Proteotoxic Stress and Cell Death in Cancer Cells

To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive responses aimed at restoring proteostasis, allowing cells to survive the stress condition. However, if the proteostasis restoration fails, a permanent and sustained proteotoxic stress can be deleterious, and cell death ensues. Many cancer cells convive with high levels of proteotoxic stress, and this condition could be exploited from a therapeutic perspective. Understanding the cell death pathways engaged by proteotoxic stress is instrumental to better hijack the proliferative fate of cancer cells.


Proteotoxic Stress: An Introduction
Proteins are key macromolecules that play fundamental roles in almost every cellular process, from gene expression to cell/tissue protection [1]. The important and relentless actions of proteins oblige cells to supervise and guarantee their correct folding and assembling. Protein homeostasis or proteostasis is the fundamental cellular effort aimed at reaching this goal. Proteostasis is governed through a complex network of regulative mechanisms and is an essential task for cell survival [2]. The vast majority of proteins need to assume a peculiar thermodynamically stable three-dimensional structure that depends on their amino acid sequence [3]. During the folding process, proteins, particularly those presenting complex domains, can often produce folded intermediates. These intermediates can expose hydrophobic amino acid residues, thus becoming more susceptible to being stacked into a misfolded condition, a circumstance that can lead to the formation of misfolded aggregates [4].
Cells use a complex network, called the proteostasis network (PN), in order to monitor protein homeostasis. PN includes molecular chaperones and proteolytic machinery. These gene families promptly cooperate to guarantee regular proteostasis. In this manner, PN coordinates protein synthesis with folding and, if necessary, it can trigger protein degradation [5][6][7]. The importance of proteostasis maintenance becomes evident in the presence of PN dysfunctions. Inefficiency in these monitoring activities is responsible for several pathologies, including neurodegenerative diseases. Frequently, these deficiencies are age-dependent, with significant social and economic costs [7][8][9][10].
Molecular chaperones supervise protein folding, a process that requires ATP hydrolysis and a high cost in terms of energy. In particular, the chaperones of the heat shock protein (HSP) family help protein folding and are fundamental when critical conditions such as heat stress, oxidative stress, or hypoxia emerge [1]. These particular proteins are defined as HSPs because their expression is dramatically upregulated when cells are exposed to high temperatures or other forms of stress. The human genome encodes for about 330 chaperones and cochaperones [11]. The most known

Cell Death Pathways Activated by Proteotoxic Stress
The induction of proteotoxic stress through the use of small compounds/drugs achieves a therapeutic interest, particularly from an antitumor perspective [93]. In order to better synergize the induction of proteotoxic stress with the available therapies, it is fundamental to dissect the molecular mechanisms controlling cell death in response to proteotoxic stress.

The Extrinsic Pathway of Caspase Activation
It is well established that proteotoxic stress engages the mitochondrial pathway of caspase activation [94]. However, proteotoxic stress is a broad and complex pro-death insult; additionally, the extrinsic pathway is involved [95]. This role was suggested by early studies reporting the upregulation of TNFRSF10B/DR5, the TRAIL receptor, in response to ER-stressors/PERK activation, UPS inhibitors, as well as the influence of caspase-8 inhibitors on proteotoxic stress-induced cell death [39, [95][96][97][98][99][100][101]. More recently, it has been proposed that UPR not only upregulates DR5 expression but misfolded proteins can directly engage with DR5 in the ER-Golgi intermediate compartment to drive the assembly of DR5 in complexes competent for caspase-8 activation ( Figure 1). This activation can occur independently from the binding of its canonical extracellular ligand Apo2L/TRAIL [102]. Although the mechanism involved in such activation is unknown, a plausible hypothesis points to the release of an autoinhibitory activity that normally prevents spontaneous activation of