the impact of the endoplasmic reticulum protein-folding environment on cancer development

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells for calcium storage and regulated release and as the entrance to the secretory pathway, for which approximately one-third of all cellular proteins traffic enroute to their proper intracellular or extra-cellular location. Numerous environmental, physio-logical and pathological insults, as well as nutrient fluctuations, disrupt the ER protein-folding environ-ment to cause protein misfolding and accumulation of misfolded proteins, referred to as ER stress. The unfolded protein response (UPR) is a collection of sig-nalling pathways that evolved to maintain a productive ER protein-folding environment. Both ER stress and UPR activation are involved with the pathology of many, if not all, degenerative diseases. Moreover, ER stress and UPR activation are documented in the development of many cancer types (TABLE1; see Supplementary infor-mation S1 (table)), and evidence suggests that they have important roles in every aspect of cancer development.

As cancer usually arises and progresses in a stressful microenvironment, transformed cells may use UPR acti-vation as a survival strategy. In fact, numerous studies demonstrate crucial roles for UPR signalling in tumour growth and chemoresistance. However, only recently has it been demonstrated that invivo UPR activation is a vital step during oncogenic transformation and cancer development. Recent studies also suggest that UPR signalling molecules interact with well-established oncogene and tumour suppressor gene networks to modulate their function during cancer development. It will be important to understand exactly how these

signalling pathways regulate each other, their inter-dependence and how interference with one affects the others. Aside from its pro-survival role, prolonged UPR activation owing to severe or unresolved ER stress leads to cell death. This greatly complicates the development of cancer therapies that target UPR signalling. In this Review, we highlight recent advances in our understanding of how UPR activation has both tumour-supporting and tumour- suppressive roles, and we discuss strategies that target UPR components for cancer treatment.

ER stress and UPR activation in cancerThe ER is the organelle in eukaryotic cells that is responsible for intracellular Ca2+ homeostasis, lipid biosynthesis and protein folding and transport. Protein folding in the ER is exquisitely sensitive to changes in the environment, such as altered Ca2+ levels, redox state, nutrient status, increases in the rate of protein synthesis, pathogens or inflammatory stimuli, which lead to disrupted protein folding to cause accumula-tion of unfolded or misfolded proteins a condition termed ER stress. Early studies demonstrated two key events that occur when proteins misfold in the ER. First, misfolded proteins bind and sequester the chaperone immunoglobulin heavy-chain binding protein (BIP; also known as GRP78 and HSP5A)1 and, second, reduc-tion in the level of free BIP activates signalling pathways to induce transcription of BIP, as well as other genes encoding protein chaperones2,3, now known as the UPR. The outcome of UPR activation involves tran-sient attenuation of protein synthesis, increased capacity

Degenerative Diseases Program, Center for Cancer Research, Sanford-Burnham Medical Research Institute, 10901 N.Torrey Pines Rd, La Jolla, California 92037, USA.Correspondence to R.J.K. e-mail: [email protected]:10.1038/nrc3800

The impact of the endoplasmic reticulum protein-foldingenvironment on cancer developmentMiao Wang and Randal J.Kaufman

Abstract | The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells for the storage and regulated release of calcium and as the entrance to the secretory pathway. Protein misfolding in the ER causes accumulation of misfolded proteins (ER stress) and activation of the unfolded protein response (UPR), which has evolved to maintain a productive ER protein-folding environment. Both ER stress and UPR activation are documented in many different human cancers. In this Review, we summarize the impact of ER stress and UPR activation on every aspect of cancer and discuss outstanding questions for which answers will pave the way for therapeutics.

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ER-associated degradation(ERAD). A process by which misfolded proteins in the endoplasmic reticulum (ER) are targeted by retrotranslocation and ubiquitylation for subsequent degradation by the proteasome.

for protein trafficking through the ER, protein fold-ing and transport, and increased protein degradative pathways, including ERassociated degradation (ERAD) and autophagy. If these adaptive mechanisms cannot resolve the protein-folding defect, cells enter apoptosis. This applies not only to normal cells but also to cancer cells. Therefore, it is not surprising that UPR activation contributes to both enhanced survival and induced

apoptosis in cancer cells depending on the context. It needs to be pointed out that much of what we under-stand about UPR activation in cancer is derived from mouse models, which have limitations (BOX1).

UPR activation in transformed cells is attributed to both intrinsic and extrinsic factors (BOX 2; BOX 3). Hyperactivation of oncogenes or loss-of-function mutations in tumour suppressor genes, such as

Table 1 | Evidence of ER stress and UPR activation in various human cancer types*

Cancer Activation of UPR components

Prognosis

Classification Site

Carcinoma Lung BIP and CHOP BIP was correlated with low tumour stage and longer survival CHOP was correlated with high tumour stage and shorter survival

BIP and GRP94 Correlated with low grade of differentiation and high tumour stage

Breast BIP Expressed more in oestrogen receptor-negative tumours than in oestrogen receptor-positive tumours

BIP and XBP1 Correlated with oestrogen receptor expression

BIP, GRP94, GRP75, HSP60 and calreticulin

NA

Colon BIP Correlated with high cell malignancy

CHOP Correlated with high tumour stage

Gastric BIP and GRP94 Correlated with tumour size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, and tumour stages, but not independent prognostic factors

BIP and GRP94 BIP was correlated with low tumour stage, high grade of differentiation and longer survival

GRP94 was correlated with low tumour stage

Pancreas BIP and HSP90 NA

Liver BIP, ATF6 and XBP1 Correlated with high histological grade

BIP, HSP27 and HSP70 Correlated with high tumour venous infiltration

BIP Correlated with CD147, which inhibits apoptosis and induces chemosensitivity

Prostate BIP Correlated with shorter survival

BIP Correlated with castration resistance, greater risk of recurrence and shorter overall survival

Kidney BIP Correlated with higher tumour grade, advanced tumour stage, lymphovascular invasion, regional nodal involvement, distant metastases and shorter survival

BIP Correlated with larger tumour size and higher tumour stage

Skin BIP Correlated with increased tumour thickness, metastases and shorter survival in patients with melanoma

Uterus BIP, ATF6 and CHOP NA

Ovary BIP Correlated with higher cell malignancy, but not associated with survival

Leukaemia Lymphoblast BIP, XBP1s and calreticulin

NA

BIP, XBP1s, CHOP and calreticulin

Correlated with lower relapse rate and longer overall and disease-free survival

Lymphoma Bcells XBP1s Correlated with higher tumour grade, therapy resistance and shorter survival

XBP1s Correlated with advanced plasma differentiation

GRP94, IRE1 and GADD34

GRP94 was correlated with therapy resistance and shorter survival

BIP Correlated with shorter overall survival and lower sensitivity to therapy

Glioma Brain BIP Correlated with shorter survival

ATF6, activating transcription factor 6; BIP, immunoglobulin heavy-chain binding protein; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; GADD34, growth arrest and DNA damage-inducible protein 34; GRP94, 94 kDa glucose-regulated protein; HSP, heat shock protein; IRE1, inositol-requiring protein 1; NA, not applicable; UPR, unfolded protein response; XBP1, X-box binding protein 1; XBP1s, transcriptionally active XBP1. *Each row of the table represents one study; see Supplmentary information S1 (table) for a version of this table with references.

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Mitochondria-associated ER membranes(MAMs). A specialized endoplasmic reticulum (ER) membrane is directly juxtaposed to the mitochondrion to coordinate efficient communication between these two organelles.

PolysomeA cluster of ribosomes translating a single mRNA molecule.

induction of oncogenic HRAS, MYC or the oncogenic latent membrane protein 1 of EpsteinBarr virus47, or loss of tumour suppressors tuberous sclerosis com-plex 1 (TSC1; also known as hamartin), TSC2 (also known as tuberin), BRCA1 or PTEN811, increase pro-tein synthesis and translocation into the ER owing to high metabolic demand during oncogenic transforma-tion. Consequently, the UPR is activated to increase the protein folding capacity. In addition, some gene mutations, such as smoothened (SMO) mutants, cause UPR activation owing to their intrinsic misfolding12. Furthermore, UPR activation is required to pro-mote ER expansion for division and transmission to daughter cells during mitosis13. Certain types of can-cer cells are highly secretory and therefore prone to constitutive UPR activation. For example, haemato-logical malignancies such as multiple myeloma and other plasma cell malignancies express high levels of immunoglobulins. Increased mucin production is also documented in many solid cancers, including pancre-atic, lung, breast, ovarian and colon cancers14. During malignant progression, cancer cells activate pathways that co-opt cells in the tumour microenvironment, such as immune cells and endothelial cells, to support tumour growth15,16, which may require UPR signal-ling to increase folding and the secretion of cytokines, metallo proteinases, angiogenesis factors and extra-cellular matrix components. Besides the intrinsic factors, rapidly proliferating cancer cells frequently encounter a hostile environment, which disrupts ER protein folding to activate the UPR (BOX2).

