Apoptosis (also known as Type I Programmed Cell Death) is a vital aspect of numerous processes, including cell turnover, immune system activity, embryonic development, and most relevant to cancer, chemical/radiation-induced cell death (Fig. 9.2). Apoptosis, an energy-dependent process, is characterized by loss of mitochondrial membrane potential, plasma membrane “blebbing, (bulging irregularly)” cell shrinkage, and nuclear fragmentation (Hotchkiss et al. 2009). There are many facets to the mechanisms of apoptosis, and expectantly, these mechanisms involve signaling cascades complete with sophisticated feedback responses. In cancer, apoptosis is particularly important and it is induced by therapeutic agents to reduce or eliminate malignant growths.

There are currently two main pathways in apoptosis: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated when specific pro-apoptotic signals such as irreparable DNA damage or other serious stresses cause mitochondrial outer membrane permeabilization (MOMP), opening protein channels to allow the release of cytochrome c, a highly soluble heme protein and electron carrier found in between the mitochondria’s inner and outer membranes employed in the electron transport chain (Tafani et al. 2002). Cytochrome c forms a wheel-like complex with apoptotic protease activating factor-1(Apaf-1) creating the “apoptosome.” Procaspase 9, the initiator caspase (family of potent cysteine-dependent, aspartate-specific proteases), is bound by the apoptosome and modified to its enzymatically active form, caspase 9. Caspase 9 will then cleave the executioner procaspases, procaspase-3, 6, and 7, thus converting them to their active caspase form. These active executioner caspases cleave cellular substrates and catalyze the fragmentation and degradation of DNA, the cytoskeleton, and nuclear proteins, as well as the formation of apoptotic bodies, cross-linking of proteins, ligand expression for phagocytic cell receptors, and finally the uptake of degraded cellular material by phagocytic cells (Elmore 2007). The intrinsic apoptotic pathway is tightly regulated by a large family of proteins known as the B cell lymphoma 2 (Bcl-2) family which consists of at least 25 genes that contain up to 4 specific Bcl-2 Homology (BH1-4) domains that mediate hetero-and homodimerization with each other. This family consists of proteins with wide variability in function, an can be arranged based on how they regulate apoptosis; whether they perform a pro-apoptotic function (i.e. Bcl-X_s, Bax, Bak, Bim) or an anti-apoptotic function(i.e. Bcl-2, Bcl-X_L, Bcl-w, Bfl-1). During homeostasis, these regulators, in tandem with other regulatory mitochondrial proteins known as small mitochondria-derived activators of caspases (SMACs) and their counterparts, inhibitors of apoptosis proteins (IAPs) (Fesik and Shi 2001), often hold each other in check. As a result, the fate of the cell is largely determined by the balance in the activation of these pro-death and pro-survival proteins (Karam et al. 2007). In many cancers that attempt to escape Bcl-2 mediated apoptosis, the pro-survival regulators will be overexpressed (Bcl-X_L) and/or pro-death regulators (Bax) will be inhibited. The expression patterns of certain regulators can even help to predict the efficacy of certain treatments; for example, expression of Bcl-2 can predict the outcome of radiotherapy in laryngeal cancer with an accuracy of 71 % (Nix et al. 2005).

The extrinsic pathway is initiated at the plasma membrane via the ligand binding and activation of cell surface death receptors (Ashkenazi and Dixit 1998). There are several death receptors (Fas, Trail-R1, TNFR), all with their own domain adaptors (FADD, TRADD) and ligands (FasL, Trail, TNFα); one main death receptor is Fas (also called FasR or apoptosis antigen 1) whose corresponding ligand and adaptor domain is FasL and Fas Associated Death Domain (FADD), respectively (Wajant 2002). These adaptors are recruited via their specific death domains to the receptor upon ligand binding and are necessary for facilitating the assembly of the Death Inducing Signaling Complex (DISC) which comprises procaspases 8 and 10, waiting to be cleaved and activated to then initiate the same execution pathway observed in the intrinsic pathway. In this pathway, FADD and DISC are homologous to Apaf-1 and the apoptosome from the intrinsic pathway respectively. Several decoy death receptors (DcRs), members of the tumor necrosis factor receptor (TNFR) superfamily can compete with the legitimate death receptors for ligand binding to avoid apoptosis (Zong et al. 2014). These decoy receptors are often overexpressed in cancer (Huang et al. 2014). Another inhibitory mechanism exists through the ability of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) to bind the death domain of FADD (and other adaptors), preventing the binding of the initiator pro-caspases 8 and 10, and thus inhibiting apoptosis (Salvesen and Walsh 2014).