the receptor. The increased levels of expression, the trapping in a particular membrane domain, and the priming effect of misfolded proteins could be the culprits [102,103]. In the receptor-independent activation of caspase-8 following ER stress, a contribution of RIPK1 (receptor interacting serine/threonine kinase 1) has also been proposed. The contribution appears indirect and is sustained by the use of Ripk1-deficient murine cells. The involvement of Ripk-1 in ER stressor-induced apoptosis is still mysterious. It is independent of the kinase activity from cIAP1/2 (BIRC1/2-baculoviral IAP repeat containing 1 and 2)-mediated ubiquitylation and does not involve the direct regulation of JNK/MAPK8 or CHOP [104]. ER stress can also promote inflammatory responses in the presence of chemotherapeutic regiments. Here, again, ER stress elicits TRAIL receptor upregulation, which results in a caspase-8/FADD/RIPK1-dependent activation of NF-κB. Similar to cell death, inflammatory cytokine production occurs in a ligand-independent manner. The importance of this response is testified by the protection observed in DR5 −/− mice from taxol-induced In the receptor-independent activation of caspase-8 following ER stress, a contribution of RIPK1 (receptor interacting serine/threonine kinase 1) has also been proposed. The contribution appears indirect and is sustained by the use of Ripk1-deficient murine cells. The involvement of Ripk-1 in ER stressor-induced apoptosis is still mysterious. It is independent of the kinase activity from cIAP1/2 (BIRC1/2-baculoviral IAP repeat containing 1 and 2)-mediated ubiquitylation and does not involve the direct regulation of JNK/MAPK8 or CHOP [104]. ER stress can also promote inflammatory responses in the presence of chemotherapeutic regiments. Here, again, ER stress elicits TRAIL receptor upregulation, which results in a caspase-8/FADD/RIPK1-dependent activation of NF-κB. Similar to cell death, inflammatory cytokine production occurs in a ligand-independent manner. The importance of this Cancers 2020, 12, 2385 6 of 22 response is testified by the protection observed in DR5 −/− mice from taxol-induced inflammation [103]. These studies confirm that similar to other observations, the engagement of DR5 can result in different cellular responses that are context-dependent [105].

The ATF Network
A huge plethora of studies have indicated that cell death induced by proteotoxic stress can follow different routes. Certainly, the foremost investigated signaling pathway linking proteotoxic stress to apoptosis regards ER stress and the consequent UPR. A key element of this pathway is represented by ATF4 (activating transcription factor 4), a TF that belongs to the cAMP response element-binding protein (CREB)-2 family [106]. As explained above, eIF2α phosphorylation results in the attenuation of the cap-dependent protein translation, as well as the specific translation of selected mRNAs, including ATF4 itself. Normally, ATF4 protein is almost undetectable due to its very short half-life and low translation efficiency [107,108]. In fact, ATF4 levels dramatically increase in response to proteasome inhibitors because of the double effect exerted by UPR activation and the suppression of its degradation [109]. ATF4 is structured into different domains, which comprise a basic/leucine zipper domain (bZIP domain) that binds DNA. ATF4 interacts with several partners that influence its variegated transactivation activities and its stability [106,107]. As a consequence, ATF4 controls the expression of a wide range of genes that play different roles in resolving proteotoxic stress. Some of these genes are directly transcribed by ATF4, others indirectly through the action of other TFs ( Figure 2). An example of a TF regulated by ATF4 is CHOP/GADD153 (CCAAT-enhancer-binding protein homologous protein), an important player of the apoptotic response [110]. Again, translation of CHOP mRNA is sustained by eIF2α phosphorylation that allows the escape from a poor translation initiation sequence [111]. Interestingly, this signaling arm is also involved in controlling ferroptosis through both GCN2-dependent and -independent mechanisms, which are elicited by cysteine depletion [112,113].
Cancers 2020, 12, x FOR PEER REVIEW 6 of 23 inflammation [103]. These studies confirm that similar to other observations, the engagement of DR5 can result in different cellular responses that are context-dependent [105].

