Therapeutic strategies involving survivin inhibition in cancer

Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.


| IAPs function and structure
The IAP family embraces a functionally and structurally related group of proteins that function as endogenous cellular inhibitors of apoptosis in response to daily stresses and insults. Due to its relevance as negative regulators of programed cell death, a dysregulation of IAPs is closely related to cancer development and drug resistance. 1 Structure of all mammalian IAPs members contains one to three defining baculovirus IAP repeat (BIR) domains encoding a zinc-finger motif typically arranged at the N-terminus of the protein ( Figure 1A). Although the BIR domain is shared by all members of the IAP family, not all BIR-containing proteins exhibit antiapoptotic functions. 2 This domain mediates protein recognition and protein-protein interactions. 3 In addition to the BIR domain, several IAP family members contain a zinc-finger domain called RING (really interesting new gene), which functions as an E3 ubiquitin ligase, an ubiquitin-associated (UBA) domain, a ubiquitin-conjugating (UBC) domain, and/or a caspaserecruitment domain (CARD).
Among all the IAPs, survivin is the smallest member of the IAP family. Encoded by BIRC5 gene (baculoviral inhibitor of apoptosis repeat-containing 5), mapped to chromosome 17q25, survivin is a 16.5 kDa protein that contains only a single N-terminus BIR domain linked to a C-terminal α-helical coiled-coil domain. The latter domain is important for the interaction and formation of the chromosomal passenger complex (CPC), proper segregation of chromosomes, and cytokinesis during cell division. 4,5 A dimerization domain at two different locations in the linear sequence of survivin allows it to form a stable homodimer that seems to carry out its mitotic activity, while the monomeric form of survivin is mostly associated with its antiapoptotic activity 6,7 ( Figure 1B and 1C). Moreover, different functions of survivin seem to be affected by differential subcellular localization, being the nuclear survivin related to cell division regulation while cytosolic survivin is believed to function as apoptotic suppressor ( Figure 2). 8 Additionally, a small pool of survivin can be found in the mitochondria, from where is rapidly released to the cytosol in response to cell death stimulation and confers resistance to apoptosis. 9 Although the overexpression of survivin inhibits both extrinsic and intrinsic apoptosis pathways contributing to cancer progression, the exact molecular mechanism remains unknown. It has been reported a direct binding of survivin to effector caspases, but a prevailing model is that survivin inhibits apoptosis by interacting with other proteins (Figure 2). One of these interactions is a complex formation between survivin and X-linked inhibitor of apoptosis protein (XIAP). 10 This IAP-IAP complex enhances XIAP stability against ubiquitin-dependent degradation, increasing in this way the ability of XIAP for inhibiting caspases. 11 Another mechanism proposed is the formation of the complex between survivin and hepatitis B X-interacting protein (HBXIP), which binds to procaspase 9 preventing its recruitment to the apoptosome and later activation. 12

