Survivin, a molecular target for therapeutic interventions in squamous cell carcinoma

Functions of survivin

In cancer cells, survivin has two major functions; 1) regulation of mitosis by forming chromosomal passenger complex (CPC) with other proteins, and 2) inhibition of apoptosis [30, 31]. As shown by embryonic lethality in mice with survivin locus disruption that it plays a critical in overall normal embryonic development [32]. In adults, survivin is absent in most of the terminally differentiated tissues as opposed to it high re-expression in malignant cells.

Role of survivin in cell division

Survivin plays an important role in the regulation of mitosis [30, 33]. It is expressed in a cell cycle dependent manner as reported mostly in G2-M phase [34]. During mitosis survivin interacts with tubulin and localizes to the mitotic spindle indicating its involvement in the regulation of mitosis [35]. It is now very well documented that survivin controls multiple facets of cell division in association with other proteins. It plays an important role in centrosome functions [21], microtubule assembly during metaphase and anaphase [36, 37], and spindle checkpoints (Fig. 1). Depletion of survivin causes defective cell division that involves activation of spindle checkpoints mediated by tumor suppressor protein p53 due to an arrest of DNA synthesis [30, 38]. Survivin-deficient cells frequently fail to complete both chromosome segregation and cytokinesis during mitosis. In the absence of survivin, sister chromatids start separating normally during anaphase, but often fail to move along with the main mass of segregating chromosomes that ultimately leads to an abnormal chromatid separation. Cytokinesis is also initiated normally, but in the absence of survivin it fails in late stages due to abnormality in spindle midzone and midbody microtubule formation [28, 39]. It has been demonstrated that these abnormalities in chromosome segregation and cytokinesis can be attributed to a defective CPC. Survivin forms a complex with Aurora B and inner centromere protein (ICP or INCENP) i.e., a characteristic of CPC [30, 35, 40]. This survivin/auroraB/ICP complex interacts with the central spindle midzone at the metaphase and anaphase chromosome, where it plays a crucial role in chromosomal segregation and cytokinesis [41] (Fig. 1). Aberrant mitosis and multi-nucleation has been observed in survivin-knockout cells [40, 42, 43]. Similar functions of survivin or its homolog have also been reported in other species. For example, in fission yeast, a survivin homolog Bir1P/Cut17P/Pbh1p forms a complex with Pic1P (an ICP homolog) and with replication initiation factor Psf2P, which regulates chromosomal segregation during mitosis [41].

Role of survivin in apoptosis

Apoptosis can be triggered with the two major types of stimuli, external and internal. The extrinsic apoptotic pathway initiates by the activation of death receptors (CD-95/Fas and TNFα receptors) through external signals following activation of initiator caspase-8 [35]. The intrinsic apoptotic pathway initiates due to intracellular signals that act through mitochondria. In response to signals, mitochondria release cytochrome-c (cyt-c) and Smac/DIABLO to form apoptosome for activating initiator caspase-9 [11]. Generally, mammalian IAPs inhibit apoptosis by direct or indirect inhibition of caspases [44] (Fig. 2).

In mammalian cells, mitochondria elicit arrays of cell death regulators [45]. Survivin is present abundantly in the inter-mitochondrial membrane space [46]. The mechanism(s) of survivin localization in mitochondria is currently unknown. However, molecular chaperone heat shock protein 90 (Hsp90) is thought to participate in importing many client proteins to mitochondria, and found to be associated with survivin and other IAPs [47, 48]. Interestingly, in normal cells survivin is not present in mitochondrial fractions [46], indicating that survivin translocation to mitochondria may be related to oncogenic transformation.

Several mechanisms have been proposed to explain anti-apoptotic activity of survivin. Some investigators have speculated that survivin may inhibit effector caspase-3 directly, even though it lacks a structural motif to bind directly with caspase-3, as is present in other IAPs. It has been shown that phosphorylation of survivin at threonine 34 (Thr34) is crucial for its anti-apoptotic functions. Another clue regarding anti-apoptotic mechanism of survivin came through its ability to interact with Smac/DIABLO suggesting that survivin may suppress activation of caspases indirectly. Smac/DIABLO acts like pro-apoptotic protein because of its participation in the formation of apoptosome and activation of caspase-9 [44]. Therefore, it is proposed that survivin most probably interferes in the down-stream steps of mitochondrial-apoptotic pathway, such as antagonizing apoptosome formation [12]. A point mutation in survivin protein at Asp-71 is sufficient to eliminate its interaction with Smac/DIABLO and anti-apoptotic function [49]. A strong inhibitor of apoptosis, XIAP interacts directly with caspases and inhibits them [49, 50]. It has been reported that Smac/DIABLO antagonizes functions of XIAP [50]. Therefore, presence of survivin may indirectly allow XIAP to function, ultimately leading to inhibition of apoptosis (Fig. 2).

