BCL-w: apoptotic and non-apoptotic role in health and disease

The BCL-2 family of proteins integrates signals that trigger either cell survival or apoptosis. The balance between pro-survival and pro-apoptotic proteins is important for tissue development and homeostasis, while impaired apoptosis contributes to several pathologies and can be a barrier against effective treatment. BCL-w is an anti-apoptotic protein that shares a sequence similarity with BCL-XL, and exhibits a high conformational flexibility. BCL-w level is controlled by a number of signaling pathways, and the repertoire of transcriptional regulators largely depends on the cellular and developmental context. As only a few disease-relevant genetic alterations of BCL2L2 have been identified, increased levels of BCL-w might be a consequence of abnormal activation of signaling cascades involved in the regulation of BCL-w expression. In addition, BCL-w transcript is a target of a plethora of miRNAs. Besides its originally recognized pro-survival function during spermatogenesis, BCL-w has been envisaged in different types of normal and diseased cells as an anti-apoptotic protein. BCL-w contributes to survival of senescent and drug-resistant cells. Its non-apoptotic role in the promotion of cell migration and invasion has also been elucidated. Growing evidence indicates that a high BCL-w level can be therapeutically relevant in neurodegenerative disorders, neuron dysfunctions and after small intestinal resection, whereas BCL-w inhibition can be beneficial for cancer patients. Although several drugs and natural compounds can bi-directionally affect BCL-w level, agents that selectively target BCL-w are not yet available. This review discusses current knowledge on the role of BCL-w in health, non-cancerous diseases and cancer.

In addition to its pro-survival function, BCL-w plays a non-apoptotic role in regulation of cell motility and senescence.
• The role of BCL-w has been demonstrated in many types of normal cells and diseases, including disorders of nervous system and cancer.
• A plethora of regulators involved in the control of BCL2L2 expression determine cellular and developmental contexts of BCL-w level and activity.

Open questions
• How unique is the apoptotic and non-apoptotic role of BCL-w compared with other members of the BCL-2 family of proteins?

Introduction
The balance between pro-survival and pro-apoptotic proteins is important for tissue development and homeostasis, while impaired apoptosis contributes to several pathologies and can be a barrier against effective treatment 1,2 . Proteins from the B-cell lymphoma-2 (BCL-2) family are essential integrators of signals that trigger cell survival or apoptosis, while cell fate depends on the abundance, localization, and interactions between particular BCL-2-like proteins 3 . The BCL-2 family members are classified based on the structure and structure-related function. Anti-apoptotic members of this family, BCL-2 itself, B-cell lymphoma-extra-large (BCL-X L ), B-cell lymphoma-w (BCL-w), BCL-2-related protein A1/BCL-2related isolated from fetal liver-11 (A1/BFL-1) and myeloid cell leukemia-1 (MCL-1) 4,5 share four BCL-2homology (BH) domains (BH1-BH4), but A1/BFL-1 and certain isoforms of MCL-1 lack the BH4 domain ( Fig. 1a) 6 . The pro-apoptotic proteins such as BCL-2associated X protein (BAX) and BCL-2 antagonist/killer (BAK) possess BH1-BH3 motifs 4,5,7 . In addition to their non-canonical roles 8,9 , BAX and BAK directly execute mitochondrial outer membrane permeabilization (MOMP), which is usually considered a point of no return in an apoptotic cascade 10 . The proteins of the third subclass (BH3-only proteins) share exclusively the BH3 domain. BCL-2-interacting mediator of cell death (BIM), p53-upregulated modulator of apoptosis (PUMA) and truncated form of BH3-interacting domain death agonist (tBID) are called 'activators' as they can bind to and provoke a conformational change of BAX and/or BAK to induce MOMP. In turn, BH3-only proteins that do not associate with BAX and BAK are named 'sensitizers' 5,11,12 . Regulation of apoptosis by the proteins of BCL-2 family relies on the balance between the activity of anti-apoptotic proteins that leash the 'activators' and MOMP-initiating molecules, and the 'sensitizers' that antagonize the prosurvival members by liberating BAX/BAK and the BAX/ BAK-activating BH3-only proteins (Fig. 1b) 4,5 . This succinct review will address the current understanding of the structure and function of BCL-w and its apoptotic and non-apoptotic role in health and disease.
