Dario C. Altieri

Control of apoptosis and mitotic spindle checkpoint by survivin

Progression of the cell cycle and control of apoptosis (programmed cell death) are thought to be intimately linked processes, acting to preserve homeostasis and developmental morphogenesis. Although proteins that regulate apoptosis have been implicated in restraining cell-cycle entry and controlling ploidy (chromosome number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive. Here we show that a new inhibitor of apoptosis (IAP) protein,, survivin, is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner. At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Disruption of survivin–microtubule interactions results in loss of survivins anti-apoptosis function and increased caspase-3 activity, a mechanism involved in cell death, during mitosis. These results indicate that survivin may counteract a default induction of apoptosis in G2/M phase. The overexpression of survivin in cancer may overcome this apoptotic checkpoint and favour aberrant progression of transformed cells through mitosis.

Validating survivin as a cancer therapeutic target

Acquisition of the ability to evade cellular suicide, or apoptosis, is one of the master switches that contributes to cellular transformation and, ultimately, to invasive cancer. Much has been learned about the molecular organization of apoptotic pathways and their regulators, but the identification and validation of translational targets for apoptosis-based cancer therapy has posed a great challenge. Survivin is an attractive candidate for cancer therapy, so what is its potential applicability in the clinic?

Survivin, cancer networks and pathway-directed drug discovery

Although there is no shortage of potential targets for cancer therapeutics, we know of only a handful of molecules that are differentially expressed in cancer and intersect multiple pathways required for tumour maintenance. Survivin embodies these properties, and orchestrates integrated cellular networks that are essential for tumour cell proliferation and viability. Pursuing the nodal functions of survivin in cancer might lead to the development of global pathway inhibitors with unique therapeutic potential.

Survivin, versatile modulation of cell division and apoptosis in cancer

Survivin is a member of the inhibitor of apoptosis (IAP) gene family that has attracted attention from several viewpoints of basic and translational research. Its cell cycle-regulated expression at mitosis and association with the mitotic apparatus have been of interest to cell biologists studying faithful segregation of sister chromatids and timely separation of daughter cells. Investigators interested in mechanisms of apoptosis have found survivin an evolving challenge: while survivin inhibits apoptosis in vitro and in vivo, this pathway may be more selective as compared to cytoprotection mediated by other IAPs. Finally, basic and translational researchers in cancer biology have converged on survivin as a pivotal cancer gene, not simply for its sharp expression in tumors and not in normal tissues, but also for the potential exploitation of this pathway in cancer diagnosis and therapy. The objective of the present contribution is to line up current evidence and emerging concepts on the multifaceted functions of survivin in cell death and cell division, and how this pathway is being pursued for novel cancer therapeutic strategies.

Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

INDUCTION OF APOPTOSIS AND INHIBITION OF CELL PROLIFERATION BY SURVIVIN GENE TARGETING

Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75–130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer.

Anti-apoptosis gene, survivin, and prognosis of neuroblastoma

Neuroblastoma may be aggressive with a dismal outcome or regress spontaneously. Prognostic factors including age, stage, and ploidy, N-myc amplification, and histology are necessary to direct treatment. Gene products controlling apoptosis may modulate neuroblastoma’s variable course. Although expression of anti-apoptotic bcl-2 has been linked to a more aggressive disease, and inversely correlated with morphological features of apoptosis, it is unclear whether inhibition or apoptosis is a predictive factor in neuroblastoma or whether it reflects the degree of cell differentiation. An apoptosis inhibitor, survivin, has recently been identified. Unlike other members of the bcl-2 and IAP gene families, survivin is selectively expressed in all the most common human cancers but not in normal adult tissues. We determined the expression of survivin in neuroblastoma and its relation to disease progression. We studied 72 cases of neuroblastoma for expression of survivin by immunohistochemistry and immunoblotting and correlated our results with clinical stage and histological classification (Shimada). Specific staining for survivin was observed in 34 cases (47%) with variable numbers of positive cells (5–90%). Cases were scored positive when more than 5% of cells reacted with the anti-survivin antibody. When stratified for favourable/unfavourable histology, expression of survivin significantly (p=0·001) segregated with the unfavourable group (17/23, 73·9%), whereas the absence of survivin coincided with favourable histology (32/49, 65%). 14 of 17 cases (82%) with favourable histology which expressed survivin also stained positive for bcl-2 and AChE activity, did not change with age, whereas mean cortical CBF values measured with IMP-SPECT significantly decreased (figure). Since K1 equals the product of extraction fraction and CBF, K1 and CBF should correlate. The estimated mean cortical K1 values, simultaneously obtained with the calculation of k3, also decrease with age in the same fashion as CBF (data not shown, Y=20·0020X+0·62, r=0·356), indicating validity of the kinetic model. Cholinergic enzyme activity levels have been reported to decrease or remain unchanged with age in studies of necropsy brain. The biochemical studies in normal human ageing with necropsy brain specimens have limited value since not only lag time from death to sampling but also duration and condition preceding death of the patients may have great influences on the results. Therefore, a problem in research on neurochemistry of human ageing is the lack of fresh non-diseased brain tissue for analysis. In vivo noninvasive measurement of enzyme activity enables us to circumvent these drawbacks and gives us useful information. The results of this study show that AChE activity of the cerebral cortex does not change with age whereas CBF decreases, suggesting a preserved ascending cholinergic system to the cerebral cortex from the basal forebrain in normal ageing. We conclude that biochemical processes in the cholinergic system underlying ageing and dementia are different.

The molecular basis and potential role of survivin in cancer diagnosis and therapy.

Despite its genetic complexity and multifactoriality, two processes appear almost universally compromised in cancer: the control of cell proliferation and the regulation of cell lifespan. Survivin is a recently described molecule that has been implicated in both processes, and is overexpressed in most human cancers. The exploitation of the survivin signaling pathway might provide important predictive and prognostic clues in cancer diagnosis, and offer new therapeutic alternatives for cancer treatment.

Control of Apoptosis during Angiogenesis by Survivin Expression in Endothelial Cells

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis

Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood. Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells. In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis. Selective targeting of survivin to mitochondria enhances colony formation in soft agar, accelerates tumor growth in immunocompromised animals, and abolishes tumor cell apoptosis in vivo. Therefore, mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression.