the

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need for Ire-1/Xbp1 for ER homeostasis and function. Interestingly, Skr proteins can also

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interact directly with Cul-1, another core component of the E3-ubiquitin ligase, to promote

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nuclear accumulation and transcriptional activity of Daf-16/FOXO. Studies in C. elegans

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have also shown that Pek-1 acts synergistically with Atf-6 to maintain ER homeostasis as an

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alternative mechanism to the Ire-1/Xbp-1 pathway. Crucially, Pek-1 and Atf-6 have both

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been found to be able to compensate for the loss of ire-1 and xbp-1, which regulate the

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same gene signatures as Daf-16/FOXO during ER stress response, suggesting a possible

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central role of Daf-16/FOXO in maintaining ER homeostasis, which involves all three major

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ACCEPTED MANUSCRIPT ER stress sensor-signalling pathways (Shen et al., 2005; Tatham et al., 2008; Zeng et al.,

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2004). Daf-16 can also function independently of the three ER stress sensor-signalling

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cascades by cooperating with Hsf-1 expression to activate chaprones such as Hsp70

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which in turn promotes longevity and ER homeostasis through regulation of protein

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folding(Hsu et al., 2003)

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Regulation of PERK by dFOXO:

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In a similar manner, a role for the fly protein kinase PERK in regulating dFOXO by

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phosphorylation has also been described, consistent with its role in mammalian cells. PERK

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constitutes another arm of the UPR, and inhibits protein biosynthesis under ER stress by

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phosphorylation of the translation initiation factor eIF2α (Malhi and Kaufman, 2011).

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However, it has been shown that PERK can also phosphorylate dFOXO at S243, promoting

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its nuclear localization and activity, enhancing the ER stress response. Interestingly, this

607

contrasts with the effect of dFOXO phosphorylation by the Drosophila Akt, which results in

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its sequestration in the cytoplasm (Zhang et al., 2013). In summary, in addition to studies in

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mammals, studies in C. elegans and Drosophila models have provided clear evidence for

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various crucial roles of the forkhead transcription factor FOXO in mediating the ER stress

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response.

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Target cancer through the UPR signalling and its FOXO link

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Accumulating evidence suggests that the ER-stress/UPR signalling network may be a

615

promising pharmacological target for targeting cancer cells and for overcoming drug

616

resistance, and several approaches are currently being pursued. The proposition that

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disrupting cellular protein homeostasis by triggering overwhelming ER stress constitutes a

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therapeutic option is supported by the success of proteasome inhibitors in some

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hematological malignancies. The ubiquitin-proteasome pathway is the main biological

620

pathway for intracellular protein degradation, and inhibition of 26S proteasome can result in

621

an accumulation of defective and unfolded proteins within the ER. Proteasome inhibitors are

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a novel class of cancer therapeutics designed to promote tumor cell specific cytotoxic effects

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via inducing proteotoxic ER stress (Auner and Cenci, 2015; McConkey, 2017). Clinical trials

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of these inhibitors, such as Bortezomib, have been conducted for a variety of malignancies,

625

resulting in drug regulatory agency approvals for the treatment of multiple myeloma and

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mantle-cell lymphoma in patients (McConkey, 2017).

627

Targeting IRE1α-XBP1:

628

As the IRE1α-XBP1 signalling axis is central to cancer development, inhibition of IRE1α-

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XBP1 pathway can provide a viable strategy for targeting cancer, and several specific

630

inhibitors are currently being explored in preclinical studies. For example, IRE1α-specific

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ACCEPTED MANUSCRIPT inhibitors have been proven to be able to trigger apoptosis of pancreatic cancer cells (Chien

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et al., 2014). Another compound STF-083010, which can block the IRE1α endonuclease

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function without influence on its kinase activity, also displays significant cytotoxic effects on

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multiple myeloma cells although its selectivity not clear yet (Papandreou et al., 2010). 3-

635

Methoxy-5,6-dibromosalicylaldehyde and an IRE1α-binding small molecule 4u8C can inhibit

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IRE1α-induced XBP1 splicing and RIDD, and sensitize the cells to ER stress and thus cell

637

death (Cross et al., 2012; Volkmann et al., 2011). The inhibitors toyocamycin and MKC-

638

3946, which target IRE1α-induced XBP1 splicing, have been demonstrated to be cytotoxic

639

for multiple myeloma cells (Mimura et al., 2012; Ri et al., 2012). In addition, inhibition of

640

IRE1α activity also enhances the efficacy of oncolytic viral therapy in vivo through the

641

induction of caspase-2-dependent cellular apoptosis (Mahoney et al., 2011). Moreover,

642

Imatinib-an anti-cancer tyrosine kinase inhibitor interrupts the e binding between ABL

643

tyrosine Kinase and IRE1α which leads to reverse type 1 diabetes in non-obese diabetic

644

(Morita et al., 2017).