The ATF Network
A huge plethora of studies have indicated that cell death induced by proteotoxic stress can follow different routes. Certainly, the foremost investigated signaling pathway linking proteotoxic stress to apoptosis regards ER stress and the consequent UPR. A key element of this pathway is represented by ATF4 (activating transcription factor 4), a TF that belongs to the cAMP response element-binding protein (CREB)-2 family [106]. As explained above, eIF2α phosphorylation results in the attenuation of the cap-dependent protein translation, as well as the specific translation of selected mRNAs, including ATF4 itself. Normally, ATF4 protein is almost undetectable due to its very short half-life and low translation efficiency [107,108]. In fact, ATF4 levels dramatically increase in response to proteasome inhibitors because of the double effect exerted by UPR activation and the suppression of its degradation [109]. ATF4 is structured into different domains, which comprise a basic/leucine zipper domain (bZIP domain) that binds DNA. ATF4 interacts with several partners that influence its variegated transactivation activities and its stability [106,107]. As a consequence, ATF4 controls the expression of a wide range of genes that play different roles in resolving proteotoxic stress. Some of these genes are directly transcribed by ATF4, others indirectly through the action of other TFs ( Figure 2). An example of a TF regulated by ATF4 is CHOP/GADD153 (CCAAT-enhancer-binding protein homologous protein), an important player of the apoptotic response [110]. Again, translation of CHOP mRNA is sustained by eIF2α phosphorylation that allows the escape from a poor translation initiation sequence [111]. Interestingly, this signaling arm is also involved in controlling ferroptosis through both GCN2-dependent and -independent mechanisms, which are elicited by cysteine depletion [112,113].
ATF4 can also trigger cell death independently from CHOP. It can promote the downregulation of the IAP family member XIAP (X-linked inhibitor of apoptosis) in a still-undefined manner. These proteins can bind and block caspase activities but can also, through a RING zinc finger domain with E3 ubiquitin ligase activity, promote ubiquitylation and the subsequent proteasomal degradation of their substrates, including caspases [114].  ATF4 can also trigger cell death independently from CHOP. It can promote the downregulation of the IAP family member XIAP (X-linked inhibitor of apoptosis) in a still-undefined manner. These proteins can bind and block caspase activities but can also, through a RING zinc finger domain with E3 ubiquitin ligase activity, promote ubiquitylation and the subsequent proteasomal degradation of their substrates, including caspases [114]. CHOP supervises the expression of a collection of genes. Interestingly, some of these genes are shared with ATF4, thus suggesting the existence of a feed-forward mechanism to sustain proteotoxic-dependent gene expression [115]. Similarly, the control operated by ATF6 on CHOP transcription can be viewed as a cooperative mechanism to resolve proteotoxic stress [116]. A gene under the direct transcriptional control of CHOP is DR5 [103,117,118]. A CHOP-binding site is present in the 5 -flanking region (position -281 and -216 from TSS) of the DR5 gene [117]. Moreover, ATF3, another ATF/CREB family TF that facilitates apoptotic cell death, is involved in the ER stress-mediated DR5 induction in human p53-deficient colorectal cancer cells [119,120]. TRAIL-R1/DR4 is also engaged by ER stress, although with less relevance. CHOP/ATF4 can also promote DR4 upregulation, although with differences among models and cell lines and via both transcriptional and post-transcriptional mechanisms [121,122].
ATF5 is another ATF/CREB family member under CHOP/ATF4 control ( Figure 2). Transcriptional upregulation occurs via the direct binding of CARE elements in the ATF5 promoter [115,123]. Similar to ATF4 and CHOP, ATF5 is preferentially translated once eIF2 is phosphorylated. Among the ATF5-dependent genes involved in apoptosis, the BH3-only protein NOXA/PMAIP1 can be found [123]. Experimental downregulation of each of these TFs (ATF3, ATF4, ATF5, and CHOP) results in the abrogation of NOXA induction in response to proteotoxic stress. Hence, they all contribute to sustaining the feed-forward loop that drives apoptosis [115,123,124].

