Survivin is a member of the endogenous inhibitor-of-apoptosis (IAP) family. Several alternative Survivin splice variants have been described and while their function is not fully understood, several have been shown to have diagnostic and prognostic significance. We have identified two novel alternative splice variants, Survivin-3γ and Survivin-3g-V that differ by 4 bases resulting in proteins with very different C-termini. We characterized their expression in normal hematopoietic stem and progenitor cells (HSPC) and in cancer cells and evaluated their anti-apoptotic function. Both splice variants are highly expressed in primary umbilical cord blood CD34+ HSPC. While growth factor stimulated proliferation of CD34+ cells is associated with increased wild-type Survivin expression, the expression of the Survivin-3g+3gV splice variants decrease relative to Survivin-WT, suggesting that they may be associated with HSPC maintenance/quiescence. In contrast to neoplastic cell lines and most cancers, where Survivin-WT is the predominant transcript, Survivin-3g+3gV are expressed at higher levels in benign prostate hyperplasia tissue and at equivalent levels in normal breast tissue. In prostate and breast cancer cells where Survivin-WT expression increases with stage of disease, expression of Survivin-3g and Survivin-3gV expression does not change with increasing severity of disease. Over expression of Survivin-3g and Survivin-3gV enhances resistance to the chemotherapeutic agents Taxol and Etoposide, and imparts growth factor independent growth to normal HSPC, suggesting that the Survivin-3g and Survivin-3gV splice variants possess similar anti-apoptotic function as Survivin-WT

•    Survivin
•    Splice variants
•    Apoptosis
•    Drug resistance
 

Sampath J, Smith LM, Fukuda S, Pelus LM (2017) Identification and Characterization of Novel Survivin Splice Variants with Anti-Apoptotic Activities. J Immunol Clin Res 4(1): 1039.

WT: Wild Type; MDM2: Murine Double Minute 2; HI-FBS: Heat Inactivated Fetal Bovine Serum; SNP: Single Nucleotide Polymorphism; UCB-Umbilical Cord Blood

Survivin is a multifunctional protein linked to apoptosis, angiogenesis and cell growth and division [1,2]. While primarily developmentally expressed, it is over-expressed in virtually every human cancer. Survivin transcription is primarily cell cycle regulated, peaking during G2 /M phase [3], however cell cycleindependent Survivin gene expression has been noted [1]. Survivin interacts with prominent signaling pathways associated with cell growth and proliferation, tumor suppressor genes, oncogenes, stress response proteins and growth stimulatory cytokines. It is one of the few genes actively repressed by wild-type p53 [4,5]. Survivin over-expression is linked to resistance to apoptosis, bypass of cell cycle checkpoints and resistance to chemotherapy and numerous molecular profiling studies and retrospective analysis of patient responses have identified Survivin as a risk factor for disease progression and poor prognosis [1,6-9]. As a result of its nodal properties and expression patterns, Survivin has been pursued as a therapeutic target [1,2,6].

While Survivin’s main transcript is the predominant transcript in most cancers, alternatively spliced Survivins having both similar and opposing function have been identified and linked to differential diagnosis and prognosis [10, 11]. In this report, we describe the alternative splice variants Survivin-3γ and -3γV and characterize their biological activity relative to wild-type Survivin. Both variants have anti-apoptotic activity similar to Survivin-WT, imparting resistance to chemotherapy in neoplastic cells and growth proliferation in non-transformed cells. Both are expressed at higher levels than Survivin-WT in primary HSPC and do not increase with cell proliferation, suggesting a role in stem cell maintenance

Cell lines, primary cells and plasmids

HeLa cells were grown in DMEM with 10% HI-FBS (Hyclone Sterile Systems, Logan, UT). Murine Yac-1 lymphoma cells, HL-60 human promyelocytic leukemia cells and K562 malignant multipotent hematopoietic cells were maintained in RPMI-1640 with 10% HI-FBS. Mo7e-bcr-abl megakaryocytic leukemia cells were maintained in the presence of 100 units GM-CSF. Survivin-WT, -Flag-tagged WT, -3γ and -3γV were amplified by PCR and cloned into MIEG-3 12. All plasmids were transfected into phoenix cells 13 and the retrovirus produced used to infect Yac-1 cells. After 48 hours infection, GFP+ expression was reconfirmed. Primary umbilical cord blood cells were obtained under IRB approval.

