Browsing by Author "Strotbek, Michaela"

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Open Access
A global microRNA screen identifies regulators of the ErbB receptor signaling network
(2015) Bischoff, Annabell; Bayerlová, Michaela; Strotbek, Michaela; Schmid, Simone; Beissbarth, Tim; Olayioye, Monilola A.
Background: The growth factor heregulin (HRG) potently stimulates epithelial cell survival and proliferation through the binding of its cognate receptor ErbB3 (also known as HER3). ErbB3-dependent signal transmission relies on the dimerization partner ErbB2, a receptor tyrosine kinase that is frequently overexpressed and/or amplified in breast cancer cells. Substantial evidence suggests that deregulated ErbB3 expression also contributes to the transformed phenotype of breast cancer cells. Results: By genome-wide screening, we identify 43 microRNAs (miRNAs) that specifically impact HRG-induced activation of the PI3K-Akt pathway. Bioinformatic analysis combined with experimental validation reveals a highly connected molecular miRNA-gene interaction network particularly for the negative screen hits. For selected miRNAs, namely miR-149, miR-148b, miR-326, and miR-520a-3p, we demonstrate the simultaneous downregulation of the ErbB3 receptor and multiple downstream signaling molecules, explaining their efficient dampening of HRG responses and ascribing to these miRNAs potential context-dependent tumor suppressive functions. Conclusions: Given the contribution of HRG signaling and the PI3K-Akt pathway in particular to tumorigenesis, this study not only provides mechanistic insight into the function of miRNAs but also has implications for future clinical applications.
Open Access
MicroRNAs to boost the productivity of Chinese hamster ovary producer cells
(2013) Strotbek, Michaela; Olayioye, Monilola A. (Prof. Dr.)
MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate the expression of different target genes and, thus, potentially offer the opportunity to engineer networks of genes in order to achieve complex phenotypic changes in mammalian cells. The goal of this thesis was to explore whether this feature of miRNAs could be exploited as a strategy to improve therapeutic protein production processes by increasing the viable cell densities and/or productivity of mammalian producer cells. To identify miRNAs that increase the productivity of producer cells, a genome-wide functional miRNA screen was established and performed in Chinese hamster ovary (CHO) cells stably expressing an IgG1 that were grown in suspension in chemically defined medium. Using this approach nineteen human miRNAs were identified that significantly improved IgG titers in the primary screen. Almost half of these miRNAs could be validated to significantly increase the IgG concentrations and/or specific productivity when transiently introduced into CHO producer cells in a secondary screen. The increased titers of recombinant human serum albumin analyzed as a second secreted model protein pointed to product independent effects of most of the validated miRNAs. In addition, two of the validated miRNAs, hsa-miR-557 and hsa-miR-1978, also increased the secretion of an endogenously as well as a transiently expressed model protein in human cells, indicating that these miRNAs manipulate the cellular machinery by a universal mechanism. The strongest impact on the specific productivity of CHO cells was observed by a dead entry and thus artificial miRNA (hsa-miR-1978), which may represent a promising molecular tool for future synthetic biology approaches aimed at optimizing producer cells. For further studies, the two miRNAs hsa-miR-557 and hsa-miR-1287, positively impacting the viable cell density and specific productivity, respectively, were selected. Transient experiments supported the idea of combining these two miRNAs to further boost cellular productivity. Using a vector-based expression system, CHO pools stably overexpressing these human miRNAs were successfully generated allowing the study of miRNA-engineered producer cells under industrially relevant culture conditions such as fed-batch cultivation. Importantly, three independent cell pools stably coexpressing miR-557 and miR-1287 gave rise to significantly increased IgG titers by 30% in fed-batch cultures whilst product quality was conserved, proving the transferability of the transient results to a stable setting. Taken together, these results demonstrate that miRNA-based cell line engineering is an attractive approach toward the genetic optimization of CHO host cells for industrial applications.