The BCL2 Family Members
NOXA/PMAIP1 is a BCL-2 proapoptotic family member that plays important roles in different apoptotic responses. NOXA is the smallest of BH3-only proteins (54 residues), and its expression is dramatically upregulated after proteotoxic stress [125]. Initially identified as a TP53 target gene [126], further studies have demonstrated that its transcription can be potently upregulated by TP53-independent mechanisms under different stress conditions, including oncogenic transformation and proteotoxic stress [127][128][129][130]. NOXA depletion impairs apoptosis in response to proteotoxic stress. NOXA can act as either sensitizer and activator by virtue of its BH3 domain, which is inserted into the hydrophobic-binding groove of multidomain proapoptotic or antiapoptotic BCL2 family members. As a sensitizer, it interacts with MCL1, BCLXL, and BCL2A1 ( Figure 1). In this manner, NOXA interrupts the sequestration operated by these antiapoptotic proteins against multidomain proapoptotic proteins such as BAX and BAK. As a consequence, NOXA unleashes the pro-death activities (oligomerization and channel formation) of BAX/BAK. In contrast, as an activator, NOXA directly binds and activates BAX/BAK [131][132][133][134]. Curiously, murine Noxa contains two BH3 domains (A and B, encoded by exons 2 and 3), with only the BH3 domain B conserved in humans [126].
Additional mechanisms are used by proteotoxic stress to engage the mitochondrial pathway of caspase activation. BIM/BCL2L11 and PUMA/BBC3 are other BH3-only proteins, of which upregulation was reported in several models of proteotoxic stress and, particularly, during ER stress. The ablation of these proteins influences the death response to proteotoxic stress [84,93,94]. BIM was reported as being a transcriptional target of CHOP [135]. Similarly, PUMA expression is induced through transcriptional upregulation in response to an ER stress stimulus in a variety of human cell lines [136,137]. In addition to the action on BH3-only proteins, proteotoxic stress can downregulate BCL2 at a transcriptional level through CHOP [138] (Figure 1). Moreover, JNK activation via the IRE1 pathway triggers BCL2 and BCLXL phosphorylation and their subsequent inactivation [139,140]. Among the different routes that proteotoxic stress can engage to trigger apoptosis, the regulation of BOK (BCL2 family apoptosis regulator BOK) must also be included. This proapoptotic BCL2 family member is normally expressed at low levels. In fact, it is constitutively degraded, with a short half-life of 15 min. During proteotoxic stress, E3 ligases such as gp78, which mediates BOK degradation, become saturated because of the accumulation of misfolded proteins. Hence, BOK can accumulate to favor mitochondrial outer membrane permeabilization [141]. Normally, DNAJB12 (JB12) contributes to maintain low levels of BAK and to sustain the survival of cancer cells. This chaperon is an ER-associated Hsp40 family protein that recruits Hsp70 to the ER surface in the protein quality control system [142].

Additional Cell Death Responses
When proteotoxic stress advances, UPS becomes clogged by the accumulation of polyubiquitylated proteins. Blocking the proteasome affects the expression of unstable signaling proteins and, therefore, signaling pathways controlling cell survival and cell death are modulated. Two important UPS targets, controlling the survival/death switch, are the inhibitors of NF-kB, IkBA [143], and TP53 [144]. Furthermore, elements of the apoptotic machinery, both pro-and antiapoptotic, such as NOXA, BIM, and MCL1, also accumulate in response to UPS saturation [29, 145,146]. MCL1 stabilization represents the dark side in the anticancer effect engaged by UPS inhibitors. Interestingly, multiple kinase inhibitors, such as erlotinib, rapidly enhance UPS-dependent degradation of MCL1. Erlotinib upregulates NOXA expression, which, in turn, through the action of the mitochondria-associated ubiquitin ligase MARCH5 supervises MCL1 degradation [147,148]. Similar to MCL1, other pro-survival proteins such as IAPs (XIAP, cIAP1, and cIAP2 in mammals) accumulate in response to proteotoxic stress-dependent UPS saturation [149]. The activities of these proteins can be instrumental in maintaining cell survival under stress conditions. For example, AIRAP, a proteotoxic-stress gene regulated by the master TF HSF1 (heat-shock factor 1), can regulate cell survival by controlling the levels of cIAP2 [150]. The switch between cell survival/death must also imply control over IAPs. An example is the ability of tunicamycin and thapsigargin (two ER stressors) to reduce XIAP levels in a number of mammalian cell lines [114]. XIAP translation can be reduced in a PERK-mediated manner, and ATF4 promotes its degradation, a new scenario that can contribute to reducing the threshold required for caspase activation.