| Survivin as a promising therapeutic target
Due to its dual role both in cell cycle promotion and apoptosis inhibition, survivin has been considered an ideal target for anticancer therapy. Indeed, several molecular approaches that block survivin expression and/or function are emerging as promising therapeutic strategies in cancer. 13 Remarkably, survivin is almost undetectable in most normal differentiated tissues, but prominently expressed in most cancer malignancies. 14 This expression pattern provides selectivity over the tumor cells, thus decreasing the potential side effects in treated patients. Moreover, survivin overexpression has already been correlated with tumor prognosis, being considered a biomarker with a negative correlation on patient clinical outcome and drug resistance in many cancers. 15 Altogether, survivin overexpression in tumors and its key biological roles promoting carcinogenesis and chemoresistance, makes survivin a promising therapeutic target. F I G U R E 2 Survivin subcellular functions and direct survivin inhibitors. The main mechanisms of action of direct survivin inhibitors are shown. 1, Transcriptional inhibitors decrease survivin expression levels; 2, SMAC mimetics impede that survivin inhibits caspases; 3, Hsp90 inhibitors destabilize the Hsp90-survivin complex, inducing survivin degradation. 4, Homodimerization inhibitors disassemble survivin homodimer, increasing survivin degradation. 5, Mitotic inhibitors prevent CPC complex formation and survivin interaction with other mitosis-related proteins. BIRC5, baculoviral inhibitor of apoptosis repeat-containing 5; CPC, chromosomal passenger complex; HBXIP, hepatitis B X-interacting protein; SMAC, second mitochondria-derived activator of caspase; XIAP, X-linked inhibitor of apoptosis protein [Color figure can be viewed at wileyonlinelibrary.com] Since survivin has emerged as an ideal target for cancer drug discovery, many survivin inhibitors have been reported in the literature. Some of them are specific direct inhibitors ( Figure 2 and Table 1) but others do not   directly bind and interact with survivin protein itself, instead, they usually target other biomolecules and ultimately reduce survivin expression ( Figure 3 and Table 2). In the following sections, we discuss the available information about survivin inhibitors developed so far, as well as the direct or indirect mechanisms by which they repress survivin. YM155 was the first identified small-molecule inhibitor that targets and suppresses specifically the activity of the survivin promoter, regardless of p53 status. 16 Interestingly, it suppressed survivin gene promoter and induced apoptosis in prostate cells as well as promoted tumor regression in human prostate PC3 ectopic xenograft tumors. 16 Other preclinical studies showed the promising anticancer properties of YM155 against a panel of 119 human cancer cell lines with an average inhibitory concentration (IC 50 ) of 15 nM. Moreover, continuous 3-day or 7-day YM155 infusion (1-10 mg/kg) in xenograft model demonstrated significant antitumor activity without significant toxicity, measured through bodyweight loss. 17 Phase I study reported YM155 as a well-tolerated anticancer drug that showed some efficacy against blood cancers. 18 However, in phase II studies, YM155 showed modest single-agent activity against non-small-cell lung carcinoma (NSCLC), but with a disease control rate similar to another second-line agents for advanced NSCLC. 19 The combination of YM155 with carboplatin and paclitaxel also exhibited a favorable safety profile but failed to demonstrate an improvement in response rate in advanced NSCLC. 20 However, despite having evidence supporting that YM155 can antagonize survivin expression, recent data support that YM155 is a DNA damaging agent where suppression of survivin is a secondary event, probably a consequence of transcriptional repression. 21 Similarly, other study also suggests that inhibition of survivin occurs via suppression of EGFR signaling and its downstream factors. 22 Therefore, YM155 may not be only considered a specific survivin inhibitor. In contrast, a recent study identified other compound targeting survivin by highthroughput screening of chemical libraries following in vitro and in vivo analysis. 23 This compound, named FL118, is a nonselective small-molecule inhibitor of survivin expression that structurally resembles the topoisomerase I inhibitor, irinotecan. Its antitumor activity results from inhibiting survivin promoter activity and survivin gene expression. In addition, FL118 also downregulates the expression of myeloid cell leukemia 1 (Mcl-1) and some IAPs, such as XIAP and cellular inhibitor of apoptosis 2 (c-IAP2). 23 FL118 effectively inhibited cancer cell growth at concentrations lower of 1 nM in a p53 status-independent manner. The in vivo studies revealed that FL188 has greater antitumor efficacy without significant toxicity compared with leading first-line chemotherapeutics. 23 In addition to small-molecules targeting survivin gene promoter, antisense oligonucleotides have also been developed to inhibit survivin expression. LY2181308 is one example of single-strand antisense oligonucleotide that targets survivin by binding to and degrading its mRNA preventing its translation into protein and thus, limiting survivin expression. LY2181308 showed significant reduction of both survivin mRNA and protein, as well as cell-cycle arrest, cell-death induction, and tumor growth inhibition in several tumor cell lines and human tumor xenografts. 24 Several clinical studies have been carried out showing a favorable safety profile but mixed clinical outcomes. [25][26][27] As an example, while LY2181308 showed synergistic benefits in patients with refractory or MARTÍNEZ-GARCÍA ET AL. SPC3042 (EZN-3042), another antisense oligonucleotide, was identified as a new agent with higher potency for survivin mRNA inhibition compared with former antisense agents, including LY2181308. 28 However, SPC3042 not only targets survivin mRNA but also has significant effect over B-cell lymphoma 2 (Bcl-2) mRNA. Downregulation of survivin expression using SPC3042 led to cell cycle arrest, pronounced cellular apoptosis and sensitization of prostate cancer cells to taxol treatment, both in vitro and in vivo. EZN-3042 showed, as a single agent, a 60% downmodulation of survivin mRNA in tumors of A549 and Calu-6 lung xenograft models and 37% to 45% of tumor growth inhibition. In addition, when EZN-3042 was combined with paclitaxel, 83% of tumor growth inhibition was achieved. 29 Despite these promising outcomes, phase I trial of EZN-3042 was terminated due its dose-limiting toxicity. 30 Finally, other gene therapy-based approaches are currently being studied, especially in combination with conventional chemotherapeutics. It has recently been demonstrated that small interfering RNA against survivin, combined with temozolomide or etoposide, induced a synergistic cytotoxic effect in glioblastoma cells. 31 Moreover, the combination of the microRNA miR-542-3p mimic that targets survivin in combination with paclitaxel significantly inhibited in vivo tumor growth of HER-2-overexpressing breast cancer cells, overcoming their chemoresistance. 32

