Survivin and Granzyme B-induced apoptosis, a novel anticancer therapy.

Survivin is an antiapoptotic protein highly expressed in malignant cells that confers resistance to cytotoxic therapy. Granzyme B is a potent cytotoxic protein that is released from mammalian natural killer cells and CTLs following noxious stimuli, including foreign invaders. Here, we took advantage of the properties of these two functionally divergent molecules to create a molecular agent that specifically activates Granzyme B within tumor cells. We designed Survivin and Granzyme B-induced apoptosis (SAGA), which consists of a fusion of the Survivin gene promoter to the coding sequence of active Granzyme B. In cultured human tumor cells transfected with SAGA DNA, Granzyme B is rapidly expressed and results in significant tumor cell death. In vivo, mice harboring human ovarian tumors had statistically significant clinical responses to SAGA treatment that were magnified following combination therapy with SAGA and paclitaxel. At the completion of a 3-week therapeutic trial, 3 of 15 animals were free of disease in the SAGA-treated group, and an additional eight animals had tumors that were nonpalpable and only detected on surgical resection. In contrast, 15 of 15 animals in the control and paclitaxel-only-treated groups had tumors at end of therapy. Treatment with SAGA with or without paclitaxel also prevented disease dissemination in 19 of 20 animals. These results strongly suggest that SAGA has the potential to be a potent agent for the treatment of primary and recurrent human ovarian carcinoma. Moreover, we predict that SAGA will be useful therapeutically in any human cancer that expresses Survivin.

Survivin splice variants regulate the balance between proliferation and cell death.

Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-DeltaEx3 interact within the mitochondria where they may inhibit mitochondrial-dependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.

Essential role for survivin in early brain development.

Apoptosis is an essential process during normal neuronal development. Approximately one-half of the neurons produced during neurogenesis die before completion of CNS maturation. To characterize the role of the inhibitor of apoptosis gene, survivin, during neurogenesis, we used the Cre-loxP-system to generate mice lacking survivin in neuronal precursor cells. Conditional deletion of survivin starting at embryonic day 10.5 leads to massive apoptosis of neuronal precursor cells in the CNS. Conditional mutants were born at the expected Mendelian ratios; however, these died shortly after birth from respiratory insufficiency, without primary cardiopulmonary pathology. Newborn conditional mutants showed a marked reduction in the size of the brain associated with severe, mutifocal apoptosis in the cerebrum, cerebellum, brainstem, spinal cord, and retina. Caspase-3 and caspase-9 activities in the mutant brains were significantly elevated, whereas bax expression was unchanged from controls. These results show that survivin is critically required for the survival of developing CNS neurons, and may impact on our understanding of neural repair, neural development, and neurodegenerative diseases. Our study is the first to solidify a role for survivin as an antiapoptotic protein during normal neuronal development in vivo.

Survivin 2alpha: a novel Survivin splice variant expressed in human malignancies.

BACKGROUND: Survivin and its alternative splice forms are involved in critical cellular processes, including cell division and programmed cell death. Survivin is expressed in the majority of human cancers, but minimally in differentiated normal tissues. Expression levels correlate with tumor aggressiveness and resistance to therapy. RESULTS: In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2alpha. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2alpha is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2alpha attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2alpha suggests a physical interaction with survivin. CONCLUSION: We characterized a novel survivin splice variant that we designated survivin 2alpha. We hypothesize that survivin 2alpha can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2alpha may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.

Dissecting the role of endothelial SURVIVIN DeltaEx3 in angiogenesis.