Regulation of survivin

Mechanisms of survivin regulation are still not fully understood. However, many signaling pathways and factors have been reported to activate survivin in cancer cells. It was originally thought that survivin up-regulation could be directly linked with cell proliferation, but its upregulation in non-proliferating Ki-67 MCF-7 breast cancer cells changed this concept [38]. It is now believed that overall intracellular pathways that activate survivin are more active in cancer as compared to normal cells. Inconsistent, reporter gene assays show negligible survivin promoter activity in normal cells as opposed to cancer cell lines [38], suggesting differences in regulation of survivin expression.

Several oncoproteins such as c-Myc and H-Ras that exceptionally expressed in malignant cells have been positively correlated with the upregulation of survivin. Studies show that oncoproteins, at least c-Myc and H-Ras induced survivin expression through PI3K signaling pathway, which is crucial for cancer cell survival [51, 52]. Amplification of survivin locus on 17q25 and demethylation of survivin exon 1 has been implicated in the upregulation of survivin in cancer cells [53, 54]. Importantly, mutations in retinoblastoma and p53 gene and its functional losses are often associated with the up-regulation of survivin in cancer cells. In normal cells, wild-type p53 and retinoblastoma directly or indirectly repress survivin transcription [55, 56]. Since, E2F activators can also induce survivin transcription, indicating that the retinoblastoma/E2F/p53 pathways may contribute to aberrant survivin expression. Activation of signal transducer and activator of transcription-3 (STAT-3) is associated with the up-regulation of survivin in gastric cancer, breast cancer, and primary effusion lymphoma [57–59]. In colorectal cancer, mutation in adenomatous polyposis coli (APC) tumor suppressor gene was associated with the aberrant stabilization of β-catenin and upregulation of survivin [60]. Nuclear factor-kappa B (NF-kB) is also associated with the transcriptional upregulation of survivin [61]. A p53/NF-κB crosstalk was reported to increase survivin expression in p53 mutant cells that shows strong chemoresistance [62, 63]. In myeloid leukemia, survivin expression is up-regulated in response to hematopoietic cytokines [64], suggesting that survivin expression can be controlled in autocrine or paracrine manner and hematopoietic cytokines may deliver their anti-apoptotic functions by increasing survivin. Besides upregulation, functional and structural stability of survivin in cancer cells requires post-translational modification in its interactions with other proteins. For example, survivin phosphorylation at threonine 34 by the cyclin dependent kinase 1 (CDK1) plays a crucial role in survivin function in cell division and in activation of pro-caspase 9 [65]. It has been recently shown that survivin functions is also controlled by acetylation at lysine residue K129, which directs survivin for nuclear localization [66, 67]. Survivin interacts with Hsp90, a central molecular chaperone to the cellular stress responses. This interaction involves the ATPase domain of Hsp90 and the BIR domain of survivin. Any disruption in this interaction induces proteasomal degradation of survivin [68, 69], suggesting Hsp90 protects survivin from degradation.

