NODAL/TGFβ signalling mediates the self-sustained stemness induced by PIK3CAH1047R homozygosity in pluripotent stem cells

Abstract

Activating PIK3CA mutations are known “drivers” of human cancer and developmental overgrowth syndromes. We recently demonstrated that the "hotspot" PIK3CAH1047R 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 TGFβ signalling to the stemness phenotype of homozygous PIK3CAH1047R 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/TGFβ signalling. A significant transcriptomic signature of TGFβ pathway activation in heterozygous PIK3CAH1047R was observed but was modest and was not associated with the stemness phenotype seen in homozygous mutants. Notably, the stemness gene expression in homozygous PIK3CAH1047R iPSCs was reversed by pharmacological inhibition of NODAL/TGFβ 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/TGFβ 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.

Transcriptomically-inferred PI3K activity and stemness show a counterintuitive correlation withPIK3CAgenotype in breast cancer

“…Disentangling the apparent biphasic relationship between single versus multiple copies of PIK3CA mutation and stemness scores will require direct experimentation, but is likely to reflect context-dependent feedback loops within the intracellular signalling networks. Such feedback loops can result in non-intuitive and discontinuous outcomes upon different levels of activation of the same pathway, as demonstrated in our isogenic iPSC system with heterozygous and homozygous PIK3CA H1047R expression [8,40]. In general, our observations caution against the use of a binary PIK3CA-mutant-centric approach to predict PI3K pathway activity outcomes.…”

“…a module previously shown to be active in various cancers and predictive of cancer outcome [46]. Moreover, computational analyses of iPSCs with homozygous PIK3CA H1047R expression identified MYC as a central hub connecting the PI3K, TGFb and pluripotency networks in these cells [40]. Recently, PIK3CA H1047R /KRAS G12V double knock-in breast epithelial cells were also shown to exhibit a high MYC transcriptional signature, when compared to single-mutant counterparts [47].…”

“…Surprised by this observation, we next asked whether the biphasic relationship between PIK3CA genotype and transcriptionally-derived PI3K/stemness scores could be recapitulated in a controlled cellular model. We turned to human induced pluripotent stem cells (iPSCs) that we engineered previously to harbour heterozygous or homozygous PIK3CA H1047R alleles, the only reported cellular models of heterozygous and homozygous PIK3CA H1047R expression on an isogenic background to date [40]. Using our previously reported high-depth transcriptomic data on PIK3CA WT/H1047R and PIK3CA H1047R/H1047R iPSCs [40], we performed conventional gene set enrichment analysis (GSEA) with the two gene set signatures used for PI3K and stemness score calculations in the breast cancer setting (MSigDB "HALLMARK_PI3K_AKT_MTOR_SIGNALING" and PluriNet, respectively).…”

“…We turned to human induced pluripotent stem cells (iPSCs) that we engineered previously to harbour heterozygous or homozygous PIK3CA H1047R alleles, the only reported cellular models of heterozygous and homozygous PIK3CA H1047R expression on an isogenic background to date [40]. Using our previously reported high-depth transcriptomic data on PIK3CA WT/H1047R and PIK3CA H1047R/H1047R iPSCs [40], we performed conventional gene set enrichment analysis (GSEA) with the two gene set signatures used for PI3K and stemness score calculations in the breast cancer setting (MSigDB "HALLMARK_PI3K_AKT_MTOR_SIGNALING" and PluriNet, respectively). In line with their established biochemical and cellular phenotypes [8,40], homozygous PIK3CA H1047R iPSCs showed strong positive enrichment for both PI3K and stemness gene signatures (Fig.…”

“…Using our previously reported high-depth transcriptomic data on PIK3CA WT/H1047R and PIK3CA H1047R/H1047R iPSCs [40], we performed conventional gene set enrichment analysis (GSEA) with the two gene set signatures used for PI3K and stemness score calculations in the breast cancer setting (MSigDB "HALLMARK_PI3K_AKT_MTOR_SIGNALING" and PluriNet, respectively). In line with their established biochemical and cellular phenotypes [8,40], homozygous PIK3CA H1047R iPSCs showed strong positive enrichment for both PI3K and stemness gene signatures (Fig. 4D).…”