Refining Pathways: A Model Comparison Approach

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Abstract

Cellular signalling pathways consolidate multiple molecular interactions into working models of signal propagation, amplification, and modulation. They are described and visualized as networks. Adjusting network topologies to experimental data is a key goal of systems biology. While network reconstruction algorithms like nested effects models are well established tools of computational biology, their data requirements can be prohibitive for their practical use. In this paper we suggest focussing on well defined aspects of a pathway and develop the computational tools to do so. We adapt the framework of nested effect models to focus on a specific aspect of activated Wnt signalling in HCT116 colon cancer cells: Does the activation of Wnt target genes depend on the secretion of Wnt ligands or do mutations in the signalling molecule β-catenin make this activation independent from them? We framed this question into two competing classes of models: Models that depend on Wnt ligands secretion versus those that do not. The model classes translate into restrictions of the pathways in the network topology. Wnt dependent models are more flexible than Wnt independent models. Bayes factors are the standard Bayesian tool to compare different models fairly on the data evidence. In our analysis, the Bayes factors depend on the number of potential Wnt signalling target genes included in the models. Stability analysis with respect to this number showed that the data strongly favours Wnt ligands dependent models for all realistic numbers of target genes.

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A shrinkage approach to large-scale covariance matrix estimation and implications for functional genomics.

Juliane Schäfer, Korbinian Strimmer (2005)

Inferring large-scale covariance matrices from sparse genomic data is an ubiquitous problem in bioinformatics. Clearly, the widely used standard covariance and correlation estimators are ill-suited for this purpose. As statistically efficient and computationally fast alternative we propose a novel shrinkage covariance estimator that exploits the Ledoit-Wolf (2003) lemma for analytic calculation of the optimal shrinkage intensity. Subsequently, we apply this improved covariance estimator (which has guaranteed minimum mean squared error, is well-conditioned, and is always positive definite even for small sample sizes) to the problem of inferring large-scale gene association networks. We show that it performs very favorably compared to competing approaches both in simulations as well as in application to real expression data.

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Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells

Oksana Voloshanenko, Gerrit Erdmann, Taronish Dubash … (2013)