As indirect consequences elicited by proteotoxic stress can favor cell death, the accumulation of ROS and the alterations of calcium homeostasis must be mentioned. These cofactors can be the deleterious corollaries of the progressive impairments in the clearance capacities normally operated by UPS and autophagy. Accumulation of unfolded proteins and aggregates impact ER and mitochondrial functions, thus leading to alterations in ROS and calcium levels that, in turn, engage further signaling events leading to cell death. How these events integrate with classic apoptotic signaling is not clear. In some studies, induction of oxidative stress can be observed in the initial phases of proteotoxic stress [84,151,152]. Certainly, the augmented levels of ROS and calcium can be responsible for the induction of alternative forms of death in response to the proteotoxic stress observed in different studies [153,154]. In general, the appearance of nonapoptotic or alternative forms of cell death in response to proteotoxic stress is a less investigated item [155][156][157]. Frequently, these necrotic-like responses appear when apoptosis is defective. Interestingly, in a model of toxicity elicited by mutant Huntingtin, a new hypothesis to explain the apoptotic/necrotic switch has been proposed. If the sequestered mutant protein is soluble, cells are characterized by hyperpolarized mitochondrial membrane potential and increased levels of reactive oxygen species, and cell death occurs via apoptosis. Instead, when mutant Huntingtin is present as aggregates, where other cellular proteins can be sequestered, a collapse in mitochondrial potential, cellular quiescence, and deactivated apoptosis occur. Overall, this response curtails cellular metabolism and leads to a slow death by necrosis [158]. Clearly, this model must be verified with general inducers of proteotoxic stress, but it is an interesting hypothesis that deserves further study. Necrotic proteotoxicity can be hampered by NRF2, possibly through the formation of autophagosomes aimed at decreasing the ubiquitylated protein aggregates [159]. Finally, in the necrotic arena, a new role of NOXA cannot be excluded, since its mitochondrial targeting domain (MTD) can trigger mitochondrial fragmentation and necrosis [160].
Necroptosis is a specific form of cell death activated through the serine/threonine kinases RIPK1 and RIPK3 and the pseudokinase MLKL [161]. Compounds that trigger necroptosis can also activate UPR [162,163]. This observation suggests some links between proteotoxic stress and necroptosis. However, as similarly discussed below for ferroptosis, it is not simple to discriminate if UPR engagement is within a pro-survival effort rather than an effective contribution to the cell death process. Importantly, a study aimed at investigating the involvement of UPR in the "classical necroptosis" induced by TNF-α discovered that two commonly used PERK inhibitors, GSK2606414 and GSK2656157, are indeed potent RIPK1 inhibitors [164]. Certainly, RIPK1, in its pleiotropic activities, can also antagonize proteotoxic stress-induced cell death. Overexpression of RIPK1 enhances the induction of autophagy and confers resistance of melanoma cells to ER stress-induced cell death [165]. Finally, during UPR and ER stress induced by hypoxia, which characterize preeclampsia, the contribution of necroptosis has been excluded. Instead, pyroptosis linked to the activation of the NLRP3 inflammasome, through the activity of thioredoxin-interacting protein (TXNIP), has been proposed [166].
Ferroptosis is a specific form of iron-dependent cell death, characterized by the accumulation of lipid peroxides due to the failure of glutathione-dependent antioxidant defenses [167,168]. Few data are available about the implications of ferroptosis in the proteotoxic stress-induced cell death. It is possible that connections exist, as recently discussed [169]. In particular, if we take into account that different ferroptotic agents can also trigger UPR [170,171], the involvement of UPR, at least in the initial phase, can be viewed as a pro-survival strategy [172], as discussed above for necroptosis. On the other hand, ROS could be the link between ferroptosis and proteotoxic stress. For example, glutathione peroxidases can regulate ferroptosis through their ability to reduce hydroperoxy groups in complex lipids and to silence lipoxygenases. However, they can also play a part during the oxidative protein-folding control in ER by reacting with protein isomerase as an alternate substrate [173].