The intrinsic and extrinsic pathways of apoptosis are linked. While both of these pathways are initiated uniquely, they both converge on the same execution pathway. Additionally, member of the Bcl-2 family of apoptotic regulators, BH3 Interacting Domain (Bid) function in both pathways. In the extrinsic pathway, when capsase 8 becomes activated, it cleaves cytoplasmic BID into a truncated form (tBid) which translocates to the mitochondria to help facilitate the release of cytochrome c to stimulate increased apoptosis via the intrinsic pathway (Brasacchio et al. 2014).

Autophagy (Type II Programmed Cell Death) is a cannibalistic process of self-degradation of cytoplasmic materials, translating quite literally to “self-eating” (Fig. 9.3). Autophagy is identified by the swelling of the vacuole to massive proportions within the cell, loss of organelles, and the appearance of many autophagosomes (Kroemer and Levine 2008). There are three types of autophagy: chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy (the type that is most commonly referred to plainly as “autophagy”) (Jin et al. 2013). This process has long been thought to be a broad-scope, non-selective process, however, recently several forms of selective autophagy have been identified.

CMA is one such selective form of autophagy which applies most appropriately to UPR in that improperly folded proteins in the cytoplasm are systematically targeted and directly translocated into the lysosome to be degraded. This translocation is mediated by Heat Shock Cognate 70 (HSC70 aka HSPA8) as well as lysosome-associated membrane protein 2(LAMP2) (Dice 2007).

Microautophagy can be both selective and non-selective. Microautophagy describes the process by which the lysosome directly consumes and degrades cytoplasmic materials. Selective forms of this type include microautophagy of the mitochondria (micromitophagy), peroxisomes (micropexophagy), and the nucleus (Krick et al. 2008). While microautophagy and macroautophagy employ separate signaling pathways, some of the autophagy-related proteins are required in both processes (Li et al. 2012).

Macroautophagy (referred to hereafter simply as, autophagy) requires the formation of multi-membrane organelles known as autophagasomes to engulf cytoplasmic materials. These autophagasomes will transport the cellular material destined to be degraded to the lysosome whereby the autophagasome will fuse with and disperse its contents into the lysosome. While non-selective forms of autophagy exist, there are many forms of selective autophagy including: ribophagy (ribosomes), mitophagy (mitochondria), pexophagy(peroxisomes), glycophagy (glycogen), lipophagy (lipid droplets), xenophagy(foreign pathogens), zymophagy (secretory molecules), and perhaps most relevant to this chapter on UPR, ER-phagy. ER-phagy is induced to prevent the excessive distension of the ER during stress caused by the aggregation of unfolded proteins (Yorimitsu and Klionsky 2007). Indeed, researchers have observed the formation of autophagasomes simultaneously with the induction of UPR (Bernales et al. 2006). Interestingly, there has been research that shows that the degradation of cellular materials during autophagy occurs in a certain order; starting with cytosolic and proteosomal proteins proceeding further to organelles (Kristensen et al. 2008).

In general, the events that require the formation of autophagosomes, engulfing of cellular material, and docking to the lysosomes are controlled through the unimaginatively termed, autophagy-related proteins (ATGs), encoded via the ATG genes (Maiuri et al. 2007). The initiation of autophagy is controlled by ATG1, ATG13, and ATG17; the induction of this process requires a signal from the nutrient sensing protein, mTOR, which catalyzes the dephosphorylation of ATG13 which forms a complex with ATG1 and ATG17. The formation of the autophagosome proceeds through to vesicle nucleation and the formation of an isolation membrane controlled via ATG6 (aka Beclin-1). Once this occurs, mTOR and BCL-2 in the ER must be suppressed before ATG6 and ATG13 can form the autophagosome. The vesicle then elongates to consume target cytoplasmic materials; this is achieved through the action of several ATG proteins including: ATG3 ATG4, ATG5, ATG7, ATG10, ATG12, and ATG16. After the autophagosome has been assembled, the autophagosome will dock with the lysosome, fusing membranes and allowing the contents of the autophagosome to enter the lysosome to be degraded by lysosomal enzymes (Maiuri et al. 2007).

