Endoplasmic Reticulum Stress, the Unfolded Protein Response, Autophagy, and the Integrated Regulation of Breast Cancer Cell Fate

Abstract

How breast cancer cells respond to the stress of endocrine therapies determines whether they acquire a resistant phenotype or execute a cell death pathway. A successfully executed survival signal then requires determination of whether or not to replicate. How these cell fate decisions are regulated is unclear but evidence suggests that the signals determining these outcomes are highly integrated. Central to the final cell fate decision is signaling from the unfolded protein response, which can be activated following the sensing of stress within the endoplasmic reticulum. Duration of the response to stress is partly mediated by the duration of inositol requiring enzyme-1 (IRE1; ERN) activation following its release from heat shock protein A5 (HSPA5). The resulting signaling appears to use several B-cell lymphoma-2 (BCL2) family members to both suppress apoptosis and activate autophagy. Changes in metabolism induced by cellular stress are key components of this regulatory system, and further adaptation of the metabolome is affected in response to stress. Here we describe the unfolded protein response, autophagy and apoptosis, and how their regulation is integrated. Central topological features of the signaling network that integrate cell fate regulation and decision execution are discussed.

Introduction

Cell fate primarily incorporates the decision to live or die. If the decision is to live, the cell must then decide whether to differentiate, to arrest growth, or to enter the cell cycle. If the decision is to die, the cell must activate a programmed cell death (PCD) pathway such as apoptosis (PCD1), autophagy (PCD2), or necrosis (PCD3). Appropriate regulation of these cell fate decisions is often critical during normal development, tissue differentiation, and response to stress. The breast provides a useful example, where normal breast function includes periods of proliferation and differentiation in preparation for lactation, followed by the PCD that occurs during involution as the post-lactational breast returns to a resting state. Inappropriate activation/repression of cell fate decisions can have major consequences; the loss of regulation of cell cycling, and inappropriate cell survival, are common characteristics of neoplasia. How cells integrate complex cell fate signaling, and how it may differ between normal and neoplastic breast cells, remain unclear. For example, during lactation the normal breast must balance the extensive production of milk proteins with the risk that an excessive load of these proteins could result in endoplasmic reticulum (EnR) stress and induction of the unfolded protein response (UPR). In breast tumors, stress that induces UPR can arise from the nutrient deprivation and hypoxia induced by inadequate vascularization and from the application of cytotoxic and endocrine therapeutic interventions. Since the UPR can be either prodeath or prosurvival, both the lactating and neoplastic breast must maintain prosurvival UPR, perhaps using many of the same regulatory mechanisms.

Signaling initiated within the UPR leads to changes in the levels and activities of key regulators of cell survival, with the integration of both prodeath and prosurvival signals and functions determining cell fate. Determinants in this process include signals that crosstalk among the plasma membrane, EnR, mitochondria, cytosol, and nucleus, leading to the eventual induction or repression of apoptosis and/or autophagy and the changes in cellular metabolism necessary to enable execution of these decisions. In the breast, central molecular players in this orchestration include members of the BCL2 and autophagy-related (ATG) gene families, estrogen receptor-alpha (ERα; ESR1), NFκB (RELA), and components of the UPR including X-box binding protein-1 (XBP1) and its unconventional splicing.

Precisely how cancer cells die following either endocrine or cytotoxic interventions is unclear. Several independent but potentially interrelated cell death mechanisms are known (Fig. 1); for example, mitotic catastrophe may be important in response to therapies that target microtubules (1). For endocrine therapies, the extent to which necrotic cell death occurs is uncertain (2,3), but cell death by apoptosis (4-7) and autophagy (8,9) are consistently reported in vitro.

Figure 1. Mechanisms of programmed cell death.

A. Apoptosis is an ATP dependent process characterized by organized chromatin condensation and fragmentation of the nucleus, DNA cleavage, formation of apoptotic bodies, cell shrinkage, and plasma membrane ruffling (102-104). The intrinsic (mitochondrial) pathway is regulated by BCL2 family members and involves changes in mitochondrial membrane permeability, release of cytochrome c, exposure of phosphatidylserine, and loss of plasma membrane integrity (105). The extrinsic (cell surface receptor) pathway is dependent upon extracellular signals including tissue necrosis factor-α (TNFα), Fas ligand, and TNF-related ligand TRAIL (103,104).

B. The three forms of autophagy (macroautophagy, microautophagy, chaperone-mediated autophagy) each involve the degradation of cellular contents by lysosomal hydrolases. Macroautophagy (the focus of this review) requires the formation of double membrane structures called autophagosomes or autophagic vacuoles (106), for which accumulation of autophagosomes and cleavage of the microtubule-associated protein LC3 are characteristic but often not definitive (47).

