Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these... Show moreScreening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established. Show less
Herpers, B.H.A.; Wink, S.; Fredriksson, L.; Di, Z.; Hendriks, G.; Vrieling, H.; ... ; Water, B. van de 2016
Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions... Show moreDrug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines. Show less
Herpers, B.H.A.; Wink, S; Fredriksson, L.; Di, Z.; Hendriks, G.; Vrieling, H.; ... ; Water, B. van de 2016
Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions... Show moreDrug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines. Show less
Di, Z.; Herpers, B.H.A.; Fredriksson, L.; Yan, K.; Water, B. van de; Verbeek, F.J.; Meerman, J.H.N. 2012
Nuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear... Show moreNuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g. upon drug exposure. Furthermore, there is significant heterogeneity in individual cell response upon stress signaling. In order to systematically determine factors that define NF-κB translocation dynamics, high-throughput screens that enable the analysis of dynamic NF-κB responses in individual cells in real time are essential. Thus far, only NF-κB downstream signaling responses of whole cell populations at the transcriptional level are in high-throughput mode. In this study, we developed a fully automated image analysis method to determine the time-course of NF-κB translocation in individual cells, suitable for high-throughput screenings in the context of compound screening and functional genomics. Two novel segmentation methods were used for defining the individual nuclear and cytoplasmic regions: watershed masked clustering (WMC) and best-fit ellipse of Voronoi cell (BEVC). The dynamic NFκB oscillatory response at the single cell and population level was coupled to automated extraction of 26 analogue translocation parameters including number of peaks, time to reach each peak, and amplitude of each peak. Our automated image analysis method was validated through a series of statistical tests demonstrating computational efficient and accurate NF-κB translocation dynamics quantification of our algorithm. Both pharmacological inhibition of NF-κB and short interfering RNAs targeting the inhibitor of NFκB, IκBα, demonstrated the ability of our method to identify compounds and genetic players that interfere with the nuclear transition of NF-κB. Show less