BCL-w as a pro-survival member of the BCL-2 family of proteins BCL-w is an anti-apoptotic protein that shares the highest sequence similarity (51%) with BCL-X L in comparison to other pro-survival molecules 13 . BCL-w interacts with BAX and BAK, and several BH3-only proteins such as BAD, tBID, BIM, PUMA, BMF, and BIK as shown by co-immunoprecipitation [14][15][16] . Results of the isothermal titration calorimetry indicate a preferential binding of BCL-w to BAX in comparison to its binding to BAK, with K D = 22.9 nM and K D = 114 nM, respectively 17 . The precisely regulated interactions between pro-and anti-apoptotic proteins are possible due to the spatial architecture of the BH1-3 domains. They form the hydrophobic groove responsible for the sequestration capability of pro-survival molecules (Fig. 1a), and structure of the binding site dictates the repertoire of interacting proteins 4,5 . For example, it has been demonstrated that a Gly94 residue within the BH1 domain of BCL-w is critical for BAX inhibition 18 , and a G94E substitution in BCL-w abolishes its cytoprotective function in response to interleukin-3 (IL-3) deprivation 14 . In addition, FXXRXR and R/KXV/IXF motifs in BCL-w enables interaction with protein phosphatase 1α (PP1α) 19 . Consequently, BCL-w forms a complex with PP1α and BAD, which leads to dephosphorylation of BAD upon interleukin-4 (IL-4) deprivation 19 . The interactions between BCL-w and poorly characterized members of the BCL-2 family, BFK 20 and BOP 21 have also been reported. Moreover, α1/2 and α5/6 loops of BCL-w can associate with p53 through p53 DNA-binding domain, which contributes to transcription-independent regulation of cell death 22 . BCL-w also interacts with BH3-like domain of Beclin-1, an autophagy-related protein 23 . Interactions between anti-and pro-apoptotic proteins can be precisely quantified as recently demonstrated by using a fluorescence resonance energy transfer (FRET) assay 24 .
The pro-survival BCL-2-like proteins normally associate with the lipid bilayer of mitochondrial, endoplasmic reticulum (ER) and nuclear envelope membranes via their hydrophobic domains (Fig. 1a) 25 . Accordingly, confocal microscopy and cell fractioning have revealed that BCL-w associates with intracellular membranes 26 , and these interactions are strengthened under stress 27 . It has been demonstrated that in unstressed cells the C-terminal domain of BCL-w is folded back within the hydrophobic pocket, and remains only loosely attached to the mitochondrial membrane. When an apoptotic signal is received, C-terminal arm of BCL-w is released by a ligation of pro-apoptotic BH3-only protein, which consequently promotes a tight interaction between BCL-w and mitochondrion [28][29][30] . Notably, it has been demonstrated that the membrane-inserted pool of BCL-w interacts with BH3-only proteins, whereas BCL-w molecules loosely attached to the mitochondrial membrane are associated with MOMP-inducing proteins 28 . Deletion of C-terminal α-helix increased BCL-w binding affinity for BID-derived BH3 peptide, which indicates that this helix modulated interactions of BCL-w with pro-apoptotic partners by competing for peptide binding to the hydrophobic pocket 27 . More recent study that involved BCL-w in complex with designed ankyrin repeat proteins (DARPins) has revealed, however, greater structural similarity of BCL-w to ligand-free BCL-X L than it was primarily thought 31 . In addition, the BCL-X L C-terminus has also been shown to interact with a hydrophobic groove in the water-soluble form of the protein, however, the Cterminal tail in BCL-X L did not trigger a conformational change and did not contribute to the formation of a tightly bound structure as observed in BCL-w 32 . It has been suggested that increased flexibility of the BCL-w groove area is not determined by the hinge regions, but by the weaker interactions between the α3-α4 and the α5-α6 helical hairpins of BCL-w 31 . Consequently, crucial interactions identified in the ligand-binding area of BCL-X L are weakened or lost in BCL-w 31 , which is in line with previous observations showing weaker interactions between the BH1 domain of BCL-w and BID or BIM in comparison to BCL-X L /BID and BCL-X L /BIM complexes 33 . Identification of BCL-w homodimer has further envisaged a high conformational flexibility of BCL-w. The X-ray crystallography structure has revealed that helices α3 and α4 hinge away from the core of one molecule to cross into another BCL-w protomer. This conformation results in the dimerization-specific exposition of helices α5 and α6 while remaining BH3-binding pocket intact. BCL-w homodimer retains selectivity of binding to BH3only proteins, but the affinity is lower than for monomeric BCL-w as exemplified for BAD binding with K D = 150 nM and K D = 14 nM, respectively 34 . Further research is necessary to delineate how the conformational flexibility of BCL-w is unique compared with other members of the BCL-2 family of proteins, and how it can be exploited in the development of the BCL-w-selective inhibitors.

