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

REVIEWS The impact of the endoplasmic reticulum protein-folding environment on cancer development Miao 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.

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: rkaufman@ sanfordburnham.org doi:10.1038/nrc3800

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 en route 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 environment to cause protein misfolding and accumulation of misfolded proteins, referred to as ER stress. The unfolded protein response (UPR) is a collection of signalling 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 (TABLE 1; see Supplementary information 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 activation 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 in vivo 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 tumoursuppressive roles, and we discuss strategies that target UPR components for cancer treatment.

ER stress and UPR activation in cancer The 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 accumulation 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, reduction in the level of free BIP activates signalling pathways to induce transcription of BIP, as well as other genes encoding protein chaperones 2,3, now known as the UPR. The outcome of UPR activation involves transient attenuation of protein synthesis, increased capacity

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

Activation of UPR components

Prognosis

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

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

BIP

Correlated with high cell malignancy

CHOP

Correlated with high tumour stage

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

BIP

Correlated with shorter survival

BIP

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

BIP

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

Classification Site Carcinoma

Lung

Breast

Colon Gastric

Prostate

Kidney

Leukaemia

Lymphoma

Glioma

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

Lymphoblast

BIP, XBP1s and calreticulin

NA

BIP, XBP1s, CHOP and calreticulin

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

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

BIP

Correlated with shorter survival

B cells

Brain

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.

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 folding and transport, and increased protein degradative pathways, including ER‑associated 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 understand about UPR activation in cancer is derived from mouse models, which have limitations (BOX 1). 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

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REVIEWS 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 24 hours 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.

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.

Polysome A cluster of ribosomes translating a single mRNA molecule.

induction of oncogenic HRAS, MYC or the oncogenic latent membrane protein 1 of Epstein–Barr virus4–7, or loss of tumour suppressors tuberous sclerosis complex 1 (TSC1; also known as hamartin), TSC2 (also known as tuberin), BRCA1 or PTEN8–11, increase protein synthesis and translocation into the ER owing to high metabolic demand during oncogenic transformation. 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 misfolding 12. Furthermore, UPR activation is required to promote ER expansion for division and transmission to daughter cells during mitosis13. Certain types of cancer 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 pancreatic, 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 signalling to increase folding and the secretion of cytokines, metalloproteinases, 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 (BOX 2).

The role of the UPR in tumorigenesis The UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK; also known as eIF2AK3)– eukaryotic translation initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α; also known as ERN1)–X‑box binding protein 1 (XBP1) and activating transcription factor 6α (ATF6α) (FIG. 1). Emerging evidence suggests that UPR activation is required for oncogenic transformation. As the UPR exerts both protective and deleterious effects on cell survival upon ER stress, UPR activation may facilitate, as well as suppress, malignant transformation (FIG. 2). Therefore, there would be a selective advantage for premalignant cells harbouring gene mutations that suppress UPR-induced apoptosis or senescence. The PERK–eIF2α pathway in ER stress. PERK is a type I transmembrane protein enriched at mitochondriaassociated ER membranes (MAMs)17 with a cytosolic serine/threonine kinase domain. Under non-stress conditions, 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 eIF2α (a subunit of the heterotrimeric eIF2 complex) at S51 (REF. 21) to attenuate translation initiation due to limiting amounts of the eIF2–GTP–tRNAmet ternary complex. The ternary complex binds the 40S ribosome to generate a 43S species that binds the 5ʹ end of the mRNA to initiate scanning downstream. When the 43S species encounters an AUG codon in an optimal context 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 eIF2α greatly increases the affinity of eIF2 for GDP, thereby preventing the eIF2B‑catalyzed exchange reaction and sequestering eIF2B with eIF2 in an inactive complex 24, as well as inhibiting the antiexchange activity of eIF5 (REF. 23). As the eIF2B/eIF2 ratio is generally less than 1 (approximately 1/7 in rabbit reticulocyte lysate and 1/2 in Ehrlich ascites cells25), it was proposed that small increases (as little as ~20%) in the amount of eIF2α phosphorylation could shut down general protein synthesis26. The transient inhibition of protein synthesis probably promotes polysome disassembly to increase the number of ribosomes available to bind newly transcribed mRNAs, which encode UPR adaptive functions. Besides eIF2α, other PERK substrates have been suggested to affect cell survival, function and differentiation, 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 eIF2α mutation at the PERK phosphorylation site30, the physiological importance of these alternative substrates remains in question.