Reverse transcription and PCR

Total RNA was obtained using the absolutely RNA purification kit (Stratagene, La Jolla, CA). A constant amount of RNA was reverse transcribed with random primers (Promega, Madison, WI) and MMLV-reverse transcriptase (Promega) in 50µl with 1mM dNTPs and RNase inhibitor [12]. Primers for SYBR Green RT-PCR were designed to produce an amplicon size of 75-150 bp. PCR was performed using Platinum SYBR Green qPCR supermix UDG with Rox (Invitrogen, Carlsbad, CA) in an ABI-7000 (Applied Biosystems, Carlsbad, CA), with an activation step of 50o C for 2 minutes, denaturation at 95o C for 2 minutes and amplification for 45 cycles at 95o C-15 sec, 50o C-30 sec, 72o C-30 sec. Total RNA from HL60 cells was reverse transcribed using oligo-dT primers (Roche, Indianapolis, IN) and the cDNA library used as a template to amplify full-length cDNA using the primers: FlagSur-Forward 5’-GGAATTCGCCGCCACCATGGACTACAAAGACGATGACGACAAGGG ATCCATGGGTGCCCCGACG-3’ and Sur-3BReverse-5’-CCGCTCGAGCTATCATTCTGCTAACAGAGC-3’and Pfu Turbo polymerase (Stratagene) and the products cloned into MIEG-3; (restriction site is shown in italics, Flag-tag sequence is underlined and sequence present in Survivin-WT is shown in bold) Transformed E.colic lones were picked and sequenced. To confirm variant sequence, specific primers were synthesized and PCR was carried out on cDNA. PCR products were gel purified by Qiaquick gel purification kit (Qiagen, Valencia, CA) and cloned into Zero blunt Topo PCR cloning kit (Invitrogen) and sequenced

Rapid amplification of cDNA ends (RACE)

To obtain the complete 5’ and 3’ Survivin-3γ, RACE was performed using the SMART RACE cDNA amplification kit (Clonetech, Mountain view, CA) as recommended by the manufacturer. PCR products were cloned and sequenced as described above.

Expression analysis using breast and prostate cancer arrays

Survivin-3γ+3γV expression in breast and prostate cancers was evaluated using a tissue scan real-time array (OriGene Tech-nologies, Rockville, MD). SYBR Green RT-PCR was carried out using primers recognizing only the intended Survivin variant and hypoxanthine phosphoribosyl transferase (HPRT). Relative expression levels of 3γ+3γV were calculated and expressed relative to Survivin-WT (2-??Ct method).

Cell lysis and western blot analysis

Western blot onYac-1 cell lysates was performed as we described [14]. Membranes were probed using rabbit anti-SurvivinpAb (Novus Biologicals, Boulder, CO) or mouse anti-Survivin mAb (DAKO, Tucson, AZ) and peroxidase conjugated secondary antibody. Bands were visualized using ECL-plus reagent (Amersham, Piscataway, NJ).

Cytotoxicity assays

Log phase Yac-1 cells were plated at 1x105 cells/well and treated with Paclitaxel (Sigma) for 96 hours or Etoposide (Sigma) for 24 hours. Following incubation, MTS reagent (Promega) was added, incubated at 37o C for ~ 3 hours and absorbance (OD 490nm) measured.

While amplifying the Survivin-3B variant from HL-60 leukemia cells, we observed a slower migrating band, which we excised, cloned and sequenced (Figure 1A), revealing a full length protein with a unique C-terminal 13 amino acids, including the stop codon (Figure 1B). We amplified the cDNA using a polydT reverse-transcribed cDNA library from three hematopoietic leukemia cell lines as previously described [15], resolved the products on an agarose gel (Figure 1C) and excised, cloned and sequenced the ~ 400bp band. Analysis showed a novel splice variant, which we named Survivin-3γ, and unexpectedly, an alternative variant that differed by 4 bases introduced at the beginning of the novel-3γ exon (shown in larger font in Figure 1D) that we named Survivin-3γV.

