Genome-Wide RNAi Screen Reveals Disease-Associated Genes That Are Common to Hedgehog and Wnt Signaling

Jacob, Leni S.; Wu, Xiaofeng; Dodge, Michael; Fan, Chih Wei; Kulak, Ozlem; Chen, Baozhi; Tang, Wei-Jen; Wang, Baolin; Amatruda, James F.; Lum, Lawrence

doi:10.1126/scisignal.2001225

Cited by 90 publications

(83 citation statements)

References 42 publications

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“…Upon further validation of siRNA specificity, and a demonstration of function in additional trailblazer populations, one or more of these 8 genes may prove to also be a bona fide regulator of invasion. In addition, negative results in siRNA experiments may be a consequence of incomplete knockdown of gene expression or the kinetics of loss of expression (48). Also, siRNAs targeting 3 genes highly expressed in trailblazer cells enhanced invasion in at least 2 experiments.…”

Section: Trailblazer Cells Induce Opportunistic Collective Invasion Imentioning

confidence: 99%

An epigenetically distinct breast cancer cell subpopulation promotes collective invasion

et al. 2015

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induced the invasion of non-trailblazer cells, thus revealing a new type of commensal relationship among naturally existing tumor subpopulations. Together, these results demonstrate how the epigenetic alteration of the signaling circuitry in a subpopulation of tumor cells can promote collective invasion through cellautonomous and non-cell-autonomous mechanisms. Results A distinct subpopulation of trailblazer cells has enhanced invasive ability.To begin defining the molecular traits that confer tumor cells with invasive ability, we analyzed spheroid invasion in an organotypic culture system that reconstitutes key features of collective invasion that are conserved in vivo (9, 13). Normal mammary epithelial cells form duct-like spheroids in this system (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI77767DS1), indicating that our model was testing for unique traits of tumor cells that promote cell-autonomous invasion, potentially during the transition from ductal carcinoma in situ (DCIS) to invasive breast cancer (13). To begin defining traits that promote collective invasion, we determined the percentage of invasive trailblazer spheroids that were formed in 7 different breast cancer cell lines that represent key known features of intertumor molecular diversity. Invasive spheroids were detected in 3 of the 7 cell lines evaluated, with the percentage of invasive spheroids ranging between 8% and 75% of the total population ( Figure 1, A and B). None of the cell lines contained a 100% pure population of invasive spheroids ( Figure 1B). The 3 cell lines that contained invasive spheroids were derived from patients with TNBC (no detectable estrogen receptor [ER], progesterone receptor, or human epidermal growth factor receptor 2 [HER2] expression) ( Figure 1A). TNBC accounts for 10% to 20% of diagnosed breast cancers and has a relatively worse outcome compared with that of ER + breast cancer (21). Importantly, the strand-like organization of the collectively invading cells observed in organotypic culture was also detected in primary breast tumors ( Figure 1C). Thus, our results indicate that there can be a distinct subpopulation within a community of tumor cells that has an enhanced capacity to lead collective invasion. We refer to this intrinsically invasive subpopulation as trailblazer cells to distinguish them from other types of leader cells, such as KRT14-expressing breast cancer cells, that are unable to invade under these conditions. The noninvasive subpopulation, which may require additional extrinsic factors to invade, is referred to as "opportunist" cells herein.Immunofluorescence analysis and time-lapse imaging showed that the leader trailblazer cells formed long cellular protrusions (LCPs) into the ECM before invading away from the main mass of cells (Figure 1, D and E, and Supplemental Video 1), similar to previous reports (9, 10). Additional trailblazer cells could then migrate into the space within the ECM created by the first invading cell, indicating tha...

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Section: Trailblazer Cells Induce Opportunistic Collective Invasion Imentioning

confidence: 99%

An epigenetically distinct breast cancer cell subpopulation promotes collective invasion

et al. 2015

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“…These high-throughput lossand gain-of-function studies quantitated the response of exogenous Wnt pathway reporters under conditions of elevated signaling (Anton et al, 2011;Caspi and RosinArbesfeld, 2008;DasGupta et al, 2005;Firestein et al, 2008;Groenendyk and Michalak, 2011;Jacob et al, 2011;James et al, 2009;Kategaya et al, 2009;Major et al, 2008;Miller et al, 2009;Port et al, 2011;Tang et al, 2008). In this study, we have performed a comprehensive genome-wide in vivo RNAi screen for kinases and phosphatases in Drosophila to build a phospho-regulatory network of the Wnt pathway.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide identification of phospho-regulators of Wnt signaling inDrosophila

2015

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Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase-and phosphatase-mediated reversible phosphorylation of proteins. In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 highconfidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phosphoregulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. We identified 29 dual regulators that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, we established that Cdc37 and Gilgamesh/CK1γ inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease.

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“…Some kinases, such as casein kinase 1ε (CK1ε), casein kinase 2 (CK2), and PAR-1, which can directly phosphorylate Dvl, were reported to play positive roles in canonical Wnt signaling (7,16,38,40). Furthermore, some negative regulators of the canonical Wnt signaling pathway were shown to inhibit the phosphorylation of Dvl (15,17,42). All of these results suggest that phosphorylation of Dvl is required for Wnt signaling; however, the regulation of the activity and stability of Dvl in Wnt signaling is still incompletely understood.…”

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confidence: 99%

The E3 Ubiquitin Ligase ITCH Negatively Regulates Canonical Wnt Signaling by Targeting Dishevelled Protein

Wei

Wang

et al. 2012

Molecular and Cellular Biology

126

142

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Genome-Wide RNAi Screen Reveals Disease-Associated Genes That Are Common to Hedgehog and Wnt Signaling

Cited by 90 publications

References 42 publications

An epigenetically distinct breast cancer cell subpopulation promotes collective invasion

An epigenetically distinct breast cancer cell subpopulation promotes collective invasion

Genome-wide identification of phospho-regulators of Wnt signaling inDrosophila

The E3 Ubiquitin Ligase ITCH Negatively Regulates Canonical Wnt Signaling by Targeting Dishevelled Protein

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