A final important point concerns the heterogenous response of cell populations to proteotoxic stress. It is well known that although exposed to the same intensity of proteotoxic stress, some cells die while others survive. Clearly, the availability of a pool of chaperones is a critical condition. Particularly, for ER stress, the ER resident chaperone BIP is a key factor during the switch from proteostasis to proteotoxicity [174]. HSF1 is the master regulator of chaperone expression in response to proteotoxic stress. Under stress conditions, HSF1 is phosphorylated and it trimerizes and binds regulative elements in chaperone genes, thus driving their transcription [175]. Recently, a model has been proposed where membrane-less organelle foci of HSF1 regulate the cell decision in terms of survival/death. In the presence of prolonged stress, the biophysical properties of HSF1 foci can undergo a change. Small, fluid condensates enlarge into indissoluble gel-like arrangements, where HSF1 is immobilized. Consequently, chaperone gene expression decreases, leading to cell death by apoptosis [176].

Proteotoxic Stress in Cancer Cells
For a detailed discussion on proteotoxic stress and cancer, we refer to previously published reviews, some of them cited thereafter. In this section, we would like to provide only a general overview of this topic.
The protein synthesis process is intrinsically prone to errors. It has been estimated that in mammalian cells, more than 30% of newly synthesized proteins are degraded by the proteasome within minutes from their translation [177]. These quickly degraded proteins are called defective ribosomal proteins (DRiPs) or rapidly degraded polypeptides (RDPs). If not removed, DRiPs can increase proteasome loading and the consequent induction of proteotoxic stress [178]. Cancer cells generally boost protein synthesis and, therefore, DRiPs accumulate more rapidly than in normal cells [93,179]. For example, cancer cells frequently overactivate the mTORC1 pathway. This pathway is required to promote elevated levels of protein synthesis, a condition that obliges cancer cells to pay tribute to the proteasome to avoid the accumulation of misfolded proteins. This dependence from the proteasome has been exploited to kill cancer cells via small compounds blocking UPS [36, [180][181][182]. The fundamental role of the proteostasis in cancer cells is further underlined by the formation of immunoproteasomes as a secondary mechanism to manage the increased proteotoxic stress arising in mutated cells for RAS, PTEN, TSC1, or mTORC1 [180,183]. Environmental conditions, which are commonly exacerbated in tumors, such as hypoxia, oxidative stress, and nutrient deprivation, are additional inducers of protein misfolding and proteotoxic stress [84,94,178].
A still poorly explored aspect of proteotoxic stress is its connection with cellular metabolism [184,185]. It seems that the switch towards an oxidative metabolism rather than glycolysis renders cancer cells resistant to the UPS inhibitor bortezomib. The regulation of the mitochondrial state could represent an additional mechanism of adaptation to proteotoxic stress that could be addressed from a therapeutic perspective [186].
In addition to the amplified levels of protein synthesis and the environmental conditions, genetic alterations accumulated in cancer are other sources of proteotoxic stress. Aneuploidy, copy number variations, and point mutations are common genetic alterations in cancers that can induce proteotoxic stress [187][188][189][190][191]. Aneuploidy is also associated with many types of stresses in cancer cells, which include both metabolic and oxidative stresses [192]. In aneuploid cells, protein complex stoichiometry imbalances are important causes of protein aggregation and proteotoxic stress induction. The uncoordinated expression of a single subunit of protein complexes, encoded on excess chromosomes, leads to its aggregate state. The excess subunits are degraded, or they aggregate, with protein aggregation nearly as effective as protein degradation for lowering the levels of excess proteins [193]. In aneuploid cells, the induction of HSF1 is also, in some way, compromised. This deficit is transduced in the impaired expression of HSP90, accumulation of misfolded proteins, and the appearance of proteotoxic stress [194]. Similarly, overexpression of genes, as well as the accumulation of mutations in coding regions, can alter normal proteostasis [195]. These mutations would produce protein variants that are more prone to misfolding, degradation, and aggregation [191].
