β-catenin-independent regulation of Wnt target genes by RoR2 and ATF2/ATF4 in colon cancer cells

Abstract

Wnt signaling is an evolutionarily conserved signaling route required for development and homeostasis. While canonical, β-catenin-dependent Wnt signaling is well studied and has been linked to many forms of cancer, much less is known about the role of non-canonical, β-catenin-independent Wnt signaling. Here, we aimed at identifying a β-catenin-independent Wnt target gene signature in order to understand the functional significance of non-canonical signaling in colon cancer cells. Gene expression profiling was performed after silencing of key components of Wnt signaling pathway and an iterative signature algorithm was applied to predict pathway-dependent gene signatures. Independent experiments confirmed several target genes, including PLOD2, HADH, LCOR and REEP1 as non-canonical target genes in various colon cancer cells. Moreover, non-canonical Wnt target genes are regulated via RoR2, Dvl2, ATF2 and ATF4. Furthermore, we show that the ligands Wnt5a/b are upstream regulators of the non-canonical signature and moreover regulate proliferation of cancer cells in a β-catenin-independent manner. Our experiments indicate that colon cancer cells are dependent on both β-catenin-dependent and -independent Wnt signaling routes for growth and proliferation.

Figure 1

Identification of Evi/Wls-regulated Wnt non-canonical target genes. (a) Schematic representation of Evi/Wls-regulated genes and their separation regarding β-catenin dependency and independency. Rows represent diversity of target genes (E_n) and columns indicate different knockdown conditions using siControl/Ctrl and siRNAs targeting EVI/WLS, APC or CTNNB1/β-catenin. (b) The steps of bioinformatical analyses for identification of meaningful biological clusters on the basis of RNAi RNAseq HCT116 data. (c,d) Representative clusters generated by ISA (Iterative Signature Algorithm), which include all 4 siRNAs. Upper (c) cluster shows β-catenin dependent Evi/Wls-regulated genes. (d) This cluster consists of β-catenin independent Evi/Wls-regulated target genes.

Figure 2

Non-canonical Wnt target genes are regulated by Evi/Wls but not by β-catenin or APC. (a) Silencing of Evi/Wls reduces both canonical and non-canonical target genes, whereas β-catenin and APC silencing effects the level of AXIN2, the classical canonical Wnt target gene. HCT116 cells were reverse transfected with the indicated siRNA and assessed for the expression of the indicated genes by qPCR 72 hours later. (b) Induction of canonical β-catenin signaling by GSKβ inhibitor XVI (CHIR99021) induces AXIN2 but does not change mRNA levels of identified non-canonical target genes. HCT116 cells were treated with indicated amounts of the inhibitor for 24 hrs, then indicated genes were analyzed by qPCR. (c) Downregulation of non-canonical Wnt target genes is a specific effect of Evi/Wls silencing. Overexpression of Evi-V5 partly rescues the phenotype induced by siEvi#3. HCT116 cells were reverse transfected with indicated siRNAs and 24 hours later cells were transfected with pcDNA Ctrl versus Evi-V5. 48 hrs after the plasmid transfection the indicated genes were analyzed by qPCR. (d) HCT116 cells were treated with LGK974 for 96 hrs and identified non-canonical genes were analyzed by qPCR. AXIN2 was used as positive control. (a-d) Every independent experiment was normalized to siCtrl (siRNA silencing experiments) or DMSO/Ctrl (drug treatment) and log₂ transformed of relative quantitation (RQ). Results of 3–6 independent experiments are shown as mean ± s.e.m. p values of every condition compared to control using single samples t-test (a,b,d) or paired Student’s t-test (c) as well as values of independent experiments are shown in the Table S2.

Figure 3

Non-canonical Wnt genes are regulated by Evi/Wls and porcupine in different cellular systems. (a,b) HT29 colon cancer cells were treated with the indicated amounts of LGK974 porcupine inhibitor for 96 hrs (a) or with 5 μM GSK3β inhibitor XVI (CHIR99021) for 24 hrs. (c,d) Primary colon cancer cells HROC69 (passages 31–34) or HROC40 (p29–34) were treated with porcupine inhibitor LGK974 for 96–120 hrs or with 5 μM GSK3β inhibitor XVI (CHIR99021) for 24 hrs. (e) MEF Evi^Fl/Fl cells were transduced with a Cre-expressing construct and selected with hygromycin. 3 days after selection MEFs were collected for analyses of the indicated genes by qPCR. (f) MEF cells were treated with 5 μM GSK3β inhibitor XVI for 24 hrs. (a–f) After treatment cells were analyzed for expression of the indicated Evi/Wls target genes by qPCR. Relative data to control treatment were log₂ transformed. Results of 3–6 independent experiments are shown as mean ± s.e.m. p values of every condition compared to control using Student’s t-test as well as values of independent experiments are shown in the Table S2.

Figure 4

Wnt5a/b regulates identified Evi/Wls dependent non-canonical genes. (a) Silencing of Wnt5b in colon cancer cells leads to downregulation of Evi/Wls dependent target genes. HCT116 cells were reverse transfected with the indicated siRNAs and then analysis of the indicated genes by qPCR was performed. (b) Addition of Wnt5a partly rescues downregulation of non-canonical Wnt genes upon Wnt5b silencing. HCT116 cells were reverse transfected with siWnt5b#1 or siCtrl. 24 hrs later, medium from L cells (parental and mouseWnt5a) was added. Indicated non-canonical Wnt target genes were analyzed by qPCR. (a,b) Relative data to control treatments (siCtrl (a) or siCtrl Ctrl-medium (b)) were log₂ transformed. Results of 4–8 independent experiments are shown as mean ± s.e.m. p values of every condition compared to control using Student’s t-test as well as values of independent experiments are shown in the Table S2.

Figure 5

Wnt5a is overexpressed in colorectal cancer and influences the growth behavior of cancer cells. (a) WNT5A mRNA is overexpressed in colorectal cancer. RNAseq expression data (TCGA data set, 2017) were analyzed for expression of WNT5A in normal colon/rectum (healthy) versus colorectal cancer samples (tumor). n – number of cases used in the analysis. Significance of the differences in expression was calculated using the Student’s t-test. (b–e) Wnt5a/b is required for the survival of colon cancer cells. Cells were reverse transfected with the indicated siRNAs. In (e) 24 hrs later medium from L parental or Wnt5a cells was added. Then cells were monitored using the Incucyte Live Cell Imaging system and growth curves were plotted in relative units according to the manufacturer’s program. Representative experiments from 3 independent experiments are shown.

Figure 6

Evi/Wls regulated target genes are dependent on RoR2/Dvl2/ATF non-canonical Wnt pathway. (a,c) Silencing of RoR2, Dvl2, ATF2 and ATF4 leads to downregulation of the Wnt non-canonical target genes. HCT116 cells were reverse transfected with indicated siRNAs for 72 hrs and then expression of the indicated target genes was analyzed by qPCR. Relative data to siCtrl were log₂ transformed. Results of 5–7 independent experiments are shown as mean ± s.e.m. p values of every condition compared to control using Student’s t-test as well as values of independent experiments are shown in the Table S2. (b) Silencing of RoR2 inhibits proliferation of HCT116 cells. Cells were reverse transfected with the indicated siRNAs. Then cells were monitored using the Incucyte Live Cell Imaging system and growth curves were plotted in relative units according to the manufacturer's program. Representative experiment from 3 independent ones is shown.

Figure 7

Wnt non-canonical target gene expression correlates with the mRNA level expression of Evi/Wls in human colon tumors. Microarray expression data of colorectal carcinoma was downloaded from the TCGA data portal. (a) Separation of microarray data in samples with low Evi/Wls expression and high Evi/Wls expression. Samples, which differ more than one standard deviation from the median Evi/Wls expression over all samples, were separated in samples with low Evi expression (<−1SD) and samples with high Evi expression (>+1 SD). 20 samples could be assigned to high Evi/Wls expression and 23 samples to low Evi/Wls expression. Median Evi expression between both groups has a fold change of 3 (p-Value: 1.0e⁻¹³). (b) Evi/Wls dependent Wnt non-canonical target genes correlate with Evi/Wls expression in human colon tumors. The percentage of identified Wnt non-canonical genes, which are significantly downregulated in the tumorsubset with lower expression of Evi/Wls, compared to the set of all genes, which were identified upon RNA sequencing of HCT116 cells (significance level α = 0.05; p = 3.9e⁻⁸, hypergeometric test).