SMAC mimetics
Second mitochondria-derived activator of caspase (SMAC/DIABLO) is a proapoptotic protein released from mitochondria upon apoptotic stimulation and promotes cytochrome c-dependent apoptosis by binding to and antagonizing IAPs. In this sense, SMAC/DIABLO binds to XIAP releasing caspase-9 from the complex, leading to apoptosis activation. In contrast, cytosolic survivin is able to bind to SMAC/DIABLO, through its Ala-Val-Pro-Ile (AVPI) peptide-binding region, inhibiting its proapoptotic functions. 33 Furthermore, survivin overexpression can also diminish the proapoptotic functions of SMAC/DIABLO by delaying its release from mitochondria through direct union after apoptotic stimuli. 34 Withanone, a natural product derived from roots of Withania somnifera, was studied as a possible competitor of SMAC/DIABLO for its binding site in survivin protein. Computational docking analysis showed that withanone binds to survivin BIR domain in the same hydrophobic cavity as that of SMAC/DIABLO, therefore being able to

Clinical trials References
Mitosis-related proteins inhibition LLP-3 40 interfere with its inhibitory activity against caspases. 35 Although the anticancer properties of withanone have already been studied in several cancer cell lines, 36 experimental analysis is needed to confirm whether this compound specifically binds and inhibits survivin. Similarly, analogs of the phenolic component of black pepper piperine have also been described as potential survivin inhibitors by binding to the hydrophobic cavity of the BIR domain. 37 Piperine was capable to inhibit cell growth and induce apoptosis in several types of cancer cells, such as human colon cancer cells, 38 and suppressed tumor growth and metastasis in mice models, but more studies are needed to determine whether its anticancer potential is mediated through survivin inhibition.
In a more recent work, using similarity-based virtual screening for the AVPI peptide in the survivin-SMAC crystal complex, UC-112 was identified as a potent and selective survivin inhibitor. 39  All this data, along with the promising outcomes given by other IAP-specific SMAC mimetics, 42 which are under evaluation in early clinical trials both as monotherapy or in rational combination therapies, encourage the study of compounds with this mechanism of action.

Heat shock protein 90-survivin inhibitors
Another well-studied protein interaction is the association between heat shock protein 90 (Hsp90) and survivin.
Hsp90 directly binds, through its N domain, to the BIR domain of survivin. 43 Besides this, it has been studied how the chaperone function of Hsp90 is required to preserve survivin stability in vivo and disruption of this complex by using Hps90 inhibitors triggers loss of survivin via proteasomal-dependent degradation. Blockage of survivin-Hsp90 complex formation promoted apoptosis and mitotic defects in cultured cells. Hence, molecular antagonists of survivin-Hsp90 interaction may provide another rational approach to treat cancer.
Shepherdine, a small peptidomimetic, was rationally designed for this purpose. 44

Survivin homodimerization inhibitors
It is known that monomeric and dimeric forms of survivin coexist, being the homodimeric form more related with the promotion of mitosis by enhancing tubulin stability. 6 Thus, development of specific inhibitors targeting the dimerization site of survivin may be a feasible approach to treat cancer. This strategy becomes more attractive due to studies that showed how exposition of the hydrophobic interface of a dimeric protein often cause conformational changes, which leads to destabilization and degradation of the protein. 46 Furthermore, MARTÍNEZ-GARCÍA ET AL.

