Inhibitor Of Apoptosis Protein Survivin Research Articles

Histone Deacetylase Inhibitors Enhance Lexatumumab-Induced Apoptosis via a p21Cip1-Dependent Decrease in Survivin Levels

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in malignant cells by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Several agents that therapeutically exploit this phenomenon are being developed. We investigated the anticancer activity of two novel, highly specific agonistic monoclonal antibodies to TRAIL-R1 (mapatumumab, HGS-ETR1) and TRAIL-R2 (lexatumumab, HGS-ETR2) in colon cancer cell lines. Our analyses revealed that colon cancer cells display significantly higher surface expressions of TRAIL-R2 than TRAIL-R1, and are more sensitive to lexatumumab-induced apoptosis. The proapoptotic effects of lexatumumab in TRAIL-resistant HCT8 and HT29 cells were dramatically augmented by the histone deacetylase inhibitors trichostatin A or suberoylanilide hydroxamic acid. The presence of p21, but not p53, was critical for the synergy between lexatumumab and histone deacetylase inhibitors. The absence of p21 did not interfere with the formation of the death-inducing signaling complex by lexatumumab, suggesting the involvement of other apoptotic and/or cell cycle regulators. Indeed, treatment with suberoylanilide hydroxamic acid greatly reduced the expression of the inhibitor of apoptosis protein survivin and cdc2 activity in HCT116 p21(+/+) cells but not in the HCT116 p21(-/-) cells. Inhibition of cdc2 activity with flavopiridol decreased survivin expression and sensitized the p21-deficient cells to lexatumumab-induced apoptosis. Similarly, small interfering RNA-mediated knockdown of survivin also enhanced lexatumumab-mediated cell death. Therefore, survivin expression plays a key role in lexatumumab resistance, and reducing survivin expression by inhibiting cdc2 activity is a promising strategy to enhance the anticancer activity of lexatumumab.

Identification of a promiscuous HLA DR–restricted T-cell epitope derived from the inhibitor of apoptosis protein survivin

The inhibitor of apoptosis protein survivin is a promising tumor-associated antigen specifically recognized by CD8+ cytotoxic effector T-lymphocytes (CTL). To improve current vaccines that aim to induce survivin-specific CTL, it is necessary to study the role of CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Because both TH and Treg cells recognize antigens in the context of HLA-class II molecules, identification of HLA class II-associated peptide epitopes from survivin is required. Here, we analyzed T-cell responses against survivin using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. Six peptides were shown to induce CD4+ T-cell responses, restricted by HLA-DR molecules. For one peptide epitope, SVN10, T-cell clones were demonstrated to be capable of recognizing naturally processed antigen. SVN10-specific T cells could be stimulated from the blood of healthy individuals and cancer patients with multiple HLA-DR genotypes. Thus the identified SVN10 epitope can be used to study the role of CD4+ TH and Treg cells in immune responses and possibly be included in a multivalent peptide vaccine against survivin.

Survivin is an independent predictor of short-term survival in poor prognostic breast cancer patients

Established clinico–pathological factors can place patients with breast cancer into good and poor prognostic categories, but even within these groups behaviour and response to treatment can differ. This study examined the value of cell cycle and apoptotic regulatory proteins in predicting behaviour in a poor prognostic group. A total of 165 patients, all of whom had died of breast cancer with duration of survival 12–127 months, median 38 months, were examined using immunohistochemistry for proliferation, apoptosis, p53, phosphorylated p53, p21, checkpoint kinase 2 (Chk2), bcl-2, bax, survivin and XIAP. All had received chemotherapy and/or hormonal therapy and were predominantly T2, node positive, grade III with only half oestrogen-receptor (ER) positive. High proliferation, phosphorylated p53, Chk2 and survivin expression correlated with grade III and lack of ER, whereas low proliferation, p21 and bcl-2 related to better grade and presence of ER. On univariate analysis grade, proliferation, phosphorylated p53, bcl-2, ER and survivin related to duration of survival. In multivariate analysis, grade (P=0.001) and survivin (P=0.005) were independent prognostic factors, grade III and presence of survivin relating to shorter survival. The latter was particularly for those patients receiving neoadjuvant therapy and adjuvant chemo- and hormonal therapy. The presence of the inhibitor of apoptosis protein survivin is a highly significant independent predictor of shorter duration of survival of patients with poor prognostic features, and merits investigation as a marker in other prognostic groups.

Autoantibodies to the inhibitor of apoptosis protein survivin in patients with brain tumors

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is frequently expressed in cancers, including meningiomas and gliomas. Survivin may be associated with tumor progression and poor prognosis of patients with brain tumors. Using ELISA and immunoblot analysis we asked whether survivin is capable of eliciting a humoral immune response in patients with meningiomas and gliomas. Survivin-specific antibodies were detected in 5 of 42 (11.9%) patients with meningiomas and 3 of 35 (8.6%) patients with malignant gliomas of the WHO grades 3 and 4, but not in healthy controls. Tumors of patients with detectable anti-survivin antibodies demonstrated survivin expression in at least 20% of the tumor cells as assessed by immunohistochemistry. We conclude that patients with meningiomas and malignant gliomas can mount a high-titer IgG immune response against the 'universal' tumor-associated antigen survivin. Anti-survivin antibodies may represent attractive tools for diagnosis and follow-up of brain tumors.

Relationship between survivin expression and recurrence, and prognosis in hepatocellular carcinoma

To study the expression of the inhibitor of apoptosis protein survivin in hepatocellular carcinoma (HCC), and its correlation with clinicopathological factors, cell proliferation, recurrence and prognosis after hepatectomy. Immunohistochemical staining of survivin and Ki-67 was performed by the standard streptavidin-peroxidase technique on paraffin sections of 55 cases of HCC. The positive rate of survivin in HCC was 52.7% (29/55). Significant correlation was found between survivin expression with portal vein thrombi and intrahepatic matastasistic nodes (P < 0.05). The recurrent rate in survivin-positive HCC was significantly higher than that in survivin-negative HCC after hepatectomy, the 1- and 3-year survival rate in patients with survivin-positive tumors was significantly lower than that in patients with survivin-negative tumors (58.62 and 10.34% vs 76.92 and 30.77%, P < 0.05, log-rank test). The proliferation index (Ki-67) in survivin-positive HCC (33.83% +/- 18.90%) was significantly higher than that in survivin-negative HCC (19.60% +/- 19.35%) (P < 0.05). Survivin may play an important role in progression of HCC by promoting cell proliferation, and may be positively correlated with high risk of disease recurrence and poor prognosis in HCC. Its expression may serve as a prognostic factor for patients with HCC after hepatectomy.

Survivin Regulates Proliferation of Normal Hematopoietic Stem and Progenitor Cells In Vivo.

The inhibitor of apoptosis protein Survivin is barely detectable in most normal adult tissues but is over-expressed in almost all cancers. Survivin regulates apoptosis, cell division and cell cycle, making anti-Survivin therapy an attractive cancer treatment strategy. We reported that Survivin is expressed and regulated by hematopoietic growth factors in normal human CD34+ cells and that over-expression of wild-type Survivin in bone marrow cells enhances in vitro proliferation and survival of normal hematopoietic progenitor cells, whereas disrupting Survivin reduced their proliferation and survival. These results suggest that Survivin regulates normal hematopoietic progenitor cell function. Although targeted anti-Survivin therapies for cancers demonstrate efficacy without overt toxicity in animal models, the consequences of in vivo Survivin disruption in normal hematopoietic stem and progenitor cells (HSPC) has not been determined. In order to understand the physiological roles of Survivin in normal HSPC function in vivo, we created Cre-ER™/Survivin flox/flox mice, in which the Survivin gene can be excised by Tamoxifen treatment and characterized HSPC growth following Survivin gene deletion. RT-PCR analysis showed that Survivin mRNA is expressed in freshly isolated normal mouse marrow Sca-1+, c-kit+, lin− (SKL) cells and more primitive CD34−SKL cells, which contain long term repopulating hematopoietic stem cells (HSC). Administration of 5mg of Tamoxifen for 6 days (3 days injection, 3 days off, 3 additional days and analyzed 14 days after final injection) in Cre-ER™/Survivin flox/flox mice induced Survivin gene deletion in marrow cells, but had little effect on peripheral blood cell count, marrow cellularity (3.5+/−7.1%, NS) or the proportion or total number of lineage committed cells (Gr-1+, Mac-1+, B220+, CD4+ and/or CD8+) in marrow and in peripheral blood. In contrast, short term Survivin deletion significantly decreased the frequency and the absolute number of undifferentiated linneg cells (37+/−6% reduction), c-kit+, lin− cells (35.2+/−8.4% reduction,), CFU-GM (31+/−9 % reduction), Lin−, IL7Ra−, Sca-1−, c-kit+, CD34+, Fcglow common myeloid progenitor cells (52+/−13% reduction), SKL cells (56.8+/−5.4% reduction) and CD34−SKL cells (60.6+/−5.5% reduction) in bone marrow compared to control mice. The effect of Survivin gene deletion was more dramatic on primitive hematopoietic populations compared to mature cells, which is consistent with down-regulation of Survivin in hematopoietic cells with terminal differentiation. Similarly, treatment of bone marrow cells from Cre-ER™/Survivin flox/flox mice with 1uM of Tamoxifen in vitro significantly reduced the number of CFU-GM, (c-kit+, lin−) KL, SKL and CD34−SKL cells cultured with hematopoietic cytokines and increased apoptosis measured by Annexin-V staining. These results suggest that Survivin is required and regulates normal hematopoietic stem and progenitor function in vivo and that Survivin function may be selectively essential for growth and differentiation of primitive hematopoietic cells. In addition, acute ablation of Survivin may cause adverse toxicity on HSPC that provide long term hematopoiesis in the patients receiving anti-Survivin target therapies.

Nuclear localization of Survivin renders HeLa tumor cells more sensitive to apoptosis by induction of p53 and Bax

Clinical studies have shown that nuclear expression of the inhibitor of apoptosis protein Survivin in tumor cells predicted a favorable prognosis whereas cytosolic-localized protein caused a decreased overall survival. Therefore Survivin’s subcellular localization may be important for its anti-apoptotic capacity. To address this question, we investigated localization and function of Survivin in normal human lung fibroblasts (NHLFs) and HeLa tumor cells. NHLFs of early passages expressed Survivin in the nucleus and were highly sensitive to C2 ceramide, which induces the mitochondrial apoptotic pathway. In contrast, NHLFs at higher passages relocated Survivin to the cytosol and became more resistant to C2 ceramide. Blocking nuclear export of Survivin by leptomycin B in HeLa cells increased susceptibility to C2 ceramide. In addition, transduction of HeLa cells with Survivin fused to a nuclear localization signal augmented basal expression levels of p53 and Bax and enhanced sensitivity for intrinsic apoptosis. Those findings suggest that a predominant nuclear localization of Survivin increases the sensitivity for pro-apoptotic stimuli, whereas nuclear export enables Survivin to fulfill its inhibitor of apoptosis function. A therapeutic intervention which holds Survivin in the nucleus of tumor cells might improve cancer therapy.