The addition of 4 bases (ACAG) in Survivin-3γV results in a frame shift generating a novel C-terminus, likely due to an alternate consensus 3’ splice site (“AG”) 4 bases upstream of the 3γ-exon. Survivin-3γ and -3γV nucleotide sequences are highly homologous to Alu sequences known to introduce alternate splicing signals. Moreover, Chromosome 17, where Survivin localizes, contains a high density of Alu sequences [16]. Interestingly, the base “A” in the 3’splice acceptor site of Survivin-3γ is a SNP site with “A” having a frequency of 60% and “G” 40% (refSNP ID: rs2467572). Thus, in individuals homozygous for “G”, Survivin3γ may not be made since the consensus 3’splice site “AG” would be “GG”.

Since a Survivin-3γV stop codon was not found in the sequenced region, we performed 3’ RACE using a primer that recognizes all splice variants and an anchor primer (Figure 2A). A second round of RACE was performed with a Survivin-3γ specific primer and the anchor primer, resulting in amplification of a specific ~500bp product (Figure 2B). Translation from the longest insert revealed that Survivin-3γ had 12 new amino acids at the C-terminus (Figure 2C), while Survivin-3γV was 162 amino acids long compared to 142 for Survivin-WT (Figure 2D). Secondary structure prediction using Jpred [17] suggests that Survivin-3γ is structurally similar to Survivin-WT (Figure 2E), with the new amino acids predicted to form a C-terminal helix that could form the coiled-coil domain, albeit smaller than Survivin-WT. Secondary structure prediction for Survivin-3γV (Figure 2F) showed that the terminal helix would not form, suggesting that its localization/function may differ from Survivin-WT and Survivin-3γ, which is supported by PSORT II analysis [18].

Genomic analysis indicates that ~60% of human genes have alternative splice forms [19], resulting in isoforms of different length and domains that result in altered sub cellular localization, expression patterns and interaction affinity. Five Survivin variants having similar or opposing function have been identified (see [10,20]), with additional variants found in the est database (reviewed in [10]). The identification of Survivin splice variants has sparked interest in their clinico-pathological significance, and diagnostic, prognostic and therapeutic value. While not meant to be inclusive, numerous studies on variant expression and value have been reported. The -2α variant carrying a truncated BIR domain is apoptotic and can attenuate Survivin-WT activity [21]. It is highly expressed in malignant stages of breast cancer [22] and likely a marker for breast cancer diagnosis and prognosis [23]. It has been linked to shorter disease free progression and overall survival in some cancers and childhood acute myelogenous leukemia (AML) [24]. The significance of high expression of proapoptotic -2α but association with adverse outcome is unclear. Lower expression of Survivin-2α is associated with thyroid malignancy [25].

The –?Ex3 variant with a modified C-terminus and pro-anti apoptotic activity [26] has been most widely studied. It is a dependable diagnostic marker of advanced bladder, prostate and breast cancers and of tumor invasiveness/metastasis in bladder and colorectal cancer [20]. Survivin–?Ex3 is highly expressed in thyroid carcinomas and a diagnostic marker of papillary thyroid carcinoma [27]. Survivin-2B having a truncated BIR domain has predicted attenuated anti-apoptotic function [28] and may undergo dimerization with -?Ex3 to promote apoptosis, particularly in breast cancer where it has been associated with favorable prognosis [29]. However, silencing of -2B results in apoptotic induction [30] and expression of -2B with -Ex3 is believed to be causative for some high grade carcinomas [31,32]. In contrast, Survivins-2B and -?Ex3 play no role in pituitary tumorigenesis [33]. Thus the role of these variants are not clear and nature and stage of disease are likely dependent variables. Survivin-3α is a truncated variant [34] not widely studied. It is expressed in most breast cancers, but also in benign tumors, but not in surrounding normal tissues [23]. Survivin -3B containing a complete BIR domain [35] is anti-apoptotic as expected, regulates cell cycle and augments chemoresistance [36]. Five splice variants are found in colorectal cancers with –?Ex3and -3B possibly associated with tumor progression [37] and decreased -2B linked to tumor progression [38].