Wnt signalling pathways are highly conserved across metazoans and have important roles in many biological processes, including embryonic development, tissue homeostasis and carcinogenesis1. Canonical Wnt signalling through β-catenin is of particular importance for the development of colorectal cancer, with more than 80% of tumours carrying loss-of-function mutations in adenomatous polyposis coli (APC) and about 5% carrying activating mutations in β-catenin2 3. In the normal intestinal epithelium, Wnt signalling regulates the proliferation and differentiation of intestinal stem and progenitor cells. Mutations in APC and β-catenin are among the earliest events of colon cancer development and lead to hyperplasia in the intestinal crypts4 5. Recent studies have shown that Wnt signalling activity is also required in colon cancer cells during tumour progression and metastasis6. The secretion of Wnt proteins requires Evenness interrupted/Wntless/G protein-coupled receptor 177 (Evi/Wls/GPR177)7 8 9 10 11. This multipass transmembrane protein acts as a cargo receptor for Wnt proteins, shuttling them from the Golgi to the plasma membrane where they act in an autocrine or paracrine manner to activate Wnt signalling pathways. Upon binding to receptors of the Frizzled family and their coreceptors, Wnt proteins activate different downstream signalling pathways. In the so-called canonical pathway, signals are transmitted through Dishevelled (Dvl) adaptor proteins, leading to the stabilization of cytosolic β-catenin and its translocation to the nucleus. In the absence of Wnt signalling, β-catenin degradation is initiated by the ‘destruction complex’, which comprises, among other proteins, APC, GSK3β and Axin1. APC acts as a scaffold, binding β-catenin and recruiting CK1α and GSK3β to phosphorylate β-catenin. This event marks β-catenin for ubiquitination by the SKP1–Cullin-1–F-box (SCFβKP1-) E3 ubiquitin ligase complex and subsequently triggers its proteasomal degradation12. When β-catenin translocates to the nucleus as a consequence of Wnt signalling, it acts as a cofactor for transcription factors of the T-cell factor (TCF) family, leading to the transcription of Wnt/β-catenin target genes such as AXIN2, SMAD7, CCND1/CyclinD and MYC. Given that mutations in APC and β-catenin are thought to lead to a constitutively active Wnt pathway that renders cells insensitive to upstream regulation, many drug development efforts for colon cancer have focused on downstream targets2 13. However, other experimental data challenge the assumption that mutant APC alone is sufficient to sustain Wnt pathway activity. For example, secreted Wnt antagonists such as the secreted Frizzled-related proteins (SFRPs) have been found to attenuate Wnt/β-catenin signalling in colorectal cancer cell lines carrying APC mutations14 15; this observation has also been explained by proposing novel, Wnt/β-catenin-independent functions for these factors16 17. Other studies have shown that β-catenin expression patterns across a colon tumour are heterogeneous, suggesting that the Wnt pathway activity across a tumour can be variable irrespective of the homogeneous distribution of the activated β-catenin mutant protein throughout that tumour18 19 20 21. These observations highlight the need to dissect the mechanisms that underlie the effects of APC and β-catenin mutations on the Wnt/β-catenin signalling cascade. Here we show that the levels of both Wnt3 and Evi/Wls are frequently higher in adenomas and differentiated carcinomas than in normal colon epithelium. We also demonstrate that interfering with the secretion of Wnt proteins in colon cancer cell lines that harbour APC or β-catenin mutations impairs the ability of the cells to proliferate and to form colonies in vitro and tumours in vivo. We provide evidence that autocrine Wnt secretion via Evi/Wls is required for maintaining the Wnt activity in cell lines and tumour-initiating cells (TICs). Taken together, these results support a model, wherein colon cancer cells acquire the ability to secrete canonical Wnt proteins continuously, and require such autocrine secretion for survival. Results Evi/Wls and Wnt3 are highly expressed in colon carcinomas In the normal mouse small intestinal epithelium, Wnt3 is highly expressed in Paneth cells where it has a crucial role in maintaining LGR5+ intestinal stem cells at the bottom of the crypt22 23. Here we asked whether a similar expression pattern of Wnt3 and its cargo receptor Evi/Wls can be detected in human colon tissue. To this end, we stained normal human colon and matched carcinoma tissue sections (from the same patient) for Wnt3 and Evi/Wls protein, using Keratin20 and Eph receptor B2 as markers of cell differentiation (Fig. 1a,b and Supplementary Fig. S1b)24. We found that Wnt3 and Evi/Wls proteins were low or were not detectable in enterocytes and goblet cells, whereas their expression was more prominent in the underlying lamina muscularis mucosae (Fig. 1a). In addition, high Wnt3 expression was observed in scattered cells, which showed characteristics of endocrine and Paneth-like25 cells localized at the bottom of the crypt, with eosinophilic granula in the cytoplasm and the basal nuclei. These results suggest that an important source of Wnt proteins in the human colon is located at the bottom end of the crypts. Surprisingly, in carcinoma sections from the same patients, Evi/Wls and Wnt3 staining was stronger and detectable throughout the epithelium (Fig. 1b). To confirm these data, we also stained tumour tissue arrays for Evi/Wls and Wnt3 (Fig. 1c; representative immunohistochemical stainings are shown in Supplementary Fig. S1b). Wnt3 and Evi/Wls expression in the epithelial cells of normal colon tissue (colon) was generally weaker compared with tumuor tissues. However, it was elevated in adenomas and showed strong expression in well- (G1) and moderately (G2) differentiated (adeno-) carcinomas, whereas poorly (G3) differentiated tumours showed heterogeneous Evi/Wls expression (Fig. 1c). Our expression data are also consistent with the TCGA messenger RNA expression data set (Oncomine, www.oncomine.org, TCGA)3, which shows that the Wnt3, Wnt1 and Wnt3a mRNAs (Fig. 1d and Supplementary Fig. S1c) are more highly expressed in colorectal adenocarcinomas than in normal colon/rectum. Next, we tested whether overexpressing the cargo receptor Evi/Wls in colon cancer cells leads to an increase in Wnt signalling activity, as previously shown in embryonic stem (ES) cells10. As illustrated in Supplementary Fig. S1d, the overexpression of Evi/Wls in the colon cancer cell line HCT116 led to a twofold increase in Wnt reporter activity, indicating that the increased secretion of Wnt proteins can upregulate canonical signalling. Taken together, our data provide evidence that Wnt secretion could have a functional role in colorectal carcinomas, and that colorectal carcinoma cells have acquired the ability to secrete Wnt ligands in an autocrine manner. Evi is required for high levels of Wnt pathway activation To investigate the requirements for Evi/Wls expression in colon cancer cells that harbour mutations in APC or β-catenin, we chose two colon cancer cell lines: DLD1 (APC mutation; truncated protein) and HCT116 (β-catenin mutation, loss of phosphorylation site S45). Short hairpin RNA (shRNA)-mediated silencing of the Evi/Wls significantly reduced lymphoid enhancer factor (LEF)/Wnt-reporter26 induction to levels comparable to those achieved by Bcl9 knockdown, an essential transcriptional cofactor for the expression of Wnt/β-catenin target genes (Fig. 2a and Supplementary Fig. S2a). This effect was rescued by adding recombinant Wnt3a (Supplementary Fig. S2b). Furthermore, knockdown of Evi/Wls with a doxycycline (dox)-inducible shmirRNA led to downregulation of the canonical Wnt target gene AXIN2 in both DLD1 and HCT116 cells (Fig. 2b), an effect that was likewise rescued by applying recombinant Wnt3a (Supplementary Fig. S2c,d). RNA interference (RNAi)-mediated downregulation of Evi/Wls reduced the phosphorylation of Lrp6 on Ser1490 in both DLD1 and HCT116 cells, confirming that pathway activity was reduced at the receptor level (Fig. 2c)27 28. Evi/Wls depletion in these cell lines also reduced the levels of active β-catenin (non-phosphorylated-S33/S37/T41) and of Axin2 (Fig. 2c). To confirm the specificity of knockdown, we rescued Wnt pathway activation in HEK293T cells after knockdown of Evi/Wls, using modified Evi/Wls expression constructs29 not targeted by the short interfering RNAs (siRNAs; Supplementary Fig. S3a). These findings suggest that Evi/Wls activity contributes to the activation of canonical Wnt signalling in the presence of an APC or β-catenin mutation. Next, we tested whether blocking Wnt secretion using IWP12, an inhibitor of the porcupine protein, the Wnt acyl-transferase30 31, interferes with Wnt activity in HCT116 cells. The results presented in Supplementary Fig. S3b were similar to those obtained for Evi/Wls silencing, supporting a model wherein Wnt secretion is required for the activity of the canonical Wnt pathway. Recently, it has been shown that R-spondins bind to Lgr5 to activate Wnt signalling32 33 34. Thus, we tested whether the effect of Evi/Wls silencing can be rescued by adding recombinant R-spondin1. As shown in Supplementary Fig. S3c–e, R-spondin1 could not rescue Wnt signalling in colon cancer cells when Evi/Wls was depleted, in contrast to control experiments. In summary, these results indicate that even in the presence of the APC or β-catenin mutations, canonical Wnt signalling in colon cancer cells depends on Wnt secretion. Depletion of Evi/Wls leads to an almost complete loss of canonical Wnt pathway activity, which cannot be rescued by the addition of R-spondin. Mutated APC and upstream regulation of Wnt signalling Next, we investigated whether the presence of Wnts is required for canonical signalling in colon cancer cell lines with mutant APC. RNAseq data from seven colon cancer cell lines indicated that several canonical Wnt ligands are expressed in each cell line, the most prominent among these being Wnt3 and Wnt10b (Supplementary Fig. S4a). To test whether these ligands are secreted and functional, we cocultured SW480 and DLD1 cells with a MDA-MB231 or HEK293T reporter cell line. We found that both colon cancer cell lines induced canonical Wnt signalling in a cell-number-dependent manner (Fig. 3a and Supplementary Fig. S4b,c), producing a range of reporter activity similar to that observed when recombinant Wnt3a was applied (Supplementary Fig. S4b). To exclude the possibility of confounding effects from increased reporter cell proliferation, we assessed the activity of a TCF/LEF–GFP reporter at the single-cell level by fluorescence-activated cell sorting. Consistent with our results from the luciferase assays, coculturing the reporter cell line with SW480 cells enhanced TCF/LEF–GFP fluorescence in reporter cells in a dose-dependent manner (Supplementary Fig. S5a). These experiments indicated that colon cancer cells secrete functional Wnt proteins. Next, we investigated whether the Evi/Wls phenotype is equivalent to that resulting from the loss of canonical Wnts. Depleting HCT116 cells of Wnt3 and Wnt3a, and DLD1 cells of Wnt3 (Fig. 3b), strongly reduced the reporter activity. Silencing of Wnt3a was rescued by adding recombinant Wnt3a (Supplementary Fig. S5b). Combined knockdown of both Wnt3 and Wnt3a led to a further reduction of Wnt signalling (Supplementary Fig. S5c). We also observed reduced expression of the Wnt target genes AXIN2 and SMAD7 (Supplementary Fig. S5d), a reduction of Lrp6 phosphorylation (Ser1490) and a decrease in Axin2 protein levels (Fig. 3c) after RNAi-mediated silencing of Wnt3 or Wnt3a. As expected, the ability of the Wnt ligands to activate signalling was dependent on the presence of Evi/Wls in the secreting cells (Fig. 3d). Consistent with these findings, silencing of Lrp6, Dvl1 and Dvl3 led to a decrease in TCF4/Wnt-reporter activity, despite the presence of APC and β-catenin mutations (Fig. 4a), and silencing of Dvl1 and Dvl3 led to downregulation of AXIN2 mRNA (Fig. 4b). In summary, colon cancer cell lines express canonical Wnt ligands, whose Evi/Wls-dependent secretion is regulating canonical Wnt signalling, regardless of the presence of mutated APC or β-catenin. Truncated APC can regulate canonical Wnt pathway Eighty per cent of colon cancers have biallelic APC mutations35, often with complete lack of expression from one allele and expression of a truncated from the other. Previous studies have indicated that depletion of APC in colon cancer cells can activate canonical Wnt signalling36 37 38 39. To test whether truncated APC alleles have residual ability to regulate downstream Wnt signalling components and whether such activity can be reduced further, we assessed the effect of RNAi-mediated APC silencing on TCF/LEF and TCF4/Wnt reporter levels in DLD1, HT29 and HCT116 cells (Supplementary Fig. S6a). In each of these cell lines, reducing APC expression induced reporter expression. We observed a particularly strong effect in HT29 cells, in which the truncated APC protein is longer than that in DLD1 cells (Supplementary Fig. S6a). In general, induction of AXIN2 mRNA appeared to correlate with the length of the truncated APC proteins (Fig. 5a), although we cannot completely exclude the possibility that variability in RNAi efficiency affected the results obtained for different cell lines (Supplementary Fig. S5b). APC silencing in DLD1 cells with different distinct APC siRNAs led to reproducible upregulation of AXIN2 (Supplementary Fig. S6c). These results are consistent with previous studies showing that truncated APC proteins remain capable of modulating the Wnt pathway signalling. Truncated APC retains functions in regulating Wnt signalling Our experiments indicated that colon cancer cell lines require canonical Wnt ligands for full pathway activation and cell survival, regardless of the presence of mutations in APC or β-catenin. We hypothesized that the mutant proteins might retain partial function, shifting the balance between activators and inhibitors of the Wnt/β-catenin pathway, rendering it more sensitive to changes in signal flow from upstream components. Previous work using overexpression constructs of APC fragments showed that it can retain β-catenin binding ability and reduce signalling activity40 41 42. Su et al. 43 also proposed that mutated APC has impaired function in transferring phosphorylated β-catenin for ubiquitylation. We therefore examined the biochemical effects of clinically found mutant forms of endogenous APC on destruction complex formation and Wnt/β-catenin pathway activity. As knockdown of truncated APC (1,360–1,560 aa) led to an increased expression of the Wnt target gene AXIN2 (Fig. 5a)36 37 39, we hypothesized that this protein may retain its ability to act as a scaffold for components of the destruction complex. We tested this by immunoprecipitating the destruction complex from Colo320, Caco2, DLD1 and RKO cells (truncations of 811, 1,367 and 1,430 aa, and full-length APC, respectively). As shown in Fig. 5b, endogenous APC, Axin1 and GSK3β co-immunoprecipitated with β-catenin in all cases, indicating that these truncated APC proteins retain the ability to bind β-catenin. Next, we evaluated whether the truncated APC proteins supported the formation of destruction complex and induced phosphorylation of β-catenin. We co-immunoprecipitated endogenous APC, Axin1 and GSK3β from DLD1 and SW480 cells. We detected β-catenin phosphorylation at sites S33 and S37 (as also shown in refs 43, 44), as well as the presence of the major components of the destruction complex (Fig. 6a and Supplementary Fig. S7a), indicating that truncated APC proteins are partly functional. Thus, loss of β-catenin-binding sites in colon cancer cells, such as DLD1 and SW480, leads to a reduction in APC levels rather than its complete inactivation. We also investigated whether a reduction in APC functionality, although insufficient to activate the pathway on its own, sensitizes cells to the Wnt ligands. We used RKO that expresses wild-type APC and shows only basal Wnt pathway activity as a model system. To assess the effects of a partial reduction of APC levels, we stably transduced RKO TCF4/Wnt-reporter cells with shRNA constructs that target APC at different efficiencies of knockdown, as assessed by their effects on expression of the AXIN2 mRNA (Fig. 6b,c). We found that efficient reduction of APC expression (85% reduction, shAPC#2) induced strong reporter activity, even in the absence of the Wnt3a ligand. In contrast, moderate downregulation of APC expression (50% reduction, shAPC#1) did not induce the reporter by itself, but resulted in a stronger response when combined with exposure to Wnt3a ligand (Fig. 6b). We further tested the effects of APC levels in HEK293T cells that had been transfected with various siAPCs and treated with Wnt3a. Although partial silencing of APC (using either siAPC#1 or siAPC pool) did not induce TCF4/Wnt-reporter activity on its own, we observed a stronger induction when combined with Wnt3a treatment (Supplementary Fig. S7b,c). Together, these findings indicate that colon cancer cells expressing truncated APC can assemble a partially functional destruction complex. We conclude that truncated APC does not lead to full pathway activation, but can instead sensitize cells to upstream signals. Evi is required for the proliferation of colon cancer cells To evaluate whether Wnt secretion is required for the proliferation of colon cancer cells, we stably transduced colon cancer cell lines with lentiviral shRNA constructs targeting the Wnt cargo receptor Evi/Wls. Knockdown of Evi/Wls reduced the viability of HCT116 cells compared with cells treated with control shRNAs and the cells accumulated in G2 phase, similar to cells deficient for β-catenin (Supplementary Figs S8 and S9a). Comparable viability effects were observed in cells expressing truncated forms of APC (Supplementary Fig. S9b,c). To study the effect of Evi/Wls downregulation on long-term cell survival, we performed clonogenicity assays in HCT116, DLD1 and SW480 cells. As shown in Fig. 7a, Evi/Wls knockdown decreased colony formation relative to that measured in controls. To assess the requirement of Evi/Wls in the survival of colon cancer cells in vivo, we injected shRNA-transduced HCT116 or DLD1 cells (Fig. 7b,c) subcutaneously into NOD/SCID (non-obese diabetic/severe-combined immunodeficiency) mice. Expression of the Evi shRNA delayed or abrogated tumour formation during the observation period (P=0.000201, log-rank test), an effect that was confirmed with dox-inducible shmirRNA constructs (shmirEvi#1 and #2; Fig. 7d and Supplementary Fig. S9d). These results demonstrate that silencing of the Wnt cargo receptor Evi/Wls reduced both the initial engraftment of the tumour and subsequent tumour growth. Evi is important for the survival of colon cancer stem cells Vermeulen et al. 21 showed that colon cancer stem cells are characterized by high Wnt activity, which is essential for stemness capacity of these cells. We hypothesized that Wnt signalling in these cells might be dependent on canonical Wnts and Evi/Wls expression in an autocrine or a paracrine manner, even in the presence of APC mutation. To investigate the role of Wnt secretion in TICs, we enriched these cells from primary human colorectal cancer tissue or derived metastases from three patients (P1–3) in serum-free medium in the presence of cytokines. Under these conditions, floating tumour spheroids are formed, which are enriched for self-renewing TICs and depleted of non-neoplastic cells45. P1 and P3 TICs harbour a truncated APC, whereas in P2 cells no truncations of APC was detected (Fig. 8a). We confirmed that the three TICs expressed Evi/Wls and Wnt3 on an mRNA level. We then tested whether truncated APC in colon cancer stem cells is functional as shown above for colon cancer cell lines. Consistently, we found that GSK3β and Axin1 bind to truncated APC and β-catenin in all three spheroid cultures independent of their APC mutation status (Fig. 8a). We further tested whether inhibition of Wnt secretion by a Porcupine inhibitor (IWP12) reduced the LEF/TCF–Wnt-reporter signalling in TICs with truncated APC. Similar to colon cancer cell lines, inhibition of Wnts secretion led to a reduction of Wnt signalling and reduces viability of these cells (Fig. 8b,c). Taken together, we conclude that high Wnt pathway activation in primary colon cancer cells relies on Wnt secretion, despite mutation in APC, indicating that the level of Wnt signalling is modulated by the secretion of Wnt proteins. Discussion In colon cancer, mutations in APC or β-catenin are known to be tightly associated with the transformation of normal epithelia, leading to dysplasia and early adenomas. However, the functional effects of these mutations on the Wnt/β-catenin signalling pathway and the mechanisms