Regulation of BCL-w level
BCL-w protein, 193-amino acid residues in length ( Fig.  1a) is encoded by BCL2L2, which is located on human chromosome 14 at band q11.2-q12 35 . BCL2L2 consists of two coding exons in addition to two non-coding exons located at the 5′-end 36 . The BCL2L2 promoter is highly conserved between human, mouse and rat, and the minimal promoter region lies within the non-coding exon 1a 37 . Analysis of the rat Bcl2l2 promoter by using phylogenetic approach has revealed putative binding sites for several transcription factors including myocyte enhancer factor 2 (MEF2), erythroblastosis virus E26 oncogene homolog (ETS-1 and ETS-2), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-κB) 37 . A number of signaling pathways and downstream transcription factors have been experimentally validated as the regulators of BCL2L2 expression (Fig. 2), although the contribution of different transcriptional factors largely depends on the cellular and developmental context. BCL2L2 was identified as a target of p65/NF-κB in chronic lymphocytic leukemia cells, which was confirmed in experiments involving BAY110782, an inhibitor of NF-κB 38 . In addition, p65/p52 NF-κB dimer was involved in upregulation of BCL-w in glial-cell-line-derived neurotrophic factor (GDNF)-treated dopaminergic neurons 39 . BCL2L2 transcription was also positively regulated by the β-catenin/transcription factor 4 (TCF4) complex, and overexpression of either dominant-negative TCF4 (TCF4ΔN) or wild-type β-catenin resulted in decreased or increased activity of the BCL2L2 promoter, respectively 36 . The role of secreted Frizzled-related protein 2 (sFRP2) in β-catenin-dependent expression of BCL2L2 has also been reported 40 . Increased BCL2L2 transcription was assessed after stimulation of distal axon with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), used either alone or in combination, and engaged ERK5dependent phosphorylation of MEF2D transcription factor 41 . In addition, BCL-w was the only anti-apoptotic protein regulated by neuronal differentiation 6 factor (NeuroD6/MATH-2) under non-stress conditions, and NeuroD6/MATH-2 also assisted in a proper subcellular localization of BCL-w upon serum deprivation 37 . A positive correlation between expression of BCL2L2 and MET was demonstrated, and c-MET downregulation was followed by a decrease in mRNA level of BCL-w, but not other pro-survival members of the BCL-2 family 42 . Different reports have suggested a cell type-specific contribution of cAMP responsive element binding protein (CREB) to BCL2L2 expression [43][44][45] . A temporal increase of CREB activity in adult visual neocortex was concomitant with an upregulation of anti-apoptotic molecules, including BCL-w 43 . In rat Sertoli cells, CREB was dispensable for 17-beta-estradiol-induced BCL-w expression 44 . In turn, CREB indirectly reduced the BCL-w level in colorectal cancer cells by binding to the promoter of the gene encoding ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1), which contains microRNA-603 (miR-603) within its exon 45 . A few other transcription factors can also indirectly control BCL-w level by affecting expression of miRs involved in BCL-w downregulation (Fig. 2). GATA-binding protein 4 (GATA-4) inhibited expression of miRs from the miR-15 family, including miR-15b, miR-16 and miR-195, and consequently promoted BCL-wdependent survival of mesenchymal stem cells 46 . On the contrary, c-MYC upregulated the miR-15 family members responsible for the suppression of BCL-w expression, and this effect was independent of p53 47 . An indirect role of p53 in the control of BCL-w level was, however, demonstrated during genotoxic stress as a human ortholog of males absent on the first (hMOF)-mediated acetylation of Lys120 residue in p53 was essential for p53dependent processing of miR-203, which downregulated BCL-w level 48 .
A number of other miRs were identified as negative regulators of BCL-w level by binding to the 3'-untranslated region (3'-UTR) of BCL-w transcript (Table 1). In addition, long non-coding RNA (lncRNA) RP11-436H11.5, which functions as a competitive endogenous RNA, was able to sponge miR-335-5p and in turn upregulate BCL-w level 49 . The sponging activity was also demonstrated for lncRNA FTX, which controlled miR-29b-1-5p-dependent BCL-w transcript level in mouse cardiomyocytes 50 , while a lncRNA small nucleolar RNA host gene 1 (SNHG1) sponged miR-195 in human cardiomyocytes 51 . Several splicing-and translationregulating factors have been involved in the processing of BCL-w mRNA. Upstream sequence element (USE) in the 3'-UTR of BCL-w transcript contributed to 3'-end formation via interaction with splicing factors: U2 small nuclear RNA auxiliary factor 1 (U2AF35), U2 small nuclear RNA auxiliary factor 2 (U2AF65) and heterogeneous nuclear ribonucleoprotein I (hnRNPI) 52 . In addition, downregulation of serine and arginine-rich splicing factor 2 (SRSF2) was associated with decreased BCL-w transcript level 53 . It was also demonstrated that multiple copies in T-cell lymphoma-1 (MCT-1) protein  Table 1.