To begin to understand the expression pattern of Survivin3γ+3γV we first evaluated non-hematopoietic cells. Design of primers detecting variants differing by 4 consecutive bases was not possible; therefore we designed primers that detected both variants. Amplification plots (Figures 3A,C) and dissociation curves (Figures 3B,D) confirmed primer specificity. SurvivinWT was the predominant transcript (Table 1A), with Survivin3γ+3γV transcripts present at ~0.7-6%. In breast cancer samples, increased Survivin-WT expression at stages 1, 2 and 3 was seen (Table 2A), with little change related to advanced stage. Ct values for Survivin-WT and -3γ+3γV in stage 0 samples indicated that they were expressed at the same level as Survivin-WT (29.2 ± 0.7vs 29.3 ± 1.0, n=7). However, Survivin-3γ+3γV expression remained constant at all stages, resulting in lower relative expression to Survivin-WT in stages 1 and 2. Survivin-3γ+3γV was elevated in stage 3, however this was skewed by very high expression in 1/11 samples. In prostate cancers (Table 2B), Survivin-WT expression was higher in stage 1 than benign prostate hyperplasia (BPH), however sample size was small. No clear overexpression of Survivin-WT was seen at advanced stages. The expression of Survivin-3γ+3γV was higher than SurvivinWT in BPH tissue and expression of Survivin-3γ+γV transcripts remained higher than Survivin-WT at all stages except stage 1.

In consensus with most reports, we found that Survivin-WT was the major transcript expressed. The lack of change in SurvivinWT with prostate disease severity is consistent with one study [39] where Survivin expression was detected by immune histochemistry. However, other reports show increased Survivin with disease severity either at the RNA level [40,41] or by immune histochemistry [41]. This could be due to methodology; SYBR green qRT-PCR is more sensitive and efficient than standard PCR in amplifying a ~150bp amplicon. We also show that Survivin-WT and Survivin-3γ+3γV expression were comparable in normal breast tissues and expression of Survivin-WT increased at stage 1 and remained high at all stages. This contrasts with one report [42] that Survivin increased gradually with disease severity. This difference may also reflect different methodologies. Interestingly, expression of Survivin-3γ+3γV was constant with only a minor increase in stage 1 breast cancer.

Survivin-3γ+3γV expression in UCB CD34+ cells enriched for HSPC (Table 1B) was >26-fold higher than Survivin-WT. Growth factor stimulation induced Survivin-WT expression, as we described [43-45]. In contrast, Survivin-3γ+3γV expression increased only slightly after 48 hours; however HPRT expression also increased (28.4 ± 0.2 to 24.4 ± 0.1, p < 0.0002), indicating that the relative levels of -3γ+3γV transcripts decreased ~2-fold compared to Survivin-WT. To our knowledge these are the first Survivin variants expressed higher than Survivin-WT in normal cells, suggesting they might play an important role in normal prostate tissue and HSPC. Analyses of Survivin function in vitro and in vivo clearly indicate that Survivin is required for normal hematopoietic function and maintenance [46,47].