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homodimerization interface of survivin is not shared with other IAPs, therefore inhibitors of this site may increase their selectivity against survivin.
Abbott 8 was identified using NMR and affinity-based screening as a small soluble compound that binds to the dimer interface of survivin. 47 Several analogs were further developed reaching compounds with nanomolar affinities, however, more studies are needed to evaluate their anticancer activity.
In this context, using in silico screening targeting the critical dimerization core residues of survivin, LQZ-7 was identified. 48

Mitosis-related proteins inhibitors
Survivin plays an essential role in cell mitosis, including chromosome segregation and cytokinesis, mostly as an integral component of CPC. 49 It is well documented how depletion of survivin causes cell proliferation arrest, sustained prometaphase blockade, chromosomal defects, and cytokinesis failure. 50 Additionally, a distinct pool of subcellular survivin is associated with polymerized microtubules, sustaining their stability, thereby contributing to the bipolar spindle assembly. 50 In this context and using in silico analysis, several hotspot residues related to protein-protein interaction were found in survivin, including its CPC complex interface. 51 Therefore, a pharmacophore model was derived and used In another in silico study, S12, a small molecule that targets a specific cavity adjacent to the survivin dimerization surfaces, was identified. 52 S12 showed disruption of the spindle formation, which led to mitotic arrest of cancer cells, followed by cell death with no apparent toxic effect over nonproliferating and normal resting cells.
Moreover, in vivo studies showed how S12 was able to inhibit tumor growth without producing systemic toxicity in animals. 52 Finally, the importance of the protein complex formation between survivin and the small GTPase Ran (Rasrelated nuclear protein) has also been studied. Ran is implicated in microtubule stability and mitotic spindle assembly in tumor cells. 53 The small-molecule LLP-3, derived from Abbott 8 compound, showed an effective disruption of the survivin-Ran complex, leading to survival and growth inhibition of glioma stem cells, both in vitro and in vivo. 54

| Survivin indirect inhibitors: targeting key cellular signaling pathways
It has already been shown that survivin expression, either through downregulation or upregulation, could be triggered by several signaling pathways. The most actively described are shown in Figure 3 and will be discussed below. Accordingly, inhibiting this pathway has become an important strategy in cancer treatment. In breast cancer, herceptin and lapatinib (HER-2 inhibitors) and AG1478 (EGFR inhibitor) inhibit survivin expression. [63][64][65] In ovarian cancer, similar results were observed with the EGFR inhibitors gefitinib and PD153035. 66,67 However, most reports have studied the inhibition of PI3K through LY294002 and wortmannin in several types of cancer: breast, colon, liver, ovary, lung, and in leukemia. 60,65,[67][68][69][70][71][72] Concerning the inhibition of AKT, MK-2206 strongly blocks survivin in glioma and colon cancer. 73 Moreover, SH5 inhibitor significantly reduces survivin levels in lung cancer 68 and similar results were observed with AKT inhibitor X in prostate cancer. 69 Likewise, mTOR inhibitors, more specifically rapamycin, firmly downregulate survivin in glioblastoma, multiple myeloma, and prostate cancer. 58,74 The induction of survivin was also inhibited by the NF-κB inhibitors, SN50 and BAY 11-7082 in endothelial cells. 61 downregulation of survivin was observed with panepoxydone in breast cancer, and this seems to be related with NF-кB inhibition. 76 Cyclooxygenase-2 (COX-2) induces survivin expression through AKT activation in several cancers (eg, glioblastoma, lymphoma, myeloma, breast, colon, and prostate) and this action could be reversed through its inhibitors, celecoxib and etodolac. 77

