The transcriptional coactivator WBP2 primes triple-negative breast cancer cells for responses to Wnt signaling via the JNK/Jun kinase pathway

Abstract

The transcriptional coactivator WW domain-binding protein 2 (WBP2) is an emerging oncogene and serves as a node between the signaling protein Wnt and other signaling molecules and pathways, including epidermal growth factor receptor, estrogen receptor/progesterone receptor, and the Hippo pathway. The upstream regulation of WBP2 is well-studied, but its downstream activity remains unclear. Here, we elucidated WBP2's role in triple-negative breast cancer (TNBC), in which Wnt signaling is predominantly activated. Using RNAi coupled with RNA-Seq and MS analyses to identify Wnt/WBP2- and WBP2-dependent targets in MDA-MB-231 TNBC cells, we found that WBP2 is required for the expression of a core set of genes in Wnt signaling. These included AXIN2, which was essential for Wnt/WBP2-mediated breast cancer growth and migration. WBP2 also regulated a much larger set of genes and proteins independently of Wnt, revealing that WBP2 primes cells to Wnt activity by up-regulating G protein pathway suppressor 1 (GPS1) and TRAF2- and NCK-interacting kinase (TNIK). GPS1 activated the c-Jun N-terminal kinase (JNK)/Jun pathway, resulting in a positive feedback loop with TNIK that mediated Wnt-induced AXIN2 expression. WBP2 promoted TNBC growth by integrating JNK with Wnt signaling, and its expression profoundly influenced the sensitivity of TNBC to JNK/TNIK inhibitors. In conclusion, WBP2 links JNK to Wnt signaling in TNBC. GPS1 and TNIK are constituents of a WBP2-initiated cascade that primes responses to Wnt ligands and are also important for TNBC biology. We propose that WBP2 is a potential drug target for JNK/TNIK-based precision medicine for managing TNBC.

Keywords: TNIK; WW domain–binding protein 2; Wnt signaling; axin; breast cancer; cell signaling; oncogene; precision medicine; therapy; transcription coactivator.

Figure 1.

A, analysis of WBP2 amplification (multiplication of intra-chromosomal region of 0.5 to 10 Mb), gain (increase in larger chromosomal region or intact chromosome) and deletion in 6 studies of breast cancer. About 20–40% of breast cancer patients harbor WBP2 amplification/gain. B, a heat map showing the correlation of WBP2 mRNA expression and copy number alteration. y axis refers to individual clinical samples in the TCGA breast cancer database, whereas the x axis indicates the intensity of WBP2 gene expression (left panel) or copy number levels (right panel). C, dot plot of WBP2 mRNA expression in individual clinical breast cancer samples in the TCGA database categorized according to copy number alterations. D, Kaplan-Meier plot of breast cancer patient survival (n = 273) according to transcriptomic fingerprint of WBP2 amplification: (i) up- and (ii) down-regulated.

Figure 2.

A, schematic design of the RNA-Seq analysis, showing the strategy used for exploring the role of WBP2 in the WNT3A-induced transcriptional program. B, sample QC before RNA-Seq analysis. WBP2 was depleted by lentivirus-expressed shRNAs. MDA-MB-231 cells were treated with 200 ng/ml of rWNT3A for 12 h. C, heat map of the expression pattern of the 28 Wnt/WBP2 target genes. Color intensity refers to the fold-change in mRNA level between WNT3A treatment versus control. Each column represents the mean value generated from biological triplicates. D, percentages of the Wnt/WBP2 target genes under different fold change cut-offs.

Figure 3.

A, RT-qPCR validation of the Wnt/WBP2 target genes in MDA-MB-231 cells. B, RT-qPCR showing the expression of AXIN2 regulated by Wnt/WBP2 in MDA-MB-468 (i) and BT549 (ii). WBP2 manipulation was examined by Western blotting. C, transwell cell migration (i and ii) and clonogenic assay (iii and iv) in MDA-MB-231 cells were infected by lentiviruses expressing WBP2 shRNAs and AXIN2 plasmids as indicated and stimulated with 200 ng/ml of rWNT3A. Representative images (i and iii) and corresponding quantitative analysis (ii and iv) were shown. D, (i) RT-qPCR showed the effect of WBP2 depletion on AXIN2 expression and AXIN2 rescue by WBP2 expression in MDA-MB-231 cells. WBP2 knockdown-mediated down-regulation of active β-catenin (ii) and TCF reporter activity (iii) was rescued by AXIN2 overexpression in MDA-MB-231 cells.