It is interesting that the BIR domain of Survivin is disrupted in all variants excerpt -3B. Since theSurvivin-3γ and 3γV variants were identified while attempting to clone -3B, we expected that they would show anti-apoptotic function. To investigate function we created Yac-1 lymphoma cells stably expressing human Survivin-WT with/without an N-terminal Flag-tag or Survivin-3γ or Survivin-3γV, each with N-terminal HA-tags. Since crystal structure of Survivin-WT indicates that the C-terminal helix could be involved in protein-protein interaction, whereas the N-terminus was unordered [48], we engineered N-terminal tags into all Survivinc DNAs. Western blots using a rabbit anti-Survivin polyclonal antibody that recognizes human and mouse Survivin and a selective mouse anti-human Survivin monoclonal antibody showed a background amount of murine Survivin as well as the presence of each of the transduced human Survivins, indicating that each variant was translated (Figure 3E). Survivin-WT was expressed at the highest level followed by HA-3γV and Flag-WT with HA-3γ expressed at a lower level, despite being the highest expressed transcript. Drug resistance to Etoposide and Taxol were evaluated at concentrations and duration of incubation predetermined to be optimal. Overexpression of either Survivin-WT or Flag-Survivin-WT provided 2.74- and 2.81-fold resistance to Taxol-induced cell death (Figure 3F). Ectopic expression of HASurvivin-3γ or -3γV also provided 2.59- and 2.37-fold resistance, respectively. In three experiments, the IC50’s for Survivin-WT, Flag-Survivin-WT, HA-Survivin-3γ and HA-Survivin-3γV were significantly higher than control. Similarly, Yac-1 cells expressing Survivin-WT also showed resistance to Etoposide with an IC50 of 0.27 ± 0.06nM (Figure 4G). Flag-Survivin-WT, HA-Survivin-3γ and HA-Survivin-3γV all conferred similar resistance. Expression of murine Survivin-WT and the only known murine Survivin splice variant, Survivin-121 were unchanged (not shown), indicating that changes in endogenous murine Survivins were not involved in the increased drug resistance. Since Yac-1 cells over-expressing either Survivin-3γ or -3γV showed the same level of resistance as Survivin-WT to two different classes of cytotoxic drugs, Taxol, a microtubule destabilizer, and Etoposide, a topoisomerase-II inhibitor, it is likely that Survivin-WT, -3γ and -3γV proteins possess equivalent anti-apoptotic function. To further evaluate Survivin-WT, -3γ and -3γV function, we transduced primary cord blood CD34+ cells and measured CFU-GM proliferation in vitro. Transduction with all 3 constructs significantly enhanced proliferation of HSPC, indicating equivalent growth promoting activity (Figure 4H). We have not yet evaluated the effects of Survivin-3γ and -3γV proteins on cytokinesis, cell cycle or long term HSPC function. Furthermore, we have evaluated chemotherapy resistance in murine cell lines in the context of endogenous Survivin. Future analysis in conditional Survivin deletion models, as we described for Survivin-WT [49], will provide more definitive information

Table 1: A. Expression of Survivin-WT and Survivin 3γ + 3γV in cell lines. B. Expression of Survivin-WT and Survivin 3γ + 3γV in primary hematopoietic cells

Table 2: A. Expression of Survivin-WT and Survivin 3γ + 3γV in primary breast cancer.

B. Expression of Survivin-WT and Survivin 3γ + 3γV in primary prostate cancer

We identified two Survivin splice variants with anti-apoptotic activity similar to Survivin-WT. Survivin-3γ and Survivin-3γV expression do not change with increasing severity of primary prostate and breast cancers. Unlike other Survivin splice variants, Survivin-3γ and -3γV are expressed at higher levels than Survivin-WT in primary HSPC, and their lack of increase with cell proliferation suggests that they may play a role in HSPC maintenance. Since these splice variants were cloned from and found to be highly expressed in hematopoietic tissue, it will be particularly interesting to evaluate their expression and role in hematopoietic malignancies, particularly maintenance of leukemic stem cells. In this regard, a recent report has also linked Survivin splice variant expression to pluripotency in human embryonic stem cells [50].

Supported by NIH Grants HL079654 and HL096305 (to LMP)

1. Altieri DC. Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 2008; 8: 61-70.

2. Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res. 2008; 14: 5000-5005.

3. Lens SM, Vader G, Medema RH. The case for Survivin as mitotic regulator. Curr Opin Cell Biol 2006; 18: 616-622.

4. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem. 2002; 277: 3247-3257.

5. Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene. 2002; 21: 2613-2622.

6. Ryan BM, O’Donovan N, Duffy MJ. Survivin: a new target for anti-cancer therapy. Cancer Treat Rev. 2009; 35: 553-562.

7. Span PN, Tjan-Heijnen VC, Manders P, van TD, Lehr J, Sweep FC. High survivin predicts a poor response to endocrine therapy, but a good response to chemotherapy in advanced breast cancer. Breast Cancer Res Treat. 2006; 98: 223-230. 

8. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004; 351: 2817-2826.

9. Yoshida A, Zokumasu K, Wano Y, Yamauchi T, Imamura S, Takagi K, et al. Marked upregulation of Survivin and Aurora-B kinase is associated with disease progression in the myelodysplastic syndromes. Haematologica. 2012; 97: 1372-1379.

10. Sah NK, Seniya C. Survivin splice variants and their diagnostic significance. Tumour Biol. 2015; 36: 6623-6631.

11. Sampath J, Pelus LM. Alternative splice variants of survivin as potential targets in cancer. Curr Drug Discov Technol. 2007; 4: 174-191.