REVIEWS While attenuating global mRNA translation, PERK– eIF2α 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 transcription of the growth arrest and DNA damage-inducible protein 34 (GADD34; also known as PPP1R15A) to direct eIF2α 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 in vitro and in vivo33,34. 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‑β (TRIF; also known as TICAM1) signalling through protein phosphatase 2A (PP2A)-mediated serine dephosphorylation of the eIF2B ε-subunit 36. Under conditions of chronic stress, constitutive PERK-mediated phosphorylation of eIF2α leads to apoptosis, as the IRE1α–XBP1 and ATF6α pathways are attenuated37–39. 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 the cell. 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) transcription41 and promotes the translocation of BAX to mitochondria42. 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 caspase 8 in cancer cells48. However, chromatin immunoprecipitation followed by sequencing (ChIP–seq) 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 mediated by CHOP, it is possible that, in tumour cells, different pathways are activated downstream of CHOP to regulate survival. The PERK–eIF2α pathway in tumorigenesis. As the PERK–eIF2α pathway induces either survival or apoptosis 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 susceptibility to spontaneous mammary tumorigenesis owing to increased genomic instability 52. 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 cells56,172–174. To survive the hostile environment, the unfolded protein response (UPR) is activated in cancer cells, which ameliorates ER stress to promote cell survival and growth103–105. 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 oxygendependent175. 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 HIF1α‑mediated upregulation of its downstream targets to promote cell survival93,178. However, eukaryotic translation initiation factor 2α (eIF2α) 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 ER–Ca2+ 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 eIF2α (discussed in BOX 3). Growth factors that are present in the tumour microenvironment can also contribute to UPR activation in cancer, independent of ER stress (discussed in BOX 3).



REVIEWS promotes MYC-induced cell transformation through autophagy 7,53. This may be related to PERK-mediated eIF2α 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 incidence in a KrasG12V-induced mouse model of lung cancer, suggesting a tumour-suppressive role of CHOP56. Additionally, hepatocyte-specific Chop deletion increased tumorigenesis in a mouse model of hepatocellular carcinoma57. CHOP mutations were reported in human tumours58, although it is unknown whether these mutations alter protein expression or function and whether they contribute to tumorigenesis. The IRE1α–XBP1 pathway in ER stress. Mammals have two IRE1 genes, IRE1A (also known as ERN1; which encodes IRE1α) and IRE1B (also known as ERN2; which encodes IRE1β). IRE1A is ubiquitously and constitutively expressed, whereas IRE1B expression is restricted to intestinal and lung epithelial cells.

Like PERK, IRE1α is a type I transmembrane protein with a cytosolic serine/threonine kinase domain. Under non-stress conditions, both HSP90 and HSP72 bind the IRE1α cytosolic domain to maintain its stability 18,59, while BIP binds the luminal domain of IRE1α to prevent dimerization. Upon ER stress, unfolded and misfolded proteins bind and sequester BIP, thereby releasing IRE1α for oligomerization, autophosphorylation and activation of its kinase and endoribonuclease activities19,20. Membrane fluidity also influences PERK and IRE1α oligomerization and activation60. Structural studies suggest that short peptides could interact with a major histocompatability complex class I (MHC class I)-like groove to promote dimerization in yeast IRE1 (REF. 61). Although the MHC class I‑type groove in the human IRE1α homodimer was not solventexposed62, recent studies show that it can bind some peptides 63. Activated IRE1α cleaves Xbp1 mRNA to initiate removal of a 26‑base intron in the cytoplasm 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 Cγ (PLCγ)-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 2α (eIF2α). In mammals, there are three additional kinases that also phosphorylate eIF2α 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 eIF2α kinase may have secondary effects on the cell that cause activation of other eIF2α 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 eIF2α kinase causes translation attenuation, the cellular response varies tremendously depending on which kinase phosphorylates eIF2α, the cell type and the environment. Generally, eIF2α phosphorylation that is mediated by GCN2, HRI and PKR leads to apoptosis39, whereas transient eIF2α phosphorylation by PERK promotes cell survival, and chronic eIF2α 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 eIF2α dephosphorylation. In the stressful tumour microenvironment, the level of eIF2α phosphorylation might be high, and so other factors need to be considered that impact the effect of eIF2α phosphorylation rate on protein synthesis. For example, GADD34 is a regulatory subunit of protein phosphatase 1 (PP1) that promotes eIF2α dephosphorylation181, and protein phosphatase 2A (PP2A)-mediated dephosphorylation of the ε-subunit of eIF2B activates exchange activity of eIF2B to bypass eIF2α phosphorylation36. As eIF2α 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 1α (IRE1α) 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 IRE1α prevents interaction with PP2A via its adaptor receptor for activated C kinase 1 (RACK1) to prevent IRE1α dephosphorylation and inactivation73. TRAF6 and PP2A compete for binding to the same site on IRE1α. TRAF6 binding reduces PP2A‑mediated dephosphorylation, promoting IRE1α activation74. IRE1α signalling is attenuated through proteasomal degradation that is mediated by TRAF6 ubiquitylation of IRE1α via a K48 linkage74. As TLR signalling is important in cancer development and drug resistance187, IRE1α could be one of the mediators in this process.



REVIEWS JNK IRE1α P BIP BIP

BIP

P

RIDD

RNA decay

XBP1u

XBP1s

XBP1

BIP Misfolded protein BIP

ATF6α

BIP

Survival Nucleus

Golgi BIP

Autophagy

BIP P

BIP P ER

Apoptosis

S1P S2P ATF6α

BIP

Increased ER protein folding capacity and ERAD

PERK

eIF2α

GADD34

ATF4 Translation

eIF2α P

ATF4

ROS

CHOP

Translation Nature Reviews Figure 1 | The unfolded protein response (UPR) signalling pathways. Upon endoplasmic reticulum (ER) stress, | Cancer 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 2α (eIF2α), inositol-requiring protein 1α (IRE1α)–X‑box binding protein 1 (XBP1) and activating transcription factor 6α (ATF6α). 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.

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

membrane to facilitate IRE1α interaction66. Genes that are regulated by IRE1α–XBP1 enhance protein folding, trafficking and ERAD, thereby resolving protein misfolding 38,67, 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 IRE1α–XBP1 promotes cell survival is challenged by recent findings that small molecule inhibitors of IRE1 did not sensitize cells to ER stressinduced apoptosis, but rather prevented expansion of secretory capacity 69. ER stress immediately activates IRE1α70,71, whereas IRE1α activation is mostly attenuated upon chronic ER stress71,72. It is unclear how IRE1α–XBP1 signalling is attenuated under conditions of sustained ER stress, although dephosphorylation, ubiquitylation and degradation 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 IRE1α luminal domain to attenuate signalling 75, other results show that PDIA6 is required for IRE1α activation76. Furthermore, Xbp1u can function as a negative regulator of both the XBP1s and ATF6α pathways by direct interaction to promote their degradation77, which possibly blocks survival signals during chronic ER stress.

