In this thesis, we used genetically engineered mouse models and a variety of cell-culture based assays to identify genes and pathways that are involved in the development and treatment of invasive... Show moreIn this thesis, we used genetically engineered mouse models and a variety of cell-culture based assays to identify genes and pathways that are involved in the development and treatment of invasive lobular carcinoma (ILC). To identify novel genes and pathways involved in the development of ILCs we employed a Sleeping Beauty (SB)-based insertional mutagenesis screen in conditional Cdh1 knockout mice. We show that active transposon mutagenesis drives ILC formation and analysis of common insertion sites in SB-induced tumors identified a mutually exclusive group of four genes (MYH9, MYPT1/2 and ASPP2), three of which are frequently altered in human ILCs. We then went on to show that these hits not only drive ILC development but also do so through a shared mechanism. We identified that all four hits result in actomyosin relaxation which enables E-cadherin deficient mammary epithelial cells to invade into the mammary stroma and initiate tumor development. In addition, we show that mammary epithelial cells that lose E-cadherin expression can survive in the fibrous stroma directly surrounding the mammary ducts through interactions with components of the basement membrane. Lastly, we used active mobilization of transposons to identify resistance mechanisms to the FGFR inhibitor AZD4547. Show less
Peeters, M.C.; Fokkelman, M.; Boogaard, B.; Egerod, K.L.; Water, B. van de; IJzerman, A.P.; Schwartz, T.W. 2015
Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator... Show moreAdhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer. Show less
The aim of this thesis is to address how integrin-mediated signaling regulates cellular processes that have profound effects on cell morphology, motility, cancer metastasis, and FN fibrillogenesis,... Show moreThe aim of this thesis is to address how integrin-mediated signaling regulates cellular processes that have profound effects on cell morphology, motility, cancer metastasis, and FN fibrillogenesis, and how these findings can be utilized for relevant medical purposes or advancement of drug discovery. Show less
This thesis addresses the regulation of TRPM7 channels by receptor-mediated signals and the effects of the ensuing ionic signals on the cytoskeleton. Several signaling pathways were investigated... Show moreThis thesis addresses the regulation of TRPM7 channels by receptor-mediated signals and the effects of the ensuing ionic signals on the cytoskeleton. Several signaling pathways were investigated that activate TRPM7 channel opening. Stimulation of PLC-activating receptors opens TRPM7 channels, leading to influx of extracellular Ca2+. The results contrast markedly with a report by Runnels et al. who showed inhibition of TRPM7, rather than activation by PLC. To explain this discrepancy, we explored the differences in PIP2-mediated regulation of TRPM7 measured in perforated-patch and whole-cell configuration after intracellular Mg2+ depletion. This leads us to propose that the effects of PLC activation on TRPM7 currents as detected in whole cells can best be viewed as ‘accelerated rundown’ rather than as bona fide signal transduction. Agonist-induced TRPM7 activation and subsequent Ca2+ influx affects the cytoskeleton by phosphorylation of the myosin II heavy chain to promote cytoskeletal relaxation and the conversion of focal adhesions to podosomes. Ca2+ influx appears crucial for the in vivo function of the TRPM7-kinase by triggering the association between the kinase and its substrate. A second effect of receptor-mediated Ca2+ influx can affect the cytoskeleton by translocating Rac in a PKC-dependent manner to the plasma-membrane, where it induces extensive membrane ruffling. Show less