97 Cisplatin-induced apoptosis in malignant pleural mesothelioma cells involves p53 but not p14ARF

Background: Malignant pleural mesothelioma (MPM) tumours often contain homozygous deletions in the INK4A locus that encodes p14ARF, a tumour suppressor protein that promotes p53 stability by blocking the p53 inhibitor MDM2. It has been suggested that p53 is inactive in MPM due to the absence of p14ARF expression. Methods and Results: We investigated p53 activation in p14ARF negative MPM cells or primary cultures after cisplatin treatment and found increased levels of phospho-p53 (serine-15), total p53 protein and p53 specific DNAbinding activity in the majority of cell lines. In addition, the expression levels of p53-positively controlled genes p21WAF, Bax, pig3 and MDM2 increased on incubation with cisplatin, whereas levels of the negatively regulated p53 target gene survivin decreased. Cisplatin-induced apoptosis was detected in p14ARF-deficient cells expressing wild type p53 but not in a p53 mutant cell line. Knockdown of wild type p53 with siRNA rendered MPM cells more resistant to cisplatin-induced apoptosis. Over-expression of p14ARF in p53 wild type MPM cells by transient transfection did not increase their sensitivity to cisplatininduced apoptosis, however, pre-incubation with antisense oligonucleotides targeting the inhibitor of apoptosis protein survivin lead to p53 activation and increased apoptosis sensitivity to cisplatin. Conclusions: These results suggest that p53 is functional in MPM in the absence of p14ARF, and that therapies that target the inhibitor of apoptosis protein survivin are worthy of investigation in MPM.

Downregulation of the inhibitor of apoptosis protein survivin in keratinocytes and endothelial cells in psoriasis skin following infliximab therapy

Survivin, an inhibitor of apoptosis protein (IAP), has been implicated in endothelial cell stability, through inhibition of apoptosis and in cell proliferation. To evaluate the effect of antitumour necrosis factor (TNF)-alpha therapy on survivin expression in psoriasis skin at 0, 2 and 12 weeks after infliximab therapy. Skin biopsies were obtained from 16 patients; 11 also had arthritis with active skin/joint disease. Clinical scores [Psoriasis Area and Severity Index (PASI), involved body surface area (BSA), Disease Activity Score (DAS28) and Health Assessment Questionnaire] were recorded. Inflammatory infiltration and survivin protein expression were examined and graded by immunohistochemical staining, and mRNA levels were determined by real-time polymerase chain reaction. Survivin mRNA and protein were demonstrated in all baseline lesional biopsies. Survivin mRNA and protein expression was significantly greater in lesional compared with nonlesional baseline skin (P < 0.05). Differential cellular localization of survivin was demonstrated with cytoplasmic survivin protein expression localized to the perivascular/endothelial regions and strong nuclear staining localized in the basal layer of the epidermis. Infliximab produced a dramatic clinical response in skin and joints (P < 0.05), paralleled by significant reduction in the inflammatory infiltrate and survivin protein expression (P < 0.05) which was reflected at the mRNA level where expression was significantly reduced by week 12 (P < 0.01). Survivin protein levels before and after treatment significantly correlated with PASI (r = 0.478, P < 0.05) and BSA scores (r = 0.528, P < 0.024). PASI strongly correlated with BSA (r = 0.949, P < 0.0001) and DAS28 (r = 0.717, P < 0.002) scores. Survivin correlates with disease activity in patients with psoriasis and is significantly downregulated following anti-TNF-alpha treatment. Understanding the role of IAPs in cell survival/antiapoptosis and proliferation mechanisms may provide important insights into downstream therapeutic targeting in inflammation.

Cross-talk between Epidermal Growth Factor Receptor and Hypoxia-inducible Factor-1α Signal Pathways Increases Resistance to Apoptosis by Up-regulating Survivin Gene Expression

Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.

Survivin silencing sensitizes imatinib-resistant CML cells to the cytotoxic effect of hydroxyurea

