Polycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is... Show morePolycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments. The signalling rewiring in PKD that drives cyst formation is highly complex and not fully understood. As a consequence, the effects of drugs are sometimes difficult to predict. We previously established a high-throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth. Here, we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules, including approved drugs. We identified 81 active molecules that inhibit cyst growth. Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties. Celastrol, a triterpenoid from Tripterygium wilfordii, was identified as a potent inhibitor of cyst growth in vitro. In an in vivo iKspCre-Pkd1(lox,lox) mouse model for PKD, celastrol inhibited the growth of renal cysts and maintained kidney function. Show less
Nair, N.U.; Das, A.; Rogkoti, V.M.; Fokkelman, M.; Marcotte, R.; Jong, C.G. de; ... ; Ruppin, E. 2019
The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are... Show moreThe efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells' transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor's aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival. Show less
Koedoot, E.; Fokkelman, M.; Rogkoti, V.M.; Smid, M.; Sandt, I. van de; Bont, H.J.G.M. de; ... ; Water, B. van de 2019
Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an... Show moreTtriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development. Show less
Polycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is... Show morePolycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments. The signaling rewiring in PKD that drives cyst formation is highly complex and not fully understood. As a consequence, the effects of drugs are sometimes difficult to predict. We previously established a high throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth. Here, we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules, including approved drugs. We identified 81 active molecules that inhibit cyst growth. Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties. Celastrol, a triterpenoid from Tripterygium Wilfordii, was identified as a potent inhibitor of cyst growth in vitro. In an in vivo iKspCre-Pkd1lox,lox mouse model for PKD, celastrol inhibited the growth of renal cysts and maintained kidney function. Show less
The main aim of this thesis was to unravel the signaling and regulatory networks that drive tumor cell migration during breast cancer metastasis. Understanding how tumor cells migrate, how this... Show moreThe main aim of this thesis was to unravel the signaling and regulatory networks that drive tumor cell migration during breast cancer metastasis. Understanding how tumor cells migrate, how this process is differentially regulated, and how this highly heterogeneous and plastic behavior is coordinated during metastatic dissemination, will ultimately provide novel insights in therapeutic opportunities. The work presented in this thesis includes different microscopy techniques and analytical tools to study migratory behavior (Chapters 2, 3, 4, and 5), reveals an important role for adhesion GPCRs in cell adhesion and migration (Chapter 3), provides a compendium of genes that drive tumor cell migration in breast cancer (Chapter 4), and investigates the intricate connection between adhesion dynamics, cell migration and contractility (Chapter 5). Collectively, this work provides novel insights in the molecular determinants of tumor cell migration and will help us better understand the signaling landscape that drives the formation of breast cancer metastases. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most... Show morePolycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1–/– kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD. Show less
Fokkelman, M.; Balcioglu, H.E.; Klip, J.E.; Yan, K.; Verbeek, F.J.; Danen, E.H.J.; Water, B. van de 2016
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