If IRE1α signalling is not attenuated, chronic IRE1α activation signals apoptosis. Hyperactivated IRE1α cleaves many mRNAs, in addition to Xbp1 (REFS 70,78) and its own mRNA 79, a process called regulated IRE1‑dependent decay (RIDD) 80. A recent study suggests that RIDD is dependent on the oligomeric state of IRE1α 81. RIDD also reduces the expression of some microRNAs (miRNAs), including miR‑17, miR‑34a, miR‑96 and miR‑125b, which repress caspase 2 expression82. However, the importance of caspase 2 activation in ER stress-induced apoptosis remains in question 83. Activated IRE1α 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 IRE1α–XBP1 pathway in tumorigenesis. The role of IRE1α–XBP1 in multiple myeloma has been intensively studied because mature B cell differentiation into plasma cells and B cell-mediated defence against infection require XBP1s85–87. Increased levels of XBP1s are frequently associated with human multiple myeloma, and genetically engineered mice that overe xpress XBP1s under the control of immuno globulin V H promoter and Eμ enhancer elements



REVIEWS • Hostile environment • Cancer therapy

Loss of BRCA1

Loss of TSC

MYC

Loss of PTEN

RAS

Protein misfolding in the ER

p53 and TSC downregulated

BIP induced

Decreased ATF4 and CHOP expression

Induced autophagy

Enhanced glycolysis

Oncogenic transformation

Survival

CHOP induction

Senescence or apoptosis

Tumorigenesis Nature | Cancer Figure 2 | Crosstalk between the unfolded protein response (UPR)Reviews 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 2α (eIF2α) 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 (REFS 194,195), 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.

Regulated intramembrane proteolysis A 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 myeloma 89,90 . More importantly, analysis 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 endoribonuclease function81. In addition, loss of XBP1 function promotes tumorigenesis in mouse models of intestinal cancer 92. Although these findings suggest a

tumour-suppressive role for IRE1α–XBP1, increased XBP1 mRNA splicing was observed in human triplenegative breast cancers, possibly indicating a requirement for XBP1 in cancer stem-like cells93. The ATF6 pathway in ER stress. ATF6 is a type II transmembrane protein that contains a cytosolic cAMPresponsive element-binding protein (CREB)/ATF basic leucine zipper (bZIP) domain. Under nonstressed 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 mammalian genome. Cleaved ATF6α mediates the adaptive response to ER protein misfolding by increasing the transcription of genes that increase ER capacity and the expression of Xbp1 (REFS 95,96), whereas ATF6β may function as a repressor of ATF6α‑mediated transcription and function97. Presently, no genes have been identified that require ATF6β for expression. PERK–eIF2α signalling facilitates ATF6α synthesis and trafficking to accentuate ATF6α signalling 98. To date, no substantial evidence supports a role of ATF6α in ER stress-induced apoptosis. The ATF6α pathway in tumorigenesis. Some studies suggest that ATF6α 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 susceptibility to hepatocellular carcinoma100. More importantly, BIP, a downstream transcriptional target of ATF6α, was reported to serve as a malignancy marker for cells. Upon induction of ER stress, ATF6α quickly induces BIP expression, which binds to unfolded protein and misfolded protein to ameliorate ER stress. Under normal conditions, BIP is localized to the ER lumen, but upon overexpression in many human cancers (TABLE 1; see Supplementary information S1 (table)), it becomes detectable on the cell surface 101. BIP expression not only correlates with cancer cell proliferation and histological grade but also correlates with response to therapies and prognosis102 (TABLE 1; see Supplementary information S1 (table)).

The UPR in tumour cells UPR in autonomous cancer cell survival. UPR activation protects cancer cells from stress-induced cell death103–105. Acute UPR activation enhances the protein 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 IRE1α–XBP1 and ATF6α pathways, so that the apoptotic signals dominate38. Some cancer cells, however, exhibit constitutive activation of