13119 Background: Imatinib Mesylate (IM), a semi-specific inhibitor of the BCR-ABL tyrosine kinase, is currently the treatment of choice for Chronic Myeloid Leukemia (CML). However, about one third of CML patients treated with IM develop resistance to the drug because of reactivation of BCR-ABL kinase activity. This phenomenon is usually ascribed to the amplification of the BCR-ABL gene or to the selection of leukemic clones harboring point mutations that abrogate IM binding. To identify novel anti-apoptotic signaling pathways employed by BCR-ABL and devise strategies capable of killing IM-resistant CML cells, we investigated the interplay between BCR-ABL and the Inhibitor of Apoptosis Protein Survivin. Methods: Murine hematopoietic cells (32D) transduced with p210 BCR-ABL and human cell lines either positive (K562, KCL22, KYO1 and LAMA84) or negative (HL60) for the BCR-ABL oncoprotein, were analyzed for Survivin expression by western blot before and after IM treatment. Three different pathways (MAPK, PI3K and JAK2/STA3) potentially involved in BCR-ABL-mediated induction of Survivin were studied using inhibitors specific for each signaling cascade. The effect of Survivin on the proliferation and viability of IM-sensitive and IM-resistant CML cells was investigated after silencing Survivin expression with small interfering RNAs. Results: BCR-ABL tyrosine kinase activity induced an over-expression of Survivin in both human and murine hematopoietic cell lines. This over-expression was both at the transcriptional and the translational level and required the JAK2/STAT3 pathway. Survivin silencing by siRNA increased IM cytotoxicity in IM-sensitive cells but failed to restore IM efficacy in IM-resistant cells. However, Survivin silencing sensitized CML cells to the cytotoxic effect of hydroxyurea and enhanced the efficacy of this compound on three different murine cell lines are insensitive to IM because of point mutations in the BCR-ABL kinase domain (Ba/F3p210Y253F, Ba/F3p210D276G and Ba/F3p210T315I). Conclusions: Reduction of Survivin expression improves the efficacy of IM and increases the sensitivity of IM-resistant CML cells to hydroxyurea. Survivin may represent an attractive therapeutic target for both IM-sensitive and IM-resistant CML patients. No significant financial relationships to disclose.

Caveolin-1 controls cell proliferation and cell death by suppressing expression of the inhibitor of apoptosis protein survivin

Caveolin-1 is suggested to act as a tumor suppressor. We tested the hypothesis that caveolin-1 does so by repression of survivin, an Inhibitor of apoptosis protein that regulates cell-cycle progression as well as apoptosis and is commonly overexpressed in human cancers. Ectopic expression of caveolin-1 in HEK293T and ZR75 cells or siRNA-mediated silencing of caveolin-1 in NIH3T3 cells caused downregulation or upregulation of survivin mRNA and protein, respectively. Survivin downregulation in HEK293T cells was paralleled by reduced cell proliferation, increases in G0-G1 and decreases in G2-M phase of the cell cycle. In addition, apoptosis was evident, as judged by several criteria. Importantly, expression of green fluorescent protein-survivin in caveolin-1-transfected HEK293T cells restored cell proliferation and viability. In addition, expression of caveolin-1 inhibited transcriptional activity of a survivin promoter construct in a beta-catenin-Tcf/Lef-dependent manner. Furthermore, in HEK293T cells caveolin-1 associated with beta-catenin and inhibited Tcf/Lef-dependent transcription. Similar results were obtained upon caveolin-1 expression in DLD1 cells, where APC mutation leads to constitutive activation of beta-catenin-Tcf/Lef-mediated transcription of survivin. Taken together, these results suggest that anti-proliferative and pro-apoptotic properties of caveolin-1 may be attributed to reduced survivin expression via a mechanism involving diminished beta-catenin-Tcf/Lef-dependent transcription.

High nuclear expression of the apoptosis inhibitor protein survivin is associated with disease recurrence and poor prognosis in laryngeal basaloid squamous cell carcinoma

Conclusion. Nuclear expression of survivin should be studied as a promising marker of higher-risk laryngeal basaloid squamous cell carcinomas (BSCCs), which can then be treated more aggressively and followed more closely. Objective. BSCC is an uncommon bimorphic variant of SCC. The hypothesized greater aggressiveness and poorer prognosis of head and neck BSCC compared to SCC are still under debate. The regulation of apoptotic cell death has a profound effect on the pathogenesis and progression of malignancies. Survivin is the smallest member of the inhibitor of apoptosis gene family. The aim of this study was to investigate for the first time the expression of the inhibitor of apoptosis protein survivin in laryngeal BSCCs and their neck lymph node metastases and to compare the results with those obtained with conventional SCCs. Material and methods. Immunoreactivity to survivin was determined in nine laryngeal BSCCs and nine site- and stage-matched SCCs. Results. A nuclear subcellular localization of survivin dominated in both primary laryngeal BSCCs and SCCs and in their lymph node metastases. There was no significant difference in mean survivin expression between primary laryngeal BSCCs (25.1%) and SCCs (25.6%) (p=0.86). Nuclear survivin expression was significantly higher in BSCCs associated with disease recurrence and poor prognosis (p=0.02).