C. Mitotic catastrophe produces multinucleation or the products of micronuclei. Faulty checkpoints, DNA structure checkpoints, or the spindle assembly checkpoint are key components (107,108). Disruption of the normal segregation of many chromosomes results in rapid cell death (107); when this cell death does not occur, the cell can divide asymmetrically and produce aneuploid daughter cells (109) that can become neoplastic (107,109).

D. Necrosis is induced when the intracellular concentration of ATP falls to a level incompatible with survival (110). Vacuolation of the cytoplasm, breakdown of the plasma membrane, and an induction of inflammation around the dying cell are characteristic (45). Increased membrane permeability in the absence of organized chromatin condensation and DNA fragmentation also occur (45,111). Increased cell volume causes rupturing of the plasma membrane and the disorganized breakdown of swollen organelles.

Emerging evidence has begun to define a more intimate relationship between apoptosis and autophagy, implying significant communication between these two activities. Such communication may reflect the use of similar or related signaling molecules in an integrated or even interdependent manner. For example, events within mitochondria and EnR, and their regulation by BCL2 family members, are areas of commonality in apoptosis and autophagy (10,11).

Endoplasmic Reticulum Stress and the Unfolded Protein Response

The rates of protein synthesis and secretion are tightly linked to the ability of the EnR to fold, process, and traffic newly synthesized proteins. Within the EnR, nascent proteins are appropriately folded and moved to the Golgi apparatus for further trafficking. Folding of the polypeptide chain is achieved through the action of a series of molecular chaperones and foldases, which keep the polypeptide in solution and facilitate folding into a thermodynamically favored structure. When this process is incomplete, the cell must deal with any proteins or protein subunits that remain unfolded or misfolded within the EnR, which can become characteristically distended (EnR stress). If unresolved, protein folding becomes further impaired because inappropriately folded proteins continue to sequester molecular chaperones and activate their ATPases. Continual disulfide bond reduction and reformation depletes both energy and reducing molecules such as glutathione, increases the generation or persistence of reactive oxygen species (ROS) and creates oxidative stress, further damaging existing proteins and further limiting their appropriate folding.

Up to one-third of cellular proteins are synthesized within the EnR (12). To address the adverse effects of accumulating unfolded proteins, the cell induces a series of events collectively known as the UPR (EnR stress response; Fig. 2A). The primary goal of the UPR is to eliminate inappropriately folded proteins and reduce the load of newly synthesized unfolded proteins within the EnR. These actions are accomplished by reducing the amount of mRNA template for proteins through degrading existing mRNAs and slowing the transcription/translation of new mRNA, and by reducing the influx of nascent proteins into the EnR lumen (13). Concentrations of protein folding effectors, including molecular chaperones and foldases, are also increased to process the mass of accumulated proteins. Remaining misfolded proteins are eliminated through an endoplasmic reticulum-associated degradation pathway (ERAD) (14) either by a ubiquitin/proteasome pathway ERAD(I) or an autophagic/lysosomal pathway ERAD(II) (15). Soluble targeted proteins are retro-translocated into the cytosol, ubiquitinated, and are degraded by the proteasome in ERAD(I) (16,17). Insoluble misfolded protein aggregates are degraded by autolysosomes in ERAD(II) (15,18).

Figure 2. The Unfolded protein response and cross-talk between apoptosis and metabolism.

A. The UPR is an adaptive signaling pathway where the proximal activators of each of its three arms (PERK, ATF6, IRE1α) are normally activated following their dissociation from HSPA5 (GRP78; BiP). When released from HSPA5 the N-terminal luminal domains of two PERK proteins bind together (112), the resulting dimer undergoing an activating autophosphorylation; phosphorylation of PERK tyrosine-615 is a key event (113). PERK is a type I transmembrane protein that phosphorylates the eukaryotic translation initiation factor 2 α-subunit (eIF2α) (114). Phosphorylation of eIF2α at serine-51 blocks translational initiation (115) because, as a dominant negative inhibitor of eIF2β, the EI2F recycling required for further protein biosynthesis is blocked and the rate of protein biosynthesis is reduced (113). Downstream events include the induction of ATF4 that then regulates expression of several genes including the proapoptotic DNA damage-inducible transcript 3 (DDI3; also known as CHOP or GADD153) (116). The two mammalian ATF6 alleles (ATF6α; ATF6β) encode a type II transmembrane bZIP transcription factor. HSPA5 blocks two Golgi localization signals that are exposed upon its dissociation from ATF6 (117). Following translocation to the Golgi, regulated intramembrane proteolysis by the site-1 (S1P) and -2 proteases (S2P) cleave ATF6α to its active p50 form (ATF6β plays only a minor role in the UPR). ATF6 p50 then enters the cytosol, translocates to the nucleus, and activates transcription in cooperation with the general transcription factor nuclear factor-Y (NF-Y) (118,119). Among the key genes regulated by ATF6 p50 are XBP1 (spliced in the IRE1α pathway) (120), DDI3 (also induced in the PERK pathway) (121) and HSPA5 (regulates all three pathways (122)). Activation of IRE1α and splicing of HAC1 (yeast) and XBP1, is the oldest and most conserved pathway for UPR signaling. Downstream targets of XBP1(S) include p58^IPK and several UPR chaperones (123). p58^IPK represses PERK activity (124). Thus, persistent XBP1(S) production in the face of continued EnR stress could shift UPR signaling from PERK to favor IRE1α and/or the integration of ATF6 (though increasing XBP1 transcription) and IRE1α (through increased XBP1 splicing) signaling.