Cancer cells convive with proteotoxic stress by upregulating all the possible mechanisms that are able to maintain proteostasis [196][197][198][199][200][201][202][203][204]. As a consequence, cancer cells are more dependent on the presence of HSPs from UPS for their growth and survival [205,206]. Among HSPs, HSP90s and HSP70s are critical for escaping from antiproliferative signals, resisting cell death, and evading senescence. Additionally, these chaperones are involved in many distinct tracts of cancer cells, including drug resistance, angiogenesis, and metastasis [207,208]. Clearly, impacting these adaptive mechanisms has important consequences to the survival of cancer cells [209,210]. This dependence has attracted interest in developing therapeutic approaches aimed at switching-off these adaptations and thus unleashing all the dramatic consequences of the unresolved proteotoxic stress [210][211][212][213][214][215][216][217][218]. In some circumstances, adaptations to proteotoxic stress can favor the resistance to other therapeutic regiments, as observed for HSF1 and the resistance to the receptor tyrosine kinase (RTK) inhibitor lapatinib in breast cancer [219]. Interestingly, the master regulators of ER stress and UPR (ATF3/4/5/6 and CHOP) are highly expressed in a fraction of bladder, kidney, and prostate cancers, indicative of high levels of proteotoxic stress ( Figure 3A). These subgroups of tumors exhibit aggressive behavior characterized by a reduction of overall survival ( Figure 3B). Cancers 2020, 12, x FOR PEER REVIEW 11 of 23

Conclusions
Proteostasis is a fundamental task for every cell. The evolution has sculptured elaborate interconnected mechanisms to maintain proteostasis. Some of these mechanisms have been highly conserved through evolution and, with the appearance of eukaryotic cells, each subcellular compartment has evolved a dedicated set of strategies [220,221]. Proteostasis alterations and the induction of proteotoxic stress are responsible for several pathological conditions, particularly in neurodegenerative diseases, including Huntington's, Parkinson's, amyotrophic lateral sclerosis, and Alzheimer's diseases [222]. On the other hand, small compounds that are able to trigger proteotoxic stress or target the machinery resolving proteotoxic stress are actively investigated as anticancer agents [93]. Undoubtedly, the central role played by proteotoxic stress in the cell life/death decision guarantees that by studying its regulation or developing new compounds aimed to improve or impair its appearance, benefits for the human health will be generated.  The heatmap shows the alterations in the expression levels and was generated through cBioPortal (http://www.cbioportal.org). mRNA expression z-scores are relative to diploid samples (RNA Seq V2 RSEM). (B) Kaplan-Meier survival analysis related to the alterations in the mRNA levels of the ATF network. All cases were analyzed and clustered into two groups according to ATF3/4/5/6 and DDIT3/CHOP alterations in the expression levels, as illustrated in (A). Data were generated through cBioPortal (http://www.cbioportal.org).

Conclusions
Proteostasis is a fundamental task for every cell. The evolution has sculptured elaborate interconnected mechanisms to maintain proteostasis. Some of these mechanisms have been highly conserved through evolution and, with the appearance of eukaryotic cells, each subcellular compartment has evolved a dedicated set of strategies [220,221]. Proteostasis alterations and the induction of proteotoxic stress are responsible for several pathological conditions, particularly in neurodegenerative diseases, including Huntington's, Parkinson's, amyotrophic lateral sclerosis, and Alzheimer's diseases [222]. On the other hand, small compounds that are able to trigger proteotoxic stress or target the machinery resolving proteotoxic stress are actively investigated as anticancer agents [93]. Undoubtedly, the central role played by proteotoxic stress in the cell life/death decision guarantees that by studying its regulation or developing new compounds aimed to improve or impair its appearance, benefits for the human health will be generated. Funding: This research was funded by Interreg Italia-Osterreich rITAT1054 EPIC in a grant to C.B.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.