The role of the UPR in tumorigenesisThe UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK; also known as eIF2AK3)eukaryotic translation initiation factor 2 (eIF2), inositol-requiring protein 1 (IRE1; also known as ERN1)X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) (FIG.1). Emerging evi-dence suggests that UPR activation is required for onco-genic transformation. As the UPR exerts both protective and deleterious effects on cell survival upon ER stress, UPR activation may facilitate, as well as suppress, malig-nant transformation (FIG.2). Therefore, there would be a selective advantage for premalignant cells harbouring gene mutations that suppress UPR-induced apoptosis or senescence.

The PERKeIF2 pathway in ER stress. PERK is a typeI transmembrane protein enriched at mitochondria associated ER membranes (MAMs)17 with a cytosolic serine/threonine kinase domain. Under non-stress con-ditions, heat shock protein 90 (HSP90) and BIP bind to the cytoplasmic and ER luminal domains of PERK, respectively, to stabilize and prevent activation18. Under conditions of ER stress, BIP binds to unfolded proteins and misfolded proteins, permitting the release of PERK for homodimerization and autophosphorylation, leading to its activation19,20. Activated PERK then phosphorylates eIF2 (a subunit of the heterotrimeric eIF2 complex) at S51 (REF.21) to attenuate translation initiation due to limiting amounts of the eIF2GTPtRNAmet ternary complex. The ternary complex binds the 40S ribosome to generate a 43S species that binds the 5 end of the mRNA to initiate scanning downstream. When the 43S species encounters an AUG codon in an optimal con-text for initiation, eIF5 activates eIF2-mediated GTP hydrolysis22. To perform another round of initiation, eIF2B is required to promote GTP exchange for GDP on eIF2 a reaction that is inhibited by eIF5 (REF.23). Phosphorylation of eIF2 greatly increases the affinity of eIF2 for GDP, thereby preventing the eIF2B-catalyzed exchange reaction and sequestering eIF2B with eIF2 in an inactive complex24, as well as inhibiting the anti-exchange activity of eIF5 (REF.23). As the eIF2B/eIF2 ratio is generally less than 1 (approximately 1/7 in rab-bit reticulocyte lysate and 1/2 in Ehrlich ascites cells25), it was proposed that small increases (as little as ~20%) in the amount of eIF2 phosphorylation could shut down general protein synthesis26. The transient inhibition of protein synthesis probably promotes polysome disassem-bly to increase the number of ribosomes available to bind newly transcribed mRNAs, which encode UPR adaptive functions. Besides eIF2, other PERK substrates have been suggested to affect cell survival, function and dif-ferentiation, including nuclear factor erythroid 2-related factor 2 (NRF2; also known as NFE2L2)27, forkhead box O (FOXO)28 and diacylglycerol29. However, as the PERK-dependent changes in gene expression in mouse embryonic fibroblasts (MEFs) can be prevented by eIF2 mutation at the PERK phosphorylation site30, the physiological importance of these alternative substrates remains in question.

Box 1 | Considerations in studying the UPR in cancer

The consequence of activation of the unfolded protein response (UPR) is closely associated with different types, severity and duration of endoplasmic reticulum (ER) stress, which needs consideration for data interpretation when characterizing UPR activation in cancer. Many studies are performed with cells in which genes have been deleted or knocked

down. There is a considerable difference between gene deletion versus knockdown. Both approaches require adequate controls: multiple gene-deleted lines or gene rescue experiments; for knockdowns or clustered regularly interspaced short palindromic repeat (CRISPR)-mediated deletions, elimination of off-target effects is essential.

A reduction in a protein may have a completely different effect than complete removal of a protein. It is now evident that many requirements for a protein or protein modification exhibit a bell-shaped distribution in which either higher or lower levels of protein or protein modification show similar deficiencies.

It is also necessary to carry out detailed kinetic studies because deletion of a gene may be advantageous after 24hours but extremely detrimental for long-term survival. Most studies analyse a single time point after gene knockdown. This is especially important in the analysis of growth and metastasis in xenotransplant experiments.

Most convincing experiments require genetic correction of a phenotype by altering a downstream component to demonstrate the correction is dependent on the gene that is deleted.

Most studies of ER stress use pharmacological induction of protein misfolding that is, tunicamycin, thapsigargin, dithiothreitol, and so on. As these agents have many additional effects on the cell, it is important to study how the synthesis of a misfolded protein, an increase in general protein secretion in cancer cells or other physiological challenges (hypoxia, nutrient deprivation, redox changes, reactive oxygen species (ROS), and so on) affect tumour cell survival to obtain more physiologically meaningful results.

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While attenuating global mRNA translation, PERKeIF2 activation paradoxically increases the translation of a growing number of mRNAs, including those encoding ATF4 (also known as CREB2) and ATF5 (REF.26), as well as amino acid transporters31. ATF4 enters the nucleus to activate ER stress response genes that are responsible for the antioxidant response and amino acid biosynthesis and transport to promote cell survival26. ATF4 activates tran-scription of the growth arrest and DNA damage-inducible protein 34 (GADD34; also known as PPP1R15A) to direct eIF2 dephosphorylation and restore global mRNA translation. ATF4 also activates transcription of C/EBP homologous protein (CHOP; also known as DDIT3 and GADD153)26,32, which is required for ER-stress-mediated apoptosis both invitro and invivo33,34. At early times after ER stress, PERK activation induces miR-211 expression, which represses CHOP transcription through histone methylation35. CHOP expression can also be suppressed by Toll-like receptor (TLR)TIR-domain-containing adapter-inducing interferon- (TRIF; also known as TICAM1) signalling through protein phosphatase 2A (PP2A)-mediated serine dephosphorylation of the eIF2B -subunit36. Under conditions of chronic stress, consti-tutive PERK-mediated phosphorylation of eIF2 leads to apoptosis, as the IRE1XBP1 and ATF6 pathways are attenuated3739. Therefore, PERK activation promotes both adaptive, as well as apoptotic, responses depending on the severity of the stress. It is likely that the particular response differs between cell types and environments on the basis of different thresholds for ER stress tolerance of thecell.

Although CHOP accumulation in the cell correlates with cell death, both ATF4 and CHOP mRNAs and proteins have short half-lives; therefore, a strong and chronic activation of PERK is necessary to increase steady state levels of CHOP to promote cell death37. Previous reports suggest that CHOP represses BCL-2

expression40, upregulates BCL-2-interacting mediator of cell death (BIM; also known as BCL2L11) transcrip-tion41 and promotes the translocation of BAX to mito-chondria42. CHOP was also shown to directly bind and induce the promoters of p53 upregulated modulator of apoptosis (PUMA; also known as BBC3)43, lipocalin 2 (LCN2)44, tribbles homologue 3 (TRIB3)45 and death receptor 5 (DR5; also known as TNFRSF10B)46,47. It was recently confirmed that CHOP-mediated DR5 induction is responsible for ER stress-induced apoptosis via cas-pase 8 in cancer cells48. However, chromatin immuno-precipitation followed by sequencing (ChIPseq) studies did not detect either ATF4 or CHOP occupying genes of the pro-apoptotic family upon induction of ER stress in MEFs. Instead, ATF4 and CHOP formed heterodimers that upregulated genes encoding functions in the UPR, autophagy and, surprisingly, mRNA translation, leading to increased protein synthesis, ATP depletion, oxidative stress and cell death49. Indeed, compared with wild-type cells, ER stress caused less ER protein aggregation and apoptosis in Chop-null cells, which is consistent with the idea that CHOP increases protein synthesis to cause protein misfolding and oxidative stress33,34,50,51. Therefore, although ER stress-induced apoptosis is indirectly medi-ated by CHOP, it is possible that, in tumour cells, dif-ferent pathways are activated downstream of CHOP to regulate survival.

The PERKeIF2 pathway in tumorigenesis. As the PERKeIF2 pathway induces either survival or apop-tosis upon ER stress, it may facilitate, as well as suppress, malignant transformation depending on the context. Indeed, Perk deletion delays Neu-dependent mammary tumour development and reduces lung metastases, whereas long-term PERK inactivation increases suscep-tibility to spontaneous mammary tumorigenesis owing to increased genomic instability52. PERK activation also

Box 2 | The tumour microenvironment and the induction of ER stress

Tumours (especially solid tumours) are often challenged by hypoxia and a lack of glucose, as well as other nutrients, owing to poor vascularization upon quick expansion of the tumour mass, which results in severe endoplasmic reticulum (ER) stress in cancer cells56,172174. To survive the hostile environment, the unfolded protein response (UPR) is activated in cancer cells, which ameliorates ER stress to promote cell survival and growth103105.

