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Item Open Access miR-149 suppresses breast cancer metastasis by blocking paracrine interactions with macrophages(2020) Sánchez-González, Ismael; Olayioye, Monilola (Prof. Dr.)Triple-negative breast cancer (TNBC) constitutes 20% of all breast cancer cases. TNBC is defined by the lack of estrogen and progesterone receptors, and HER2 overexpression and is characterized by its frequent recurrence and high metastatic potential. The crosstalk between cancer cells and the surrounding tumor microenvironment (TME) plays a crucial role in fostering or restraining tumor progression and metastasis. Tumor-associated macrophages (TAMs) are the predominant cellular component of the TME in several solid-cancer entities, including mammary tumors. In response to microenvironmental stimuli, TAMs polarize towards tumoricidal M1 or tumor-promoting M2 activated cells. In breast cancer, malignant cells recruit and educate macrophages into an M2 phenotype that supports the metastatic spread of cancer cells. Breast cancer progression and metastasis are driven by the epidermal growth factor (EGF) and colony stimulatory factor 1 (CSF1) positive paracrine loop established between macrophages and cancer cells. However, the molecular mechanisms involved in establishing and maintaining these signaling loops are still poorly understood. Dysregulation of microRNAs (miRNAs), a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level, has been implicated in tumor progression by remodeling the TME composition and inducing its transition into a tumor-supportive state. Considering the critical role of the TME in promoting tumor progression, a deeper understanding of the role of miRNAs in TME modulation is fundamental for developing more efficient therapies. This thesis focuses on understanding whether miR-149 downregulation in TNBC is involved in the modulation of TAMs, which may result in a suitable TME required for cancer cell invasion and subsequent metastasis. Here, CSF1 was validated as a direct target of miR-149. In silico analysis of TCGA clinical data demonstrated that in lymph node-positive TNBC tissues low miR-149 expression correlated with high CSF1 expression and M2-macrophage infiltration as well with reduced patient survival. Functional analyses using 3D co-cultures and Transwell assays demonstrated that by directly targeting CSF1, miR-149 overexpression in TNBC cell lines inhibited the interaction between macrophages and the tumor cells. In macrophages co-cultured with MDA-MB-231 cells expressing miR-149 the expression levels of two EGF receptor ligands, EGF and amphiregulin, were strongly reduced, resulting in reduced EGF receptor activation in the cancer cells. Moreover, using an in vivo mouse model, lung metastases developing from orthotopic MDA-MB-231 tumors were reduced by 75% by the ectopic miR-149 expression, and this was associated with impaired M2-macrophage infiltration of the primary tumors. Furthermore, the inhibition of DNA-methyltransferases in TNBC cell lines showed that miR-149 expression is epigenetically silenced by DNA methylation mechanisms. Taken together, the experimental studies performed in this thesis suggest that in TNBC miR-149 downregulation functionally contributes to breast tumor progression by recruiting macrophages to the tumor and facilitating CSF1 and EGF receptor crosstalk between cancer cells and macrophages.Item Open Access Targeting ErbB receptors in a three-dimensional cell culture model of K-Ras mutant colorectal cancer(2017) Möller, Yvonne; Olayioye, Monilola (Prof. Dr.)K-Ras is a key signaling molecule regulating central biological processes like proliferation, polarization and survival, and is essential in the control of tissue homeostasis. Constitutive active K-Ras mutations are found in 40% of all colorectal cancers (CRC) contributing to tumor development, progression and therapy resistance. Despite constant efforts there are no targeted therapies available for K-Ras mutated CRC thus far. To develop new, mechanism-based treatment strategies the contribution of oncogenic Ras to transformation and therapy resistance has to be understood in detail. For this purpose, the human epithelial colorectal adenocarcinoma cell line Caco-2 was used as a model in this thesis. Cultured Caco-2 cells represent an early stage of CRC. They express wild-type K-Ras and have no further mutations in downstream Ras signaling pathways. Seeded into a three-dimensional