B. Two stem-loop structures, each containing a highly conserved CNGNNG motif, are cleaved. How the two exons are ligated in mammalian cells remains unclear; this function likely being different from that described for yeast (125). XBP1 splicing may not be exclusively cytosolic but this remains controversial (126). Other nucleotide substrates may exist for IRE1α but none is known to possess the stem-loop structures evident in XBP1 and HAC1.

C. UPR modulates crosstalk between autophagy and apoptosis through various mechanisms. Stimulation of UPR results in an increase in CHOP that promotes apoptosis. Moreover, IRE1α activation promotes apoptosis by phosphorylation of JNK, directly and indirectly inactivating anti-apoptotic BCL2 proteins. UPR release of EnR Ca²⁺ also directly promotes apoptosis. UPR signaling also stimulates autophagy. Activation of PERK and the resulting phosphorylation of eIF2α promote autophagy through ATF4-mediated Atg12 transcription. Furthermore, IRE1α-mediated activation of JNK and the subsequent phosphorylation of BCL2 results in dissociation of the BCL2/BECN1 complex, promoting autophagy.

D. Low intracellular glucose concentrations result in the accumulation of unfolded proteins, stimulating the release of the three UPR signaling arms (PERK, IRE1, and ATF6) by HSPA5, and activating the UPR. UPR signaling can activate autophagy resulting in increased degradation of cellular material and release of peptides, amino-acids, and fatty acids. During metabolic stress, a major role of the peroxisome proliferator-activated receptors (PPARs) is likely their ability to ensure adequate turn-over of peroxisomes to manage a greater metabolic requirement for release of the energy stored in the longer chain fatty acids. Autophagy degradation byproducts (amino acids, carbohydrates and short chain fatty acids) promote the TCA cycle and the corresponding generation of ATP. Formation of ATP by mitochondria, using the raw material provided by autophagy, enables the cell to cope with the low glucose levels and promotes survival.

Accumulation of unfolded or misfolded proteins is detected by EnR transmembrane receptors. The three primary molecular sensors are inositol-requiring protein-1α (IRE1α; ERN1), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK; EIF2AK3) (19). In the absence of stress, each is maintained in an inactive state through its association with glucose-regulated protein 78 (GRP78; BiP; HSPA5). As unfolded proteins accumulate, HSPA5 dissociates from the molecular sensors and binds to hydrophobic domains on the surface of these unfolded proteins (20) in an attempt to affect their repair (21). All three arms of the UPR can be regulated by changes in the concentration of free HSPA5 (Fig. 2A) (22) but how this leads to stress-specific activation of selected UPR signaling is uncertain (19).

PERK Signaling in UPR

Some UPR-associated signaling may not be unique to the UPR alone. Three signaling processes have been suggested; (i) signaling through IRE1α/XBP1 and through ATF6 that are largely restricted to UPR; (ii) signaling through PERK and eukaryotic translation initiation factor-2α (eIF-2α; EIF2S3) that can be restricted to UPR, and (iii) signaling through PERK/eIF-2α and ATF6 that may be specific to UPR but that can also be induced by other stressors (23) (Fig. 2A). Activation of PERK signaling appears to be independent of signaling that involves either ATF6 or IRE1α (23) and may be the least uniquely definitive pathway of the UPR. For example, the primary target of PERK (eIF2α) is also activated by protein kinase RNA-activated (PKR), eukaryotic translation initiation factor-2α kinase 4 (EIF2AK4), and eukaryotic translation initiation factor-2α kinase 1 (EIF2AK1) (24). Recent studies indicate that protein kinase B (AKT) phosphorylates and inhibits PERK (25). AKT-mediated inhibition of PERK signaling can inhibit the downstream phosphorylation of eIF2α, preventing the cytoprotective activity of eIF2α. Inhibition of the PERK/eIF2α pathway leads to increased cell death in tumor cells in response to PI3K and AKT inhibitors, indicating a possible role of PERK/EIF2α signaling in PI3K/AKT inhibitor resistance (25). Together, these observations suggest a prosurvival role of PERK/eIF2α signaling in UPR. PERK signaling inhibits translation to reduce the protein load on the EnR and increases p53 levels through a PERK-required ribosomal-Hdm2 interaction preventing Hdm2-mediated p53 ubiquitination (26). Increases in p53 in response to UPR activation leads to cell cycle inhibition, suggesting another adaptive method for UPR-mediated cell survival.