REVIEWS the IRE1α–XBP1 pathway 106,107 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 eIF2α phosphorylation, leading to subsequent 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 pathways may also directly contribute to enhanced cancer cell survival upon stress. Some IRE1α mutants identified in human cancers showed reduced endoribonuclease function. Although still able to splice XBP1 mRNA, they cannot induce RIDD, thereby promoting 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 development 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 complex 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 signal for nutrient, energy or oxygen deprivation. On the one hand, conditions of low nutrient supply (for example, 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 nutrients (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 (BOX 2), and tumours arise at higher rates and are more malignant in a nutrient-rich environment compared with a normal environment 117,118. 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 autophagosomes 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 activates autophagy are only partly understood. Early studies showed a requirement for eIF2α phosphory lation for autophagy induction 54,120. For example, activation of the PERK–eIF2α pathway, in response to the expression of polyglutamine 72 (polyQ72) aggregates, induced ER stress, LC3 (also known as MAP1LC3A) conversion, autophagosome formation and survival120. Whether eIF2α phosphorylation is required to attenuate protein synthesis to initiate autophagy or whether events downstream of eIF2α 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 IRE1α–JNK pathway is also required for autophagy and cell survival upon ER stress121. Analysis of XBP1‑deficient mice suggested that cells compensate for decreased ERAD by activation of autophagy 122. In addition, ATF6α is also reported to be required for interferon‑γ (IFNγ)-induced autophagy 123. ER stress was also associated with activation of a novel protein kinase C (PKC) family member, PKCθ124, and activation of Ca2+/calmodulin-dependent kinase kinase‑β (CaMKKβ)125. CaMKKβ activates AMP-activated protein kinase (AMPK) while attenuating AKT–mTOR 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 activation of the PERK–eIF2α–ATF4 signalling pathway, increasing cell survival via induction of cytoprotective 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 activate 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 oxidation and/or an increased protein folding load resulting from hyperactivation of the mTOR anabolic signalling pathway. Additionally, activation of AMPK or inhibition of JNK prevented palmitate-induced ER stress and UPR activation129. In response to acute or physiological ER stress, the UPR pathways (PERK–eIF2α and IRE1α– XBP1) activate CCAAT/enhancer-binding proteins (C/EBPs), sterol regulatory element-binding transcription factor 1 (SREBP1)130 and SREBP2 (REF. 131), two transcriptional activators of fatty acid and cholesterol synthesis 132, to accommodate the need for ER expansion. SREBP-mediated lipogenic activity also maintains the balance between the saturated and



REVIEWS monounsaturated fatty acid pools to prevent lipo toxicity in tumour cells133. These findings, however, are complicated by the observation that severe and persistent ER stress (for example, as induced by tunicamycin treatment) may dampen lipogenesis and increase fatty acid oxidation through mechanisms that are dependent 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 β-cells136. Whether chronic exposure to cholesterol and glucose induce ER stress in other cell types remains unknown. Obesity and type 2 diabetes are frequently associated 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 mechanisms have been put forth to explain this connection, it is possible that excess nutrients associated with

Inflammasome A large intracellular multiprotein oligomeric complex that is activated by pattern recognition receptors to initiate an innate immune response by maturation of the inflammatory cytokines interleukin‑1 (IL‑1) and IL‑18.

eIF2α

PERK P BIP

BIP BIP

ATF4

Translation

eIF2α P

P

CHOP

BIP

ROS

BIP

BIP

P

BIP

BIP

P

IRE1α

IκB

NF-κB

JNK

AP1

Inﬂammation

NF-κB BIP

S1P S2P

BIP

Misfolded protein

ATF6α

Golgi

ER

Figure 3 | The unfolded protein response (UPR) and inflammation. The three UPR Nature Reviews | Cancer pathways augment the production of reactive oxygen species (ROS) and activate nuclear factor‑κB (NF‑κB) and activator protein 1 (AP1) pathways, thereby leading to inflammation. NF‑κB, which is a master transcriptional regulator of pro-inflammatory pathways, can be activated through binding to the inositol-requiring protein 1α (IRE1α)– TNF receptor-associated factor 2 (TRAF2) complex in response to endoplasmic reticulum (ER) stress, leading to recruitment of the IκB kinase (IKK), IκB phosphorylation (P) and degradation, and nuclear translocation of NF-κB196. Moreover, the IRE1α–TRAF2 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 2α (eIF2α) and activating transcription factor 6α (ATF6α) branches of the UPR activate NF‑κB through different mechanisms. Engaging PERK–eIF2α signalling halts overall protein synthesis and increases the ratio of NF‑κB to IκB, owing to the short half-life of IκB, thereby freeing NF‑κB for nuclear translocation198,199. ATF6α activation following exposure to the bacterial subtilase cytotoxin that cleaves immunoglobulin heavy-chain binding protein (BIP) leads to AKT phosphorylation and consequent NF‑κB activation109,200.

metabolic disorders (for example, obesity and type 2 diabetes) trigger ER stress and UPR activation in premalignant 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 alterations 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 environment 57.