The Survivin/p21WAF1/Cip1 Axis and the CXC Chemokine SDF1α Regulate Growth Factor Independent Survival of Primary Hematopoietic Progenitor Cells Induced by Internal Tandem Duplication Mutations of Flt3.

Internal Tandem Duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) and aberrant over-expression of the inhibitor of apoptosis protein Survivin, are frequently found in patients with acute myeloid leukemia (AML) and are associated with poor prognosis. We have shown that ITD-Flt3 increases expression of Survivin and the cyclin dependent kinase inhibitor p21WAF1/Cip1 in Ba/F3 cells and that Survivin regulates hematopoietic progenitor cell (HPC) proliferation through p21WAF1/Cip1 dependent and independent pathways (Fukuda et al. Blood, 2004.103:120), which functionally link Survivin and p21 downstream of ITD-Flt3 signaling. Elevated CXCR4 expression associated with ITD-Flt3 mutations has been recently reported in AML patients and may confer an adverse prognosis. Although it is not known whether ITD-Flt3 signaling interacts with the CXCR4 signaling pathway to regulate cell survival, our recent data indicate that ITD-Flt3 and SDF1α/CXCR4 signaling pathways functionally interact to enhance hematopoietic cell migration (Fukuda et al. Blood , 2005. 105:3117). This suggests that these two pathways may coordinate or regulate trafficking of transformed hematopoietic cells and may be involved in AML disease progression. In order to investigate whether Survivin, p21 and SDF1α/CXCR4 mediate enhanced HPC survival as a consequence of constitutive ITD-Flt3 signaling, we utilized marrow cells from control, p21−/− or Tamoxifen-Cre conditional Survivinfloxed/floxed knockout mice and evaluated if disrupting Survivin or p21 can block the aberrant enhanced HPC proliferation induced by ITD-Flt3 in vitro and whether SDF1α could accentuate the enhanced survival of primary mouse HPC induced by ITD-Flt3. Over-expression of human wild-type Flt3 failed to induce growth factor independent proliferation of marrow CFU-GM, however, ectopic expression of ITD-Flt3 mutants cloned from AML patients significantly enhanced survival of CFU-GM from control mice after growth factor deprivation and allowed for factor independent proliferation. In contrast, conditional deletion of Survivin or loss of p21 significantly reduced growth factor independent survival of CFU-GM induced by ITD-Flt3 (53±17% and 41±9% reduction in Survivin and p21 KO cells, respectively; P<0.05), suggesting that Survivin and p21 are required for enhanced survival of HPC induced by ITD-Flt3. Furthermore, SDF-1α dose-dependently increased the number of growth factor independent CFU-GM expressing ITD-Flt3 (40–140% increase), suggesting that SDF-1α/CXCR4 and ITD-Flt3 signaling functionally interact to enhance HPC survival, an effect consistent with the synergistic enhancement of normal HPC survival induced by the combination of Flt3 ligand plus SDF-1α and enhanced migration to SDF1α induced by ITD-Flt3. This effect is unlikely due to up-regulation of CXCR4, since CXCR4 in Ba/F3 and 32D cells expressing ITD-Flt3 was significantly down-regulated compared to wild type Flt3. These reults provide evidence that the Survivin/p21 axis and SDF1α/CXCR4 mediate resistance to apoptosis and transformation of HPC and/or cell trafficking by ITD-Flt3, and that disruption of these signaling pathways may be additional therapeutic strategies for patients with AML expressing ITD-Flt3.

