AbstractAnaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CA^E545K into the LAM1 PTC cell line, which carries a BRAF^V600E mutation. In culture, the engineered cell line (LAM1:PIK3CA^E545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1^EV). Both the LAM1^EV and LAM1:PIK3CA^E545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CA^E545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CA^E545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CA^E545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC. Graphical Abstract