The UPR in the tumour microenvironment The 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 normal and neoplastic tissues can increase gene mutation rates and overall genomic instability, promote cell proliferation, survival and invasion, induce angiogenesis, facilitate evasion from immune surveillance and render tumour cells resistant to anticancer therapies139. As shown in several pathological conditions, ER stress and UPR activation are required for the signal transduction 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 development and progression through activating inflammatory responses. ER stress is implicated in various chronic pathological conditions (for example, obesity, diabetes, inflammatory bowel diseases, atherosclerosis and neurodegenerative diseases) involving inflammation140. For example, loss of XBP1 function in Paneth cells caused spontaneous enteritis, as a consequence of IRE1α hyperactivation 141. Investigation of the pathogenic mechanisms revealed a reciprocal regulation of ER stress and inflammation; in which proinflammatory stimuli (for example, ROS, TLR ligands and cytokines) trigger ER stress, which in turn initiates or amplifies inflammatory responses142. Strikingly, all three UPR pathways lead to activation of nuclear factor‑κB (NF‑κB), a master transcriptional regulator of pro-inflammatory pathways (FIG. 3). In β-cells, ER stress triggers activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome and interleukin‑1β (IL‑1β) secretion through IRE1α and PERK-mediated induction of thioredoxininteracting protein (TXNIP), resulting in β-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 systemic 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



REVIEWS Type M2 macrophage A subset of activated macrophages that are involved in immunosuppression and tissue repair.

MHC class I pathway (Major histocompatability complex class I pathway). A pathway by which cells present peptides from cytosolic proteins to T cells.

products can, in turn, amplify inflammation to eliminate infection and restore tissue homeostasis. Upon ER stress in liver, ATF6α 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 progression 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 marrow 146. ER stress in prostate cancer cells initiates transcription of pro-inflammatory cytokines147. ER-stressed tumour cells also secrete soluble factors that initiate ER stress responses and upregulate the expression of proinflammatory 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 helper 17 (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 antigen presentation by innate immune cells for adaptive immunity, especially through the MHC class I pathway. MHC class I is synthesized and loaded with peptides inside the ER prior to trafficking to the plasma membrane. Therefore, altered ER homeostasis disrupts MHC class I antigen presentation 151. Calreticulin (CRT), an ER chaperone that is induced by ER stress, facilitates antigen processing and peptide loading of MHC class I molecules 152,153. Moreover, misfolded proteins that accumulate within the ER are translocated to the cytosolic proteasomes for degradation into peptides for antigen presentation. Phosphorylation of eIF2α upon ER stress reduces synthesis of MHC molecules, as well as the overall peptide pool for MHC loading, and consequently impairs antigen presentation. Hence, ER stress can either aid or impede antigen presentation pathways, depending on the cellular context. One hallmark of cancer is the evasion of cancer cells from immune surveillance 154. ER stressinduced 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 premalignant and neoplastic cells, ER stress and the UPR can initiate signalling cascades that function as prophagocytosis 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 endothelial cells stimulate tumour angiogenesis (FIG. 4). UPR activation not only protects cancer cells from apoptosis induced by hypoxia, as well as by lack of glucose and other nutrients (BOX 2), but also tips the balance from anti-angiogenic factors (for example, thrombospondin 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‑1β, 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 AMPK 156. The endothelial cell compartment in the tumour microenvironment also experiences ER stress and UPR activation owing to the accumulation of misfolded proteins 157 or the presence of VEGFA 158. A defective UPR is associated with reduced endothelial cell proliferation, survival and migration159. Knockdown of XBP1 or IRE1α decreases endothelial cell proliferation via suppression of AKT and glycogen synthase kinase 3β (GSK3β) phosphorylation, β‑catenin nuclear translocation and E2F2 expression 160. 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 therapy Therapeutic interference can induce severe ER stress, leading to cell death (TABLE 2; see supplementary information S2 (table)). Moreover, ER stress in the tumour microenvironment modulates the function of cancersupporting stromal cells, such as endothelial cells161, and suppresses tumour growth. However, as UPR activation 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 compromise survival of the tumour cell. Meanwhile, ER stress and UPR activation may alter the cancer cell response to adjunctive therapies, offering a target for combination therapy. Specific gene targeting experiments