IRE1 and XBP1 Signaling in UPR

How the balance between prodeath and prosurvival UPR outcomes is determined is only beginning to emerge. Using mathematical modeling, Rutkowski et al. (27) proposed a model where the prosurvival outcome is driven by the relative stability of the UPR mRNAs and proteins associated with the restoration of metabolic homeostasis, balanced by the relative instability of molecules that promote apoptosis. Lin et al. (28) showed that EnR stress activates both prosurvival and prodeath signaling, with the outcome determined by the maintenance (prosurvival) or termination (prodeath) of IRE1α activity.

Where the key activity within prosurvival UPR signaling is the duration of IRE1α activation (28), cell fate outcome is substantially mediated by its unconventional splicing of XBP1 (19,29), one of the primary regulators of the transcription network activated by the UPR (30). Conventional mRNA splicing generally occurs within spliceosomes in the nucleus. Non-spliceosomal extranuclear splicing can occur where essential components of the spliceosome are present such as in the cytoplasm of platelets (31). Unconventional splicing occurs in the cytoplasm and is largely independent of spliceosomal components. For XBP1, this splicing is performed by the endoribonuclease activity of IRE1α. Splicing removes a 26 bp sequence (Fig 2B) creating a frame-shift that encodes a larger protein XBP1(S) that can now act as a transcription factor. Regulation of transcription by XBP1(S) is a consequence of its ability to activate specific cAMP response elements (CREs) with a conserved ACGT core sequence (32,33); XBP1(S) can also regulate transcription from EnR stress response elements (ERSE1; consensus sequence CCAAT-N9-CCACG) (34). The unspliced mRNA protein product, XBP1(U), has a molecular weight of ~33 kDa and can act as a dominant negative of the spliced XBP1(S) mRNA protein product that encodes a protein of ~54 kDa (35,36). Activation of both ATF6 (induces XBP1 transcription) and IRE1α (splices XBP1) can be coordinated by their respective dissociation from HSPA5. This coordinated activation, and the eventual balance between the relative production of XBP1(U) versus XBP1(S), could have significant consequences for UPR activation, function, and cell fate.

While XBP1 transcription is increased by the UPR (30), XBP1 is also rapidly induced in breast cancer cells following 17β-estradiol (E2) stimulation (37,38). Upregulation of XBP1, by either activation of the UPR or a UPR-independent mechanism, confers antiestrogen resistance and implicates XBP1 function as an important component of breast cancer signaling (39). Moreover, expression of XBP1 mRNA is strongly associated with ESR1-positivity in breast tumors (40), and XBP1 can bind to and activate ESR1 in a ligand-independent manner (41). XBP1(S) expression is associated with acquired endocrine resistance (42); overexpression of the XBP1 cDNA in breast cancer cells produces primarily XBP1(S) and is sufficient to confer both E2-independence and antiestrogen crossresistance (39). Expression of XBP1(S) is elevated in breast tumors that respond poorly to Tamoxifen (43).

The Unfolded Protein Response and the Regulation of Autophagy and Apoptosis

UPR regulates multiple signals in its attempt to restore metabolic homeostasis, a process that could be fruitless should the cell not concurrently attempt to block cell death signaling long enough to determine whether or not the stress can be adequately resolved. The most effective means to accomplish both tasks is for their respective signaling to be integrated. This integration can be initiated within the UPR and yet concurrently regulate both autophagy and apoptosis.

Autophagy (macroautophagy) is a lysosomal degradation process where cellular components are encapsulated within autophagosomes and degraded by lysosomal hydrolases (see Cook et al. for recent review (10)). The signaling network topology associated with autophagy is complex and only beginning to emerge (44). Autophagy is generally characterized by the presence of cytoplasmic vacuoles and autophagosomes, the absence of marginated nuclear chromatin (45,46), an increase in cleavage of microtuble-associated protein 1 light chain 3 (LC3), and a reduction in p62/sequestosome-1 (p62/SQSTM1) protein levels (47). LC3 cleavage, which can require eIF2α phosphorylation by PERK within the UPR (48), may not occur with non-canonical ATG5/ATG7-independent autophagy (49). Under normal conditions, basal autophagy removes long-lived proteins and damaged organelles, releasing the degradation products into the cytosol as intermediate metabolites. Autophagic removal of specific organelles is uniquely identified; for example, pexophagy (peroxisomes), mitophagy (mitochondria), crinophagy (golgi), ribophagy (ribosomes), and reticulophagy (EnR).

The level and duration of autophagy can vary significantly and, like the UPR, autophagy is associated with both cell survival and cell death (33). Prosurvival autophagy likely depends on recycling of cellular contents to feed the cell’s basal metabolic machinery at a level sufficient for survival. An induction or persistence of autophagy, such that the minimum subcellular machinery necessary for survival is no longer maintained, could result in either an autophagic and/or apoptotic cell death. Prodeath outcomes may reflect the need to eliminate cells that cannot function normally due to the absence of key proteins, failing to secrete correctly folded proteins including essential hormones and growth factors, and/or have been subjected to excessive/irreversible oxidative stress and DNA damage (33).