Role of BCL-w in normal cells and non-cancer diseases
BCL-w has been already detected in a number of solid tissues, including testes, colon, and brain, as well as in cells of myeloid and lymphoid origin 26,35 . Mice that lacked BCL-w were viable, exerted normal appearance and most of their tissues exhibited typical histology. However, the males were infertile in contrast to female mice that could efficiently reproduce. It was observed that the seminiferous tubules of BCL-w-deficient male mice contained apoptotic cells, and the numbers of both Sertoli cells and germ cells were reduced 55,56 . Further studies confirmed the essential contribution of BCL-w to spermatogenesis 26 , and demonstrated that BCL-w was largely expressed in Sertoli cells 57,58 , Leydig cells, spermatogonia, and spermatocytes 57 . Elevated levels of BAX/BCL-w and BAK/BCL-w complexes were found in most of these types of cells 57 suggesting a functional significance of BCL-w in their survival. Accordingly, BCL-w promoted survival of mouse post-mitotic Sertoli cells by suppressing BAX-dependent apoptotic activity 59 . It was demonstrated that BCL-w-dependent survival of germ cells was regulated by stem cell factor (SCF), which simultaneously downregulated expression of pro-apoptotic members of the BCL-2 family, including BAX 60 , while decreased BCLw protein levels were assessed in testes from cigarette smoke-exposed rats 61 . Interestingly, BCL-w overexpression impaired spermatogenesis as it prevented from entering cell cycle 62 . Testes of transgenic mice that overexpressed BCL-w exhibited degeneration of spermatocytes, vacuolization of Sertoli cells and reduced number of spermatogonia 62 . This indicates that temporal and spatial expression of BCL2L2 can be essential for normal development and function of testes. BCL-w has also been shown to contribute to survival of epithelial cells in the gut 26 . BCL-w protected small intestine-and midcolon-derived epithelial cells from apoptosis induced either by 5-fluorourcil (5-FU) or gamma-irradiation, although spontaneous cell death was not substantial upon loss of BCL-w in these cells 63 . In addition, BCL-w promoted enterocyte survival after massive small bowel resection, and the role of epidermal growth factor (EGF) was implicated in this process 64 . The activation of epidermal growth factor receptor (EGFR) decreased BAX/BCL-w ratio, which shifted the balance to cell survival [64][65][66] . Accordingly, poor survival and impaired adaptation after the resection of small bowel were observed in either BCL-w null or EGFR-deficient mice 66 suggesting that manipulation of EGF-EGFR-BCL-w pathway might be therapeutically relevant in patients after massive resection of small intestine.
A stage-dependent increase in BCL-w transcript level has been reported during the development of rat brain 67 . The high levels of BCL-w were assessed in several regions of the mature brain, including cerebellum, hippocampus, and sensory neurons, whereas BCL-X L was abundantly expressed during early stages of development 67,68 . At the molecular level, both serine and glycine could selectively upregulate BCL2L2 expression in neuronal cells, while retaining BCL-X L level unaltered 69 . BCL-w also controlled the mitochondria morphogenesis and dendrite development in Purkinje cells, and was involved in synapse formation in mouse cerebellum. In this context, BCL-w did not determine number of cells in the brain, but promoted mitochondrial fission in Purkinje dendrites, which was also shown in vivo as BCL-w null mice displayed a marked increase in mitochondrial length 70 . The role of BCL-w has also been demonstrated in several disorders of neuron functions and neurodegenerative diseases. Increased expression of BCL-w was found in ischemic brain suggesting a neuroprotectant role of this protein 71,72 . Accordingly, study on the rat model revealed that overexpression of BCL-w significantly improved neurological functions after focal cerebral ischemia in up to 40% animals 73 . In this respect, also indirect manipulation of BCLw level could attenuate ischemic damage of the brain as exemplified by inhibition of miR-497 (Table 1), which was involved in downregulation of BCL-w and neuronal death after ischemia 74 . In addition, upregulation of BCL-w as a result of intravenous delivery of miR-15a/16-1 antagomir or miR-15a/16-1 knockout, reduced size of cerebral infarct and improved sensorimotor deficits in a middle cerebral artery occlusion (MCAO) mice 75 . Using a rat model of transient MCAO and oxygen-glucose deprivation in neurons, it was demonstrated that miR-29b contributed to cell death following ischemic injury as it inhibited BCL-w 76 . Protein level of BCL-w was also affected in the hippocampus after seizures 77 . BCL-w was upregulated following brief electroshock seizures, whilst it was bound to BIM and integrated in the mitochondrial membrane in damaged subfields after status epilepticus 77 . Moreover, epileptic seizures induced more significant nuclear fragmentation and hippocampal damage in BCL-w-deficient mice compared with wild-type controls 77 . In addition, an increased BCL-w protein level associated with punctate intracytoplasmic structures was found in a model of Alzheimer's disease, in contrast to low level and diffuse distribution of BCL-w in control cases 78 . Mechanistically, it was shown that overexpression of BCL-w protected neurons from β-amyloid-induced cell death by blocking mitochondrial release of Smac, as accumulation of β-amyloid has been proposed as a key factor of neuron loss in Alzheimer's disease 78,79 . In turn, β-amyloid reduced BCL-w protein level via c-JUN N-terminal kinase (JNK)-dependent mechanism 79 , whilst hyperactivation of AKT could counteract β-amyloid-mediated downregulation of BCL-w and cytotoxicity 80 . Neurotoxicity of β-amyloid was substantially attenuated through manipulation of the BCL-w level by β-asarone, a natural compound isolated from Acorus tatarinowii Schott (Table 2) 81,82 . BCL2L2 expression was also significantly higher in Parkinson's disease patient-derived dopaminergic neurons harboring mutant PARK2 83 . In turn, BCL2L2 was hypermethylated and expressed at lower levels in multiple sclerosis-affected brain samples than in controls 84 . The role of BCL-w was also implicated in the viability of nociceptors as BCL-w knockout mice developed the symptoms of small fiber sensory neuropathy, including a decline in sensitivity to thermal stimuli and reduced innervation within the epidermis 85 . BCL-w level was increased in axons of sensory neurons, and cells deprived of BCL-w exerted mitochondrial dysfunctions such as abnormal size and membrane potential, and low level of intracellular ATP 85 . A forkhead box O3 (FOXO3a)/c-JUN-dependent upregulation of PUMA followed by inhibition of BCL-w was necessary to initiate axon degeneration 86 . More recently, it was shown that BH4 domain of BCL-w interacted with inositol 1,4,5-trisphosphate receptor 1 (IP 3 R1) and protected axons from degeneration 87 . This cytoprotective mechanism could be impaired by chemotherapeutics used in the treatment of cancer patients as shown for paclitaxel. Paclitaxel diminished the level of RNA-binding protein splicing factor proline and glutamine rich (SFPQ) and reduced translation of BCL-w transcript. As a consequence, deregulation of IP 3 R1 triggered neuronal degeneration associated with mitochondrial dysfunction and calpain-dependent proteolysis, which largely contributed to the chemotherapy-induced peripheral neuropathy 87 .