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Targeting PERK-ATF4:

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In addition to IRE1α inhibitors, the realization that PERK over-activation has a significant

647

impact on development and progression of different types of cancers and the finding that

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PERK knockdown prevents tumor growth inhibition in mice (Bi et al., 2005) have also led to

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the generation of a number of small molecule inhibitors of PERK for cancer treatment (Pytel

650

et al., 2016). GSK2606414 is the first selective PERK inhibitor developed and has

651

demonstrated tumor growth inhibitory effects on human tumors in mouse xenograft models

652

(Chaturvedula et al., 2013). Hitherto, most PERK inhibitors have demonstrated therapeutic

653

potentials in restoring proteostasis, and in blocking tumor growth and angiogenesis in cell

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culture and animal models of cancer (Axten, 2017).

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Chaperones inhibitors and FOXO3:

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The Hsp70 and Hsp90 ER-related chaperones plays a key role in the ER stress response

657

and are the first lines of defence in the cellular response against proteotoxic stress

658

(McConkey, 2017). Targeting these ER-related chaperones can potentially tip the UPR from

659

promoting cell survival towards cell termination.

660

Hsp90/GRP94 inhibitor FW-04-806 has been shown to display potent anti-tumor effects in

661

HER2-positive breast cancer (Huang et al., 2015). Accordingly, FW-04-806 can block the

662

PI3K/AKT pathway and induce FOXO3 to cause proliferative arrest and apoptosis in HER2+

663

breast cancer cells (Huang et al., 2015). Interestingly, FW-04-806 also combines

664

synergistically with the EGFR/HER2 tyrosine kinase inhibitor lapatinib (Huang et al., 2015),

665

which has previously been shown to target FOXO3 to mediate its cytostatic effects (Francis

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et al., 2009; Krol et al., 2007). Moreover, in BRAF inhibitor-resistant melanoma cells, the

667

HSP90 inhibitor XL888 has also been found to prevent growth of tumor and lead to

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Consistent with this concept, a novel

ACCEPTED MANUSCRIPT degradation of many survival signalling molecules, such as IGFR1, PDGFRβ and AKT

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(Paraiso et al., 2012), which are upstream signalling repressors of FOXO3 (Myatt and Lam,

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2007). In agreement, XL888 has been shown to mediate its cytotoxic function through

671

activating FOXO3 to induce the expression of the pro-apoptotic Bim and repress the

672

expression of the anti-apoptotic Mcl-1 Bcl-2-related genes (Paraiso et al., 2012).

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Appropriately both Bim and Mcl-1 are downstream targets of FOXO3. In addition, 17-

674

Allylamino-17-demethoxygeldanamycin (17-AAG), a urinary bladder anti-cancer treatment,

675

induce apoptosis by disrupting the ability of Hsp90 to regulate its protein clients that regulate

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cancer cells survival (Karkoulis et al., 2010). Since FOXO proteins play a crucial part in the

677

UPR, targeting the FOXO proteins can prove to be an attractive strategy for tackling cancer

678

and for overcoming drug resistance. FOXO activity is modulated by acetylation, which is in

679

turn regulated by Sir2-related NAD-dependent protein deacetylases, (SIRTs or Sirtuins)

680

(Olmos et al., 2011). Research from mammalian systems and nematodes has also shown

681

that SIRT-mediated FOXO deacetylation not only limits its cytotoxic activity but also confers

682

drug resistance by directing it towards genes that have a role in ER stress and

683

chemotherapy resistance (Khongkow et al., 2013; Olmos et al., 2011; Peck et al., 2010). A

684

number of SIRT inhibitors have now been developed and these compounds have

685

demonstrated to be able to enhance the cytotoxic function of FOXO3 in cancer drug

686

treatment (Khongkow et al., 2013; Olmos et al., 2011; Peck et al., 2010). Besides targeting

687

cancer independently, these UPR signalling disruption compounds may also prove useful to

688

reinforce the cytotoxic effects of chemotherapeutic drugs, particular in drug resistance

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cancers. In addition to being anticancer therapeutic targets, these ER stress and FOXO-

690

regulated signalling and gene signatures can also be used as reliable diagnostic and

691

prognostic biomarkers and as indicators for drug responsiveness.

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Acknowledgements

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Glowi Alasiri is a recipient of a scholarship from the Saudi Arabian Cultural Bureau in

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London (MSU434). Stefania Zona is a post-doctoral research associate supported by the

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Medical Research Council (MRC) of UK (MR/N012097/1). Lavender Yuen-Nam Fan is a

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PhD student and research assistant supported by Imperial College IC Trust. Holger W.

698

Auner acknowledges the support of the Imperial College London National Institute of Health

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Research-Biomedical Research Centre (NIHR-BRC) and the Imperial College London

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Cancer Research UK Centre. Eric W.-F. Lam's work is supported by MRC (MR/N012097/1),

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CRUK (A12011), Breast Cancer Now (2012MayPR070; 2012NovPhD016), the Cancer

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Research UK Imperial Centre, Imperial ECMC and NIHR Imperial BRC.