Beclin 1 and BCL2 Interactions Determine Activation of Autophagy

Two primary regulatory activities are reported to initiate autophagosome production in canonical autophagy signaling (50,51). BECN1 acts through its ability to form the “beclin 1 complex”, which includes phosphoinositide-3-kinase class 3 (PIK3C3), Vps34, and Vps15, and activating molecule in beclin 1-regulated autophagy (AMBRA1) (Fig 1). Alternatively, derepression of ULK1 (ATG1) by either suppression of mTOR (10) or phosphorylation by AMP kinase (AMPK) (52), enables the formation of a protein scaffold for building the pre-autophagosomal structure. Signaling initiated within the UPR can affect both these autophagy initiating mechanisms.

BECN1 binds to, and is inhibited by, BCL2, BCL-X_L (BCL2L1), BCL-W (BCL2L2), and MCL1. Proteins that regulate the expression and/or interact with these BCL2 family members affect their ability to inhibit BECN1’s proautophagic function. Thus, competitive interactions by BAD, BID, BIK, BIM (BCL2L11), BNIPL, BNIP3, NOXA (PMAIP1), and PUMA (BBC3) can promote autophagy by effectively sequestering BECN1 inhibitors and releasing free BECN1 to act elsewhere (10). Phosphorylation of BECN1 by death associated protein kinase (DAPK) reduces BECN1 affinity for BCL-X_L (53), also releasing BECN1. Subcellular localization is critical. BCL2 inhibition of BECN1 is evident in the EnR but not when this interaction occurs at mitochondria (54). The apparent ability of BCL2 to sequester AMBRA1 at mitochondria can prevent formation of the beclin-1 complex at the EnR, whereas BCL2 cannot bind AMBRA1 when they are localized in the EnR (55). Once autophagy is initiated, AMBRA1 can cause BCL2 to dissociate from BECN1 (55), perhaps reflecting the binding of BCL2 and BECN1 at distinct sites on AMBRA1 (56). The relative importance of location for the action of other BECN1 interacting proteins requires further clarification.

Other key regulatory events can be initiated within the UPR and directly affect autophagy including the ability of XBP1(S) to transcriptionally induce BCL2 expression (39). Given the importance of IRE1α in affecting UPR prodeath/prosurvival outcomes (28), and by implication the importance of XBP1 splicing, the ability of XBP1(S) to regulate BCL2 expression may be one of several essential downstream activities that integrate UPR and autophagy signaling. For example, endogenous XBP1(S) is overexpressed in antiestrogen resistant breast cancer cells (42), and its overexpression increases BCL2 expression and induces antiestrogen resistance in sensitive cells (39). BCL2 inhibition can partly reverse XBP1-induced antiestrogen resistance but a greater effect is seen when both BCL2 and BCL-W are inhibited, and a further improvement is seen when BECN1 is also inhibited by either 3-methyladenine or antiBECN1 shRNA (57). XBP1 can bind ESR1 and increase its transcriptional potency (39,41). Since ESR1 can also induce BCL2 expression, XBP1 can potentially drive BCL2 through two independent mechanisms, directly through ACTG-CRE sites in the BCL2 promoter and indirectly through ESR1, providing redundancy for XBP1 regulation of BCL2 (58).

NFκB has multiple functions including regulation of the inflammatory response and apoptosis. Since EnR stress is associated with increased ROS production and oxidative stress, it is logical that NFκB and its signaling would be activated. In the context of UPR, NFκB can be activated by PERK through the action of phosphorylated eIF2α and its regulation of IκBα translation (59). In some cells, NFκB can induce BECN1 expression (60). NFκB can inhibit CHOP (GADD153) and prevent EnR-induced cell death, establishing a link between NFκB and UPR regulation (61). Importantly, endogenous NFκB expression is increased in antiestrogen resistant breast cancer cells, in part through the increased expression of p65/RELA and IKKγ (IKBKG) (42,62). While activation of PERK may contribute to increased NFκB activity, overexpression of XBP1(S) also increases endogenous NFκB transcription and activation in breast cancer cells (Hu et al. in preparation). Activation of NFκB increases BCL2 expression; inhibition of either NFκB (62) or BCL2 (57) can partly restore antiestrogen sensitivity in resistant cells.

JUN N-terminal kinase (JNK; MAPK8) is activated following the binding of IRE1α and tumor necrosis factor receptor-associated factor 2 (TRAF2), a process that often requires signal-regulating kinase-1 (ASK1; MAP3K5) (63). ASK1 is strongly implicated in EnR stress-induced autophagy, a process accompanied by IRE1α activation (64). Phosphorylation of BCL2 by JNK does not affect BCL2 binding to AMBRA1 (55) but can disassociate BCL2 from BECN1, potentially freeing BECN1 to initiate autophagosome formation. While JNK has roles in both the intrinsic and extrinsic apoptotic pathways (65), basal levels of JNK and phospho-JNK expression are increased in antiestrogen resistant cells (66), suggesting a dominant role in prosurvival UPR/autophagy rather than apoptosis. These activities may reflect release of BCL2 (antiapoptotic) and BECN1 (prosurvival autophagy) from each other.