Several reports have revealed the putative contribution of BCL-w in other types of cells. Abundant expression of BCL2L2 was found within the whole epithelium and blood vessels of pterygium, in contrast to the presence of BCL-w protein predominantly in the basal layer of epithelium in normal conjunctiva 88 . Recently, it was also shown that BCL2L2 overexpression contributed to the survival of megakaryocytes and increased formation of platelets 87 . A positive correlation between platelet numbers and BCL-w transcript levels in platelets was assessed in 154 healthy donors 89 . A fundamental role of BCL-w was also reported in the survival of B lymphocytes, as a loss of BCL-w substantially accelerated cell death upon deprivation of growth factors 47 . It was also demonstrated that BCL-w prevented from osteogenic differentiation of human mesenchymal stem cells 90 .

Contribution of BCL-w to survival of cancer cells and their response to anti-cancer drugs
Elevated level of BCL-w has been assessed in various types of cancers 91 , but survival of different types of cancer cells does not predominantly rely on BCL-w as exemplified for acute myeloid leukemia 92 and melanoma 93 . Only few genetic alterations of BCL2L2 have been detected in cancers, including copy-number variations in small 94 and non-small 95 cell lung cancer, and a 3'-UTR variant (rs1950252) that was significantly associated with the risk of oral cancer 96 . In a large-scale analysis of somatic copynumber alterations, BCL2L2 has been, however, classified as neither deleted nor amplified across different types of human cancers 97 . This suggests that increased level of BCL-w is rather a consequence of abnormal activation of cancer-related signaling pathways, and BCL-w cooperates with oncogene activation in development and progression of cancer.
A significantly higher level of BCL-w was assessed in gastric adenocarcinomas compared with normal neighboring mucosa, and BCL-w was associated with infiltrative morphotype of the tumor 98 . BCL-w level was also associated with poor survival of patients with colorectal cancer 99 . BCL-w was expressed at low levels in colorectal adenomas, while the majority (92%) of adenocarcinomas showed positive staining for BCL-w 100 suggesting the contribution of BCL-w to cancer progression. This was also supported by higher BCL-w level in samples with node involvement, and in TNM stage III tumors compared with TNM stage II specimens 100 . BCL-w inhibited cell apoptosis by precluding activation of stress-activated protein kinase (SAPK)/JNK in gastric cancer cells 98 . A high level of BCL-w in colorectal cancer cells was related to a loss of SMAD family member 4 (SMAD4) 99 . Downregulation of BCL-w increased ionizing radiation (IR)induced cytotoxicity in human colorectal cancer cell lines 45 . BCL-w conferred resistance to 5-FU 99 . BCL-w protein level was also increased in doxorubicin-resistant colon cancer cells, while the BCL-w inhibition partly reversed resistant phenotype 101 .
Hypomethylation status of BCL2L2 was frequently observed in patients with glioblastoma multiforme (GBM), which exerted a high proliferation index and low sensitivity to apoptosis 102 . Consequently, expression of BCL2L2 was significantly higher in GBM than in lowgrade gliomas 103,104 , and BCL-w was involved in an aggressive phenotype of glioblastoma cells associated with specificity protein 1 (Sp1)-dependent expression of stem cell-related markers 104 . In addition, conditioned medium from the culture of BCL-w-overexpressing cells promoted tumorigenicity of GBM, which was associated with elevated levels of SRY-box 2 (SOX-2), NANOG, octamerbinding transcription factor 4 (OCT4), Nestin, NOTCH2, Musashi and CD133 103 . Increased BCL-w level was accompanied with upregulation of platelet-derived growth factor alpha (PDGFα) 103 . BCL-w overexpression also promoted formation of neurospheres 105 . BCL-w was required for tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-dependent protection of glioblastoma cells against TRAIL and camptothecin 106 . Downregulation of BCL-w accompanied neurotensin receptor-1 (NTSR1) inhibition-induced mitochondrial apoptosis in glioblastoma cells, while restoration of BCLw expression rescued these cells to certain extent 107 .