Disruption of the Inhibitor of Apoptosis Protein Survivin Sensitizes Bcr-abl–Positive Cells to STI571-Induced Apoptosis

The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides -1,194 and -1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl-transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl-transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.

Survivin expression is up-regulated in vascular injury and identifies a distinct cellular phenotype

The healing response to vascular injury is characterized by neointimal thickening. Proliferation and phenotypic transformation of vascular smooth muscle cells (SMCs) have been implicated in this process. We sought to investigate the role of survivin, a dual regulator of cell proliferation and apoptosis, in lesion formation after diverse forms of vascular injury. Rabbits underwent either carotid interposition vein grafting (n = 17) or bilateral femoral balloon injury (BI; n = 29); some in the BI group were placed on a high-cholesterol diet. A subset of BI arteries were treated with local adenoviral gene delivery of a survivin dominant negative-mutant (AdT34A) versus vector or saline controls. Survivin expression in vessels was analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry (IHC), which also included markers of SMC differentiation. Specimens of human tissue including failed lower extremity bypass grafts and carotid plaque were also examined. RT-PCR and IHC demonstrated increased survivin expression in all experimental models, colocalizing at early times with proliferating and alpha-actin-expressing cells but was largely absent in mature, contractile SMCs. Delivery of AdT34A after BI attenuated neointimal hyperplasia. These studies provide strong evidence supporting a role for survivin in the cellular response to vascular injury. The regulation of cell proliferation, death, and phenotype after vascular interventions remains incompletely understood. We investigated the role of the inhibitor of apoptosis protein survivin in diverse models of vascular injury. The results suggest that survivin is an important modulator of the generalized vascular injury response and may represent a relevant target for therapies targeting intimal hyperplasia.

A DNA Vaccine Targeting Survivin Combines Apoptosis with Suppression of Angiogenesis in Lung Tumor Eradication

A novel strategy achieved the eradication of lung tumor metastases by joint suppression of angiogenesis in the tumor neovasculature and induction of tumor cell apoptosis. This was accomplished by CTLs induced by a DNA vaccine encoding secretory chemokine CCL21 and the inhibitor of apoptosis protein survivin, overexpressed by both proliferating endothelial cells in the tumor vasculature and tumor cells. Oral delivery of this DNA vaccine by doubly attenuated Salmonella typhimurium (dam(-) and AroA(-)) to such secondary lymphoid organs as Peyer's patches in the small intestine, elicited marked activation of antigen-presenting dendritic cells, and an effective CD8(+)T cell immune response against the survivin self-antigen. This resulted in eradication or suppression of pulmonary metastases of non-small cell lung carcinoma in both prophylactic and therapeutic settings in C57BL/6J mice. Moreover, the suppression of angiogenesis induced by the vaccine did not impair wound healing or fertility of treated mice. It is anticipated that such novel DNA vaccines will aid in the rational design of future strategies for the prevention and treatment of cancer.

Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma

To investigate expression and significance of inhibitor of apoptosis protein survivin in hepatocellular carcinoma (HCC). The expression of survivin and vascular endothelial growth factor (VEGF) was investigated in 38 cases of HCC tissues and 38 liver cirrhosis tissues by immunohistochemistry and Western blot. The relationship between the expression of survivin and clinicopathological factors of HCC was analyzed. Survivin protein was detected in 23 (60.5%) of 38 HCCs and 3 (7.9%) of 38 liver cirrhosis tissues. In 23 cases of HCC which expressed survivin, the expression of VEGF was positive in 18 cases and slight positive or negative in 5 cases. While in 15 cases of HCC which did not express survivin, 12 cases did not express or slightly expressed, and 3 cases expressed VEGF. In liver cirrhosis tissues, the expression of VEGF was as follows: 24 cases were negative, 10 cases were weak positive and 4 cases were strong positive. The expression of survivin was coincident with the expression of VEGF in HCC (P<0.01). The expression of survivin in HCC had no relationship with the patients' age, gender, tumor size and differentiation level of HCC, while it was related to the metastasis of HCC. The protein quantitative analysis by Western blot also showed that overexpression of survivin in HCC was closely correlated to the expression of VEGF (P<0.01). Furthermore, stronger expression of survivin and VEGF was also found in patients with metastasis rather than in those with no metastasis (P<0.01). Survivin plays a pivotal role in the metastasis of HCC, and it has some correlation with tumorigenesis. The expression of survivin in the primary lesion is very useful as an indicator for metastasis and prognosis of HCC. It could become a new target of gene therapy of HCC.