UPR, Autophagy, and Apoptosis Pathway Crosstalk

Many of the UPR signaling outputs associated with autophagy are also associated with the regulation of apoptosis (Fig. 2C). For example, NFκB and JNK activation contribute to the regulation of apoptosis. Both caspase-8 and apoptosis are activated when NFκB activity is inhibited in antiestrogen resistant breast cancer cells, whereas autophagy is not (66). NFκB can directly regulate BCL2 expression, partly explaining NFκB’s ability to influence both autophagy and apoptosis. Antiapoptotic BCL2 action in the mitochondria is well known, and the binding between AMBRA1 and BCL2 at mitochondria is reduced during apoptosis (55). Association of IRE1α with BAK and BAX likely also affects apoptosis (67), and the loss of IRE1α activation enables the induction of apoptosis (28). Indeed, many members of the BCL2 family, including those implicated above in sequestering BECN1 interacting proteins, are intricately involved in the functional regulation of apoptosis (11).

Antiestrogens induce both apoptosis and an apparently prodeath autophagy in sensitive cells (68). However, resistant cells that are resensitized to antiestrogens by inhibition of BCL2 and/or BCL-W do not die through apoptosis but through an autophagy-associated necrosis (57). When BECN1 is then also inhibited, necrosis (PCD-3) is no longer a dominant cell death mechanism and the cells recover the ability to die through apoptosis. Thus, the cell fate decisions associated with regulation of BCL2 family members and BECN1 are differentially regulated depending on the cellular contexts in endocrine sensitive and resistant breast cancer cells (57). Death receptor-5 (DR5; TNFRSF10B), a major component of the extrinsic apoptosis pathway, is regulated by CHOP (Gadd153; DDIT) that is activated by both PERK and ATF6 (69). CHOP also regulates BCL2 expression (70), likely concurrently affecting its role in both apoptosis and autophagy.

While strongly implicated in the regulation of apoptosis, the role of p53 in UPR-associated signaling is unclear. Limited evidence suggests a dual role for p53 with respect to autophagy. Genomic stress can induce an apparent p53-dependent autophagy and stimulate the transcription of autophagy related genes. Conversely, deletion or inhibition of p53 can also activate autophagy (71). Currently, a definitive mechanistic link has yet to be established between antiestrogens, autophagy, and p53. Studies exploring the role of antiestrogen therapies and autophagy using both MCF7 (p53 wild-type) or T47D (p53 null) breast cancer cell lines show a broadly similar activation of autophagy in response to endocrine therapy. For example, inhibition of autophagy using either RNAi or chemical inhibitors potentiates antiestrogen-mediated cell death (72), suggesting that p53 may not play a central role in mediating antiestrogen-induced autophagy.

Changes in intracellular Ca²⁺ and activity of the Ca²⁺ binding protein calmodulin are implicated in responsiveness to antiestrogens (73). Increased cytosolic Ca²⁺ induces a BECN1/ATG7-dependent, BCL2 sensitive autophagy by activating calcium/calmodulin-dependent protein kinase II beta (CAMK2B) and AMPK, which then inhibits mTOR (74). An AMPK-independent pathway involving the protein phosphatase WIP1 (PPM1D) and LC3 is also implicated in Ca²⁺-mediated autophagy (75). JNK phosphorylation of BCL2, and its consequent release from BECN1, allows BCL2 to bind and inhibit the function of inositol 1,4,5,-triphosphate receptor (IP3R; ITPR1) (76,77). IP3R controls the release of Ca²⁺ from the EnR into the cytosol, and less Ca²⁺ could delay or reduce apoptosis. This activity may be unrelated to its role in autophagy (78). Rather, the concurrent release of BECN1 is likely to be the regulator of autophagy. Cleavage of ATG5 by the calcium-dependent, non-lysosomal, cysteine protease calpain can also cause a transition from autophagy to apoptosis (79). While the precise role of Ca²⁺-mediated signaling may be complex and cell context dependent, these observations provide further evidence of how components common to UPR, apoptosis, and autophagy may coordinately affect their relative activation.