whereby they contribute to carcinogenesis are not yet fully understood. Here we provide evidence that sustained Wnt secretion is important for the proliferation and survival of colon cancer cells, both ex vivo and in vivo. Cells carrying mutations in APC or β-catenin are still dependent on the Wnt/β-catenin pathway activation upstream of the β-catenin destruction complex (Fig. 9). Blocking upstream signalling by depleting the Wnt cargo receptor Evi/Wls significantly reduces the proliferation of colon cancer cells. Our results suggest that truncations of the APC tumour suppressor do not render cells independent of exogenous Wnt ligands, but rather lower the threshold of pathway activation by Wnt proteins. Our findings are interesting with regards to the observation that extracellular inhibitors of the Wnt/β-catenin signalling, Dkk4 and Sfrps, are frequently downregulated in colorectal cancers14 15. These proteins had been proposed to have novel functions given the earlier assumption that mutations in APC or β-catenin would render cells completely independent of canonical Wnt/β-catenin signalling. Our results offer a different explanation: as Wnt/β-catenin signalling remains critical for proliferation, reduced expression of secreted Wnt/β-catenin antagonists and high expression of Wnts and Evi/Wls promotes tumourigenesis. The discovery of Evi/Wls as the cargo receptor for all Wnt proteins opened new avenues for addressing whether the production of Wnt ligands is required in cells that harbour APC or β-catenin mutations. Evi/Wls was found to be essential during development, because it is required for the export of all Wnt proteins9 11. We observed increased expression of Evi/Wls and Wnt ligands in colon cancers, and detected them throughout the whole tissue, as opposed to weak or very localized expression in healthy tissue (for example, Paneth-like cells, lamina muscularis mucosae). This suggests that colon cancer cells are not only dependent on secreted Wnt ligands but also acquire autocrine Wnt secretion capabilities. Notably, upregulation of Wnts or Evi/Wls in colorectal cancer is associated with lower overall survival. Consistently, TCGA recently showed that colon cancer (tissues) display upregulation of Fzd10 and downregulation of DKK1-4 mRNA levels3. This, as well as the silencing of SFRPs, supports a model whereby the destruction complex is regulated by upstream signals in colorectal cancers. These results also provide possible explanations for the uneven distribution of nuclear β-catenin20 46 and the clustering of mutations in APC47 in colon cancer. Mutations in APC most often occur in the mutation cluster region (1,286–1,513 aa), whereas a complete deletion of the APC locus is extremely rare36 39 48 49. The retention of truncated APC has alternatively been attributed to its role in cell division50 51, C-terminal binding protein (CtBP)-dependent oligomerization52 or to limit excessive levels of β-catenin in the nucleus53 54. It has also been proposed that certain truncations of APC are selected to ensure that the Wnt/β-catenin signalling pathway is activated to a degree optimal to support tumour growth37 47. Regulation of β-catenin in colon cancer cells could also be dependent on the APC-like protein, the closest homologue of APC55. Our results are consistent with previous studies on overexpressed APC protein fragments of different length, which were shown to retain the β-catenin-binding ability and reduce the signalling activity40 41 42. Importantly, such a model requires that mutant forms of APC remain, at least in part, a functional component of the destruction complex. Accordingly, truncating mutations in APC represent hypomorphic rather than complete loss-of-function alleles, as suggested by Gaspar and Fodde38, based on experiments in familial adenomatous polyposis (FAP) mouse models. Genetic studies in mice also demonstrated that APC truncations select for a certain amount of Wnt/β-catenin pathway activation and show a dosage effect in TopFlash reporter assays conducted in mouse ES cells47 56 57. Our study provides further mechanistic evidence that endogenous truncated APC indeed retains the ability to bind β-catenin. Notably, APC proteins have several β-catenin-binding repeats (15 amino-acid repeats (AARs) and 20 AARs), which enable the wild-type form to bind to multiple β-catenin proteins (Figs 5a and 8). Truncated APC proteins that have been associated with colorectal cancer usually retain several of these AAR repeats. Thus, they have the ability to productively engage β-catenin in the destruction complex. Further, we observed that even the shortest APC protein (in Colo320 cells) is able to bind to β-catenin despite a complete lack of known β-catenin-binding repeat regions, indicating that additional interfaces between APC and β-catenin may exist. It has recently been shown that partial silencing of APC can lead to colon cancer in the absence of biallelic mutations in APC49. This directly relates to our finding that partial reduction of APC can sensitize RKO cell to Wnt ligands, arguing that mutations in APC create a special situation of reduced β-catenin binding and processing ability. Taken together, these data support a model, whereby colon cancer cells with truncated APC remain susceptible to regulation by upstream signals. Do colon cancers create their own Wnt-dependent niche? High Wnt pathway activity is essential to maintain the stem cell and transient amplifying progenitor compartment in intestinal crypts, and elevated Wnt/β-catenin pathway activity is associated with stem cell signatures in cancer. It is tempting to speculate that colon cancer cells generate a ‘niche’ that enables them to retain an undifferentiated and stem cell-like behaviour, whereas non-cancerous colon stems cells require the crypt microenvironment to provide Wnt and other ‘stemness’ signals22. In the case of tumours, the secretion of Wnt ligands and autocrine activation of canonical receptors may have a crucial role in maintaining stem cell populations independent of signals from the microenvironment. Thus, despite the presence of pathway-activating mutations in APC or β-catenin, the survival of colon cancer cells remains dependent on secreted Wnt ligands, and thus requires the activity of the Wnt cargo receptor Evi/Wls. The expression levels of both Wnt3 and Evi/Wls correlate with the degree of colon tumour differentiation, that is, the grade, which in turn correlates with prognosis24. The development of small-molecule Wnt antagonists for the treatment of colon cancer has focused on targets downstream of, or at the same level as, the destruction complex. Large-scale sequencing of human colon cancers has revealed recurrent genetic aberrations in a number of signal transducers, including components of the canonical Wnt pathway58. In addition, functional screening has uncovered a number of potential targets that act just upstream of the destruction complex. Our results indicate that the pathway can be inhibited