Autophagy is an important albeit unclear subject in the scope of cancer research. Researchers propose that autophagy fulfils a pro-survival role due to the necessity to overcome stresses such as nutrient depletion and hypoxia (Hanahan and Weinberg 2000). However, autophagy is a cell death mechanisms responsible for the extermination of cancer cells. Autophagy can have a profound effect on the efficacy of chemotherapeutic treatments and the development of resistance to anti-cancer therapies. When autophagy is inhibited, the therapeutic responses of resistant cancer cells to chemo-, endocrine- or radio-, therapies is increased, further supporting the pro-survival role of autophagy in cancer cells (Cook et al. 2011, 2012; Schwartz-Roberts et al. 2013). Autophagy is also instrumental in the cross-talk between the tumor-microenviroment and the cancer cells themselves, specifically in the syncing of metabolic activity (Pavlides et al. 2012).

Pro-survival autophagy is closely connected to the UPR. When the integrity of protein folding is compromised, and the continuous misfolding of proteins leads to energy depletion in the cell, UPR initiates and signals an up-regulation of autophagy in an attempt to recycle proteins to generate new metabolites and ATP, ideally alleviating the stress (Clarke et al. 2011, 2012; Cook et al. 2011, 2012). It is important to note that the nature of the autophagic response is not so dramatic as to be switched-on or switched-off, rather it is gradual; the level of autophagy increases or decreases to appropriate itself with the current level of stress (Tyson et al. 2011). However, what complicates autophagy in cancer is that although it is generally accepted as a pro-survival mechanism, excessive or prolonged stress will inevitably trigger cell death by arresting autophagy, which can in turn induce apoptosis or necrosis (Crawford et al. 2010; Schwartz-Roberts et al. 2013). An intricate relationship is emerging between autophagy and apoptosis, but unfortunately, there is little consensus among researchers. For instance, it is unclear whether what we describe as autophagic cell death is a truly novel form of cell death executed solely through autophagy, or if instead we are witnessing a tandem effort of autophagy and apoptosis in which the former simply initiates the latter. There are several hypotheses based on different cell lines and models. In the case of true autophagic cell death, the total volume of the autophagic vesicles would be equal or greater than the amount of free cytosol, thus a huge proportion of cytoplasmic material as well as organelles would be destroyed; this would cause irreversible damage to the cell, inducing atrophy and failure of cellular functions still intact leading inevitably to the death of the cell (Lum et al. 2005). In the scenario where autophagy triggers other forms of cell death, autophagy is first initiated as a response to stress stimuli, however, when the stress is not able to be resolved, apoptosis or necrosis is triggered (Espert et al. 2006).

Necrosis (Type III Programmed Cell Death) is a much less selective process, and is commonly associated with injury induced by physical trauma. Necrosis causes inflammation in groups of cells and is found commonly in the center of solid tumors. It is characterized by organelle and plasma membrane swelling, and rupture (Hotchkiss et al. 2009). It was long thought that necrosis was not regulated through signaling pathways and was only a passive form of cell death, however, research in the last decade has proved otherwise. Necroptosis, a programmed form of necrosis dependent on receptor-interacting protein kinase-3 (RIPK3), has been identified and is seen to be mediated in the cell through the binding of Tumor Necrosis Factor (TNF) to its cellular receptor (TNFR) which binds to and activates specific cell death receptors(Galluzzi and Kroemer 2008). Precise mechanism of the regulatory pathway of necroptosis remain largely undiscovered, however, recent research suggest that it is related to the other forms programmed cell death since necroptosis can be induced when both autophagy and apoptosis are blocked (White 2008).