Janus kinase/signal transducer and activator of transcription
The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a critical role in cell proliferation, differentiation, migration, apoptosis, and immunity. 78 A wide range of cytokines, hormones, and growth factors, and their respective receptors stimulate the JAK/STAT pathway. 78 JAK activation results in the recruitment and activation of STAT proteins that will then translocate to the nucleus and induce the transcription of target genes, such as survivin. 78 It is known that dysregulation of JAK/STAT pathway participates in cancer development and metastasis. 78 Interleukin 6 (IL-6), induces STAT3 activation leading to an increase on survivin expression in breast cancer cells. 79 In leukemia, the upregulation of survivin involving the JAK/STAT signaling pathway is dependent on GM-CSF 80 and breakpoint cluster region protein (BCR)-Abelson murine leukemia viral oncogene homolog (ABL). 81 Interestingly, after blocking JAK2 kinase with the TG101209 inhibitor, a decrease of survivin is observed in leukemia. 81 Another JAK2 inhibitor known as AG490 82 has shown to downregulate survivin in lymphoma as well as leukemia. Similar results were reported with the JAK2 inhibitor SD-1029 for breast and ovary cancers. 83 Our group has also shown that novel indol-based tambjamine analogs were able to reduce survivin levels by inhibiting JAK/ STAT pathway through an unknown mechanism of action, 84 and unpublished data . Epidermal growth factor (EGF) induces the activation of MAPK/ERK signaling and increases survivin levels. 85 Cytokines such as GM-CSF and the BCR-ABL tyrosine kinase could induce survivin expression via MAPK/ERKdependent mechanism in leukemia. 86 Moreover, HER-2 upregulation of survivin is regulated by MAPK/ERK signaling, apart from the PI3K/AKT contribution in breast cancer. 70 Similar results were observed with c-Harvey rat Abbreviations: ABL, Abelson murine leukemia viral oncogene homolog; AKT, protein kinase B; BCR, breakpoint cluster region protein; CBP, CREB-binding protein; CDK, cyclin-dependent kinases; COX-2, cyclooxygenase-2; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HER-2, human epidermal growth factor receptor 2; JAK, Janus kinase; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; NF-κB, nuclear factor κB ; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; mTOR, mammalian target of rapamycin; STAT, signal transducer and activator of transcription; TGF-β, transforming growth factor-β.

Mitogen-activated protein kinase/extracellular signal-regulated kinase
Correlative numbers correspond to those depicted in Figure 3.
sarcoma viral oncogene homolog (c-H-RAS), where both MAPK/ERK and PI3K/AKT are participating in survivin overexpression. 72 Interestingly, MEK inhibitors significantly reduce survivin levels. In particular, PD98059 in breast cancer 65 and leukemia, and similar results were described with CI1040 in leukemia. 86 Besides this, PD0325901 (a CI1040 derivative) prevented the placental lactogen induction of survivin. 66 The U0126 inhibitor strongly blocks MAPK induction of survivin in leukemia, 86 breast cancer, 70 and keratinocytes. 72 In addition, BCR-ABL inhibition by imatinib significantly hampers MAPK/ERK survivin expression in leukemia cells. 86

| Cell cycle regulation
Survivin is expressed in a cell cycle-dependent way and localizes in the mitotic apparatus. The cyclin-dependent kinases (CDKs) 2/4 are active in both G1 and S phases and phosphorylate retinoblastoma (Rb) relieving it from the E2F transcription factors to allow the cell cycle transition. Survivin is positively regulated by E2F1, E2F2, and E2F3 through cell cycle-dependent element (CDE)/cell cycle genes homology region (CHR) dependent mechanism while is negatively regulated by E2F4 and E2F5 repressors. 87 Moreover, the CDK1/cyclin-B1 pair phosphorylates survivin on Thr34, contributing to its stability and its association with caspases. 88 Some CDKs inhibitors, in addition to arresting the cell cycle, have an interesting ability to induce apoptosis. In addition, CDK inhibitors abrogate survivin phosphorylation on Thr34 leading to its degradation. 89 The specific CDKs inhibitor (purvalanol A) downregulates survivin through JAK/STAT inhibition in gastric cancer. 79 A broad spectrum CDK inhibitor known as flavopiridol inhibits survivin phosphorylation on Thr34, reducing its protein levels in cervical cancer and has shown its antitumor activity in several phase II clinical trials. 90,91 The specific CDK2 inhibitor NU6140 also diminishes survivin expression in cervical and ovary cancers. 88 Finally, cephalochromin is a selective bacterial inhibitor that reduces the levels of CDK2/cyclin-E and CDK4/cyclin-D1 pairs producing cell cycle arrest (G 0 /G 1 phase) and consequently decreasing survivin expression and inducing apoptosis. 92

