Thesis: Rabeah Amre Almhassneh

PhD supervisor: Dr. María Ángeles Juanes Ortiz

Title: Understanding the molecular mechanisms of APC C-terminal missense mutations in young colorectal cancer patients

Abstract: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with an increasing incidence among young patients (less than 50 years old), a condition known as early-onset colorectal cancer (EOCRC). The tumour suppressor adenomatous polyposis coli (APC) is essential for epithelial homeostasis and is one of the earliest driver genes altered in CRC. Although APC truncating mutations have been extensively studied, particularly in the context of the Wnt/β-catenin pathway, the contribution of C-terminal APC missense mutations to EOCRC remains poorly understood. This thesis establishes a previously unrecognized role for C-terminal APC missense mutations in regulating the cytoskeleton–integrin axis and identifies a potential mechanism underlying their pathogenicity in EOCRC.
Two pathogenic APC missense mutations identified in young patients were characterized and shown to impair cell migration and invasion in 2D and 3D environments. Mechanistically, these mutations were found to disrupt APC-dependent actin regulation. Using purified APC C-terminal fragments, impaired actin bundling and nucleation were demonstrated through co-sedimentation, bulk pyrene, and TIRF assays. These defects were recapitulated in cells. Fluorescence polarization microscopy further revealed altered actin filament organization at focal adhesions, while focal adhesion dynamics remained unaffected. Furthermore, both mutations reduced β1-integrin activation, a defect that was restored by MnCl₂ treatment. Importantly, restoration of β1-integrin activation also rescued the motility defect in one of the mutant cell lines, providing functional evidence for a link between APC-dependent cytoskeletal regulation and integrin signalling.
Neither mutation altered β-catenin localization or cell proliferation, indicating that the phenotypes occur independently of canonical Wnt signalling. Collectively, this thesis demonstrates that C-terminal APC missense mutations can drive cellular phenotypes through disruption of actin organization and cytoskeleton- integrin signalling rather than through the canonical Wnt/β-catenin pathway. These findings provide new mechanistic insight into the contribution of APC missense mutations to EOCRC and establish cytoskeletal regulation as a potential therapeutic vulnerability in this disease.