culture system, Caco-2 cells are able to polarize, partly recapitulating the morphological features of the normal colorectal epithelium. By introducing an oncogenic K-Ras variant (G12V) in this organotypic model system, using an inducible expression system, two new aspects of oncogenic Ras signaling could be described for the first time: Firstly, the acute expression of K-RasG12V disrupted polarized morphogenesis of Caco-2 grown in 3D culture. I was able to identify a novel autocrine signaling loop that mediated the hyperproliferation and loss of cell polarity induced by K-RasG12V expression, which involved the receptor tyrosine kinase ErbB3 and transcriptional upregulation of its ligand heregulin (HRG). Secondly, in Caco-2 3D cultures, K-RasG12V expression led to resistance against a targeted single-chain TRAIL molecule (Db-scTRAIL) comprising an ErbB1 blocking moiety derived from cetuximab and three TRAIL monomers. Here, I identified a resistance mechanism triggered by K-RasG12V involving the upregulation of the anti-apoptotic proteins cIAP1/2. The combination of Db-scTRAIL with a new Smac mimetic (SM83) was able to override this resistance not only in the Caco-2 model but also in additional Ras-mutated CRC cell lines. Taken together these findings provide the basis for a new rational approach: combining ErbB3 blockade in Ras mutant CRC with an apoptosis inducing TRAIL molecule plus a sensitizing Smac mimetic. This combination might efficiently block the autocrine Ras-HRG-ErbB3 loop and therefore suppress transformation whilst simultaneously inducing apoptosis.Item Open Access IL-15-based trifunctional antibody-fusion proteins with costimulatory TNF-superfamily ligands for cancer immunotherapy(2018) Beha, Nadine; Kontermann, Roland (Prof. Dr.)IL-15 shows great potential to support an antitumor immune response and emerges as a promising agent in cancer immunotherapy. However, the systemic application of IL-15 is associated with toxicity and, as a monotherapy the efficacy of IL-15 is still limited. This study focusses on the development of novel trifunctional fusion proteins enforcing the activity of IL-15 with costimulatory ligands of the TNF superfamily and targeting the therapeutic activity to the tumor site by an antibody moiety. The homotrimeric trifunctional fusion proteins of the first generation was comprised of an antibody moiety (scFv), IL-15 fused to the extended sushi domain of the IL-15Rα chain (RD), and the extracellular domain (ECD) of 4-1BBL. Non-covalent trimerization of the ECD of 4-1BBL led to a homotrimeric fusion protein with three antibody moieties and three RD_IL-15 units. Based on the first generation trifunctional fusion protein, a novel second generation trifunctional fusion protein incorporating the ligand of the TNF superfamily in the single-chain format, i.e. genetic fusion of three extracellular domains by linkers on the same polypeptide chain, was generated, resulting in a monomeric trifunctional fusion protein with only one functional unit of each component. Similar T cell stimulation in a non-targeted setting, even improved capacity to enhance T cell stimulation when target bound and a clear antitumor effect in a mouse model in vivo was observed for the novel trifunctional fusion protein in the single-chain format. Furthermore, OX40L and GITRL were successfully incorporated into the novel trifunctional fusion protein in the single-chain format demonstrating stable protein configuration. Advantageous costimulatory properties in comparison to the combination of the respective bifunctional fusion proteins were observed for all trifunctional fusion proteins. Strongest synergistic effects were shown for RD_IL-15_scFvFAP_scGITRL in terms of enhancing the cytotoxic potential of CD8+ T cells and enhanced proliferation of CD4+ T cells. Finally, in a syngeneic lung tumor mouse model evaluating the antitumor potential of RD_IL-15_scFvFAP_scGITRL revealed a strong, targeting-dependent antitumor response. Additionally, the effect of an EGFR-directed trifunctional fusion protein on Trastuzumab-mediated ADCC was evaluated. Strong enhancement of the ADCC was achieved by the trifunctional fusion protein RD_IL-15_scFvEGFR_sc4-1BBL and the bifunctional fusion protein RD_IL-15_scFvEGFR. Thus, the trifunctional fusion protein format incorporating the ligand of the TNF superfamily in the single-chain format appears as a promising platform with versatile opportunities for further development.