Edinburgh Research Explorer NODAL/TGF signalling mediates the self-sustained stemness induced by PIK3CAH1047R homozygosity in pluripotent stem cells

Transcriptomic and proteomic analyses of iPSCs with allele-dose dependent expression of the PIK3CA oncogene confirm network rewiring in homozygous mutants, with self-sustained stemness likely driven by constitutive TGF  /NODAL pathway activation. Abstract Activating PIK3CA mutations are known “drivers” of human cancer and developmental overgrowth syndromes. We recently demonstrated that the "hotspot" PIK3CA H1047R variant exerts unexpected allele dose-dependent effects on stemness in human pluripotent stem cells (hPSCs). In the present study, we combine high-depth transcriptomics, total proteomics and reverse-phase protein arrays to reveal potentially disease-related alterations in heterozygous cells, and to assess the contribution of activated TGF  signalling to the stemness phenotype of homozygous PIK3CA H1047R cells. We demonstrate signalling rewiring as a function of oncogenic PI3K signalling strength, and provide experimental evidence that self-sustained stemness is causally related to enhanced autocrine NODAL/TGF  signalling. A significant transcriptomic signature of TGF  pathway activation in heterozygous PIK3CA H1047R was observed but was modest and was not associated with the stemness phenotype seen in homozygous mutants. Notably, the stemness gene expression in homozygous PIK3CA H1047R iPSCs was reversed by pharmacological inhibition of NODAL/TGF  signalling, but not by pharmacological PI3Kα pathway inhibition. Altogether, this provides the first in-depth analysis of PI3K signalling in human pluripotent stem cells and directly links strong PI3K activation to developmental NODAL/TGF  signalling. This work illustrates the importance of allele dosage and expression when artificial systems are used to model human genetic disease caused by activating PIK3CA mutations. Given the unexpected and surprisingly mild phenotype caused by heterozygous PIK3CA H1047R expression in iPSCs, we reasoned that more sensitive assays would allow us to discern small but disease-relevant alterations in these cells. Thus, in this study, we first applied high depth transcriptomics, and proteomics to seek evidence of disease-related phenotypes in heterozygous cells, and to investigate how high-dose PI3K signalling leads to self-sustained stemness in homozygous PIK3CA H1047R iPSCs. We demonstrate that heterozygous cells do exhibit significant transcriptomic changes, although these are a weak echo of the widespread changes seen in homozygous cells. The mild transcriptional consequences of heterozygous expression of disease-relevant PIK3CA mutations were also validated in additional model systems and contrast with previous findings of major transcriptional rewiring in immortalised, non-transformed breast epithelial cells (Hart We demonstrate that the stemness phenotype of PIK3CA iPSCs is maintained by self-sustained NODAL/TGF  signalling, in line with increased PIK3CA -mediated NODAL expression, and that it is not reversible by PI3K  -specific inhibition. This work provides in-depth characterisation of the near-binary PI3K signalling effects seen in hPSCs, and provides evidence for PI3K  induced NODAL/TGF  signalling as the mechanism for self-sustained stemness in homozygous PIK3CA H1047R iPSCs. We discuss the implications of our findings for understanding and modelling developmental disorders and cancers driven by genetic PI3K activation.