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Mol Cancer
2015 Dec 08;14:206. doi: 10.1186/s12943-015-0475-1.
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A functional connectome: regulation of Wnt/TCF-dependent transcription by pairs of pathway activators.
Freeman J
,
Smith D
,
Latinkic B
,
Ewan K
,
Samuel L
,
Zollo M
,
Marino N
,
Tyas L
,
Jones N
,
Dale TC
.
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BACKGROUND: Wnt/β-catenin signaling is often portrayed as a simple pathway that is initiated by Wnt ligand at the cell surface leading, via linear series of interactions between 'core pathway' members, to the induction of nuclear transcription from genes flanked by β-catenin/TCF transcription factor binding sites. Wnt/β-catenin signaling is also regulated by a much larger set of 'non-core regulators'. However the relationship between 'non-core regulators' is currently not well understood. Aberrant activation of the pathway has been shown to drive tumorgenesis in a number of different tissues.
METHODS: Mammalian cells engineered to have a partially-active level of Wnt/β-catenin signaling were screened by transfection for proteins that up or down-regulated a mid-level of TCF-dependent transcription induced by transient expression of an activated LRP6 Wnt co-receptor (∆NLRP).
RESULTS: 141 novel regulators of TCF-dependent transcription were identified. Surprisingly, when tested without ∆NLRP activation, most up-regulators failed to alter TCF-dependent transcription. However, when expressed in pairs, 27 % (466/1170) functionally interacted to alter levels of TCF-dependent transcription. When proteins were displayed as nodes connected by their ability to co-operate in the regulation of TCF-dependent transcription, a network of functional interactions was revealed. In this network, 'core pathway' components (Eg. β-catenin, GSK-3, Dsh) were found to be the most highly connected nodes. Activation of different nodes in this network impacted on the sensitivity to Wnt pathway small molecule antagonists.
CONCLUSIONS: The 'functional connectome' identified here strongly supports an alternative model of the Wnt pathway as a complex context-dependent network. The network further suggests that mutational activation of highly connected Wnt signaling nodes predisposed cells to further context-dependent alterations in levels of TCF-dependent transcription that may be important during tumor progression and treatment.
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Fig. 1. A schematic overview of the screening and pairwise assays
Fig. 2. Identification of novel Wnt regulators. a Dose-dependent induction of TCF dependent transcription in 7df3 cells by constitutively active LRP6 (∆NLRP). b and c Examples of the 45 inducers (b) and 96 inhibitors (c) of TCF-dependent transcription identified from a cDNA library screen of 9000 Xenopus tropicalis cDNAs (see Additional file 1: Figure S1). d Prune siRNA reduced h-Prune protein levels. FLAG-tagged h-Prune was transfected into MDA-MB231 breast cancer cells 24 h after transfection of either Renilla luciferase or Prune siRNA. Expression was detected with an anti-FLAG antibody. e Knockdown of Prune by siRNA reduced the level of active (de-phosphorylated) β-catenin in MDA-MB231 cells. Blot shown is representative of four separate experiments. f Levels of active β-catenin after siRNA transfection in 4 separate experiments were quantified. Knockdown of Prune reduces levels of de-phosphorylated β-catenin to a level that was not significantly different from the knockdown of β-catenin (Student’s T-test, p < 0.05)
Fig. 3. Transcription regulation in colon cancer cells and Xenopus laevis. a Example ‘inducer’ cDNAs effect on TopFlash reporter when co-transfected into HCT116 cells (see Additional file 4: Table S3 for complete list). Comparison with the mutant FopFlash reporter demonstrates high basal levels of β-catenin/TCF-dependent transcription. Results are from triplicate wells across different plates. Stars represent statistical significantly different values from TopFlash (Benjamini-Hochberg corrected p < 0.01). b Expression of a subset of ‘inducers’ activated transcription of the endogenous Wnt target genes Xnr3 and Siamois in animal cap assays. AC; uninjected animal caps. ODC; ornithine de-carboxylase control. c and d TopFlash reporter activity from a subset of cDNAs co-transfected into SW480 cells (c) or HCT116 cells (d). (see Additional file 3: Table S2 for complete a list). The data shown are from triplicate wells across different plates. Stars represent statistically significantly different values from TopFlash (Benjamini-Hochberg corrected p < 0.01)
Fig. 4. A network of functional interactions that modulate TCF-dependent transcription. a Reporter activity of cDNAs individually transfected into 7df3 cells. Green circles represent constitutively active versions of âcoreâ pathway members. The 5/45 cDNAs that were able to activate TCF dependent transcription to a level greater than background (Kolmogorov-Smirnov test, pâ<â0.01) are shown in blue. All other cDNAs were unable to activate TCF dependent transcription when individually transfected and are shown in black. ÎNLRP levels of activation were defined as 100. b Heat map displaying functional interactions. Red and blue squares indicate positive or negative interaction respectively. The opacity of the colour indicates the strength of the interaction. c Network visualisation of functional interactions. Positive (red) and suppressive (blue) interactions whose strength is represented by line opacity are displayed. Core nodes are coloured green, activator nodes are blue whilst enhancers are black. The core pathway members tend to be the most highly connected nodes. d and e Synergistic interactions were observed using promoter-reporters driven by the endogenous Wnt target genes Myc (d) or Siamois (e) in HEK293 cells. All combinations with the exception of Ras/CK1δ and the Siamois promoter showed (multiplicative) synergistic responses
Fig. 5. Functional interrogation of interacting pairs. a h-Prune synergy with HRAS was reduced following mutation of h-Prune catalytic residues (Prune 4Ddelta) [40]. Activation by cDNAs pairs (b) and inhibition by anti-Wnt signaling compounds (c) Compounds were added at 5 times their IC50 48Â h after transfection. Luciferase assays were carried out 24Â h later and were normalised to CMV-lacZ levels
Fig. 6. Comparison of functional network structure in 7df3 and HCT116 cells. a Pattern of functional interaction between a subset of cDNAs in 7df3 TCF-luciferase reporter cells. The thickness of the line represents the relative strength of the synergy. b Pattern of functional interaction in HCT116 cells. Functional interaction between cDNAs is indicated by a line between the relevant nodes when the activity of cDNA pairs exceeded the product of the reporter activity for each cDNA when expressed individually (Studentâs t-test, pâ<â0.01). c Functional interaction between HMGB3/NUCKS1, and IRX3/HDGF in HCT116 cells
Fig. 7. Non-overlapping interaction patterns and protein interaction networks. a Data from a subset of the 7df3 network displayed in more detail to highlight an important feature of the network; that functional co-operativity is limited to selected enhancer combinations. b Further examples of simple non-overlapping sub-networks. c Overlay of the âfunctional connectomeâ on a Wnt PIN. Twenty protein communities (coloured nodes with grey links indicating protein interaction) were identified by modularity maximisation using the Louvain method. To guide the eye the layout algorithm emphasises these communities and only positive functional connections with a link weight greater than 1.5 are shown with opacity reflecting the strength of interaction
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