BCL-w mRNA level was also significantly higher in breast cancer specimens than in adjacent normal cells 103,108 . In addition, the level of BCL-w transcript was higher in plasma of patients with metastatic disease compared to that of patients with primary tumors 103 . BCL-w facilitated proliferation of breast cancer cells through a mechanism involving lncRNA HOX transcript antisense RNA (HOTAR)-dependent sequestration of miR-206, which downregulated BCL-w 108 . Moreover, BCL-w was implicated in resistance of breast cancer cells to radiotherapy. BCL-w was induced in response to IR via a mechanism involving hypermethylation of CpG islands within miR-205-5p promoter, which resulted in the upregulation of BCL2L2 in both in vitro and in vivo models 103 . IR-induced BCL-w contributed to mesenchymal traits of cancer cells, and supported different phenotypes, including angiogenic, migratory, and stem cell-like phenotype 103 .
BCL-w promoted survival of non-small cell lung cancer cells 109 , and its overexpression was significantly associated with advanced tumor stage 95 . BCL-w level was higher in paclitaxel-resistant than in paclitaxel-sensitive non-small cell lung cancer cells, and miR-107-dependent downregulation of BCL-w sensitized resistant cells to the drug 110 . BCL-w level also determined the extent of lung cancer cell response to cyramza, a drug used for inhibition of vessel formation 111 . This might be related to the role of BCL-w in tumor angiogenesis. It was demonstrated in a mouse model of melanoma that blood vessel formation was enhanced upon interactions between endothelial cells (ECs) and pericytes as pericytes promoted EC survival via paracrine integrin α V -and NF-κB-dependent regulation of gene expression in endothelial cells, including BCL-w 112 . In addition, knockdown of BCL-w increased sensitivity of melanoma cells to tetrathiomolybdate (TTM), which is a copper chelator 113 .
BCL-w has been associated with malignancy of urinary system. BCL-w protein level was substantially higher in bladder tumor cells than in adjacent normal cells 114 , which was also confirmed in a cohort of 41 bladder cancer samples 115 . High level of BCL-w accompanied bladder cancer progression, and downregulation of BCL-w sensitized cells to cisplatin 116 . Notably, BCL2L2-PABPN1 chimeric RNA, which was generated by cis-splicing of adjacent genes, was detected at significantly higher level in bladder cancer specimens than in normal cells. Additionally, BCL2L2-PABPN1 RNA was preferentially detected in the nuclear fraction suggesting the role as a lncRNA 117 . BCL-w also showed significantly higher expression in metastatic clear cell renal cell carcinoma than in primary tumor cells 118 , which is consistent with the study demonstrating that overexpression of BCL-w increased the proliferation rate and invasion of these cancer cells 119 . The role of BCL-w has been also implicated in survival of other types of cancers and their response to drugs. Expression of BCL2L2 was significantly higher in cervical tumor samples compared with normal cervix tissue 120 . Downregulation of BCL-w reduced cell survival and attenuated resistance of cervical cancer cells to cisplatin 120 , and accelerated paclitaxel-induced mitotic cell death in vitro 121 . BCL2L2 was selectively upregulated in samples of endometrial cancer representing G2 histological stage 122 . Downregulation of BCL-w enhanced serum deprivation-induced apoptosis in osteosarcoma cells 123 , while increased level of BCL-w protein accompanying overexpression of miR-196a promoted survival of osteosarcoma cells in vitro 124 . A significantly higher BCL-w protein level was also assessed in leiomyosarcomas in comparison to benign uterine smooth muscle tumors, and BCL-w expression reversely correlated with overall patient survival 125 . BCL-w protein level was also increased in advanced prostate cancer cell lines, which might result from epigenetic silencing of miR-205 expression, and conferred resistance to cisplatin and docetaxel 126 . In addition, a high expression of BCL-w rendered resistance of ovarian cancer cells to cisplatin, and BCL-w knockdown significantly reduced size of tumor derived from cisplatin-resistant cells 127 . Downregulation of BCL2L2 resensitized ovarian cancer cells resistant to etoposide (VP-16) 128 . Recently, BCL2L2 has been correlated with drug resistance of high-grade serous ovarian cancer (HGSOC) cells 129 . BCL-w protein level was also markedly higher in