Coordination of Cellular Metabolism and Cell Fate

Appropriately activated UPR can eliminate EnR stress, restore correct protein folding, and allow the cell to function normally (prosurvival function). UPR activation of ERAD may support the “recycling” of material recovered from the degradation of misfolded proteins, which could also allow cells to survive when extracellular nutrient sources are limited. A link between cell fate and UPR is consistent with the use of ERAD(II) to eliminate insoluble misfolded proteins through an autophagic process. The eventual dissolution of autolysosomes during autophagy releases the degraded or partially degraded macromolecules from damaged or unnecessary organelles and cytosolic contents for subsequent reuse. Autophagy can be initiated by several stressors including the persistent nutrient deprivation that may arise from inadequate vascularization and/or loss of stimulation by growth factors including the insulin-like growth factors (IGFs). However, precisely how nutrient deprivation is sensed is not entirely clear. mTOR can integrate signaling from insulin, growth factors including IGF-1 and IGF-2, and amino acids (80,81). Nutrient/energy deprivation regulated signaling may also include activation of AMPK by an increased AMP:ATP ratio (ATP depletion), or induction of REDD by HIF1 in response to hypoxia/oxidative stress, which can lead to inactivation of the TORC1 complex and release its repression of autophagy (82,83).

p53 is altered in 20-40% of all breast carcinomas (84) and has recently been implicated in the regulation of metabolism. For example, decreased oxygen consumption and increased glycolytic activity occurs in p53^−/− mutant mice, with no overall change in total ATP production. Altered metabolism is linked to p53-mediated transcriptional regulated targets such as mitochondrial cytochrome oxidase c (COX)-complex, with an observed increase in lactate accumulation (85). Low pH can stimulate AMPK and p53 expression, resulting in a high glycolytic flux and inhibiting apoptosis through increased expression of BCL2 and p53 (TP53)-induced glycolysis and apoptosis regulator (TIGAR) (86). p53-induced TIGAR expression protects cells against oxidative stress and regulates glycolysis (87). With the high frequency of p53 mutations observed in breast cancer, the role of p53 in the possible coordination between UPR signaling, antiestrogen resistance, and metabolism clearly requires further study.

In cancer cells, insufficient glucose or other energy substrates may create low intracellular ATP concentrations. Moreover, as intracellular glucose levels fall, members of the glucose-regulated protein family are activated (88). This family includes HSPA5, and low glucose can result in the release of HSPA5 from the UPR sensor proteins and activation of the UPR. Thus, activation of glucose regulated proteins represents another general means to sense nutrient insufficiency and induce a UPR-regulated autophagy. Whatever the upstream activation, once initiated, autophagy can enable metabolite recycling and contribute to the restoration of metabolic homeostasis.

Further study is needed to determine precisely how the contents released from autolysosomes feed into a cancer cell’s energy metabolism, which generally has a high glycolytic demand from the Warburg effect, or into its intermediate metabolism to maintain/replace basic cellular components. Intermediate metabolism may be largely intact, and the reuse of amino acids, peptides, carbohydrates, and small fatty acids may ultimately feed into the TCA cycle in adequately functional mitochondria. Larger fatty acids are probably metabolized in peroxisomes, as would also be the case in most cells. During metabolic stress, a major role of the peroxisome proliferator-activated receptors (PPARs) may be their ability to ensure adequate turn-over of peroxisomes to manage a greater metabolic requirement for release of the energy stored in the longer chain fatty acids that are provided by autophagy (Fig. 2D). This is likely to be a dominant role for PPARs during stress. Similarly, the primary roles of insulin and the IGFs may be their effects on autophagy and basal survival metabolism including regulation of glucose metabolism, with their ability to increase proliferation only possible if cellular metabolism permits. Whether or not growth factors or other mitogens activate proliferation is probably a secondary concern for the cancer cell, since the ability to survive, even if in an essentially dormant (non-replicative) state, is likely preferable to death. Thus, it is not surprising that growth factors, hormones, and other mitogenic signals involve coordinated regulation of metabolism, cell survival, and cell cycling. We propose that this regulation is often hierarchical, or at least appears so. Since both survival and programmed cell death mechanisms are energy dependent and the choice to live or die may be determined by metabolic status, the hierarchical importance for cellular decision making may be signaling to regulate metabolism (highest priority) → survival → proliferation (lowest priority). As such, the frequent focus on therapeutically targeting replication may miss the potential of targeting metabolism, provided this can be done in a manner that does not also adversely affect non-cancer cells to induce excessive toxicity.

UPR and the Tumor Microenvironment

While this review clearly highlights the relevance of UPR-mediated control of autophagy and apoptosis to regulate tumor cell fate, recent studies also implicate UPR signaling in affecting interactions within the tumor microenvironment. A transgene-induced mammary tumor model in HSPA5 heterozygous knockout mice exhibits decreased angiogenesis and tumor microvessel density (89). In a syngeneic breast tumor model, wild-type tumor cells implanted into a HSPA5 heterozygous mouse showed decreased angiogenesis in early but not late phase tumor growth. The number of metastatic lesions was also reduced in the HSPA5 heterozygous animals (90). Knockdown of HSPA5 in endothelial cells decreased their proliferation, survival, and migration, implicating UPR in angiogenesis within the tumor microenvironment (90). In contrast, increased expression of HSPA5, GRP94 (HSP90B1), and protein disulfide isomerase (PDI) is detected in the circulating progenitor/cancer stem cells of patients with breast cancer (90). Since UPR signaling may be important in both the tumor cells and other cells in the tumor microenvironment, the UPR coordination of cell fate proposed in this review may be broadly applicable to many different cell types.