further upstream, for example, through inhibitors of Wnt production acting at the level of Porcupine or the Wnt cargo receptor Evi/Wls to regulating Wnt signalling in colon tumours at the site of production. Methods Cell culture All cell lines were obtained from ATCC. HEK293T, MDA-MB231 (DMEM; Gibco), HCT116, HT29 (McCoy’s; Gibco), LoVo, SW480 and DLD1 (RPMI; Gibco) cells were cultured without antibiotics, unless they were stably transfected or transduced. All media were supplemented with 10% fetal bovine serum (PAA). HCT116 TCF4/Wnt cells were cultured in the presence of 50 μg ml−1 hygromycin B, and HCT116 TCF/LEF cells were cultured in the presence of 10 μg ml−1 puromycin. Primary colon cancer-initiating cells Primary human colorectal cancer or derived metastasis were obtained from the University Hospital Heidelberg in accordance with the Declaration of Helsinki. From each patient, informed consent on tissue collection as approved by the University Ethics Review Board was available. Primary human tissue was minced and digested using dispase (BD Biosciences), filtered and cells were seeded in ultralow attachment in cancer stem cell (CSC) media under serum-free conditions with the addition of fibroblast growth factor (FGF) basic (10 ng ml−1) and epidermal growth factor (EGF) (20 ng ml−1; both from R&D Systems) to allow spheroid formation45. For experimental seeding, spheroid cultures derived from three individual patients (P1 (K-Ras, p53 mutant), P2 (p53 mutant) and P3 (K-Ras, PI3CA, p53 mutant)) were dissociated to a single-cell solution by mechanical disruption via pipetting or by accutase treatment. To generate stable LEF/TCF–Wnt-reporter spheroid cultures, P3 spheroids were dissociated, lentivirally transduced using Viromag R/L beads (OZbiosciences) and subsequently cultured in the presence of 5 μg ml−1 puromycin to select for transduced cells. Quantitative PCR Complementary DNA was prepared from 0.5 or 2 μg total RNA, using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific). Subsequently, the cDNA was diluted to 5 ng μl−1 and used for quantitative PCR on the Lightcycler 480 (Roche), using the universal probe library system (Roche) in a 384-well format. GAPDH and UBC were used as reference genes for relative quantification. Oligos for quantitative PCR are shown in Supplementary Table S1. Cell-based assays and recombinant proteins siRNAs were obtained from Ambion (Applied Biosystems). Cells were transfected using a total of 0.1% DharmaFECT 1 (Thermo Fischer Scientific) or RNAiMax (Invitrogen). For plasmid transfection, cells were transfected with 10–60 ng DNA (total) per 384-plate well using TransIT reagent (VWR). Cell viability was determined using the CellTiterGlo assay (Promega) according to the manufacturer’s protocol. Recombinant mouse Wnt3a(315-20) was obtained from PeproTech and mouse recombinant R-spondin1 (3474-RS) from R&D Systems. siRNA sequences are shown in Supplementary Table S2. Full sized scans of all western blots are provided in Supplementary Figure S10. Generation of stable cell lines To generate stable Evi, control, CTNNB1 knockdown pools or TCF/LEF or LEF–GFP Wnt-luciferase reporter cell lines, we used lentiviral transduction with the indicated constructs. Viral particles were produced using the lentiviral packaging vectors pMDM2-VSVG, psPAX2 and the vector of interest transfected in the ration 3:1:2 into HEK293T cells. Supernatants were collected and filtered (45 μm) 36–42 h post transfection. Two hundred to 500 μl of the supernatant and 2.5 μg ml−1 of polybryne were added to the six-well plate/T125 flask of cells, plate/flask was centrifuged at 2,000 g for 1 h and then incubated for 24–48 h in standard conditions. Stable cell lines were selected in the presence of 20 μg ml−1 puromycin for 72–120 h. Information on sh/shmirRNAs, constructs and antibodies are shown in Supplementary Tables S3–S5. Mouse xenograft experiments NOD/SCID mice (7–8 weeks, female) were subcutaneously injected with low-passage 3 × 105 or 10 × 105 (dox experiment) human HCT116 or DLD1 cells lentiviraly transduced with the indicated constructs. shRNA experiments were performed in blinded manner. For induction, shmirEvi/Ctrl mice were treated with 1 mg ml−1 of dox in the drinking water starting from day 18, when the tumour volumes reach around 125–200 mm3. Tumour appearance was monitored once or twice per week by palpating the injection area. Experiments were terminated when tumours in control group(s) reached 1,200–1,500 mm3. Results for tumour appearance were analysed using R programme and plotted as Kaplan–Meier plot. P-values were counted with the help of the same programme by log-rank test. P-values for difference in tumour volumes was calculated by Student’s t-test. Animal welfare and experimental procedures were performed in accordance with German animal protection law and were approved by the Regierungspräsidium Karlsruhe, Germany (35-9185.81). Immunohistochemistry Immunohistochemical studies were performed on paraffin-embedded slides/sections, and on a Colon-Grade Array with 150 biopsies, including 30 sections each of normal colon, adenoma, and colon cancer grade 1, 2 and 3 tissues. In addition, serial sections from five adenocarcinoma and five normal colon biopsies were analysed. All samples were analysed as approved by the local institutional ethics boards. The samples were kindly provided by the NCT tumour bank for tissue samples. Expression data analysis The data from TCGA ( http://cancergenome.nih.gov/) colorectal carcinoma data set (array) was analysed and visualized by Oncomine (Compendia Bioscience, Ann Arbor, MI, USA; http://www.oncomine.org). Microarray data are preprocessed by Oncomine in a standardized way by log2 transformation and scaling the median value per microarray to 0. Fold change is calculated as the difference in the means of the normal colon versus adenocarcinoma samples. Cell cycle analysis Cells were seeded 16 h before analysis (20% of confluence), collected and stained with 200 μg ml−1 of propidium iodide, 0.1% sodium azide, 0.1% Triton-X100 and 10 μg ml−1 RNAses for 2–4 h. Single cells were analysed for subG1, S and G2 peaks by fluorescence-activated cell sorting array (BD Bioscience). Analysis was performed with FlowJo. Author contributions M.B., O.V. and G.E., designed the study; O.V. and G.E. performed experiments; I.A. performed ICH; M.M. analysed ICH data; H.G., T.D.D. and C.B. designed, performed and analysed the primary cell experiments; G.K. and S.L. provided sequencing data; T.S. helped to analyse experiments; G.M., C.H., B.A. and R.S. contributed the computational analysis; C.B. contributed essential reagents. All authors contributed and commented on the manuscript. Additional information How to cite this article: Voloshanenko, O. et al. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. Nat. Commun. 4:2610 doi: 10.1038/ncomms3610 (2013). Supplementary Material Supplementary Information Supplementary Figures S1-S10 and Supplementary Tables S1-S5

