Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumor-associated angiogenesis.
OBJECTIVE
Molecular antagonists of the inhibitor of apoptosis protein survivin have shown promise as novel anticancer strategies for triggering tumor cell apoptosis, dysregulating mitotic progression, and inhibiting tumor growth in preclinical models. However, how survivin couples to the cell death machinery has remained elusive, and the relevant cellular targets of survivin antagonists have not been completely elucidated.


METHODS
Human umbilical vein and dermal microvascular endothelial cells were infected with replication-deficient adenoviruses encoding survivin (pAd-Survivin), green fluorescent protein (pAd-GFP), or a phosphorylation-defective survivin Thr(34)-->Ala (pAd-T34A) dominant negative mutant. The effect of wild-type or mutant survivin was investigated on capillary network stability, endothelial cell viability, and caspase activation in vitro and on kinetics of tumor growth and development of angiogenesis in a breast cancer xenograft model in vivo. The cell death pathway initiated by survivin targeting was mapped with respect to cytochrome c release, changes in mitochondrial transmembrane potential, and apoptosome requirements using mouse embryonic fibroblasts deficient in Apaf-1 or caspase-9.


RESULTS
Adenoviral transduction of endothelial cells with pAd-Survivin inhibited growth factor deprivation- or ceramide-induced apoptosis, reduced caspase-3 and -7 generation, and stabilized three-dimensional capillary networks in vitro. Conversely, expression of pAd-T34A caused apoptosis in umbilical vein and dermal microvascular endothelial cells and resulted in caspase-3 activity. Cell death induced by survivin targeting exhibited the hallmarks of mitochondrial-dependent apoptosis with release of cytochrome c and loss of mitochondrial transmembrane potential and was suppressed in Apaf-1 or caspase-9 knockout mouse embryonic fibroblasts. When injected in human breast cancer xenografts, pAd-T34A inhibited growth of established tumors and triggered tumor cell apoptosis in vivo. This was associated with a approximately 60% reduction in tumor-derived blood vessels by quantitative morphometry of CD31-stained tumor areas, and appearance of endothelial cell apoptosis by internucleosomal DNA fragmentation in vivo.


CONCLUSIONS
Survivin functions as a novel upstream regulator of mitochondrial-dependent apoptosis, and molecular targeting of this pathway results in anticancer activity via a dual mechanism of induction of tumor cell apoptosis and suppression of angiogenesis.
Blanc-Brude
Olivier P
OP
Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Mesri
Mehdi
M
Wall
Nathan R
NR
Plescia
Janet
J
Dohi
Takehiko
T
Altieri
Dario C
DC
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D002196:Capillaries / Q000166:cytology / Q000378:metabolism; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000096:biosynthesis / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006801:Humans; D020287:In Situ Nick-End Labeling; D051034:Inhibitor of Apoptosis Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008833:Microcirculation; D008869:Microtubule-Associated Proteins / Q000378:metabolism / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism*; D008938:Mitosis; D009154:Mutation; D009363:Neoplasm Proteins; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000378:metabolism*; D009389:Neovascularization, Pathologic; D010766:Phosphorylation; D000077022:Survivin; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D000077022:Survivin; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases