Functional analysis and targeting of the survivin pathway in tumor angiogenesis
Final Report Abstract
Our proposal sought to gain novel insights into the molecular mechanisms how Survivin/CPC proteins influence (non)-tumor cells in mitosis and interphase and thus, contribute to tumor angiogenesis and progression. First, we analyzed the expression and localization of Survivin/CPC proteins in cancer and non-malignant cell types (WP1). We could for the first time identify formation of Survivin/CPC protein-containing nuclear foci in interphase cells. Survivin/CPC foci already assembled at the beginning of S-phase and their localization could be narrowed down to (peri)centromeric heterochromatin. Foci formation mechanistically mostly resembles mitotic CPC assembly, including phosphorylation of Survivin on e.g. T117 by Aurora B kinase. However, in contrast to mitosis CPC foci colocalize with heterochromatin protein HP1 in interphase cells. Remarkably, we could identify PCNA as a possible interaction partner of Survivin/CPC proteins during replication, which is subject to current investigation. Our data further indicate a possible role of Survivin in DNA damage repair in the non-homologous end-joining (NHEJ) pathway, although Survivin/CPC foci did not colocalize with bona fide members of DNA repair machinery including yH2AX, ATM or DNA-PK. Moreover, we could show that depletion of Survivin resulted in a senescent phenotype, potentially counteracting tumor angiogenesis. To identify involved signaling pathways (WP2), we isolated pericytes and fibroblasts from patient material and analyzed possible correlations of NO- or VEGF-induced signal transduction pathways and Survivin expression. Here, we found p38 MAP kinase pathway activation in pericytes isolated from a tumor sample as well as in comparable co-culture experiments, which was however not dependent on continuous Survivin expression. VEGF-A treatment increased Survivin levels without affecting p38 MAP kinase signaling. We showed that in microvascular endothelial cells Caveolin-1 transcriptionally downregulates Survivin via interaction with ß-Catenin. As an additional approach to target (tumor) angiogenesis, we developed chemico-genetic targeted interference strategies aiming particularly at the cytoprotective activity of Survivin/CPC proteins (WP3). We identified novel compounds interfering with Survivin's nuclear export activity using cellular high throughput assays, capable of inhibiting Survivin function in cancer and endothelial cell, finally inducing apoptosis. In addition, we identified miRNA-542 as a direct regulator of Survivin in neuroblastoma and HNSCC. Cellular delivery of miR-542-3p by novel calciumphosphate/PGLA nanoparticles decreased cell proliferation and induced apoptosis in neuroblastoma xenograft tumors. To address Survivin and the CPC by the modulation of posttranslational modifications (PTMs), we showed that acetylation of Survivin at K129 but not at K90 impairs dimerization and function, also as part of the CPC. Survivin acetylation now also opens up a novel interference approach by addressing Histone-deacetylases (HDACs) as key regulatory enzymes in follow-up projects. The relevance of Survivin and its nucleo-cytoplasmic transport in cancer patients with high nuclear Survivin levels was further evaluated (WP4) by identifying NES-inactivating Survivin mutations in the tumor cells, but not in corresponding normal tissues, including endothelial cells. Collectively, results generated in this project not only increase our knowledge on Survivin function in tumor and non-malignant cells but also form the basis for future targeted interference strategies on multiple levels.
Publications
- Functional characterization of novel mutations affecting Survivin (BIRC5)-mediated therapy resistance in head and neck cancer patients. Human Mutation 2012, 34(2):395-404
Knauer S.K., Unruhe B., Karczewski S., Hecht R., Fetz V., Bier C., Friedl S., Wollenberg B., Pries R., Habtemichael N., Heinrich U.R., Stauber R. H.
(See online at https://doi.org/10.1002/humu.22249) - Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(D,L-lactide-co-glycolide acid) nanoparticles for intracellular delivery. Journal of Materials Chemistry B 2014, 2:7250-59
Dördelmann G., Kozlova D., Karczewski S., Lizio R., Knauer S.K., Epple M.
(See online at https://doi.org/10.1039/c4tb00922c) - Relevance of Survivin acetylation for its biological function. Dissertation 2014
Unruhe B.
- miR-542-3p exerts tumor suppressive functions in neuroblastoma by downregulating Survivin. International Journal of Cancer 2015, 136(6):1308-20
Althoff K., Lindner S., Odersky A., Mestdagh P., Beckers A., Karczewski S., Molenaar J., Bohrer A., Knauer S.K., Speleman F., Epple M., Kozlova D., Yoon S., Baek K., Vandesompele J., Eggert A., Schramm A., Schulte J.
(See online at https://doi.org/10.1002/ijc.29091) - An old flame never dies - Survivin in cancer and cellular senescence. Gerontology 2016, 62(2):173-81
Unruhe B., Schröder E., Wünsch D., Knauer S.K.
(See online at https://doi.org/10.1159/000432398) - Analysis of HDACi-induced changes in chromosomal passenger complex localization. Methods in Molecular Biology MIMB 2017, 1510:47-59
Unruhe-Knauf B., Knauer S.K.
(See online at https://doi.org/10.1007/978-1-4939-6527-4_4) - (2018) Translocation Biosensors—Versatile Tools to Probe Protein Functions in Living Cells. In: Johnston P., Trask O. (eds) High Content Screening. Methods in Molecular Biology, vol 1683. Humana Press, New York, NY. S. 195-21
Fetz, Verena; Stauber, Roland H.; Knauer, Shirley K.
(See online at https://doi.org/10.1007/978-1-4939-7357-6_12)