Head and neck SCC

We have reported that survivin is overexpressed in majority of OSCC tissues, and in ~50% premalignant tissues [74], pointing out its early involvement in OSCC progression. As reported, accumulation of mutated p53 is considered a factor for survivin up-regulation [55, 56], we observed a positive correlation between survivin and p53 expression in premalignant and malignant OSCC tissues [74]. Studies have established a correlation between survivin status and oral cancer aggressiveness [75]. For examples, survivin significantly segregated with high-grade and undifferentiated tumors and invariably associated with lymph node metastasis (indicators of tumor aggressiveness) [76, 77], and ultimately this affects patient’s survival rate. These findings suggest that the cases of OSCC with more aggressive and invasive phenotype may identify on the basis of survivin expression, and therefore, could influence the decision for the therapy at the time of diagnosis. As compared to cytoplasmic survivin, lower nuclear expression of survivin has been shown a strong predictor for relapse-free survival in the oral cancer patients [78], suggested survivin as an early predictive marker for disease outcome. A meta-analysis study from 15 published articles (1040 cases), in which survivin expression was determined either by immunohistochemistry or RT-PCR in OSCC, found a positive correlation between survivin expression and lymph node metastasis and clinical stage [79]. However, analysis did not find an association between survivin expression and tumor differentiation grade, and depth of invasion. Other meta-analysis study from 610 esophageal cancer patients revealed a significant correlation between survivin over expression and poor overall survival [71].

By using hamster buccal-pouch mucosa experimental model for oral carcinogenesis, Dr. Chen and colleague found survivin up-regulation in all 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch squamous-cell carcinomas. They also found demethylation of survivin allele in DMBS-induced OSCC, suggested gene expression may be modulated by an epigenetic mechanism [80]. As survivin regulates cell division, a positive correlation has been observed between survivin expression other chromosomal passenger proteins, such as Ki-67 and Aurora-B expression [81], which involves in chromosome segregation. Tumors with increased nuclear survivin and Aurora-B expression exhibited marked malignant behaviors [81]. Dr. Keller and colleagues were able to distinguish between human papilloma virus (HPV) positive vs. negative HNSCC samples on the basis of survivin level [82]. The results show that HPV-negative tumors have high level of survivin and poorer prognosis than HPV-positive HNSCC. Tumors with less than a median level of survivin expression were associated with improved patient survival as compared to tumors with more than a median level of survivin [82], proving survivin as a marker for improved survival.

Esophageal SCC

ESCC is one of the most malignant tumors. Survivin is overexpressed in most of the esophageal cancer [83]. Malignant tissues showed significantly higher level of survivin as compared to non-malignant tumors [83, 84]. The different splice variants of survivin were found to be associated with diverse tumor clinicopathological variables. For example, a high cytoplasmic survivin correlated with histological differentiation and invasion, and a high survivin 2B splice variant was associated with poor prognosis in esophageal cancer patients [85]. High nuclear level of survivin also correlated with poor prognosis [86]. In esophageal cancer, survivin overexpression provides a resistant phenotype, as indicated by increased rate of tumor recurrence and lower patient survival in the case of high expression of survivin as compared to low survivin expression [83, 84, 86]. There is an inverse correlation between miR-214-3p and survivin expression with the re-expression of miR-214-3p down-regulate survivin expression via RNA-binding protein (RBP) CUG-BP1 leading to reduction of chemotherapy resistance in ESCC [87]. Case controls studies from different populations showed that single nucleotide polymorphisms in survivin gene, for example -31G/C influence the susceptibility to esophageal cancers in Indian [88] and Chinese population [89].

Lung SCC

Non-small cell lung carcinoma (NSCLC) is a major class of lung cancer. Most of the NSCLC (80-90%) shows overexpression of survivin [90, 91]. Studies show a significantly higher level of survivin in SCC as compared to adenocarcinoma of lung, [92–94], where it contributes to poor prognosis and decreased patient survival [90, 95]. Higher nuclear survivin has been identified as an independent prognostic factor for lung SCC [95]. In lung SCC, survivin up-regulation is associated with increased tumor angiogenesis and metastasis [96]. Study suggested an early detection of survivin can be considered as useful diagnostic tool for the detection of lymph node micrometastasis for stage I NSCLC patients [97]. Since p53 is a regulator for survivin, mutation in p53 gene has been positively correlated with the up-regulation of survivin in lung SCC [93]. In addition, polymorphisms in the survivin gene have been found to influence survivin production and thereby modulate susceptibility to lung cancer [98, 99].

