An AXIN2 Mutant Allele Associated With Predisposition to Colorectal Neoplasia Has Context-Dependent Effects on AXIN2 Protein Function

Abstract

Heterozygous, germline nonsense mutations in AXIN2 have been reported in two families with oligodontia and colorectal cancer (CRC) predisposition, including an AXIN2 1989G>A mutation. Somatic AXIN2 mutations predicted to generate truncated AXIN2 (trAXIN2) proteins have been reported in some CRCs. Our studies of cells from an AXIN2 1989G>A mutation carrier showed that the mutant transcripts are not significantly susceptible to nonsense-mediated decay and, thus, could encode a trAXIN2 protein. In transient transfection assays, trAXIN2 was more abundant than wild-type AXIN2 protein, and in contrast to AXIN2, glycogen synthase kinase 3β inhibition did not increase trAXIN2 levels. Like AXIN2, the trAXIN2 protein interacts with β-catenin destruction complex proteins. When ectopically overexpressed, trAXIN2 inhibits β-catenin/T-cell factor–dependent reporter gene activity and SW480 CRC cell colony formation. These findings suggest the trAXIN2 protein may retain some wild-type functions when highly expressed. However, when stably expressed in rat intestinal IEC-6 cells, the trAXIN2 protein did not match AXIN2’s activity in inhibiting Wnt-mediated induction of Wnt-regulated target genes, and SW480 cells with stable expression of trAXIN2 but not AXIN2 could be generated. Our data suggest the AXIN2 1989G>A mutation may not have solely a loss-of-function role in CRC. Rather, its contribution may depend on context, with potential loss-of-function when AXIN2 levels are low, such as in the absence of Wnt pathway activation. However, given its apparent increased stability in some settings, the trAXIN2 protein might have gain-of-function in cells with substantially elevated AXIN2 expression, such as Wnt pathway–defective CRC cells.

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs

“…HEK293T and HEK293E (a generous gift from Dr. Diane Fingar, University of Michigan) cells ( Tee et al, 2002 ) were cultured in DMEM with 10% fetal bovine serum (FBS) and 100 units/ml penicillin/streptomycin (Invitrogen). Human breast cancer cell line: MDA-MB-231 cells (kindly provided by Dr. Shaomeng Wang, University of Michigan) ( Lu et al, 2008 ), human colon cancer cell line: SW480 cells (kindly provided by Dr. Eric Fearon, University of Michigan) ( Mazzoni et al, 2015 ), and human prostate cancer cell lines: LNCaP, 22RV1 and PC3 and benign prostate cell lines: PNT2 and RWPE (generous gifts from Dr. Arul Chinnaiyan, University of Michigan) ( Ateeq et al, 2011 ; Helgeson et al, 2008 ; Tomlins et al, 2007 ) were cultured in RPMI 1640 with 10% FBS and 100 units/ml penicillin/streptomycin and RWPE cells were cultured in keratinocyte serum-free medium (Invitrogen) with Supplements for Keratinocyte-SFM (Invitrogen). Primary prostate cell line: PrEC was purchased from LONZA and cultured in the PrEGM media (LONZA).…”

Wnt, RSPO and Hippo Signalling in the Intestine and Intestinal Stem Cells

“…Correspondingly, conditional inactivation of Apc using the Cre/loxP system in LGR5 + cells initiates neoplastic growth in epithelia of the small intestine and colon [ 58 ]. Another example of genetic changes altering the Wnt pathway components and that have been identified in CRC, are missense mutations in the Ctnnb1 gene (encodes β-catenin) that stabilize the protein or mutations inactivating the additional component of the β-catenin destruction complex AXIN2 [ 5 , 59 ].…”

Variations in AXIN2 predict risk and prognosis of colorectal cancer

“…From the study of Lamni et al, 4 many investigations relating dental agenesis with predisposition to cancer, primarily Colorectal cancer (CRC), have arisen. 5–7 This association is supported by the molecular events that keep homeostasis of morphogenesis and tissue regeneration. CRC is the fourth most common cause of cancer-related deaths in the world 8 and it has been reported that colorectal carcinogenesis is associated with alterations in Wnt signaling.…”