hepatocellular carcinoma (HCC) cells resistant to 5-FU compared with matched drug-sensitive cells 130 . miR-122dependent downregulation of BCL-w rendered HCC cells sensitive to adriamycin and vincristine 131 , while inhibition of BCL-w and BCL-2 as a result of cyclooxygenase-2 (COX-2) silencing potentiated TRAIL-mediated apoptosis in HCC cells 132 . In head and neck squamous cell carcinoma cells, cisplatin-induced miR-630-dependent downregulation of BCL-w was reported 133 . A high expression of BCL2L2 was assessed in diffuse large B-cell lymphoma (DLBCL) and in almost 90% of patients with Burkitt lymphoma (BL). BCL-w knockdown induced apoptosis in Burkitt lymphoma cells whilst BCL-w overexpression conferred resistance to ABT-737 and ABT-263, BH3 mimetics targeting BCL-2-like proteins 47 . Downregulation of BCL-w markedly delayed MYC-mediated development of B-cell lymphoma 47 . In another report, however, BCL-w was expressed at high level only in a subset of BL and DLBCL cell lines. Moreover, CRISPR/ CAS9 gene editing or RNA interference leading to downregulation of BCL2L2 expression did not sensitize lymphoma cells to apoptosis, even when these cells were exposed to BH3 mimetics 134 . It has been also demonstrated that BCL-w, in addition to BCL-2 and BCL-X L , played a minor role in the development of sarcoma and thymic lymphoma in p53-deficient mice 135 . BCL-w was highly expressed in patient-derived B-cell chronic lymphocytic leukemia (B-CLL) cells in comparison to normal peripheral blood lymphocytes 136 . BCL-w was also involved in autocrine exosome-mediated regulation of chronic myeloid leukemia cell survival 137 .

Role of BCL-w in migratory and invasive potentials of cancer cells
Pro-survival proteins from the BCL-2 family have been shown to contribute to migratory and invasive capabilities of normal and cancer cells 138,139 , and the role of BCL-w to this process has been delineated. It was reported that ectopic BCL2L2 expression almost fully nullified the inhibitory effect of miR-335 on migration and invasion of ovarian cancer cells 140 . BCL-w potentiated mesenchymal phenotype of GBM cells 141,142 , and regulated the invasion capability of human gastric cancer cells 143 . BCL-w enhances the migratory and invasive potentials of gastric cancer cells by facilitating the production of several types of extracellular matrix (ECM)-degrading proteinases 126 . Secreted matrix metallopeptidase-2 (MMP-2) and urokinase plasminogen activator surface receptor (uPAR) have been demonstrated to activate focal adhesion kinase (FAK), which acts as an executioner of BCL-w-dependent invasive phenotype of gastric cancer cells 144 . Mechanistically, BCL-w increases the level of mitochondriaderived reactive oxygen species (ROS), which is followed by SRC-mediated phosphorylation of EGFR 145 , and the activation of PI3K/AKT/Sp1 signaling pathway to increase MMP2 expression in GBM and gastric cancer cells 18,104,146 . BCL-w promotes activation of MMP-2 and FAK via PI3K/AKT/β-catenin signaling pathway in GBM cells 105,142 , while BCL-w-induced nuclear accumulation of β-catenin contributes to the upregulation of vimentin ( Fig. 3) 141,142 . Notably, BCL-w-mediated BAX inhibition is essential for cell invasion as a variant of BCL-w (BCLw G94A ) that does not bind to BAX failed to stimulate ROS production and cell invasion 18 as well as cancer cell intravasation in an in vivo model of lung cancer 147 .
On the contrary, several mechanisms to counteract BCL-w-dependent cell invasion and motility have been evidenced. The PI3K/AKT/MMP-2 signaling pathway involved in cell invasion-promoting activity of BCL-w is inhibited by brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like (Bmal1) in GBM and lung cancer cells 148 . In addition, cytosolic p53 liberates BAX from BCL-w and suppresses non-small cell lung cancer cell invasion by attenuation of ROS production 147 . This is driven by BAX-dependent inhibition of NADH: ubiquinone oxidoreductase core subunit 5 (ND5), a subunit of respiratory complex I 147 . Simultaneously, nuclear p53 augments the pool of BAX molecules via executing transcription of BAX 147 . The inhibitory role of p21 in the regulation of BCL-w-dependent lung cancer, colon cancer, and neuroblastoma cell invasion has been demonstrated in addition to p53 149 . Although p53 and p21 can bind to BCL-w independently, the triple p53/p21/BCL-w complex is required for BAX release from BCL-w and suppression of cell invasion (Fig. 3) 149 .