12. Fukuda S, Mantel CR, Pelus LM. Survivin regulates hematopoietic progenitor cell proliferation through p21WAF1/Cip1-dependent and -independent pathways. Blood. 2004; 103: 120-127.

13. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987; 7: 2745-2752.

14. Wang Z, Fukuda S, Pelus LM. Survivin regulates the p53 tumor suppressor gene family. Oncogene. 2004; 23: 8146-8153.

15. Wang Z, Sampath J, Fukuda S, Pelus LM. Disruption of the inhibitor of apoptosis protein survivin sensitizes Bcr-abl-positive cells to STI571- induced apoptosis. Cancer Res. 2005; 65: 8224-8232.

16. Yulug IG, Yulug A, Fisher EM. The frequency and position of Alu repeats in cDNAs, as determined by database searching. Genomics. 1995; 27: 544-548.

17. Cuff JA, Clamp ME, Siddiqui AS, Finlay M, Barton GJ. JPred: a consensus secondary structure prediction server. Bioinformatics. 1998; 14: 892-893.

18. Nakai K, Horton P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci. 1999; 24: 34-36. 19.Modrek B, Lee C. A genomic view of alternative splicing. Nat Genet. 2002; 30: 13-19.

20. Necochea-Campion R, Chen CS, Mirshahidi S, Howard FD, Wall NR. Clinico-pathologic relevance of Survivin splice variant expression in cancer. Cancer Lett. 2013; 339: 167-174.

21. Caldas H, Honsey LE, Altura RA. Survivin 2alpha: a novel Survivin splice variant expressed in human malignancies. Mol Cancer. 2005; 4: 11.

22. Wajapeyee N, Britto R, Ravishankar HM, Somasundaram K. Apoptosis induction by activator protein 2alpha involves transcriptional repression of Bcl-2. J Biol Chem. 2006; 281: 16207-16219.

23. Moniri JS, Gharechahi J, Hosseinpour Feizi MA, Montazeri V, Halimi M. Transcriptional expression analysis of survivin splice variants reveals differential expression of survivin-3alpha in breast cancer. Genet Test Mol Biomarkers. 2013;17: 314-320.

24. Wagner M, Schmelz K, Wuchter C, Ludwig WD, Dorken B, Tamm I. In vivo expression of survivin and its splice variant survivin-2B: impact on clinical outcome in acute myeloid leukemia. Int J Cancer. 2006; 119: 1291-1297.

25. Kyani K, Babaei E, Feizi MA, Vandghanooni S, Montazeri V, Halimi M. Detection of survivin 2alpha gene expression in thyroid nodules. J Cancer Res Ther. 2014; 10: 312-316.

26. Mahotka C, Wenzel M, Springer E, Gabbert HE, Gerharz CD. SurvivindeltaEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res. 1999; 59: 6097-6102.

27. Waligorska-Stachura J, Andrusiewicz M, Sawicka-Gutaj N. Survivin delta Ex3 overexpression in thyroid malignancies. PLoS One. 2014; 9: 100534.

28. Zhu N, Gu L, Findley HW, Li F, Zhou M. An alternatively spliced survivin variant is positively regulated by p53 and sensitizes leukemia cells to chemotherapy. Oncogene. 2004; 23: 7545-7551.

29. Zheng WY, Kang YY, Li LF, Xu YX, Ma XY. Levels of effectiveness of gene therapies targeting survivin and its splice variants in human breast cancer cells. Drug Discov Ther. 2011; 5: 293-298.

30. Vivas-Mejia PE, Rodriguez-Aguayo C, Han HD, Shahzad MM, Valiyeva F, Shibayama M, et al. Silencing survivin splice variant 2B leads to antitumor activity in taxane--resistant ovarian cancer. Clin Cancer Res. 2011; 17: 3716-3726.

31. Vegran F, Boidot R, Bonnetain F, Cadouot M, Chevrier S, Lizard-Nacol S. Apoptosis gene signature of Survivin and its splice variant expression in breast carcinoma. Endocr Relat Cancer. 2011; 18: 783-792.