Figure 4.

A, (i) RT-qPCR analysis of AXIN2 mRNA levels upon WNT3A stimulation at the indicated durations in MDA-MB-231 cells. (ii) RT-qPCR analysis of AXIN2 mRNA levels. MDA-MB-231 cells were transfected with WBP2 shRNAs for 48 h and treated with control serum-free medium (O) or 200 ng/ml of rWNT3A for 3 h to examine the early-phase transcription of AXIN2 (top). IB analysis of the depletion of WBP2 is shown in the bottom. B, (i) analysis strategy to identify WBP2-regulated genes. (ii) Heat map of the expression pattern of the 86 WBP2-regulated genes. (iii) Top enriched pathways of WBP2 target genes. The number in each bar represents number of WBP2 targets involved in this pathway. C, (i) heat map of the expression pattern of the 23 WBP2-regulated proteins. (ii) Protein-mRNA expression association analysis of the WBP2-regulated proteins, 7 proteins with significant mRNA change were indicated in black. GPS1 was highlighted in red.

Figure 5.

A, qPCR (i) and IB (ii) analysis of TNIK expression upon WBP2 manipulation. MDA-MB-231 cells were infected with lentiviruses expressing WBP2 shRNAs and transfected with vector or WBP2 plasmid for 72 h before harvest. (iii) Analysis of WBP2 and TNIK up-regulation in breast cancer patients were performed on cBioPortal (www.cbioportal.org/) (77, 78). The odd ratios are originally generated as log odd ratios that were subsequently transformed by the natural exponential function. B, WBP2/WNT3A-induced up-regulation of TCF reporter activity (i) as well as AXIN2 expression and active β-catenin level (ii) were abolished by the TNIK inhibitor, NCB-0846 treatment in MDA-MB-231 cells. DMSO was used as the vehicle control.

Figure 6.

A, (i) enriched c-Jun/AP-1–binding sites in the promoter region of TNIK. The image displays the most relevant transcription factor–binding sites in this gene promoter as predicted by SABiosciences Text Mining Application and the UCSC Genome Browser. IB analysis of JNK/c-Jun expression/phosphorylation upon WBP2 exogenous expression (ii) or depletion (iii) in MDA-MB-231 cells. (iv) WBP2/WNT3A-induced up-regulation of TCF reporter activity were abolished by the JNK inhibitor JNK-IN-8 treatment in MDA-MB-231 cells. DMSO was used as the vehicle control. (v) JNK1 + 2 siRNAs knockdown combined with JNK inhibitor JNK-IN-8 abolished the WBP2/WNT3A-induced TNIK and AXIN2 expression and active β-catenin level in MDA-MB-231 cells. (vi) TNIK inhibition via NCB-0846 treatment abolished the WBP2/WNT3A-induced phosphorylation of JNK/c-Jun in MDA-MB-231 cells. B, IB analysis of GPS1 protein expression upon WBP2 depletion (i) or overexpression (ii) in MDA-MB-231 cells. WBP2-induced up-regulation of TCF reporter activity (iii) and AXIN2/active β-catenin level (iv) were abolished by GPS1 siRNA knockdown in MDA-MB-231 cells. C, (i) IB analysis of JNK/c-Jun and TNIK expression/activity upon GPS1 siRNA knockdown in MDA-MB-231 cells. (ii) WBP2-induced up-regulation of TNIK and JNK/c-Jun expression/phosphorylation were abolished by GPS1 siRNA knockdown.

Figure 7.

A, (i) WBP2-induced 2D proliferation of MDA-MB-231 could be partially abolished by TNIK siRNA knockdown. The anchorage-dependent 2D (ii) and -independent 3D (iii) cell proliferation of MDA-MB-231 were decreased by WBP2 siRNA knockdown and this could be partially rescued by exogenous TNIK expression. B, WBP2/WNT3A-induced 2D proliferation of MDA-MB-231 could be significantly abolished by JNK inhibition via (i) JNK-IN-8 and (ii) TNIK inhibition via NCB-0846. C, (i) dose-response 2D and 3D growth curve for TNIK inhibition via NCB-0846 in MDA-MB-231 upon WBP2 exogenous expression or siRNA/shRNA knockdown. (ii) Tabulated IC₅₀ in summary for TNIK inhibitor, NCB-0846 for MDA-MB-231 with WBP2 OE or KD in 2D or 3D growth. The p value is calculated by the extra sum-of-squares F test.