REVIEWS Tumour Hostile environment

Tumour microenvironment Cancer therapy

Cytokines, IL-6, TNF, etc. Macrophage

Cancer cell

UPR activation

M2 phenotype Suppressed T cell function

UPR activation

• Cytokines • Growth factors

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

T cell DC

UPR activation

Endothelial cell

UPR activation

• Survival in hostile environment • Compromised immunosurveillance • Therapy resistance • Metastasis

Tolerogenic DC

• Proliferation↑ • Survival↑ • Metastasis↑ • Maintains VEGFA levels

Angiogenesis

Tumour cell survival and growth

Figure 4 | The cancer-supporting role of the unfolded protein response (UPR). In most cases, the activation of the Reviews | Cancer UPR supports tumour survival and growth. On the one hand, UPR activation adapts cancer cellsNature 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 class I; ROS, reactive oxygen species; TNF, tumour necrosis factor; VEGFA, vascular endothelial growth factor A.

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 extrinsic 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 inhibitors correlates with the amount of immunoglobulin subunits that are retained within multiple myeloma cells163, and low XBP1 (or XBP1s) or ATF6α levels predict poor response to bortezomib in patients with multiple myeloma164. It was recently shown that the loss of IRE1α or XBP1 function causes resistance to proteasome 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 therapeutic 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 cancer types (TABLE 2; see Supplementary information S2 (table)). Some new drugs under study are designed to target specific UPR pathways to inhibit UPR activation, thereby augmenting ER stress in cancer cells (TABLE 2; see Supplementary information S2 (table)). For example, 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 environment 165. However, the effects of GSK2656157 are not solely dependent on PERK and eIF2α phosphorylation166. An IRE1α 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 requirement 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 normal cells. Another important finding, which shows that targeting the UPR is a promising approach for cancer therapy, is that BIP is expressed on the surface of



REVIEWS 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 immunoproteasome, leading to accumulation of ubiquitylated proteins and ER stress-mediated apoptosis

Bortezomib

• Inhibits IRE1α–XBP1 pathway • Suppresses activation of NF‑κB pathway • Induces NOXA expression • Triggers immunogenic cell death

FDA-approved for multiple myeloma and mantle cell lymphoma; Phase 1/2 in solid tumours

Carfilzomib

• Promotes atypical activation of NF‑κB • Promotes upregulation of pro-apoptotic BIK and anti-apoptotic MCL1 • Induces complete autophagic flux

Phase 1/2 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 HIF1α expression

Phase 1/2 in solid tumours and multiple myeloma

Marizomib

Induces caspase 8 and ROS-mediated apoptosis

Phase 1 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‑κB signalling pathway

Phase 1 in solid tumours; Phase 1/2 in haematopoietic malignancies; Phase 3 in multiple myeloma

NPI‑0052

Blocks NF‑κB signalling


Falcarindiol

Interferes with proteasome function; mechanisms remain unclear

Preclinical phase

DHA

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

Phase 2/3 in solid tumours

PAT‑SM6

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

Phase 1 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

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

Phase 1/2 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 eIF2α activation by PERK • Prevents BIP accumulation

Phase 1/2 in solid tumours and haematopoietic malignancies; Phase 3 in gastrointestinal stromal tumours

Ganetespib

• Inhibits AKT signalling • Reduces expression levels of HIF1α (but not HIF2α) and STAT3

Phase 1/2 in solid tumours and haematopoietic malignancies; Phase 3 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- κB signalling • Reduces the expression of anti-apoptotic protein RAF1