The identification of alternative splice variants of Survivin that possess distinct functions from those originally identified for the main Survivin isoform has greatly increased the complexity of our understanding of the role of Survivin in different cells. Previous functional studies of the Survivin splice variants have been performed almost exclusively in cancer cells. However, Survivin has increasingly been implicated in other normal physiologic and pathophysiologic processes, including angiogenesis. In this study, we dissect the involvement of Survivin DeltaEx3 in angiogenesis. We show by confocal microscopy that a pool of endothelial Survivin DeltaEx3 is localized to membrane ruffles. We also demonstrate that Survivin DeltaEx3 is the Survivin splice variant responsible for modulating angiogenesis in vitro, in tube formation assays, and in vivo, in an in vivo angiogenesis assay. Our data indicate that Survivin DeltaEx3 may regulate angiogenesis via several mechanisms including cell invasion, migration, and Rac1 activation. Our findings identify a novel pathway regulating angiogenesis through Survivin DeltaEx3 and a novel mechanism for Rac1 activation during angiogenesis. In conclusion, our results provide new insights into the regulation of endothelial cell homeostasis and angiogenesis by the Survivin proteins.

Survivin: an inhibitor of apoptosis in pediatric cancer.

Survivin is an inhibitor of apoptosis protein (IAP) expressed in a large number of adult malignancies. Its expression levels correlate with more aggressive disease and poor clinical outcome in many of these tumors. As its expression is restricted in normal adult differentiated tissues, it has become of great interest as both a tumor prognostic marker and as a potential biologic target for future anti-cancer therapies. Survivin expression and Survivin-based therapies have been examined in many of the more common pediatric malignancies. We present an overview of Survivin function and current research exploring its biologic and therapeutic roles in pediatric tumors.

Placental defects are associated with male lethality in bare patches and striated embryos deficient in the NAD(P)H Steroid Dehydrogenase-like (NSDHL) Enzyme.

NSDHL is a 3beta-hydroxysterol dehydrogenase that is involved in the removal of C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the Nsdhl gene are associated with the X-linked male lethal mouse mutations bare patches (Bpa) and striated (Str), as well as with most cases of human CHILD syndrome. To begin to examine the pathogenesis of these disorders, we have determined that affected male embryos for several Nsdhl alleles die in midgestation, between E10.5 and 13.5, while the majority of affected male embryos for the most severe allele, Nsdhl(Bpa1H), die prior to E9.5. Although no consistent anomalies were identified in affected male embryos themselves, the labyrinth layer of the fetal placenta was always thinner, with fewer fetal vessels and decreased proliferation of labyrinth trophoblast cells. X-inactivation is non-random in females in most lineages of the rodent placenta with preferential inactivation of the paternal X chromosome. For primary defects involving these extraembryonic lineages, heterozygous females with a mutant maternal X chromosome would be expected to have an identical placental phenotype to that found in affected male embryos. We hypothesize that abnormalities in cells of the allantoic mesoderm that undergo random X-inactivation and form the endothelial lining of the fetal vessels of the labyrinth are associated with the male lethality, perhaps through disruption of an as yet unidentified signaling pathway.

NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes and on the surface of lipid droplets.

NSDHL, for NAD(P)H steroid dehydrogenase-like, encodes a sterol dehydrogenase or decarboxylase involved in the sequential removal of two C-4 methyl groups in post-squalene cholesterol biosynthesis. Mutations in this gene are associated with human CHILD syndrome (congenital hemidysplasia with ichthyosiform nevus and limb defects), an X-linked, male lethal disorder, as well as the mouse mutations bare patches and striated. In the present study, we have investigated the subcellular localization of tagged proteins encoded by wild-type and selected mutant murine Nsdhl alleles using confocal microscopy. In addition to an ER localization commonly found for enzymes of post-squalene cholesterol biosynthesis, we have identified a novel association of NSDHL with lipid droplets, which are endoplasmic reticulum (ER)-derived cytoplasmic structures that contain a neutral lipid core. We further demonstrate that trafficking through the Golgi is necessary for ER membrane localization of the protein and propose a model for the association of NSDHL with lipid droplets. The dual localization of NSDHL within ER membranes and on the surface of lipid droplets may provide another mechanism for regulation of the levels and sites of accumulation of intracellular cholesterol.