How do environmental factors induce ER stress in cancer cells? Some environmental factors, such as hypoxia, directly impact on protein modification in the ER, leading to accumulation of misfolded or unfolded protein. A major post-translational modification of proteins synthesized in the ER is disulphide bond formation, which is catalysed by the family of disulphide isomerases. A recent finding showed that disulphide bonds that are formed during protein synthesis are oxygen-independent, but those formed during post-translational folding or isomerization in the ER are oxygen-dependent175. This provides insight into how hypoxia causes ER stress and UPR activation. Hypoxia also increases the stability of some UPR components, such as activating transcription factor 4 (ATF4), possibly because ATF4 is degraded by proline hydroxylation, similar to hypoxia-inducible factors (HIFs)176,177. Both ATF4 and X-box binding protein 1 (XBP1) further augment HIF1-mediated upregulation of its downstream targets to promote cell survival93,178. However, eukaryotic translation initiation factor 2 (eIF2) phosphorylation is more important than HIF signalling in promoting survival of therapy-resistant cancer cells104. Blocking UPR activation significantly increases cancer cell death under hypoxic conditions179. Glucose deprivation, often coinciding with hypoxia, also disrupts protein folding in the ER. Glucose metabolism supplies tumour cells with energy in the form of ATP, building blocks for biosynthesis, and functions as a donor for asparagine-linked glycosylation. Glucose shortage leads to disturbed ERCa2+ homeostasis that is mediated by deficient sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity owing to a reduced energy supply180 and protein misfolding caused by improper protein glycosylation. Some other environmental factors indirectly induce ER stress and UPR activation. Amino acid deprivation activates general control nonderepressible 2 (GCN2; also known as eIF2K4) to phosphorylate eIF2 (discussed in BOX3). Growth factors that are present in the tumour microenvironment can also contribute to UPR activation in cancer, independent of ER stress (discussed in BOX3).

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promotes MYC-induced cell transformation through autophagy7,53. This may be related to PERK-mediated eIF2 phosphorylation and the resulting increase in ATF4, CHOP and factors that activate transcription of many autophagy genes54,55. However, it was reported that CHOP induction in response to prolonged ER stress causes death of premalignant cells to prevent neoplastic progression. Deletion of Chop increased tumour inci-dence in a KrasG12V-induced mouse model of lung can-cer, suggesting a tumour-suppressive role of CHOP56. Additionally, hepatocyte-specific Chop deletion increased tumorigenesis in a mouse model of hepatocellular car-cinoma57. CHOP mutations were reported in human tumours58, although it is unknown whether these muta-tions alter protein expression or function and whether they contribute to tumorigenesis.

The IRE1XBP1 pathway in ER stress. Mammals have two IRE1 genes, IRE1A (also known as ERN1; which encodes IRE1) and IRE1B (also known as ERN2; which encodes IRE1). I RE1A is ubiquitously and constitutively expressed, whereas IRE1B expres-sion is restricted to intestinal and lung epithelial cells.

Like PERK, IRE1 is a typeI transmembrane protein with a cytosolic serine/threonine kinase domain. Under non-stress conditions, both HSP90 and HSP72 bind the IRE1 cytosolic domain to maintain its stabil-ity18,59, while BIP binds the luminal domain of IRE1 to prevent dimerization. Upon ER stress, unfolded and misfolded proteins bind and sequester BIP, thereby releasing IRE1 for oligomerization, autophosphoryla-tion and activation of its kinase and endoribonuclease activities19,20. Membrane fluidity also influences PERK and IRE1 oligomerization and activation60. Structural studies suggest that short peptides could interact with a major histocompatability complex classI (MHC classI)-like groove to promote dimerization in yeast IRE1 (REF.61). Although the MHC classI-type groove in the human IRE1 homodimer was not solvent-exposed62, recent studies show that it can bind some peptides63. Activated IRE1 cleaves Xbp1 mRNA to initiate removal of a 26-base intron in the cyto-plasm to produce a translational frame-shift creating a transcriptionally active form (Xbp1s) that enters the nucleus to regulate target genes64,65. To expedite the response, Xbp1u (unspliced) mRNA localizes to the ER

Box 3 | UPR activation independent of ER stress

A recent study revealed that vascular endothelial growth factor A (VEGFA) activates the unfolded protein response (UPR) via phospholipase C (PLC)-mediated crosstalk with mTOR complex 1 (mTORC1) in endothelial cells, in the absence of endoplasmic reticulum (ER) stress158. Thus, it is possible that growth factors in the tumour microenvironment activate the UPR in tumour cells. Although this hypothesis needs further testing with cancer cells and other growth factors, it is possible that UPR activation in cancer cells and cells in the tumour microenvironment, such as endothelial cells and macrophages, is ER stress-independent under certain conditions.

Greater evidence suggests that UPR signalling can be activated in the absence of ER stress. This is particularly evident in the regulation of protein synthesis through phosphorylation of eukaryotic translation initiation factor 2 (eIF2). In mammals, there are three additional kinases that also phosphorylate eIF2 S51: general control nonderepressible 2 (GCN2; also known as eIF2K4) induced by amino acid deprivation, the heme-regulated inhibitor kinase (HRI; also known as eIF2K1) induced by oxidative stress or heme deprivation and the double-stranded RNA (dsRNA)-activated protein kinase (PKR; also known as eIF2AK2) activated by dsRNA as part of the interferon antiviral response. Importantly, the stress conditions that activate any single eIF2 kinase may have secondary effects on the cell that cause activation of other eIF2 kinases. For example, ER stress activates PKR, as well as PRKR-like ER kinase (PERK), and ultraviolet (UV) light activates both GCN2 and PERK. Although activation of any eIF2 kinase causes translation attenuation, the cellular response varies tremendously depending on which kinase phosphorylates eIF2, the cell type and the environment. Generally, eIF2 phosphorylation that is mediated by GCN2, HRI and PKR leads to apoptosis39, whereas transient eIF2 phosphorylation by PERK promotes cell survival, and chronic eIF2 phosphorylation by PERK promotes apoptosis. The mechanism for the different outcomes remains unknown but may involve decreased synthesis of inhibitors of apoptosis that have short half-lives or different levels of growth arrest and DNA damage-inducible protein 34 (GADD34) to direct eIF2 dephosphorylation. In the stressful tumour microenvironment, the level of eIF2 phosphorylation might be high, and so other factors need to be considered that impact the effect of eIF2 phosphorylation rate on protein synthesis. For example, GADD34 is a regulatory subunit of protein phosphatase 1 (PP1) that promotes eIF2 dephosphorylation181, and protein phosphatase 2A (PP2A)-mediated dephosphorylation of the -subunit of eIF2B activates exchange activity of eIF2B to bypass eIF2 phosphorylation36. As eIF2 phosphorylation attenuates mRNA translation, less misfolded proteins accumulate in the ER21,26, which promotes cell survival.

Similar to PERK, the activation of GCN2, HRI and PKR are associated with cancer. PKR has both tumour-supportive and tumour-suppressive roles in cancer development182. GCN2 is upregulated in cancers and its inhibition delays tumour growth in xenograft models183. GCN2 upregulation is also required for angiogenesis by augmenting amino acid deprivation-induced expression of VEGFA. More importantly, this effect does not depend on PERK activation184. It was also reported that HRI expression is reduced in ovarian epithelial cancer185, although its impact in cancer remains unclear.

Inositol-requiring protein 1 (IRE1) can also be activated by Toll-like receptor (TLR) signalling, independent of ER stress186, through recruitment of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6). Interaction of TRAF6 with IRE1 prevents interaction with PP2A via its adaptor receptor for activated C kinase 1 (RACK1) to prevent IRE1 dephosphorylation and inactivation73. TRAF6 and PP2A compete for binding to the same site on IRE1. TRAF6 binding reduces PP2A-mediated dephosphorylation, promoting IRE1 activation74. IRE1 signalling is attenuated through proteasomal degradation that is mediated by TRAF6 ubiquitylation of IRE1 via a K48 linkage74. As TLR signalling is important in cancer development and drug resistance187, IRE1 could be one of the mediators in this process.

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Nature Reviews | Cancer

Misfoldedprotein

ER

BIP

PERK

IRE1

ATF6

ATF6

P

BIP

BIP BIP

BIP

BIP BIP

BIP

eIF2

eIF2

GADD34

ATF4

ATF4

Translation

Translation

Autophagy

ROS

Apoptosis

CHOP

JNK

RIDD RNA decay

XBP1u XBP1s XBP1

S1PS2P

Increased ER proteinfolding capacity and ERAD

Survival

Golgi Nucleus

P

P

BIP

BIP P

P

Regulated IRE1-dependent decay(RIDD). A process in which activated inositolrequiring protein 1 (IRE1) induces cleavage and degradation of microRNAs and of mRNAs encoding membrane and secreted proteins.

membrane to facilitate IRE1 interaction66. Genes that are regulated by IRE1XBP1 enhance protein fold-ing, trafficking and ERAD, thereby resolving protein misfolding38,67, and forced XBP1s expression inhibits CHOP expression, thereby promoting cell survival68. In addition, overexpression of XBP1 induces many genes involved in secretory pathways and physically expands the ER, which results in the characteristic phenotype of professional secretory cells67. However, the idea that IRE1XBP1 promotes cell survival is challenged by recent findings that small molecule inhibitors of IRE1 did not sensitize cells to ER stress-induced apoptosis, but rather prevented expansion of secretory capacity69.

ER stress immediately activates IRE170,71, whereas IRE1 activation is mostly attenuated upon chronic ER stress71,72. It is unclear how IRE1XBP1 signalling is attenuated under conditions of sustained ER stress, although dephosphorylation, ubiquitylation and deg-radation are probably involved73,74. Although protein disulphide isomerase family A member 6 (PDIA6), a resident ER protein, forms a disulphide bond with C148 in the IRE1 luminal domain to attenuate sig-nalling75, other results show that PDIA6 is required for IRE1 activation76. Furthermore, Xbp1u can function as a negative regulator of both the XBP1s and ATF6 pathways by direct interaction to promote their degra-dation77, which possibly blocks survival signals during chronic ERstress.

If IRE1 signalling is not attenuated, chronic IRE1 activation signals apoptosis. Hyperactivated IRE1 cleaves many mRNAs, in addition to Xbp1 (REFS70,78) and its own mRNA79, a process called regulated IRE1dependent decay (RIDD)80. A recent study suggests that RIDD is dependent on the oli-gomeric state of IRE181. RIDD also reduces the expression of some microRNAs (mi RNAs), includ-ing miR-17, miR-34a, miR-96 and miR-125b, which repress caspase 2 expression82. However, the impor-tance of caspase 2 activation in ER stress-induced apoptosis remains in question83. Activated IRE1 kinase also binds TNF receptor-associated factor 2 (TRAF2), which recruits apoptosis signal-regulating kinase 1 (ASK1; also known as MAP3K5) and JUN N-terminal kinase (JNK)84, leading to activation of BIM and inactivation of BCL-2. However, the importance of JNK activation in ER stress-induced apoptosis has not been demonstrated.

The IRE1XBP1 pathway in tumorigenesis. The role of IRE1XBP1 in multiple myeloma has been intensively studied because mature Bcell differen-tiation into plasma cells and Bcell-mediated defence against infection require XBP1s8587. Increased levels of XBP1s are frequently associated with human mul-tiple myeloma, and genetically engineered mice that over express XBP1s under the control of immuno-globulin VH promoter and E enhancer elements

Figure 1 | The unfolded protein response (UPR) signalling pathways. Upon endoplasmic reticulum (ER) stress, unfolded and misfolded proteins bind and sequester immunoglobulin heavy-chain binding protein (BIP), thereby activating the UPR. The UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK)eukaryotic translation initiation factor 2 (eIF2), inositol-requiring protein 1 (IRE1)X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6). The outcome of UPR activation increases protein folding, transport and ER-associated protein degradation (ERAD), while attenuating protein synthesis. If protein misfolding is not resolved, cells enter apoptosis. CHOP, C/EBP homologous protein; GADD34, growth arrest and DNA damage-inducible protein 34; JNK, JUN N-terminal kinase; P, phosphorylation; RIDD, regulated IRE1-dependent decay; ROS, reactive oxygen species; XBP1s, transcriptionally active XBP1; XBP1u, unspliced XBP1.

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Protein misfolding in the ER

Hostile environment Cancer therapy

Loss ofBRCA1

Lossof TSC MYC

Loss ofPTEN RAS

p53 and TSCdownregulated

BIPinduced

DecreasedATF4 and CHOPexpression

Inducedautophagy

Enhancedglycolysis

CHOPinduction

Survival OncogenictransformationSenescenceor apoptosis

Tumorigenesis

Regulated intramembrane proteolysisA process in which endoplasmic reticulum (ER) transmembrane transcription factors are cleaved within the plane of the membrane to release cytosolic fragments that enter the nucleus to regulate gene transcription.

exhibit features reminiscent of multiple myeloma transformation88. IRE1A and XBP1 mutations have been identified in tumour cells from patients with multiple myeloma89,90. More importantly, analy-sis of human multiple myeloma tumour lines that are resistant to proteasome inhibition identified loss-of-function mutations in either IRE1A or XBP190. Apparently, proteasome inhibitors select for cells that do not require ERAD: that is, multiple myeloma cells that lose immunoglobulin expression and display pre-plasmablast characteristics90. Mutations in IRE1A were also reported in other human cancers58,91, some of which lose or reduce kinase and/or endoribonu-clease function81. In addition, loss of XBP1 function promotes tumorigenesis in mouse models of intes-tinal cancer 92. Although these findings suggest a

tumour-suppressive role for IRE1XBP1, increased XBP1 mRNA splicing was observed in human triple- negative breast cancers, possibly indicating a requirement for XBP1 in cancer stem-like cells93.

The ATF6 pathway in ER stress. ATF6 is a typeII trans-membrane protein that contains a cytosolic cAMP-responsive element-binding protein (CREB)/ATF basic leucine zipper (bZIP) domain. Under non-stressed conditions, ATF6 is retained in the ER through interaction with BIP. Upon accumulation of misfolded protein, ATF6 is released from BIP and traffics to the Golgi apparatus for processing by the proteases S1P (also known as MBTPS1) and S2P (also known as MBTPS2)94 to generate an active transcription factor, in a process termed regulated intramembrane proteolysis. There are two homologues of ATF6 in the mamma-lian genome. Cleaved ATF6 mediates the adaptive response to ER protein misfolding by increasing the transcription of genes that increase ER capacity and the expression of Xbp1 (REFS95,96), whereas ATF6 may function as a repressor of ATF6-mediated transcrip-tion and function97. Presently, no genes have been iden-tified that require ATF6 for expression. PERKeIF2 signalling facilitates ATF6 synthesis and trafficking to accentuate ATF6 signalling98. To date, no substantial evidence supports a role of ATF6 in ER stress-induced apoptosis.

The ATF6 pathway in tumorigenesis. Some studies suggest that ATF6 promotes hepatocarcinogenesis by regulation of target genes99. A missense polymorphism in ATF6 that increases mRNA expression of ATF6 and its downstream genes was associated with susceptibil-ity to hepatocellular carcinoma100. More importantly, BIP, a downstream transcriptional target of ATF6, was reported to serve as a malignancy marker for cells. Upon induction of ER stress, ATF6 quickly induces BIP expression, which binds to unfolded protein and misfolded protein to ameliorate ER stress. Under nor-mal conditions, BIP is localized to the ER lumen, but upon overexpression in many human cancers (TABLE1; see Supplementary information S1 (table)), it becomes detectable on the cell surface101. BIP expression not only correlates with cancer cell proliferation and his-tological grade but also correlates with response to therapies and prognosis102 (TABLE1; see Supplementary information S1 (table)).

The UPR in tumour cellsUPR in autonomous cancer cell survival. UPR acti-vation protects cancer cells from stress-induced cell death103105. Acute UPR activation enhances the pro-tein folding capacity to meet the need for increased protein synthesis, which benefits cancer cell survival. Where chronic ER stress kills normal cells, tumour cells use strategies that neutralize apoptosis when challenged with ER stress. In response to chronic stress, normal cells usually attenuate the IRE1XBP1 and ATF6 pathways, so that the apoptotic signals dominate38. Some cancer cells, however, exhibit constitutive activation of

Figure 2 | Crosstalk between the unfolded protein response (UPR) components and oncogene or tumour suppressor gene networks in cancer cells. Either hyperactivation of oncogenes or loss of tumour suppressor genes can activate the UPR, promoting cell survival, oncogenic transformation or cell senescence or apoptosis, depending on gene mutations and the cellular context. The loss of BRCA1 function upregulates immunoglobulin heavy-chain binding protein (BIP) expression to survive chronic endoplasmic reticulum (ER) stress10, although the mechanism is unclear. The loss of tuberous sclerosis complex (TSC) function increases protein synthesis and the requirement for ER protein folding, thereby causing ER stress and UPR activation, but expression of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) are mostly compromised9. Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is required for the anti-proliferative and pro-apoptotic effects of PTEN190. Loss of PTEN induces UPR activation and increases aerobic glycolysis (known as the Warburg effect), which is associated with transformation11,191. Downregulation of tumour suppressor candidate 3 (TUSC3), which affects N-linked glycosylation, also causes ER stress to activate the UPR and increase the malignancy of prostate cancer cells192. Furthermore, UPR activation, on the one hand, increases expression of genes that are involved in tumour initiation and progression, such as the a disintegrin and metalloproteinase (ADAM) family, to facilitate tumorigenesis193. On the other hand, the UPR decreases the expression of some tumour suppressors, including p53 (REFS194,195), TSC1 and TSC2 (REF.126), to promote cell survival and oncogenic transformation. In other cases, sustained UPR activation in response to prolonged ER stress causes death of premalignant cells to prevent neoplastic progression. For example, HRAS induces UPR-mediated cell senescence in premalignant cells4. Red boxes indicate oncogenes and green boxes indicate tumour suppressors.

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the IRE1XBP1 pathway106,107 or overexpression of BIP10,108, which are anti-apoptotic. Furthermore, CHOP, induced by chronic ER stress, activates transcription of the AKT inhibitor TRIB3, which blocks mTOR pathways45,109,110 to inhibit proliferation and activate autophagy. UPR activation also represses cyclin D1 translation due to global transient translation inhibition induced by eIF2 phosphorylation, leading to subse-quent cell cycle arrest in the G1 phase111. This increases dormancy of the cancer cells, permitting survival in the stressed environment until more favourable conditions are encountered. In addition, oncogene and tumour suppressor gene mutations inhibit ER stress-induced apoptosis machinery (FIG.2). Mutations in UPR path-ways may also directly contribute to enhanced cancer cell survival upon stress. Some IRE1 mutants iden-tified in human cancers showed reduced endoribo-nuclease function. Although still able to splice XBP1 mRNA, they cannot induce RIDD, thereby promot-ing cell survival81. Hence, cancer cells escape from ER stress-induced apoptosis.

By contrast, persistent ER stress or UPR activation (particularly by pharmacological intervention) induces cancer cell death through similar apoptosis pathways that are used in normal cells (FIG.1). Therefore, chronic ER stress or UPR activation-induced cell death pathways are intact in at least some tumour cells. It will be of great interest to determine whether persistent ER stress or UPR activation can induce tumour cell death through other mechanisms. It will also be important to predict whether particular tumour types are dependent on UPR signalling for survival.

UPR, autophagy and cell metabolism in cancer develop-ment and progression. Early studies showed that protein folding and processing in the ER and trafficking to other organelles and the cell surface require a series of com-plex energy-requiring reactions112,113. As a result, protein folding in the ER is susceptible to energy fluctuations in the cell and protein misfolding may serve as a sig-nal for nutrient, energy or oxygen deprivation. On the one hand, conditions of low nutrient supply (for exam-ple, glucose deprivation or hypoxia) induce ER stress and UPR activation, to improve protein folding and transport, restore energy homeostasis and render cells resistant to cell death114. On the other hand, excess nutri-ents (fatty acids, cholesterol and glucose) also induce ER stress and UPR activation115,116. The integration of the UPR with cell metabolism is of special importance to the cancer biology field, as tumour cells display ER stress, UPR activation and nutrient shortage, which are probably due to poor vascular supply and rapid cell proliferation (BOX2), and tumours arise at higher rates and are more malignant in a nutrient-rich environment compared with a normal environment117,118.

Faced with a lack of nutrients and an inadequate ER protein folding environment, cells activate autophagy a stress-adaptive self-eating process in which cell-ular components are encapsulated within autophago-somes and degraded by lysosomal hydrolases to remove misfolded proteins, restore ER homeostasis

and supply cells with essential nutrients. Similar to the UPR, autophagy can lead to both cell death and survival119. The mechanisms by which the UPR acti-vates autophagy are only partly understood. Early studies showed a requirement for eIF2 phosphory-lation for autophagy induction54,120. For example, activation of the PERKeIF2 pathway, in response to the expression of polyglutamine 72 (polyQ72) aggregates, induced ER stress, LC3 (also known as MAP1LC3A) conversion, autophagosome forma-tion and survival120. Whether eIF2 phosphorylation is required to attenuate protein synthesis to initiate autophagy or whether events downstream of eIF2 phosphorylation are required for autophagy remains a major question. Recent studies showed that ATF4 and CHOP function independently, as well as together, to induce a large set of autophagy genes55. One study suggested the IRE1JNK pathway is also required for autophagy and cell survival upon ER stress121. Analysis of XBP1-deficient mice suggested that cells compen-sate for decreased ERAD by activation of autophagy122. In addition, ATF6 is also reported to be required for interferon- (IFN)-induced autophagy123. ER stress was also associated with activation of a novel protein kinase C (PKC) family member, PKC124, and activa-tion of Ca2+/calmodulin-dependent kinase kinase- (CaMKK)125. CaMKK activates AMP-activated pro-tein kinase (AMPK) while attenuating AKTmTOR signalling to enhance autophagy 126. However, it remains poorly understood whether the crosstalk between the UPR and autophagy contributes to cancer development and metabolism. A pilot study recently demonstrated that oncogenic ER stress induces acti-vation of the PERKeIF2ATF4 signalling pathway, increasing cell survival via induction of cytoprotec-tive autophagy and enhanced MYC-driven tumour transformation and growth7. Another study indicated that PERK, ATF4 and CHOP protect human tumour cells during hypoxia through autophagy 127. More studies are needed to elucidate the complex crosstalk between these processes and reveal their requirement in all stages of cancer development and progression.

Excess nutrients also induce ER stress and acti-vate the UPR. Exposure of cells to increased levels of free fatty acids (for example, palmitate and stearate) induces ER stress115, probably through aberrant protein palmitoylation128, increased accumulation of reactive oxygen species (ROS) due to elevated fatty acid oxida-tion and/or an increased protein folding load resulting from hyperactivation of the mTOR anabolic signalling pathway. Additionally, activation of AMPK or inhibi-tion of JNK prevented palmitate-induced ER stress and UPR activation129. In response to acute or physiological ER stress, the UPR pathways (PERKeIF2 and IRE1XBP1) activate CCAAT/enhancer-binding proteins (C/EBPs), sterol regulatory element-binding tran-scription factor 1 (SREBP1)130 and SREBP2 (REF.131), two transcriptional activators of fatty acid and cho-lesterol synthesis132, to accommodate the need for ER expansion. SREBP-mediated lipogenic activity also maintains the balance between the saturated and

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Translation

Misfoldedprotein

ER

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PERK

IRE1

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P

P

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P

InflammasomeA large intracellular multiprotein oligomeric complex that is activated by pattern recognition receptors to initiate an innate immune response by maturation of the inflammatory cytokines interleukin1 (IL1) and IL18.

monounsaturated fatty acid pools to prevent lipo-toxicity in tumour cells133. These findings, however, are complicated by the observation that severe and persis-tent ER stress (for example, as induced by tunicamycin treatment) may dampen lipogenesis and increase fatty acid oxidation through mechanisms that are depend-ent on UPR activation134. Besides fatty acids, cholesterol loading of macrophages induces ER stress and elicits inflammatory responses135, which promotes cancer development. Moreover, chronic exposure to elevated glucose levels triggers ER stress and glucotoxicity in cultured -cells136. Whether chronic exposure to cho-lesterol and glucose induce ER stress in other cell types remains unknown.

Obesity and type2 diabetes are frequently associ-ated with higher levels of free fatty acids, cholesterol and glucose in the circulation and overt ER stress in multiple tissues, as well as a pro-inflammatory state. These parameters are associated with higher risks of developing cancer, and the tumours generated are generally more malignant. Although multiple mecha-nisms have been put forth to explain this connection, it is possible that excess nutrients associated with

metabolic disorders (for example, obesity and type2 diabetes) trigger ER stress and UPR activation in pre-malignant and transformed cells as well as stromal cells in the tumour microenvironment, which affects cancer development and tumour cell metabolism. For example, ER stress in cells produces ROS49,50,137,138, which not only promotes genetic and epigenetic altera-tions in cells but also induces inflammatory responses (discussed below). Indeed, recent findings suggest that ER stress alone is sufficient to generate pre-oncogenic cells, which leads to hepatocellular carcinoma under conditions of a high-fat-diet-induced inflammatory environment57.

The UPR in the tumour microenvironmentThe UPR: another connection between inflammation and cancer. Chronic inflammation is associated with and can contribute to all stages of cancer development and progression. The inflammatory milieus of nor-mal and neoplastic tissues can increase gene mutation rates and overall genomic instability, promote cell pro-liferation, survival and invasion, induce angiogenesis, facilitate evasion from immune surveillance and ren-der tumour cells resistant to anticancer therapies139. As shown in several pathological conditions, ER stress and UPR activation are required for the signal transduc-tion and transcriptional regulation of inflammatory mediators. It is therefore anticipated that ER stress and UPR activation, aside from the effects on tumour cell survival and proliferation, promote cancer develop-ment and progression through activating inflammatory responses.

ER stress is implicated in various chronic patho-logical conditions (for example, obesity, diabetes, inflammatory bowel diseases, atherosclerosis and neurodegenerative diseases) involving inflamma-tion140. For example, loss of XBP1 function in Paneth cells caused spontaneous enteritis, as a consequence of IRE1 hyperactivation141. Investigation of the pathogenic mechanisms revealed a reciprocal regu-lation of ER stress and inflammation; in which pro- inflammatory stimuli (for example, ROS, TLR ligands and cytokines) trigger ER stress, which in turn initi-ates or amplifies inflammatory responses142. Strikingly, all three UPR pathways lead to activation of nuclear factor-B (NF-B), a master transcriptional regula-tor of pro-inflammatory pathways (FIG.3). In -cells, ER stress triggers activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome and interleukin-1 (IL-1) secretion through IRE1 and PERK-mediated induction of thioredoxin-interacting protein (TXNIP), resulting in -cell death143,144. By contrast, ER stress seems to activate the NLRP3 inflammasome in macrophages via a UPR-independent mechanism145.

The acute phase response (APR) is an innate sys-temic defence to infection or injury. Pro-inflammatory cytokines (for example, IL-1, IL-8 and tumour necrosis factor (TNF)) that are released by local inflammatory cells travel through the blood and stimulate hepatocytes to synthesize and secrete APR products. These APR

Figure 3 | The unfolded protein response (UPR) and inflammation. The three UPR pathways augment the production of reactive oxygen species (ROS) and activate nuclear factor-B (NF-B) and activator protein 1 (AP1) pathways, thereby leading to inflammation. NF-B, which is a master transcriptional regulator of pro-inflammatory pathways, can be activated through binding to the inositol-requiring protein 1 (IRE1)TNF receptor-associated factor 2 (TRAF2) complex in response to endoplasmic reticulum (ER) stress, leading to recruitment of the IB kinase (IKK), IB phosphorylation (P) and degradation, and nuclear translocation of NF-B196. Moreover, the IRE1TRAF2 complex can recruit apoptosis signal-regulating kinase 1 (ASK1) and activate JUN N-terminal kinase (JNK), increasing the expression of pro-inflammatory genes through enhanced AP1 activity197. The PRKR-like ER kinase (PERK)eukaryotic translation initiation factor 2 (eIF2) and activating transcription factor 6 (ATF6) branches of the UPR activate NF-B through different mechanisms. Engaging PERKeIF2 signalling halts overall protein synthesis and increases the ratio of NF-B to IB, owing to the short half-life of IB, thereby freeing NF-B for nuclear translocation198,199. ATF6 activation following exposure to the bacterial subtilase cytotoxin that cleaves immunoglobulin heavy-chain binding protein (BIP) leads to AKT phosphorylation and consequent NF-B activation109,200.

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Type M2 macrophageA subset of activated macrophages that are involved in immunosuppression and tissue repair.

MHC classI pathway(Major histocompatability complex classI pathway). A pathway by which cells present peptides from cytosolic proteins to Tcells.

products can, in turn, amplify inflammation to eliminate infection and restore tissue homeostasis. Upon ER stress in liver, ATF6 and CREBH (also known as CREB3L3) are proteolytically released from the membrane, traffic into the nucleus and form homodimers or heterodimers to induce expression of APR genes, including C-reactive protein (CRP) and serum amyloid P component (SAP; also known as APCS)142. These findings were important because they were the first to show a link between ER stress and inflammation. This is a poorly studied subject that needs further investigation.

Although this scenario that ER stress and UPR activation promotes cancer development and progres-sion through modulating inflammatory responses remains mostly unexplored, a few studies support this idea. It was recently reported that ER stress shortens the lifespan of myeloid-derived suppressor cells in the periphery and promotes their expansion in bone mar-row146. ER stress in prostate cancer cells initiates tran-scription of pro-inflammatory cytokines147. ER-stressed tumour cells also secrete soluble factors that initiate ER stress responses and upregulate the expression of pro-inflammatory cytokines in macrophages15. ER stress in macrophages promotes the type M2 macrophage phenotype148 that in turn supports tumour growth. In addition, ER stress-induced expression of CHOP, in combination with TLR agonists, enhances dendritic cell expression of IL-23 (REF.149) that favours development of T helper17 (TH17) cell-mediated inflammation and tumour growth150.

The UPR in immune defence. Immune effector cells with high protein secretion capacity, as well as high protein synthesis and turnover rates owing to rapid cell proliferation, are prone to ER stress. As a consequence, the UPR is required for immune cell differentiation and function. ER function is also essential for anti-gen presentation by innate immune cells for adaptive immunity, especially through the MHC classI pathway. MHC classI is synthesized and loaded with peptides inside the ER prior to trafficking to the plasma mem-brane. Therefore, altered ER homeostasis disrupts MHC class I antigen presentation151. Calreticulin (CRT), an ER chaperone that is induced by ER stress, facilitates antigen processing and peptide loading of MHC classI molecules152,153. Moreover, misfolded proteins that accumulate within the ER are translo-cated to the cytosolic proteasomes for degradation into peptides for antigen presentation. Phosphorylation of eIF2 upon ER stress reduces synthesis of MHC mol-ecules, as well as the overall peptide pool for MHC loading, and consequently impairs antigen presenta-tion. Hence, ER stress can either aid or impede anti-gen presentation pathways, depending on the cellular context.

One hallmark of cancer is the evasion of can-cer cells from immune surveillance154. ER stress-induced inflammation and perturbed ER homeostasis in immune cells may interfere with the function of immune cells to combat cancer. Although this hypothesis requires further testing, recent studies

suggest a novel, tumour-suppressive mechanism of ER stress and UPR activation in tumour cells. In pre-malignant and neoplastic cells, ER stress and the UPR can initiate signalling cascades that function as pro-phagocytosis and immunogenic signals for clearance of cancer cells by the immune system155. In response to ER stress caused by physiological conditions or pharmacological intervention, several ER proteins, including CRT, ERp57 (also known as PDIA3) and HSPs are translocated to the plasma membrane prior to cancer cell death. Cell surface exposure of these ER proteins can lead to activation of antitumour immune responses and the repression of tumour growth155.

The UPR stimulates tumour angiogenesis. ER stress and UPR activation in both tumour cells and endothe-lial cells stimulate tumour angiogenesis (FIG.4). UPR activation not only protects cancer cells from apop-tosis induced by hypoxia, as well as by lack of glucose and other nutrients (BOX2), but also tips the balance from anti-angiogenic factors (for example, throm-bospondin 1 (THBS1), CXC chemokine ligand 14 (CXCL14) and CXCL10) to pro-angiogenic factors (for example, vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), IL-1, IL-6 and IL-8)16. ATF4 and XBP1s directly bind to the VEGFA promoter to initiate VEGFA transcription. VEGFA mRNA stability is also increased in response to UPR activation, via activation of AMPK156. The endothelial cell compartment in the tumour micro-environment also experiences ER stress and UPR activation owing to the accumulation of misfolded proteins157 or the presence of VEGFA158. A defec-tive UPR is associated with reduced endothelial cell proliferation, survival and migration159. Knockdown of XBP1 or IRE1 decreases endothelial cell pro-liferation via suppression of AKT and glycogen synthase kinase 3 (GSK3) phosphorylation, -catenin nuclear translocation and E2F2 expres-sion160. Moreover, heterozygous ablation of Bip in the tumour microenvironment substantially inhibits tumour growth and angiogenesis161; meanwhile, BIP also confers endothelial cell chemoresistance162.

The UPR in cancer therapyTherapeutic interference can induce severe ER stress, leading to cell death (TABLE2; see supplementary infor-mation S2 (table)). Moreover, ER stress in the tumour microenvironment modulates the function of cancer-supporting stromal cells, such as endothelial cells161, and suppresses tumour growth. However, as UPR acti-vation has both pro-survival and anti-survival effects on cells, caution is necessary in the design of therapies that target UPR components and in the interpretation of the results. It is possible that tumour cells require optimal UPR signalling for survival and that either increased or decreased UPR signalling may compro-mise survival of the tumour cell. Meanwhile, ER stress and UPR activation may alter the cancer cell response to adjunctive therapies, offering a target for combi-nation therapy. Specific gene targeting experiments

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http://www.nature.com/nrc/journal/v14/n9/full/nrc3800.html#supplementary-informationhttp://www.nature.com/nrc/journal/v14/n9/full/nrc3800.html#supplementary-informationsekhar8421

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Hostile environment

Cancertherapy


Tumour Tumour microenvironment

Cancer cell

UPR activation

Cytokines Growth factors

Increased protein folding capacity Decreased ROS Reduced proliferation Decreased MHC I expression Induced angiogenic switch Increased stemness

Survival in hostile environment Compromised immunosurveillance Therapy resistance Metastasis

Macrophage UPR activation M2 phenotype

Cytokines, IL-6, TNF, etc.

T cell

Suppressed T cell function

DC UPR activation

UPRactivation

TolerogenicDC

Endothelialcell

Proliferation Survival Metastasis Maintains VEGFA levels

Angiogenesis

Tumour cell survival and growth

are required to dissect the requirement for different UPR transduction pathways in the tumour and the microenvironment.

Targeting the UPR through monotherapy. Owing to pre-existing ER stress induced by intrinsic and extrin-sic factors in cancer cells as discussed above, agents that augment ER stress should tip the balance towards apoptosis. Indeed, bortezomib, the first proteasome inhibitor for cancer therapy to be approved by the US Food and Drug Administration (FDA) owing to the success in treating multiple myeloma and mantle cell lymphoma, functions as an ER stress inducer. More importantly, the sensitivity to proteasome inhibi-tors correlates with the amount of immunoglobulin sub units that are retained within multiple myeloma cells163, and low XBP1 (or XBP1s) or ATF6 levels pre-dict poor response to bortezomib in patients with mul-tiple myeloma164. It was recently shown that the loss of IRE1 or XBP1 function causes resistance to protea-some inhibitors owing to selection for cells that do not synthesize high levels of immunoglobulin that is, pre-plasmablasts90. This suggests that UPR activation can also function as a prognostic indicator of thera-peutic outcomes. These findings indicate that highly secretory cancer cells, such as multiple myeloma cells, will have a lower threshold for ER stress-induced cell apoptosis, which suggests that inducers of ER stress

may provide efficient cancer therapies in these can-cer types (TABLE2; see Supplementary information S2 (table)).

Some new drugs under study are designed to tar-get specific UPR pathways to inhibit UPR activation, thereby augmenting ER stress in cancer cells (TABLE2; see Supplementary information S2 (table)). For exam-ple, the PERK inhibitor GSK2656157 inhibits growth of multiple human tumour xenografts in mice owing to its direct impact not only on tumour cells but also on the tumour environment165. However, the effects of GSK2656157 are not solely dependent on PERK and eIF2 phosphorylation166. An IRE1 RNase inhibitor (B-109) suppresses leukaemic progression in a mouse model167. Importantly, the UPR signalling pathways have not evolved to be constitutively activated. They function as an adaptive response to a transient require-ment to expand ER protein folding capacity, whether in the context of cell differentiation or as a response to an insult (pathogen, toxin, inflammation, and so on). Therefore, compounds that target UPR components will selectively kill cells that experience ER stress and require a functional UPR for survival. If this is correct, there may be selective toxicity of cancer cells to UPR antagonists compared to normalcells.

Another important finding, which shows that tar-geting the UPR is a promising approach for cancer therapy, is that BIP is expressed on the surface of

Figure 4 | The cancer-supporting role of the unfolded protein response (UPR). In most cases, the activation of the UPR supports tumour survival and growth. On the one hand, UPR activation adapts cancer cells to the hostile environment and/or to cancer therapies. On the other hand, UPR activation in cells in the tumour environment, such as endothelial cells and immune cells, can also facilitate tumour growth. DC, dendritic cell; IL-6, interleukin-6; MHC I, major histocompatibility complex classI; ROS, reactive oxygen species; TNF, tumour necrosis factor; VEGFA, vascular endothelial growth factor A.

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Table 2 | Strategies to target the UPR components for cancer treatment*

Strategy Drugs Other involved mechanisms Clinical trials as cancer therapy

Proteasome inhibitors: target the chymotrypsin-like subunits in both the constitutive proteasome and the immuno- proteasome, leading to accumulation of ubiquitylated proteins and ER stress-mediated apoptosis

Bortezomib Inhibits IRE1XBP1 pathway Suppresses activation of NF-B pathway Induces NOXA expression Triggers immunogenic cell death

FDA-approved for multiple myeloma and mantle cell lymphoma; Phase1/2 in solid tumours

Carfilzomib Promotes atypical activation of NF-B Promotes upregulation of pro-apoptotic BIK and anti-apoptotic MCL1 Induces complete autophagic flux

Phase1/2 in haematopoietic malignancies and lung cancer; Phase 3 in multiple myeloma

Nelfinavir Inhibited HSP90 function Induced upregulation of SREBP1 and ATF6 results from inhibition of S2P Activates caspase 3, caspase 7 and caspase 8 Inhibits AKT signalling, resulting in downregulation of VEGFA and

HIF1 expression

Phase1/2 in solid tumours and multiple myeloma

Marizomib Induces caspase 8 and ROS-mediated apoptosis Phase1 in solid tumours and haematopoietic malignancies

MLN9708 Induces activation of caspase 3, caspase 8 and caspase 9 Increases p53, p21, NOXA, PUMA, and E2F expression Inhibits NF-B signalling pathway

Phase1 in solid tumours; Phase1/2 in haematopoietic malignancies; Phase 3 in multiple myeloma

NPI-0052 Blocks NF-B signalling Phase1 in solid tumours and haematopoietic malignancies

Falcarindiol Interferes with proteasome function; mechanisms remain unclear Preclinical phase

BIP inhibitors: inhibit BIP expression

DHA Inhibited total and surface GRP78 expression Augments the expression of the ER resident factors ERdj5 and inhibits

PERK

Phase2/3 in solid tumours

PAT-SM6 A monoclonal IgM antibody with high avidity of its interaction with multiple BIP on cancer cell surface

Phase1 in multiple myeloma

Arctigenin Specifically blocks the transcriptional induction of BIP and GRP94 under glucose deprivation

Blocked the activation of AKT induced by glucose deprivation Suppressed both constitutively activated and IL-6-induced STAT3

phosphorylation and subsequent nuclear translocation

Preclinical phase

HSP90 inhibitors: disrupt HSP90 function

Tanespimycin Suppression of chymotryptic activity in the 20S proteasome Downregulated BRAF, leading to decreased cell proliferation Inhibited FGF2 and VEGFA-induced HUVEC proliferation and resulted

in apoptosis

Phase1/2 in solid tumours and haematopoietic malignancies; Phase 3 in multiple myeloma

IPI-504 Interacts with the HSP90 conserved ATP-binding site Inactivates the transcription factors XBP1 and ATF6 and blocks the

tunicamycin-induced eIF2 activation by PERK Prevents BIP accumulation

Phase1/2 in solid tumours and haematopoietic malignancies; Phase3 in gastrointestinal stromal tumours

Ganetespib Inhibits AKT signalling Reduces expression levels of HIF1 (but not HIF2) and STAT3

Phase1/2 in solid tumours and haematopoietic malignancies; Phase3 in non-small-cell lung cancer

AUY922 Suppresses the activity of AKT and ERK in PTEN-null oesophageal squamous cancer cells, but not in PTEN-proficient ones

Inhibits NF- B signalling Reduces the expression of anti-apoptotic protein RAF1

Phase1/2 in solid tumours and haematopoietic malignancies

AT13387 Induces cellular senescence Reduces expression of oncoproteins EGFR, AKT, CDK4 Restores the expression of p27

Phase1/2 in solid tumours

SNX-5422 NA Phase1 in solid tumours and haematopoietic malignancies; Phase2 in HER2-positive cancers

PU-H71 Reduces expression levels of AKT, ERK, RAF1, MYC, KIT, IGF1R, TERT and EWSFLI1 in Ewing sarcoma cells

Promotes degradation of IKK and activated AKT and BCL-XL

Phase1 in solid tumours and haematopoietic malignancies

XL888 Promotes degradation of CDK4 and WEE1 Inhibits AKT signalling Increases BIM expression and decreases MCL1 expression

Phase1 in melanoma

DS-2248 NA Phase1 in solid tumours

Debio 0932 NA Phase1 in solid tumours and haematopoietic malignancies

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cancer cells but not normal cells101. Overexpression of BIP in cancer cells correlates with chemotherapy resistance (TABLE 1; see Supplementary informa-tion S1 (table)), which can promote cancer cell survival by inhibiting p53-mediated expression of pro- apoptotic BCL-2-antagonist/killer (BAK) and NOXA (also known as PMAIP1)168. In addition, expression of BIP seems to be increased in the tumour vascu-lature, suggesting that targeting BIP (TABLE2; see supplementary information S2 (table)) will have an impact on both cancer cells and the tumour microenvironment162.

Targeting the UPR in combination cancer therapies. One therapeutic rationale is to induce ER stress and UPR activation to activate death pathways in cancer cells. Alternatively, preventing UPR activation could sensitize cancer cells to other therapies, as the UPR pro-motes adaptation and drug resistance103105,169. An IRE1 inhibitor sensitized resistant human glioblastoma cells to oncolytic virus therapy both invitro and invivo170. Inhibition of PERK kills hypoxic tumour cells that are radioresistant invivo104. Besides the effects on the actively proliferating cancer cells, a recent report showed ER stress to be a mechanism for cancer therapy-induced senescence that acquired the senescence-associated secretory phenotype171, which indicates that blocking UPR activation can be an effective therapy for cancer cells undergoing senescence. Furthermore, inhibition

of UPR signalling may also sensitize cancer-supporting stromal cells, such as endothelial cells, to traditional cancer therapies. Therefore, combination therapies that include drugs targeting ER stress and UPR activation may be one of the most promising anticancer approaches.