32. Mishra R, Palve V, Kannan S, Pawar S, Teni T. High expression of survivin and its splice variants survivin DeltaEx3 and survivin 2 B in oral cancers. Oral Surg Oral Med Oral Pathol Oral Radiol. 2015; 120: 497- 507.

33. Waligórska-Stachura J, Andrusiewicz M, Sawicka-Gutaj N, Kubiczak M, Jankowska A, Liebert W, et al. Evaluation of survivin splice variants in pituitary tumors. Pituitary. 2015; 18: 410-416.

34. Mola G, Vela E, Fernandez-Figueras MT, Isamat M, Munoz-Marmol AM. Exonization of Alu-generated splice variants in the survivin gene of human and non-human primates. J Mol Biol 2007; 366: 1055-1063.

35. Badran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T, et al. Identification of a novel splice variant of the human anti-apoptopsis gene survivin. Biochem Biophys Res Commun. 2004; 314: 902-907.

36. Végran F, Mary R, Gibeaud A, Mirjolet C, Collin B, Oudot A, et al. Survivin-3B potentiates immune escape in cancer but also inhibits the toxicity of cancer chemotherapy. Cancer Res. 2013; 73: 5391-5401.

37. Antonacopoulou AG, Floratou K, Bravou V et al. The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival. Anal Cell Pathol (Amst). 2010; 33: 177-189.

38. Cho GS, Ahn TS, Jeong D. Expression of the survivin-2B splice variant related to the progression of colorectal carcinoma. J Korean Surg Soc. 2011; 80: 404-411.

39. Kaur P, Kallakury BS, Sheehan CE, Fisher HA, Kaufman RP Jr, Ross JS. Survivin and Bcl-2 expression in prostatic adenocarcinomas. Arch Pathol Lab Med. 2004; 128: 39-43.

40. Kishi H, Igawa M, Kikuno N, Yoshino T, Urakami S, Shiina H. Expression of the survivin gene in prostate cancer: correlation with clinicopathological characteristics, proliferative activity and apoptosis. J Urol. 2004; 171: 1855-1860.

41. Krajewska M, Krajewski S, Banares S, Huang X, Turner B, Bubendorf L, et al. Elevated expression of inhibitor of apoptosis proteins in prostate cancer. Clin Cancer Res. 2003; 9: 4914-4925.

42. Sohn DM, Kim SY, Baek MJ. Expression of survivin and clinical correlation in patients with breast cancer. Biomed Pharmacother. 2006; 60: 289-292.

43. Fukuda S, Pelus LM. Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34(+) cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis. Blood. 2001; 98: 2091-2100.

44. Fukuda S, Foster RG, Porter SB, Pelus LM. The antiapoptosis protein survivin is associated with cell cycle entry of normal cord blood CD34 (+) cells and modulates cell cycle and proliferation of mouse hematopoietic progenitor cells. Blood. 2002; 100: 2463-2471.

45. Fukuda S, Pelus LM. Elevation of Survivin levels by hematopoietic growth factors occurs in quiescent CD34+ hematopoietic stem and progenitor cells before cell cycle entry. Cell Cycle. 2002; 1: 322-326.

46. Fukuda S, Pelus LM. Survivin, a cancer target with an emerging role in normal adult tissues. Mol Cancer Ther. 2006; 5: 1087-1098.

47. Leung CG, Xu Y, Mularski B, Liu H, Gurbuxani S, Crispino JD. Requirements for survivin in terminal differentiation of erythroid cells and maintenance of hematopoietic stem and progenitor cells. J Exp Med. 2007; 204: 1603-1611.

48. Chantalat L, Skoufias DA, Kleman JP, Jung B, Dideberg O, Margolis RL. Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions. Mol Cell. 2000; 6: 183-189.

49. Fukuda S, Hoggatt J, Singh P. Survivin modulates genes with divergent molecular functions and regulates proliferation of hematopoietic stem cells through Evi-1. Leukemia. 2015; 29: 433-440.

50. Mull AN, Klar A, Navara CS. Differential localization and high expression of SURVIVIN splice variants in human embryonic stem cells but not in differentiated cells implicate a role for SURVIVIN in pluripotency. Stem Cell Res. 2014; 12: 539-549