Skin SCC

Squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma are three major type of skin cancers. BCC and SCC are sometime called non-melanoma cancer [100]. Survivin is overexpressed in both melanoma and non-melanoma skin cancers [76, 101–103]. It is overexpressed in 64-92% skin SCC compared to normal skin [76, 104]. The level of survivin is more in high grade and undifferentiated tumors with lymph node metastasis indicating tumor aggressiveness and invasive behavior [76, 105]. The expression of survivin was also present in high percentage of premalignant lesions of Bowen’s disease (SCC in situ) and hypertrophic actinic keratosis (HAK), suggesting that its appearance occurs early during keratinocyte transformation [105]. Dallaglio et al. [103] analyzed intracellular localization of survivin and its correlation with keratinocytes differentiation and SCC. They found marked increases of nuclear survivin (not cytoplasmic) in actinic keratosis and in SCC in situ, and that was highest in poorly differentiated SCC. They found survivin mostly localizes in the deep infiltrating areas of tumors that associated with increased cell migration [103]. In skin, it is quite established that genetic alterations in keratinocyte stem cells (KSC) gives rise to SCC-derived Stem-like Cells (SCC-SC) [106]. Survivin overexpression is a key factor in the transformation of KSC to SCC-SC, and tumor-producing KSC can be isolated on the basis of survivin expression [107]. Since, survivin support maintaining SCC cancer stem cells [101, 108], it is one of the key factor for tumor recurrence and poor clinical outcome in skin cancer

Cervical SCC

Many studies have found survivin overexpression in cervical SCC as compared to the normal tissues [109–112], and its expression associated positively with lesion size, lymphovascular invasion and poor prognosis [111], tumor grade and clinical stages [110, 112]. HPV infection is a leading risk factor for the development cervical cancer [113]. Studies found a positive correlation between survivin expression and HPV infection in cervical carcinoma [113, 114]. In contrary to other SCCs (in which nuclear survivin is associated with poor prognosis), cytoplasmic survivin expression is associated with poor prognosis in cervical carcinoma [115]. All these findings suggest survivin participate in the onset and progression of cervical carcinoma.

Diagnostic potential

Survivin or its auto-antibodies have been found in biological fluids of cancer patients that may be used for cancer diagnosis. For example, sensitive diagnostic tests have been developed that are based on the presence of survivin in urine of bladder cancer patients [116, 117]. Similarly, presence of survivin auto-antibodies in the saliva of OSCC patients provides a novel and practicable approach for OSCC screening [118, 119]. In addition, anti-survivin antibodies were also detected in the circulating blood of cancer patients [120], which can be exploited as a cancer diagnostic tool.

Antisense technology

Antisense technologies are proving a useful tool for cancer therapeutics. These include uses of antisense oligos, siRNA and shRNA techniques, which specifically suppressed expression of target genes (Fig. 3). Antisense-RNAs can be expressed directly in cells by delivering a plasmid or viral vectors or it can be synthesized chemically and transfected into cells. Since survivin is overexpressed in many cancers, its down-regulation by antisense-oligos could be of therapeutic use. Indeed, anti-survivin oligos have been evaluated in many cancers to suppress survivin and its effects on cell death. Deliveries of these oligos in cancer cells induce apoptosis and also increase anti-cancer effects of other therapies such as chemotherapy and radiotherapy. Dr. Olie and colleagues [121] have tested many anti-survivin oligonucleotides in study on NSCLC [121]. Out of many designed oligonucleotides, 4003 was reported to be most effective in suppressing NSCLC growth. We are conducting studies to investigate the role of survivin in HNSCC resistance to conventional drugs. Our results have shown that survivin overexpression in HNSCC cells provide resistance against conventional drugs. siRNA-mediated suppression of survivin significantly inhibits HNSCC cell proliferation and also increases response of chemotherapy and radiotherapy [122, 123]. For pre-clinical studies, we have developed lentivirus vector to deliver survivin-siRNA [124]. A significant growth reduction was observed in human HNSCC tumor xenograft in mouse model with survivin knockdown-using lentivirus-siRNA therapy [125]. Furthermore, a high efficacy was observed when we used a combination of lentivirus-siRNA and chemotherapy or radiotherapy. Many other in vitro and in vivo studies have been conducted in which anti-survivin techniques were used either alone or in combination with conventional drugs to control cancer cell proliferation including most of the SCCs, such as oral [126], laryngeal [127, 128], head and neck [129], skin [103], esophageal [87, 130], and lung [131]. Wen et al. [132] investigated inhibitory function of survivin in laryngeal SCC cell lines GRIM-19 and Hep-2 using plasmid-based survivin-specific shRNA. During proliferation of laryngeal cancer cell lines undergoing transfection with p-siRNA, survivin was markedly inhibited (79%). In vivo study also showed a significant suppression of Hep-2 tumor growth and apoptosis induction due siRNA-mediated silencing of survivin. Survivin inhibition by shRNA abrogated radiation-induced G2 phase arrest and amplified radiation-induced apoptosis [128]. Stoleriu et al. [133] successfully tested multimodality therapy regimen to treat chemoresistant NSCLC cell lines. siRNA-mediated knockdown of survivin along with other genes in these cell lines sensitized them to chemotherapies and significantly induced apoptosis. Over-expression of survivin in OSCC makes it a potential gene therapy target. A lentivirus vector encoding shRNA targeting survivin was used to suppress the survivin gene in a study [134]. Inhibition of survivin reduced proliferation of tumor cells in vitro and sensitized cells to radiation and vincristine. In the OSCC xenograft model, both tumor development and growth of established tumors was inhibited with survivin-lentivirus therapy. Similar growth inhibitory effects of survivin have been observed by using anti-survivin siRNA in esophageal [87, 130] and skin SCC tumor xenograft models [103].

As in SCCs, inhibition of survivin showed anti-proliferative effects in many other malignancies. An antisense oligo that targets 232 to 251 nucleotide sequence of survivin showed a significant killing of mesothelioma cancer cells and also sensitized them to chemo-radiotherapy [135]. Growth of lymphoma cells was also arrested by using anti-survivin oligos [136]. In a study, adenoviral antisense vector targeting survivin (pAd-CMV-SAS) was used to treat colon cancer, which resulted in an cell cycle arrest of cancer cells in Go/G1 phase and induced chemotherapy-mediated cells death [137]. PC-3 prostate cells showed nuclear fragmentation, hypodiploidy, activation of caspase-3, all apoptotic signatures when treated with antisense survivin cDNA [138]. Similar observations were made in human neuroblastoma cells [139]. Survivin knockdown by adenoviral vector (Adv-siSurv) expressing multiple anti-survivin oligos induced apoptosis in many different cancer cell lines, in vitro [140, 141]. Intratumoral injection of this adeno-vector in human tumor xenograft mouse model significantly suppressed tumor growth. A shRNA containing two reverse repeat motifs was designed to target survivin gene. Treatment of liver cancer cell lines with this vector efficiently down-regulated expression of survivin and induced apoptosis [142]. These observations suggest that antisense technology targeting of survivin could be a potential selective cancer therapy.

Dominant negative mutants

In this technique, a nonfunctional protein is formed due to the replacement of an essential amino acid by another amino acid. Due to having the same targets, these nonfunctional proteins compete with normal protein and dilute its function. Quite few dominant negative mutants have been designed and tested for the inhibition of survivin, out of which T34A mutant is well studied, in which threonine is replaced with alanine at amino acid 34 (Fig. 3). Transduction of many different cancer cell types including lung, breast, cervical, prostate, colorectal, liver, and skin with adenovirus vector (pAd-T34A) encoding a non-phosphorylated T34A mutant of survivin, induced apoptosis by increasing cyt-c release from mitochondria and activation of procaspase-3. This treatment also sensitized cancer cells to chemotherapeutic agents such as taxol and adriamycin [143]. Transfection of malignant HeLa cells with T34A survivin mutant could reverse the malignant phenotype [144]. Intratumor injection of pAd-T34A in a tumor bearing mouse significantly suppressed pre-established tumor size by inducing apoptotic cell death [143]. Injection of pAd-T34A in peritoneal cavity significantly reduced tumor growth of breast cancer cells in immunodeficient mice. Similar results were observed in mice bearing NSCLC tumor [145]. Interestingly this treatment did not cause any visible effects on normal cell viability and systemic toxicity. In a similar in vivo study, intratumoral injection of pAd-T34A suppressed tumor growth in prostate cancer mouse model [146] and enhanced anti-androgen sensitivity [147]. These results suggest that the uses of pAd-T34A may selectively target tumor cells with very limited normal tissue toxicity.

Another interesting survivin-mutant (C84A) was constructed by replacement of cystein residue at amino acid 84 with alanine. In gastric cancer cell lines, transfection with plasmid expressing C84A mutant decreased cell growth and induced spontaneous apoptosis [148]. In prostate cancer cell lines, C84A treatment is able to induce all apoptotic hallmarks including hypoploid DNA, activation of caspases, and cleavage of caspase substrates such as Poly (ADP-ribose) polymerase. Similar results were obtained with cutaneous SCC, where C84A transfection resulted in spontaneous apoptosis even without adding any other genotoxic stimuli. Due to this treatment, cells were arrested in sub-G0/G1 phase that correspond to apoptosis with a 4 N DNA content [105]. Injection of adeno-associated virus in colon cancer mice model encoding C84A mutant suppressed angiogenesis and tumor growth without causing normal cell toxicity [149]. In large-cell lymphomas, injection of survivin mutant C84A reduced tumor cell growth and enhanced cell death by increase of tumor-specific cytotoxic T lymphocytes [150] (Fig. 3).

Interestingly combined therapy using survivin negative dominant mutants and chemo-radiotherapies showed better anti-tumor effects. Mostly the effects of survivin mutants combined with other drugs were reported to be synergistic (more than additive). For instance, combined treatment of NSCLC cells with T34A and radiation induced more cell death than single drug treatments, and this effect was more than additive, suggesting that the inhibition of survivin by T34A mutant could sensitize NSCLC cells to radiation treatment [145]. In another study, lyposome complex was used to deliver survivin T34A mutant with or without cisplatin. An intravenous injection of lyposome-T34A in mice, tumor volume was reduced significantly. The antitumor effect of lyposome-T34A combination with cisplatin was greater than their anticipated additive effects, suggesting a synergistic interaction. In vivo studies also showed anti-angiogenesis effects of survivin dominant negative constructs [151]. Zhang et al. [152] constructed a double dominant negative mutant of survivin (T34A-C84A) for understanding whether it could have better potential to kill cancer cells. Treatment of hepatocellular cancer cells with the adenoviruses expressing this double mutant (Ad-T34A-C84A) showed much stronger cell killing as compared to single survivin mutants T34A or C84A alone [152].

Ribozyme technique

Ribozyme (ribonucleic acid enzyme) is a new approach to degrade RNA in cells for therapeutic purposes [153]. Several ribozyme molecules have been developed, of which hammerhead ribozyme is best studied [154]. Hammerhead is the smallest ribozyme containing a highly conserved core residue required for RNA cleavage and three base-paired stems required for identifying the target site. It cleaved target mRNA just after NUH sequence (N can be any nucleotide, and H can be any nucleotide except G) [155]. The specificity of target mRNA cleavage by ribozyme is much higher than siRNA approaches, which often produce off-target effects. Paired flanking regions of cleavage site determine the specificity of cleavage (Fig. 3).

Several ribozymes have developed to inhibit survivin in cancer cells. Choi et al. [156] have designed two hammerhead ribozymes (RZ1and RZ2) to target human survivin mRNA. These ribozymes cleaved survivin mRNA at nucleotide positions +279 and +289. For functional study, an MCF-7 breast cancer cell line was transduced with adenoviral vector encoding these ribozymes, which significantly reduced expression of survivin, and consequently induced apoptosis[156]. In prostate cancer cell lines (PC-3 and DU145), infection with adenoviral vector encoding a ribozyme targeting 3’-end of the survivin-mRNA induced apoptosis [157]. Transduction of melanoma cancer cells with adenovirus-vector expressing ribozyme increased sensitivity of cancer cells to chemo-radiotherapy treatment [157, 158]. Four hammerhead ribozymes (R1 to R4) to suppress survivin gene were designed by Fei et al. [159]. Adenoviruses encoding these ribozymes have been tested in vitro and in vivo for controlling cancer cell growth. Results showed that inhibition of survivin deregulates mitotic cell division and induces caspase-3-dependent apoptosis of cancer cells. Injection of these ribozyme adenoviruses also suppressed tumor growth in a xenograft mouse model of hepatocellular carcinoma. Study further demonstrated that the combination of these ribozymes can give even better clinical outcomes, as reflected by higher cancer cell death in a combination treatment of R1, R3 and R4 as compared to single ribozyme treatment. All forms of survivin were cleaved during this combination treatment, which gives a very strong anti-cancer effect [159]. Despite high substrate cleavage efficiency, clinical application of ribozyme is still limited due to misfolding and RNA degradation of ribozyme, when fused to a carrier. An innovative chimeric ribozyme was constructed by Liu et al. (2007). This chimeric ribozyme derived from a motor pRNA of phi29 bacteriophage, shows enhanced stability and robustness in folding [160]. In a variety of in vitro and in vivo cancer models, treatment with this chimeric ribozyme has been found to suppress survivin mRNA and protein efficiently, and strongly induced apoptosis with very limited normal cell cytotoxicity. These studies clearly indicate that ribozyme-targeting of survivin in cancer cells could be of therapeutic use.