Internal Tandem Duplication of Flt3 (ITD-Flt3) Bypasses Growth Factor Mediated Survivin Expression, while Disruption of Survivin Accelerates Cell Death in Hematopoietic Cells Expressing ITD-Flt3.

The inhibitor of apoptosis protein Survivin is barely detectable in normal adult tissues but is over-expressed in almost all cancers and hematopoietic malignancies and confers resistance to apoptosis and aberrant cell proliferation. We have shown that hematopoietic growth factors induce Survivin expression in normal CD34+ cells in both cell cycle dependent and independent fashion and that oncogenic Ras can enhance Survivin expression independent of growth factor stimulation. In addition, over-expression of Survivin blocks apoptosis of hematopoietic progenitor cells through a p21WAF1/Cip1 (p21) dependent mechanism. However the mechanisms responsible for Survivin overexpression in malignant hematopoietic cells is poorly understood. Flt3 is a type III tyrosine kinase receptor expressed mainly in the primitive hematopoietic compartment. Internal tandem duplication (ITD) mutations of the Flt3 gene that activate the Flt3 tyrosine kinase are frequently found in patients with AML and associated with poor prognosis. Furthermore, their ectopic expression in IL-3 dependent Ba/F3 cells results in IL-3-independent proliferation and a myeloproliferative disease in mice. In order to understand the mechanism by which ITD-Flt3 enhances cell survival, we examined whether ITD-Flt3 can bypass Survivin up-regulation independent of hematopoietic growth factors and if Survivin lies down stream of ITD-Flt3 in prolonging cell survival. Incubation of primary umbilical cord blood CD34+ cells with Flt3 ligand induced Survivin expression, indicating that Survivin lies down stream of Flt3 signaling pathways in CD34+ cells. While Survivin expression was comparable in Ba/F3 cells expressing ITD-Flt3 (W51, W73 and W78 mutants, kindly provided by Dr. DG Gilliland) and wild-type Flt3 when maintained in the presence of IL-3, ITD-Flt3 prevented down-modulation of Survivin expression induced by IL-3 withdrawal even during G0/G1 phase of cell cycle, suggesting that up-regulation of Survivin is not a consequence of cell cycle progression associated with ITD-Flt3 expression. The increase in Survivin by ITD-Flt3 was associated with reduced active caspase-3 and prolonged cell survival coincident with induction of p21 expression and Tyrosine 15 phosphorylation of CDK1 (Cdc2), which have been implicated in anti-apoptosis. These results are consistent with a p21 dependent anti-apoptosis effect of Survivin in primary hematopoietic progenitor cells and suggest the involvement of Survivin-p21 and CDK1 in ITD-Flt3 mediated cell survival. Overexpression of dominant negative T34A Survivin inhibits the enhanced survival induced by ITD-Flt3 in Ba/F3 cells following IL-3 withdrawal, suggesting that the survival effect of ITD-Flt3 is mediated by Survivin. Similar to ITD-Flt3, overexpression of ectopic wild-type Survivin results in enhanced cell survival in Ba/F3 cells but failed to induce cell cycle progression and IL-3-independent proliferation, indicating that Survivin alone is not sufficient for induction of growth factor independent proliferation and the cell cycle enhancing effect of ITD-Flt3. In summary, our findings indicate that ITD-Flt3 up-regulates Survivin expression independent of hematopoietic growth factors and that the ITD-Flt3/Survivin axis provides a survival advantage in transformed hematopoietic cells. Disruption of these pathways may increase apoptosis of malignant hematopoietic cells and provide therapeutic benefit.

Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2-overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2-overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.