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Survivin-directed RNA interference cocktail is a potent suppressor of tumour growth in vivo.

ABSTRACT: BACKGROUND: Survivin is proposed to play a central role in the progression and resistance to therapy of diverse tumour types. High levels of this molecule in tumour cells also correlate with loss of the TP53 tumour suppressor gene, suggesting a molecular connection between TP53 loss and transcriptional induction of Survivin. Patients with TP53 germline mutations, such as those with Li-Fraumeni syndrome, are particularly susceptible to sarcomas, including rhabdomyosarcomas. Our study aimed to identify rhabdomyosarcoma tumours that express Survivin, in order to test novel Survivin-targeted therapies in these tumours. METHODS: Tumour microarray slides composed of 63 primary rhabdomyosarcoma tumours were stained with a polyclonal antibody to Survivin to identify tumours expressing Survivin. Subcutaneous tumours were then established in NOD/SCID mice using RH30(red) cells, a red fluorescent clone of the RH30 human alveolar rhabdomyosarcoma cell line. Tumours were treated by hydrodynamic injection with a cocktail of Survivin-shRNA-encoding plasmids for a period of 2 weeks. RESULTS: Over 80% of primary rhabdomyosarcoma tumours expressed Survivin. Treatment of rhabdomyosarcoma xenografts showed greater than 70% reduction in growth when compared with control injected tumours at study completion (average tumour sizes: 1683 v 304 mm3, p<0.05). CONCLUSIONS: Our findings support a role for Survivin in rhabdomyosarcoma biology and provide preliminary evidence for the therapeutic use of Survivin-targeted RNA interference for human tumours that express high levels of this molecule.

SUBMITTER: Caldas H

PROVIDER: S-EPMC2564625 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Survivin-directed RNA interference cocktail is a potent suppressor of tumour growth in vivo.

Caldas H H Holloway M P MP Hall B M BM Qualman S J SJ Altura R A RA

Journal of medical genetics 20050520 2

<h4>Background</h4>Survivin is proposed to play a central role in the progression and resistance to therapy of diverse tumour types. High levels of this molecule in tumour cells also correlate with loss of the TP53 tumour suppressor gene, suggesting a molecular connection between TP53 loss and transcriptional induction of Survivin. Patients with TP53 germline mutations, such as those with Li-Fraumeni syndrome, are particularly susceptible to sarcomas, including rhabdomyosarcomas. Our study aimed ...[more]

PMID: 15908567

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Project description:Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G(2)/M fraction consistent with a mitotic defect; 4',6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G(2)/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall.

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Survivin-directed RNA interference cocktail is a potent suppressor of tumour growth in vivo.

Publications

Survivin-directed RNA interference cocktail is a potent suppressor of tumour growth in vivo.

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