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Nested effects models for high-dimensional phenotyping screens.

Florian Markowetz, Olga G Troyanskaya, Dennis Kostka … (2007)

In high-dimensional phenotyping screens, a large number of cellular features is observed after perturbing genes by knockouts or RNA interference. Comprehensive analysis of perturbation effects is one of the most powerful techniques for attributing functions to genes, but not much work has been done so far to adapt statistical and computational methodology to the specific needs of large-scale and high-dimensional phenotyping screens. We introduce and compare probabilistic methods to efficiently infer a genetic hierarchy from the nested structure of observed perturbation effects. These hierarchies elucidate the structures of signaling pathways and regulatory networks. Our methods achieve two goals: (1) they reveal clusters of genes with highly similar phenotypic profiles, and (2) they order (clusters of) genes according to subset relationships between phenotypes. We evaluate our algorithms in the controlled setting of simulation studies and show their practical use in two experimental scenarios: (1) a data set investigating the response to microbial challenge in Drosophila melanogaster, and (2) a compendium of expression profiles of Saccharomyces cerevisiae knockout strains. We show that our methods identify biologically justified genetic hierarchies of perturbation effects. The software used in our analysis is freely available in the R package 'nem' from www.bioconductor.org.

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Author and article information

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Hong Wang: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2016

Publication date (Electronic): 1 June 2016

Volume: 11

Issue: 6

Electronic Location Identifier: e0155999

Affiliations

[1 ]Department of Statistical Bioinformatics, Institute of Functional Genomics, University of Regensburg, Regensburg, Germany

[2 ]Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Department of Cell and Molecular Biology, Faculty of Medicine Mannheim, Heidelberg University, Heidelberg, Germany

Rutgers, the State Univesity of New Jersey, UNITED STATES

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: MB RS. Performed the experiments: OV GE. Analyzed the data: GM CH. Wrote the paper: GM RS. Developed the statistical method: GM RS MJS.

* E-mail: giusi.moffa@ 123456gmail.com

Article

Publisher ID: PONE-D-15-36807

DOI: 10.1371/journal.pone.0155999

PMC ID: 4889067

PubMed ID: 27248690

SO-VID: de59f72c-1d13-4b2a-aaa5-79364ad4be18

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 20 August 2015

Date accepted : 6 May 2016

Page count

Figures: 5, Tables: 0, Pages: 13

Funding

Work in the group of R.S. was supported by the NGFN-Project FKZ01GS08183 - Verbund Kolonkarzinom and the BioSysNet-Project - Bavarian Research Network for Molecular Biosystems. Work in the laboratory of M.B. was supported by grants from the BMBF (NGFN-plus 01GS08181-6) and BMBF GERF.

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Data Availability ArrayExpress database with accession number E-MTAB-651 http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-651/.

Refining Pathways: A Model Comparison Approach

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A shrinkage approach to large-scale covariance matrix estimation and implications for functional genomics.

Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells

Nested effects models for high-dimensional phenotyping screens.

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