Oncogenic Mutations in Armadillo Repeats 5 and 6 of β-Catenin Reduce Binding to APC, Increasing Signaling and Transcription of Target Genes

Abstract

Supplementary Figure 4. AXIN2 RNA expression in b-catenin S37F and K335I knock-in clones. The top panels show the AXIN2 expression levels determined by qRT-PCR (mean ± standard deviation, n ¼ 5) for all analyzed clones. The bottom panels show the results for K335I knock-in clones only. All expression levels are depicted relative to the housekeeping gene GAPDH. Data are presented as mean ± standard deviation. **P < .01; ***P < .001; ****P < .0001. CTR, control. March 2020 Armadillo Repeat 5/6 Mutations of b-Catenin 1043.e6 IgG, immunoglobulin G.

Evaluation of AXIN1 and AXIN2 as targets of tankyrase inhibition in hepatocellular carcinoma cell lines

“…Interestingly, these same groups also show an enrichment in CTNNB1 mutations within armadillo repeats 5 and 6 (i.e. K335, W383, N387), for which we have recently unequivocally shown that they are efficient inducers of HCC development in mice, associated with a weak but significant activation of β-catenin signaling 8 . Expression profiling of tumor tissues has the inherent shortcoming that average expression is determined of a heterogeneous group of cells, including contaminating normal cells.…”

“…AXIN1-mutant HCCs predominantly show an exclusive membranous β-catenin staining 10 – 12 , 15 , indeed suggesting that there is no nuclear signaling. However, this is also observed in tumors carrying oncogenic CTNNB1 mutations within the armadillo repeats and a subset of S45-mutant cancers 8 , 43 , while these clearly lead to enhanced signaling. Thus, although nuclear accumulation of β-catenin is a reliable predictor of active signaling, its absence does not exclude that a low level of biologically relevant signaling is active 44 .…”

“…RNA isolation and qRT-PCR analysis were performed as described previously 8 , 14 , 50 . In short, RNA was isolated with a Machery-NucleoSpin RNA II kit (Bioké, Leiden, The Netherlands) according to the manufacturer’s instructions, and quantified using a Nanodrop ND-1000 (Wilmington, DE, USA).…”

“…Following Wnt stimulation, the multiprotein complex dissociates resulting in the accumulation of cytosolic and nuclear β-catenin, which in turn triggers the transcription of specific target genes. Aberrant activation of Wnt/β-catenin signaling in HCC has been mainly attributed to activating somatic mutations in the CTNNB1 gene coding for β-catenin (20–25%) 3 , 5 – 8 , for which it is well-accepted that they support tumor growth by enhancing β-catenin signaling in a dominant fashion.…”

Drug Discovery in Liver Disease Using Kinome Profiling

“…Despite evident promise, immune therapy for this disease remains unsatisfactory [ 47 , 48 , 49 , 50 ]. Importantly, the genetic basis of the disease is now partially understood [ 51 , 52 , 53 ] as is the resulting cell biology. Intrinsic factors such as stem cell markers (i.e., LGR5 [ 54 ]) and extrinsic factors including the surrounding microenvironment (e.g., bacterial and toxic drivers of mutagenesis [ 55 ] and cancer associated fibroblasts supporting cancer growth [ 56 ]) contribute to hepatocellular carcinoma growth.…”