Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification.

Abstract

Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (<i>Adenomatous polyposis coli</i>, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (<i>RNF43</i>, <i>RSPO</i>-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of <i>RSPO</i> may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (<i>AXIN2</i>, <i>NKD1</i>) occurring even in CIMP-negative LD cancers. <i>AXIN2</i> mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential <i>AXIN2</i> expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.