| Cellular stress: p38MAPK
The p38MAPK signaling is involved in inflammation, cell cycle, cell death, development, differentiation, and senescence. 93 Environmental stresses (eg, UV and heat) and inflammatory cytokines trigger this pathway by phosphorylation of MAPKKKs. These kinases signal the cascade through MKK3/MKK6/p38MAPK which will finally activate transcription factors (eg, p53) that will regulate several target gene expression. 93 Activation of the p38MAPK route by the COX-2 inhibitor celecoxib induces survivin downregulation in colon cancer cells. 94 Likewise, oxaliplatin, a chemotherapeutic drug, leads to colon cancer cell death through survivin downregulation via p38MAPK pathway and via proteasomal degradation. 95

| Development and differentiation
Transforming growth factor-β Transforming growth factor-β (TGF-β) signaling controls several cellular functions including cell growth, proliferation, differentiation, development, and migration. Upon ligand binding, constitutively active TGF-β receptor II (TβRII) recruits and activates by phosphorylation the TβRI. Due to this activity, Smad proteins, SMAD2 and SMAD3, are phosphorylated and form a complex with co-Smad (SMAD4) in the cytoplasm, which is then translocated to the nucleus where it regulates gene expression, including survivin. 96 Although TGF-β is an important tumor suppressor, cancer cells subvert this pathway by taking control of the tumor-promoting arm. 96 Interestingly, an overexpression of TGF-β1 was described in various types of cancer. In addition, inactivating mutations on SMAD2 and SMAD4 were reported in hepatocellular, colorectal, and lung cancer. 96 The activation of the TGF-β pathway in colon cancer causes the hypophosphorylation of Rb via Smad3dependent mechanism, which causes the association of Rb/E2F4 repressive complex to CDE/CHR elements of survivin gene promoting its downregulation. 71,97 Similarly, TGF-β inhibition of PI3K/AKT signaling through protein kinase A (PKA)/A-kinase anchoring protein (AKAP149)/protein phosphatase 2 (PP2A) may also reduce the survivin levels. 71,97 Furthermore, TGFβ activation of PKA, leads also to the phosphorylation of survivin in Ser20 inducing its degradation. 97 Wnt/β-catenin A variety of Wnt and APC mutations and alterations are frequently observed in cancer. 98 It has been reported that TCF/β-catenin induces survivin expression in lung, colorectal, and breast cancers. 63,98,99 Drugs binding to HER-2 receptor, such as Herceptin (trastuzumab) in breast cancer, compromise β-catenin stabilization, and consequently decrease survivin expression. 63 Furthermore, an antibody against Wnt-2 protein, Wnt-2 Ab, induces apoptosis inhibiting this process in lung cancer. 98 Downregulation of β-catenin and survivin expression by ICG-001 (a β-catenin/CBP disruptor) was observed in colon cancer. 100

Notch
Notch signaling is involved in important cellular processes such as proliferation, development, differentiation, and homeostasis. 101 103 In addition, HIF-1α promotes Notch induction of survivin, suggesting that HIF-1-α interacts with NICD facilitating the CSL transcription factor association with a RBP-Jĸ site in the survivin promoter. 102 Aberrant Notch signaling has been associated with tumorigenesis and cancer progression in lung, pancreas, and breast cancer models. In fact, mutations on Notch receptors and consequently nuclear overexpression of NICD were described in leukemia and lymphoma. 101 Under hypoxia, both echinomycin (HIF-1α inhibitor) and inhibitors of γ-secretase (MRK-003 and a specific peptide inhibitor), decrease survivin expression in lung and breast cancers. 102

