Browsing by Author "Olayioye, Monilola A."

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Open Access
EGFR-targeted TRAIL and a Smac mimetic synergize to overcome apoptosis resistance in KRAS mutant colorectal cancer cells
(2014) Möller, Yvonne; Siegemund, Martin; Beyes, Sven; Herr, Ricarda; Lecis, Daniele; Delia, Domenico; Kontermann, Roland; Brummer, Tilman; Pfizenmaier, Klaus; Olayioye, Monilola A.
TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed Db_Î±EGFR-scTRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of Db_Î±EGFR-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward Db_Î±EGFR-scTRAIL in these 3D cultures. We show that the antibody moiety of Db_Î±EGFR-scTRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing Db_Î±EGFR-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects Db_Î±EGFR-scTRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-Ras^G12V. In the presence of doxycycline, these cells showed increased resistance to Db_Î±EGFR-scTRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and Flip_S. Co-treatment of cells with the Smac mimetic SM83 restored the Db_Î±EGFR-scTRAIL-induced apoptotic response. Importantly, this synergy between Db_Î±EGFR-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that Db_Î±EGFR-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.
Open Access
The GEF‐H1/PKD3 signaling pathway promotes the maintenance of triple‐negative breast cancer stem cells
(2019) Lieb, Wolfgang S.; Lungu, Cristiana; Tamas, Raluca; Berreth, Hannah; Rathert, Philipp; Storz, Peter; Olayioye, Monilola A.; Hausser, Angelika
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
Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport
(2023) Lungu, Cristiana; Meyer, Florian; Hörning, Marcel; Steudle, Jasmin; Braun, Anja; Noll, Bettina; Benz, David; Fränkle, Felix; Schmid, Simone; Eisler, Stephan A.; Olayioye, Monilola A.
The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase‐activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy‐based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP‐RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.
Open Access
Proteasomal turnover of the RhoGAP tumor suppressor DLC1 is regulated by HECTD1 and USP7
(2022) Frey, Yannick; Franz-Wachtel, Mirita; Macek, Boris; Olayioye, Monilola A.
The Rho GTPase activating protein Deleted in Liver Cancer 1 (DLC1) is frequently downregulated through genetic and epigenetic mechanisms in various malignancies, leading to aberrant Rho GTPase signaling and thus facilitating cancer progression. Here we show that in breast cancer cells, dysregulation of DLC1 expression occurs at the protein level through rapid degradation via the ubiquitin-proteasome system. Using mass spectrometry, we identify two novel DLC1 interaction partners, the ubiquitin-ligase HECTD1 and the deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7). While DLC1 protein expression was rapidly downregulated upon pharmacological inhibition of USP7, siRNA-mediated knockdown of HECTD1 increased DLC1 protein levels and impaired its degradation. Immunofluorescence microscopy analyses revealed that the modulation of HECTD1 levels and USP7 activity altered DLC1 abundance at focal adhesions, its primary site of action. Thus, we propose opposing regulatory mechanisms of DLC1 protein homeostasis by USP7 and HECTD1, which could open up strategies to counteract downregulation and restore DLC1 expression in cancer.
Open Access
Repeat DNA methylation is modulated by adherens junction signaling
(2024) Brenner, Lisa-Marie; Meyer, Florian; Yang, Haiqian; Köhler, Anja R.; Bashtrykov, Pavel; Guo, Ming; Jeltsch, Albert; Lungu, Cristiana; Olayioye, Monilola A.
Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.
Open Access
Spatiotemporal control of intracellular membrane trafficking by Rho GTPases
(2019) Olayioye, Monilola A.; Noll, Bettina; Hausser, Angelika
As membrane-associated master regulators of cytoskeletal remodeling, Rho GTPases coordinate a wide range of biological processes such as cell adhesion, motility, and polarity. In the last years, Rho GTPases have also been recognized to control intracellular membrane sorting and trafficking steps directly; however, how Rho GTPase signaling is regulated at endomembranes is still poorly understood. In this review, we will specifically address the local Rho GTPase pools coordinating intracellular membrane trafficking with a focus on the endo- and exocytic pathways. We will further highlight the spatiotemporal molecular regulation of Rho signaling at endomembrane sites through Rho regulatory proteins, the GEFs and GAPs. Finally, we will discuss the contribution of dysregulated Rho signaling emanating from endomembranes to the development and progression of cancer.
Open Access
Trispecific eFab-eIg T-cell engagers targeting HER2 and HER3
(2025) Löffler, Ann-Kathrin; Huber, Annika; Olayioye, Monilola A.; Kontermann, Roland; Seifert, Oliver
Trispecific antibodies have emerged as molecules for enhanced cancer immunotherapy by addressing the complexity of cancer cell biology and anti-cancer immune responses. Here, we present a novel approach to generate trispecific antibodies based on the previously developed eIg technology. These trispecific antibodies comprise one Fab and two eFab moieties, fused to obtain an asymmetric eFab-eIg molecule. The design principle employs two different eFab building blocks, characterized by divergent arrangements of heterodimerizing hetEHD2 domains. Specifically, the first (inner) eFab arm comprises the hetEHD2-1 domain in the heavy chain and the corresponding hetEHD2-2 domain in one of the light chains, while in the second eFab (outer) this arrangement is reversed. The feasibility of this approach was demonstrated for a trispecific eFab-eIg T-cell engager (TCE) targeting HER2, HER3, and CD3. Importantly, the trispecific TCE retained binding activity for all three antigens and was capable of recruiting T-cells to HER2 and/or HER3-expressing cancer cells and mediating effective cancer cell killing, as shown in 2D and 3D model systems. Due to the modular architecture, this approach should be suitable to generate trispecific antibodies of any specificity and for a multitude of applications.