Phase 1/2 in solid tumours and haematopoietic malignancies

AT13387

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

Phase 1/2 in solid tumours

SNX‑5422

NA

Phase 1 in solid tumours and haematopoietic malignancies; Phase 2 in HER2‑positive cancers

PU‑H71

• Reduces expression levels of AKT, ERK, RAF1, MYC, KIT, IGF1R, TERT and EWS–FLI1 in Ewing sarcoma cells • Promotes degradation of IKKβ and activated AKT and BCL‑XL


XL888

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

Phase 1 in melanoma

DS‑2248

NA

Phase 1 in solid tumours

Debio 0932

NA


BIP inhibitors: inhibit BIP expression

HSP90 inhibitors: disrupt HSP90 function



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

Drugs

Other involved mechanisms

Clinical trials as cancer therapy

PERK inhibitors: inhibit PERK activation and eIF2α phosphorylation

6‑shogaol

The effect on IRE1 and ATF6 was not obvious

Preclinical stage

GSK2656157

• An ATP-competitive inhibitor of PERK • Also has eIF2α phosphorylation-independent effects

Preclinical stage

GSK2606414

Binds to PERK active site

Preclinical stage

IRE1α inhibitors: inhibit IRE1 endonuclease activity

STF‑083010

Inhibits IRE1 endonuclease activity without affecting its kinase activity

Preclinical stage

MKC‑3946

Inhibits IRE1α 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, IRE1α phosphorylation, eIF2α phosphorylation, ATF4 and CHOP • Increases the pro-apoptotic BIK, BIM, BAX, and BAK levels, as well as caspase 7 activity

Phase 1/2 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; Phase 1/2 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; eIF2α, eukaryotic translation initiation factor 2α; 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; IKKβ, IκB kinase; IL‑6, interleukin‑6; IRE1, inositol-requiring protein 1; MCL1, induced myeloid leukaemia cell differentiation protein; NA, not applicable; NF‑κB, nuclear factor‑κB; 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.

cancer cells but not normal cells101. Overexpression of BIP in cancer cells correlates with chemotherapy resistance ( TABLE 1 ; see Supplementary information S1 (table)), which can promote cancer cell survival by inhibiting p53‑mediated expression of proapoptotic BCL‑2‑antagonist/killer (BAK) and NOXA (also known as PMAIP1)168. In addition, expression of BIP seems to be increased in the tumour vasculature, suggesting that targeting BIP ( TABLE 2 ; see supplementary information S2 (table)) will have an impact on both cancer cells and the tumour microenvironment 162. 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 promotes adaptation and drug resistance103–105,169. An IRE1α inhibitor sensitized resistant human glioblastoma cells to oncolytic virus therapy both in vitro and in vivo170. Inhibition of PERK kills hypoxic tumour cells that are radioresistant in vivo104. 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 prospects We 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 (BOX 4). There is an extremely strong pressure for a cancer cell to survive hostile environments or chemotherapy. The long-term approach will probably involve combinatorial therapies that attack the tumour at multiple levels. Anti-angiogenesis therapies are not adequate alone, but they may show synergy in combination with antiUPR agents. It is also essential to identify the driver mutations for individual cancer types to design selective targeting agents. We know that mutations in BRAF generate melanoma and that BRAF inhibitors generate resistance. It is necessary to inactivate processes 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



REVIEWS 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 2α (eIF2α) 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?

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 adaptive pathways will provide one asset of the armamentarium 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 recognizing the potential for increased toxicities and drug

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Acknowledgements

The authors apologize to those whose work could not be cited owing to length restraints. R.J.K. is supported by US National Institutes of Health (NIH) grants DK042394, DK088227 and HL052173.

Competing interests statement

The authors declare no competing interests.

DATABASES ClinicalTrials.gov: http://www.clinicaltrials.gov

SUPPLEMENTARY INFORMATION See online article: S1 (table) | S2 (table) ALL LINKS ARE ACTIVE IN THE ONLINE PDF


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