Role of BCL-w in cellular senescence
Cellular senescence is a form of cell cycle arrest that can develop in response to DNA damage, nutrient deficiency, telomere shortening, oxidative stress, and oncogene activation. Senescence induction is often executed as a barrier against tumorigenesis, but senescent cells can produce growth factors and cytokines, collectively named as the senescent-associated secretory phenotype (SASP), which can promote tumor development 150 . It was demonstrated that co-inhibition of BCL-w and BCL-X L by specific siR-NAs or by a BH3 mimetic (ABT-737) induced apoptosis in senescent human fibroblasts in vitro 151 . This observation was further validated in an in vivo model, and ABT-737 efficiently eliminated epidermal cells exhibiting senescent features triggered by DNA damage or p14 ARF -p53 activation 151 . More recently, BCL-w contribution to senescent phenotype has also been evidenced in GBM and lung cancer cells 152 . BCL-w promotes senescenceassociated β-galactosidase (SA-β-gal) activity and trimethylation of histone H3, as well as expression of genes encoding senescence-related proteins including p53, p21, and p16 152 . It has also been shown that overexpression of miR-93-5p in GBM and lung cancer cells is sufficient to prevent from premature senescence through downregulation of BCL-w and p21 152 .

Concluding remarks and future perspectives
BCL-w with diverse functions in development, health, and disease, can play both positive and negative roles in the particular process or cellular context. BCL-w is an attractive therapeutic target as its inhibition might be relatively well-tolerated in patients. This is supported by studies showing that loss of BCL-w was associated with defects in spermatogenesis and small intestine cells in mice but had no deleterious effects in the majority of other tissues 56,58,63 . The contribution of BCL-w to differentiation of lymphocytes has appeared questionable as BCL2L2-knockout mice exhibited unaffected lymphoid development 55 , probably as a result of low level of BCL-w in normal and malignant lymphoid cells 26 . Further research is necessary to determine an unequivocal role of BCL-w in these cells in the light of conflicting results of more recent reports 47,134 . Notably, the redundant role of BCL-w is in sharp contrast to other pro-survival members of the BCL-2 family that have been shown essential during embryogenesis, development of nervous system and hematopoiesis as exemplified especially by BCL-2, MCL-1 and BCL-X L 153-155 . Thus, observations from experiments using knockout mice have provided an overview of the loss-of-function phenotypes that may have an impact on prediction of clinical applications of the drugs that inhibit Cell motility and invasion are promoted by BCL-w-mediated inhibition of BAX that enables the mitochondrial complex I to produce ROS (green background). ROS activate signaling cascade involving SRC-EGFR-PI3K/AKT, which engages transcriptional regulators, β-catenin/TCF4 and Sp1, to upregulate expression of genes encoding pro-invasive and pro-migratory molecules, including MMP-2, uPAR, and vimentin (vim). On the contrary, cell motility and invasion are attenuated upon substitution of BAX for cytosolic p53 and p21 in the BAX/BCL-w complex (red background). BAX resides in the outer mitochondrial membrane, and the topology allows C-terminal four residues of BAX (KKMG) to interact with a subunit of complex I in the inner membrane of mitochondria, which is followed by inhibition of ROS production. BAX-dependent attenuation of ROS synthesis is potentiated by nuclear p53-driven transcription of BAX that contributes to increased BAX protein abundance.
activity of specific pro-survival proteins. Consequently, while tissue-specific BCL-w inhibition can be beneficial to overcome therapy resistance of cancer patients, increasing BCL-w level might be therapeutically relevant in a number of neurological disorders and after small intestinal resection (Fig. 4). In addition, the role of BCL-w in sustaining the survival of senescent cells suggests that manipulating BCL-w can be an useful approach in agerelated disorders. To not disturb overall organismal homeostasis and limit unwanted drug cytotoxicity, it is essential to define actual cell dependence on specific antiapoptotic protein eg., BCL-w. In this respect, BH3 profiling can be used to identify protein(s) that must be inhibited to efficiently execute MOMP 156 while Dynamic BH3 profiling, which has been established more recently as an alternative functional approach, allows to measure cell dependence that can be altered in response to drugs 157 . For the time being, there are no drugs that selectively affect BCL-w level, which might be associated with a high conformational flexibility of this protein.
Several drugs and natural compounds have been shown to affect BCL-w level in in vitro and in vivo models of different diseases (Table 2), however, BCL-w is not their exclusive target. Two BH3 mimetics, ABT-737 and its orally bioavailable derivative ABT-263, represent agents that inhibit BCL-w activity 5,158 . As both compounds mimic BAD, they neutralize BCL-2 and BCL-X L in addition to BCL-w 5,159 . Moreover, it has been demonstrated that ABT-737 displaces BIM from BCL-w with much lower efficiency than from other pro-survival proteins 160,161 suggesting that cellular effects induced by ABT-737/ABT-263 could predominantly result from BCL-2 and BCL-X L inhibition. For that reason, further research directed to the development of selective drugs either upregulating or inhibiting BCL-w is still needed. Fig. 4 An overview of cell type-specific roles of BCL-w. BCL-w is broadly expressed in many types of normal cells as well as diseased cells, in which either increased (red background) or decreased (green background) BCL-w levels are assessed. In addition to the pro-survival role exerted in health and disease, BCL-w regulates additional cell programs and functions (color frames). Consequently, drugs that either decrease or increase BCL-w level and activity can exhibit therapeutic relevance against different disorders.