| Others
Survivin promoter has several sites for the transcription factor Sp1 and mutations on some of those sites reduce survivin expression by 60% to 80% in cervical cancer. 104,105 In contrast, Sp1 could also recruit transcriptional repressors such as p53, DNMT1, and HDAC and consequently suppress survivin promoter activity. 106 Interestingly, Sp1 inhibition by mithramycin A or oxaliplatin significantly decreased survivin promoter activity in colon and esophageal cancers. 106,107 Moreover, the small molecule terameprocol significantly decreased survivin levels by hampering Sp1 binding to survivin promoter and through the downregulation of CDK1 that leads to survivin degradation. This effect was observed in colorectal, prostate, hepatic, erythroleukemia, and breast cancers. 108 Survivin has yet binding sites to other transcription factors as we have mentioned previously. Several of these transcription factors are important survivin inductors, like the GATA-1, 109 c-myc, 110 and DEC1 111 transcription factors. KLF5 binds directly to survivin promoter on three specific sites inducing its expression, but could also interact with p53 and block its repression on survivin promoter. 112 In addition, it has been reported that survivin is targeted by GLI2 transcription factor, a Hedgehog signaling effector. Defective activation of hedgehog signaling has been described in many cancers. 11 GLI binding sites were found in survivin promoter. Inhibiting Hedgehog signaling through SMO inhibitor cyclopamine strongly reduces survivin promoter activity in melanoma. 105 Besides, a specific inhibitor of GLI known as GANT61 also significantly inhibits survivin promoter activity and consequently reduces survivin protein in melanoma, colon, lung, pancreatic, and adrenal gland cancers. 105 Furthermore, palmatine has recently been described to inhibit GLI/ collagen type 1 α-1 (COL1A1) in stellate cells and survivin in cancer cells. 113 Other molecules have also shown to reduce survivin levels in several cancers: an inhibitor of CRM1 (KPT-185), a protein that plays a critical role in the nuclear export of proteins; SF002-96-1 (natural lactone) through indirect inhibition of transcription factors, STAT3 and NF-кB 114 and several small molecules (eg, GDP366, the HIV protease inhibitor Nelfinavir and the derivative of retinoic acid tretinoin) through an unknown mechanism of action. 115-117

| CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Many preclinical studies have extensively demonstrated that survivin has a relevant role in promoting tumor growth as well as inducing resistance to chemotherapy in several neoplasms. Moreover, survivin shows a differential expression, being preferentially and abundantly expressed in tumors but not in adult differentiated tissues. Altogether, these features make survivin a promising therapeutic target to treat cancer. However, early clinical data indicates that the first direct inhibitors of survivin available show modest activity as single agents and, in some cases, dose-limiting toxicities. These are the cases of LY2181308 oligonucleotide or the small-molecule inhibitor YM155. This limited response may be due to deficiencies in complete and selective survivin inhibition in patients and to tumor heterogeneity. Therefore, inhibitors with more specific mechanisms targeting survivin, such as SMAC mimetics or survivin homodimerization inhibitors, are expected to show better clinical results. In contrast, several indirect survivin inhibitors are currently in clinical trials, such as flavopiridol or lapatinib, although their effects may be due to the combination of survivin inhibition together with other mechanisms of action. 64,91 Additionally, efforts directed at identification of specific biomarkers useful for the selection of patients benefiting from the treatment should be made.
Immunotherapy is another therapeutic approach under current evaluation. It is focused on targeting survivin through the stimulation of the immune system to provoke a direct response against the tumor. Some clinical trials, like vaccination with survivin-2B80-88 peptide, 118 have shown limited clinical responses. In contrast, the good tolerability demonstrated offer promise to optimize this therapeutic approach. Indeed, there is some expectation in several phases I and II clinical trials using survivin vaccines (NCT03029403, NCT03349450, among others), which are currently recruiting patients to evaluate its safety, tolerability, and effectiveness in combination with other treatments.
Finally, it is anticipated that the maximum therapeutic effect of survivin inhibitors will be completely achieved in combination regimens. Since targeting survivin sensitizes cancer cells to apoptosis, 119  | 903 demonstrated that these combinations are able to enhance cancer cells sensitivity to chemotherapeutics 31 and overcome chemoresistance in certain types of cancers. 32 The reviewed results underscore survivin as an attractive and promising cancer drug target. Future efforts may be focused on the development of synergistic combinations between direct or indirect survivin inhibitors, or gene therapy-based approaches, together with chemotherapeutic drugs to efficiently treat cancer.

ACKNOWLEDGMENTS
Authors want to thank Dr. Martin Kotev for his technical assistance in Figure 1. Partial financial support provided by the Spanish government and the EU (Instituto de Salud Carlos III, FIS PI18/00441) as well as FEDER and the Consejería de Educación, Junta de Castilla y León (BU092U16) is gratefully acknowledged.