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Figure.1 Accumulation of misfolded protein induces UPR activity and auto-

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phosphorylation of PERK. Activation of PERK attenuates the global translation by

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phosphorylating the α subunit of eIF2 to decrease the load of protein folding in ER. As a

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result, the specifically translated ATF4 will induce transcription of pro-survival genes for

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antioxidant response, amino acid synthesis, ER Chaperones, autophagy and combine with

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FOXO to regulate glucose level under acute stress. Upon chronic stress, ATF4 transcribes

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CHOP which in turn will induce expression of ROS synthesis and pro-apoptotic genes.

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Moreover, CHOP will promote transcription of GADD34 to dephosphorylate eIF2α and

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restore the global mRNA translation. (ATF4 = Activating transcription factor 4, CHOP =

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CCAAT/enhancer-binding (C/EBP) homologous protein, eIF2 = Eukaryotic translation

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initiation factor-2, ER = Endoplasmic reticulum, GADD34 = Growth arrest and DNA-damage-

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inducible protein 34, PERK = PKR-like endoplasmic reticulum kinase, ROS = Reactive

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oxygen species, UPR = Unfolded protein response).

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Figure. 2 ER stress signalling through IRE1α and ATF6. Upon accumulation of misfolded

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proteins, BiP disassociates from IRE1α and ATF6. IRE1α dimerizes and induces its

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endoribonuclease activity. Under survival condition, introns on XBP1 mRNA will be spliced

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to form XBP1s that works as transcription factors to induce the expression of genes for ER

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chaperones, lipid synthesis and ERAD process. ATF6 will be cleaved at Golgi by SP1 and

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SP2 proteases to form transcription factor ATF6α which increases the transcription of genes

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for XBP1, ER chaperones and ERAD process. However, IRE1α activation causes

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degradation of mRNA by the RIDD process to decrease protein folding and apoptosis.

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Moreover, IRE1α mediates phosphorylation of JNK which in turn induces FOXO3 to send

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cells for either autophagy or apoptosis. (ATF6 = Activation factor 6, BiP = Immunoglobulin

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heavy-chain binding protein, ER = Endoplasmic reticulum, ERAD = ER-associated

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degradation, GRP94 = 94 kDa glucose-regulated protein, FOXO3 = Forkhead box O3,

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IRE1α = Inositol requiring enzyme 1α, JNK = c-JUN N-terminal kinase, RIDD = IRE1α-

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dependent decay, SP1 = Site-1 proteases, SP2 = Site-2 proteases, XBP1 = X-box binding

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protein 1).

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Figure.3 The role of PERK in FOXO3 regulation and ER stress signalling.

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regulates FOXO3 directly by phosphorylating five sites, including S261, S298, S301, S303

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and S311, which induces its activity and translocation to nucleus. However, PERK inhibits

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FOXO3 activity to induce insulin resistance, obesity, adipocytic differentiation and cancer

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progression through mTOR/AKT. PERK induces mTOR to phosphorylate AKT on S473

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which causes relocation of FOXO3 to cytoplasm and degradation. Moreover, CHOP,

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expression. Furthermore, CHOP mediates ROS activity by induce ERO1α to activate JNK

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which in turn phosphorylates FOXO3 and induces apoptosis. (ATF4 = Activating

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Transcription Factor 4, AKT = Protein Kinase B, CHOP = CCAAT/enhancer-binding (C/EBP)

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homologous protein, eIF2 = eukaryotic translation initiation factor-2, ER = endoplasmic

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reticulum, ERO1α = ER oxidoreductin 1α, FOXO3 = Forkhead box O3, JNK = c-Jun N-

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terminal kinases, mTOR = mechanistic/ mammalian target of rapamycin, PERK = PKR-like

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endoplasmic reticulum kinase, ROS = Reactive oxygen species).

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Table 1 Functional human orthologues of C. elegans ER stress-related genes and

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their functions and relationship with Daf-16/FOXO. A role in the regulations of ER stress

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response to promote ER stress resistance and longevity. In C. elegans, ER stress

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responses, particularly the unfolded protein response (UPR), are mainly dependent on the

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Ire-1/Xbp-1 pathway. In fact, the downstream gene targets of the ire-1/xbp-1 pathway

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accounts for the majority of UPR genes which are implicated in protein folding and

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endoplasmic reticulum-associated protein degradation (ERAD), such as cul-1, cul-2, skr,

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and cup-2. Interestingly, xbp-1 was found to collaborate with daf-16 to activate transcription

1334

of these downstream targets. Studies of C. elegans also showed that pek-1 acts

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synergistically with atf-6 to maintain ER homeostasis as an alternative mechanism to the ire-

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1/xbp-1 pathway. Furthermore, pek-1 and atf-6 are both found to interact with daf-16 during

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ER stress response, suggesting a role of daf-16 in maintain ER homeostasis that is

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independent of the ire-1/xbp-1 signaling pathway

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ACCEPTED MANUSCRIPT Highlights

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A comprehensive analysis of the main ER stress players and signalling pathways. New insights into the ER stress activation and its role in cancer initiation and progression. FOXO3 as a new key molecule within the ER stress network. Regulation of FOXO3 by PERK and CHOP and its implications in cancer progression and resistance. Significance of FOXO3 in the UPR signalling and new strategies to overcome drug resistance.

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