Immunotherapy

Enhancing immune response against cancer cells represents a fascinating approach to cancer treatment. Tumor cell specific antigens can be recognized by host immune system as short peptides bind with MHC (major histocompatibility complex) molecules [161]. As an exclusive tumor marker, survivin evidently activates T-cell immune response. Several survivin epitopes have been identified, which can induce cytotoxic T-lymphocyte (CTL) activity against cancer cells [162] (Fig. 3). In an interesting experiment, dendritic cells were infected with adenovirus-expressing survivin in the hope that endogenous overexpression of survivin may display some immunogenic short survivin peptides as human leukocyte class I or II antigens (HLA-I or II), and for avoiding any pro-oncogenic side effects, survivin dominant negative mutant was used [163]. Three HLA-A2 matching peptides of survivin have been identified against which T-cell immune response was induced by immunizing with these dendritic cells, and this CTL activity was found to be against cancer cells overexpressing survivin, such as MCF-7 breast cancer cell line [163]. Another study showed that CTL activity can be induced against B-cells transfected with survivin-expressing vector, which produced survivin-epitopes on the cell surface [164].

HLA-I restricted T-cell epitopes of survivin were also found in cancer patients that induced CTL response [165, 166]. Survivin-induced CTL have been tested against different malignancies, which are found to kill different HLA-matched tumors [167, 168]. A survivin-2B-derived HLA-24-restricted immunogenic peptide (aa-80-88: AYACNTSTL) has been identified that is recognized by CD8⁺ CTL [102]. On the basis of this peptide, a vaccine was developed and tested in a phase-I clinical trial on advanced stage patients with lung, breast and colorectal cancer, who were found to overexpress survivin-2B splice variant [169]. Another phase I trial of survivin-2B80-88 peptide vaccination has been started on HLA-A24-positive patients with advanced or recurrent OSCC, in which vaccine is being administered subcutaneously or intratumorally. Initial results have demonstrated the safety and marginal clinical effectiveness of this vaccine alone (UMIN000000976) [170]. However, subsequent clinical trials of survivin-based vaccination in combination with other drugs could be a promising therapeutic strategy to tackle advanced cancers (Table 2).

Survivin-specific antibodies have been found in the blood samples of cancer patients [164] and also reported in the tumors, but are absent in healthy specimens. This can be used for inducing humoral immune response. Purification of antibodies gives an opportunity to rationally design survivin-epitopes and consequently to develop cancer vaccines. A specific CTL response can be induced by presenting processed survivin-epitopes on dendritic cells [171]. Another approach to detect a specific T-cell response in cancer patients is based on the identification of tumor-specific survivin epitopes using ELISPOT assay [172, 173]. The presence of these survivin-epitope specific T-cells in tumor lesions indicated its tumor specificity [172]. Dr. Hooijberg and colleagues are exploring the possibility of using vaccination for the treatment of HNSCC patients based on dendritic cells targeting survivin [174]. His team was able to measure survivin-specific T cells ex vivo in peripheral blood and draining lymph node derived from HNSCC patients by using tetramer and ELISPOT analysis. Recently, a vaccine named SurVaxM is a peptidomimetic of survivin, which has entered in clinical trials (NCT01250470) (Table 2). These findings suggest that immunotherapy based on survivin may provide a novel approach for cancer treatment.

Small molecule inhibitors

Small molecules targeting cancer signaling pathways offer an attractive strategy for controlling tumor growth. A range of small molecules and peptides has been identified that control tumor cell proliferation by targeting survivin (Fig. 3). Increasing understanding of molecular mechanisms that regulate survivin expression and function is providing opportunities for designing new molecules, which selectively intercept survivin functions in cancer cells.