Future prospectsWe have come a long way in understanding the genetic defects that contribute to cancer; however, we have a long way to go to translate these findings into clinical advances, and many questions remain (BOX4). There is an extremely strong pressure for a cancer cell to survive hostile environments or chemotherapy. The long-term approach will probably involve combinato-rial therapies that attack the tumour at multiple levels. Anti-angiogenesis therapies are not adequate alone, but they may show synergy in combination with anti-UPR agents. It is also essential to identify the driver mutations for individual cancer types to design selec-tive targeting agents. We know that mutations in BRAF generate melanoma and that BRAF inhibitors generate resistance. It is necessary to inactivate pro-cesses of drug resistance, which often involve DNA damage and repair pathways, as well as eliminating adaptive survival pathways, which provide potential for the outgrowth of drug-resistant cells. Ever since the discovery of gene amplification as a mechanism for methotrexate resistance, it is evident that therapies

PERK inhibitors: inhibit PERK activation and eIF2 phosphorylation

6-shogaol The effect on IRE1 and ATF6 was not obvious Preclinical stage

GSK2656157 An ATP-competitive inhibitor of PERK Also has eIF2 phosphorylation-independent effects

Preclinical stage

GSK2606414 Binds to PERK active site Preclinical stage

IRE1 inhibitors: inhibit IRE1 endonuclease activity

STF-083010 Inhibits IRE1 endonuclease activity without affecting its kinase activity

Preclinical stage

MKC-3946 Inhibits IRE1 endonuclease domain, and significantly enhances apoptosis induced by bortezomib and 17-AAG, associated with increased levels of CHOP

Preclinical stage

WNT signalling inhibitors

Pyrvinium Suppresses the transcriptional activation of BIP and GRP94 induced by glucose deprivation or 2-deoxyglucose; other UPR pathways (for example, XBP1 and ATF4) were also found to be suppressed

FDA-approved classical anthelmintic; preclinical stage as cancer therapy

Pan-deacetylase inhibitors

Panobinostat Increases the levels of BIP, IRE1 phosphorylation, eIF2 phosphorylation, ATF4 and CHOP

Increases the pro-apoptotic BIK, BIM, BAX, and BAK levels, as well as caspase 7 activity

Phase1/2 in solid tumours and haematopoietic malignancies; Phase 3 in haematopoietic malignancies

Anti-diabetic biguanides

Metformin Inhibition of XBP1 and ATF4 expression during glucose deprivation FDA-approved anti-diabetes drug; Phase1/2 in solid tumours and haematopoietic malignancies; Phase 3 in solid tumours

ATF6, activating transcription factor 6; BIK, BCL-2-interacting killer; BIM, BCL-2-interacting mediator of cell death; BIP, immunoglobulin heavy-chain binding protein; CDK4, cyclin-dependent kinase 4; CHOP, C/EBP homologous protein; EGFR, epidermal growth factor receptor; eIF2, eukaryotic translation initiation factor 2; ER, endoplasmic reticulum; FDA, US Food and Drug Administration; FGF2, fibroblast growth factor 2; FLI1, Friend leukaemia integration 1 transcription factor; GRP, glucose-regulated protein; HER2, human epidermal receptor 2; HIF, hypoxia-inducible factor; HSP90, heat shock protein 90; HUVEC, human umbilical vein endothelial cell; Ig, immunoglobulin; IGF1R, insulin-like growth factor 1 receptor; IKK, IB kinase; IL-6, interleukin-6; IRE1, inositol-requiring protein 1; MCL1, induced myeloid leukaemia cell differentiation protein; NA, not applicable; NF-B, nuclear factor-B; PERK, PRKR-like ER kinase; ROS, reactive oxygen species; SREBP1, sterol regulatory element binding transcription factor 1; STAT3, signal transducer and activator of transcription 3; TERT, telomerase reverse transcriptase; UPR, unfolded protein response; VEGFA, vascular endothelial growth factor A; XBP1, X-box binding protein 1. *See Supplementary information S2 (table) for a version of this table with references. #See also ClinicalTrials.gov.

Table 2 (cont.) | Strategies to target the UPR components for cancer treatment*

Strategy Drugs Other involved mechanisms Clinical trials as cancer therapy

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http://www.nature.com/nrc/journal/v14/n9/full/nrc3800.html#supplementary-informationhttp://www.clinicaltrials.gov

need to be generated to hit the cancer cells hard, at high dose and at multiple targets to eliminate the potential for drug resistance. Targeting the UPR adap-tive pathways will provide one asset of the armamen-tarium in these strategies, but they are unlikely to be successful on their own. It is important that clinical avenues are encouraged for testing of multiple agents in single clinical studies, while at the same time recog-nizing the potential for increased toxicities and drug

interactions. Certainly, with the advent of personal-ized medicine and genome sequencing, therapeutic strategies will become more patient-specific and could increase the success of remission, especially in those cancers that are presently refractory to any treatment. We have come a long way, and this is most evident by remission rates in chronic myelogenous leukaemia (CML), but we have a long distance to run to win therace.

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Box 4 | Ten unresolved questions regarding the impact of ER stress on cancer development

The unfolded protein response (UPR) functions as a double-edged sword by supporting or repressing cancer initiation and progression. In premalignant cells, UPR activation prevents oncogenic transformation by inducing cell death in response to sustained endoplasmic reticulum (ER) stress and facilitates clearance of damaged cells by the immune system. In cancer, UPR activation provides a survival strategy for cells to thrive in a stressful environment. Activation of the UPR may inhibit cancer progression through induction of tumour cell death, in which UPR inhibition may reduce tumour angiogenesis. Despite recent advances in the field, many questions remain to be answered to encourage therapeutic testing of UPR-directed agents for chemotherapy: Is protein misfolding oncogenic? Protein misfolding in the ER induces oxidative stress33,34,50,51. It is unknown whether this

oxidative stress is sufficient to cause DNA mutations to activate oncogenes or inactivate tumour suppressor genes. Recent studies suggest that protein misfolding in hepatocytes can be an initiating event for hepatocellular carcinoma57.

UPR activation is both adaptive and pro-apoptotic. Why does UPR activation promote cell survival under certain circumstances, whereas persistent activation of the same UPR signalling pathway causes cell death under other conditions?

Some UPR components are independent prognostic indicators of therapeutic outcome. What is the status of the ER stress sensors and downstream targets in a broad range of human patient cancer samples compared to healthy adjacent tissue? Can the status of ER stress and UPR activation serve as a prognostic indicator of outcome?

What are the exact roles of individual UPR components in cancer incidence and progression? Studies of transgenic and knockout mice and characterization of specific small molecule inhibitors or activators in mouse models are needed to determine whether activation of a specific UPR signalling pathway is a rate-limiting primary step or a secondary event during cancer initiation and/or progression.

Cancer progresses in the presence of a stressful microenvironment. How do cancer cells evade cell death upon chronic UPR activation? Can UPR-targeted therapeutics be designed to separate pro-survival and apoptotic responses?

Solid tumours are highly heterogeneous. Does the status of UPR activation reflect such heterogeneity? Could UPR-mediated pro-survival and pro-death signals coexist in different regions of the same tumour?

UPR signalling pathways do not function in isolation. How do different survival or death signalling pathways integrate with each other to control the fate of tumour cells under unfavourable conditions?

Cancer stem cells (CSCs) were recently identified to be responsible for cancer metastasis. Indeed, X-box binding protein 1 (XBP1) is possibly required for the maintenance of this population in triple-negative breast cancer93, whereas PRKR-like ER kinase (PERK)eukaryotic translation initiation factor 2 (eIF2) is activated during epithelial-to- mesenchymal transition, which is required for invasion and metastasis188. The PERK pathway is also required for rapid induction of detachment-induced autophagy, which is crucial for the survival of detached cancer cells189. However, more questions need be addressed. Does UPR activation promote CSC-like properties and CSC-niche interactions to augment metastasis? Which UPR component (or components) is essential for CSC survival and differentiation?

Different tumour cells exhibit different degrees of protein secretion. Does the level of protein secretion of a tumour cell correlate with dependence on specific UPR pathways for survival? Can protein secretion rate be used to stratify tumour cell sensitivity to UPR-targeting agents?

Mutations in several UPR components have been identified in human cancers. Are any of these driver mutations?

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the impact of the endoplasmic reticulum protein-folding environment on cancer development

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