Conclusions and Future Prospects

Signaling initiated from within the UPR actively participates in autophagy and both the intrinsic and extrinsic apoptosis pathways. The latter is logical since EnR stress can result from internal or external stressors. Inappropriate activation of UPR, whether the effect on cell fate is prodeath or prosurvival, can be problematic. Failure to eliminate stressed cells, particularly in cells with damaged DNA, could lead to cancer. UPR activation leading to a prosurvival outcome in pre-existing cancer cells would clearly be detrimental to the host. Activation of UPR may be more likely for those cancers that arise from normal cells with a significant secretory function, where UPR activation may be a common occurrence. Cancers of the breast, prostate, immune system, and pancreas are among the most common cancers and are strong candidates to exhibit a central role for UPR activation as a cell survival mechanism. Nonetheless, this general function is conserved in evolution and may be active in most cancers. Cancer cells generally experience multiple cellular stressors associated with UPR including nutrient deprivation from inadequate vascularization (91) or exposure to either endogenous and/or treatment induced oxidative stress (92,93).

Signaling initiated within the UPR, or external to the UPR but using some of its signaling components, can influence the initiation of both apoptosis and autophagy and contribute to the cell fate decision process. Integration of this signaling is critical if the cell is to use UPR and autophagy first to determine if it should or can survive. Initiating a stress response pathway to attempt resolving the stress would be pointless if an irreversible cell death signal was concurrently activated. Thus, cell signaling appears to be wired so that the same molecules, such as BCL2 family members, can concurrently repress one function (such as prodeath) while activating an opposing activity (prosurvival). For example, the association of IRE1α with the proapoptotic BAK and BAX affects UPR (67), suggesting one mechanism by which apoptosis could be inhibited while the cell tries to use a UPR-mediated autophagy to recover.

With UPR and autophagy playing integrated and perhaps interdependent functions, it is not surprising that both can be associated with prodeath or prosurvival outcomes. How these interactions differ between the cancer and normal, or drug sensitive and drug resistant, phenotypes is an area for research. Moreover, while we have chosen to use the widely described PCD2 for autophagy in the context of cell death, very recent data studying autophagic flux in response to chemically-induced stress suggests that the process usually thought of as an autophagic cell death may actually be a very rare cell fate outcome (94). This intriguing observation requires additional study but it may also require a revision in how we think of autophagy as a mechanism for executing cell death (94). Whether or not autophagic cell death occurs, the plasticity of cell fate decision making and the importance of cellular context are already evident. Plasticity and context can both arise from within one integrated signaling network, each being explained by the presence of an adaptive network topology. For example, nodes of the signaling network that determines cell fate may be largely maintained but the frequency, strength, and direction of their interactions (edges) are changed (58). Such topological changes could be further modified by perturbations in the set-points required to activate irreversible decisions (95). Also, the relative importance of a node or edge could be modified by a change in sequence (mutation; splicing), transcription, translation, posttranslational modification, and/or subcellular localization.

Cellular signaling occurs in the context of interactive networks (58,96) and there is considerable integration and communication among the signaling associated with the UPR, autophagy, and apoptosis. Representing, understanding, and exploring such complexity are unlikely to be adequately served by attempting to capture knowledge in static wiring diagrams such as we have used here to illustrate some signaling transduction. Such diagrams are necessarily simple and there are many potential nodes already evident for inclusion in a model that might explain cell fate decisions, such as those activated in response to endocrine therapies in breast cancer (97,98). Rather, a systems approach using both computational and mathematical modeling may be required to construct hypotheses that will better identify the most important and informative experiments, and ultimately enable the testing of predictions as to how the system responds to stress and makes irreversible cell fate decisions (58).

Despite the many challenges of working in these data spaces (96), initial computational models derived from analyses of high dimensional transcriptome data have begun to identify small topological features of this system (44,99). A framework for mathematical modeling of cell fate decision making in the context of responsiveness to endocrine therapies in breast cancer has been recently proposed (100). The model incorporates modules for cell cycle, apoptosis, autophagy, and UPR. Models for some individual modules including UPR have been proposed by others (27). However, current models are generally high level and there is a notable paucity of data needed to define the parameters and construct informative and sufficiently robust mathematical models for any of these critical functions and their regulatory components. Finally, it is evident that studies involving therapy responsiveness and cell fate decisions require careful consideration of the integrated role of UPR, autophagy, apoptosis, and necrosis. It also seems likely that novel therapeutic targets reside within this network (101). It remains to be seen how these opportunities can be identified and used to good effect in the attempt to eradicate breast and other cancers.