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

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

Inactivation of class II PI3K-C2 alpha induces leptin resistance, age-dependent insulin resistance and obesity in male mice

AIMS/HYPOTHESIS: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS: All reagents are available upon request

Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca H1047R hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA H1047R mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution

A New TIPE of Phosphoinositide Regulator in Cancer

Specific phosphoinositide lipids promote cell growth and cancer. In this issue of Cancer Cell, Fayngerts and colleagues demonstrate that the TIPE3 protein enhances PtdIns(4,5)P2 and PtdIns(3,4,5)P3, is overexpressed in certain cancers, and promotes tumorigenesis. TIPE3 can act as a lipid transfer protein and may constitute a novel phosphoinositide metabolism regulator

Nek10 Mediates G2/M Cell Cycle Arrest and MEK Autoactivation in Response to UV Irradiation▿

Appropriate cell cycle checkpoint control is essential for the maintenance of cell and organismal homeostasis. Members of the Nek (NIMA-related kinase) family of serine/threonine protein kinases have been implicated in the regulation of various aspects of the cell cycle. We explored the cellular functions of Nek10, a novel member of the Nek family, and demonstrate a role for Nek10 in the cellular UV response. Nek10 was required for the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling upon UV irradiation but not in response to mitogens, such as epidermal growth factor stimulation. Nek10 physically associated with Raf-1 and MEK1 in a Raf-1-dependent manner, and the formation of this complex was necessary for Nek10-mediated MEK1 activation. Nek10 did not affect the kinase activity of Raf-1 but instead promoted the autophosphorylation-dependent activation of MEK1. The appropriate maintenance of the G2/M checkpoint following UV irradiation required Nek10 expression and ERK1/2 activation. Taken together, our results uncover a role for Nek10 in the cellular response to UV irradiation