Dataset Information

High-dose oncogenic PIK3CA drives constitutive cellular stemness through increased NODAL/TGFb signaling

ABSTRACT: Oncogenic PIK3CA mutations activate phosphoinositide 3-kinase (PI3K) and are among the commonest somatic mutations in cancer and mosaic, developmental overgrowth disorders. We recently demonstrated that the ‘hotspot’ variant PIK3CAH1047R exerts striking allele dose-dependent effects on stemness in human induced pluripotent stem cells (iPSCs), and moreover demonstrated multiple oncogenic PIK3CA copies in a substantial subset of human cancers. To identify the molecular mechanism underpinning PIK3CAH1047R allele dose-dependent stemness, we profiled isogenic wild-type, PIK3CAWT/H1047R and PIK3CAH1047R/H1047R iPSCs by high-depth transcriptomics, proteomics and reverse-phase protein arrays (RPPA). PIK3CAH1047R/H1047R iPSCs exhibited altered expression of 5644 genes and 248 proteins, whereas heterozygous hPSCs showed 492 and 54 differentially-expressed genes and proteins, respectively, confirming a nearly deterministic phenotypic effect of homozygosity for PIK3CAH1047R. Pathway and network-based analyses predicted a strong association between self-sustained TGFb/NODAL signaling and the ‘locked’ stemness phenotype induced by homozygosity for PIK3CAH1047R. This stemness gene signature was maintained without exogenous NODAL in PIK3CAH1047R/H1047R iPSCs and was reversed by pharmacological inhibition of TGFb/NODAL signaling but not by PIK3CA-specific inhibition. Analysis of PIK3CA-associated human breast cancers revealed increased expression of the stemness markers NODAL and POU5F1 as a function of disease stage and PIK3CAH1047R allele dosage. Together with emerging realization of the link between NODAL re-expression and aggressive cancer behavior, our data suggest that TGFb/NODAL inhibitors warrant testing in advanced breast tumors with multiple oncogenic PIK3CA copies.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell

DISEASE(S): Breast Cancer

SUBMITTER: Franziska Voellmy

LAB HEAD: Robert Semple

PROVIDER: PXD014719 | Pride | 2021-03-03

REPOSITORIES: Pride

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Publications

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

Madsen Ralitsa R RR Longden James J Knox Rachel G RG Robin Xavier X Völlmy Franziska F Macleod Kenneth G KG Moniz Larissa S LS Carragher Neil O NO Linding Rune R Vanhaesebroeck Bart B Semple Robert K RK

Disease models & mechanisms 20210311 3

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 this 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 sign ...[more]

PMID: 33514588

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Project description:Rationale: A critical step in heart development is the coordinated formation of nodal myocardium and cushion tissue from the atrioventricular canal (AVC). After its specification, the myocardium of the AVC aligns the chambers, forms the AV node, and induces the formation of the mesenchymal AV cushions, primordia of valves and septa, while it resists working myocardial differentiation. Objective: To assess what roles Tbx2 and Tbx3, two closely related T-box transcription factors expressed in the AVC, play in these processes. Methods and Results: We analyzed mice ectopically expressing Tbx3 in the atrial myocardium by genome-wide microarray and expression analysis. We found a prominent role for Tbx3 in defining the nodal phenotype by repressing working myocardial genes (sarcomeric, mitochondrial, fast conduction) and cell proliferation regulators, and in inducing node-associated genes. Moreover, there was a striking induction of genes associated with endocardial cushions and mesenchyme. Using gain-of-function models, we found that in the developing heart both Tbx2 and Tbx3 induce ectopic Bmp2 and Tgfb2 expression and endocardial cushion formation. Analysis of compound Tbx2/Tbx3 mutant embryos revealed that upon loss of more than two functional alleles, expansion of the AV myocardium does not occur and AV cushions fail to form. Conclusions: Tbx2 and Tbx3 locally stimulate development of the AVC myocardium, induce the AV nodal phenotype therein and trigger AV cushion formation from the overlying AV endocardium, providing a mechanism for the colocalization and coordination of these two important processes in heart development. Nppa-Cre4 (Cre4) mice were crossed with CT mice to obtain efficient activation of Tbx3 in atria of double transgenic Cre4-CT mice, as previously described (Hoogaars et al., 2007). To investigate the gene expression profile of atria of Cre4-CT mice, we performed whole genome microarray analysis using Sentrix Mouse-6 oligonucleotide beadchips. We compared the atrial gene expression profiles of six male double transgenic Cre4-CT mice and six male Cre4 control mice.

Project description:Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes1-3. Pik3ca and p53 are the two most frequently mutated genes and are associated with different types of human breast cancers4. The cellular origin and the mechanisms leading to Pik3ca-induced tumour heterogeneity remain unknown. Here, we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and its impact on tumour heterogeneity. Surprisingly, oncogenic Pik3ca-H1047R expression at physiological levels5 in basal cells (BCs) using K5CREERT2 induced the formation of luminal ER+PR+ tumours, while its expression in luminal cells (LCs) using K8CREERT2 gave rise to luminal ER+PR+ tumours or basal-like ER-PR- tumours. Concomitant deletion of p53 and expression of Pik3ca-H1047R accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca-H1047R in unipotent BCs gave rise to luminal-like cells, while its expression in unipotent LCs gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that have undergone cell fate transition upon Pik3ca-H1047R expression in unipotent progenitors demonstrate a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures, characteristic of the different cell fate switches that occur upon Pik3ca-H1047R expression in BC and LCs, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca-H1047R activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation. Luminal and basal cells, or tumour cells, from mice in which expression of PIK3CA-H1047R and YFP (and in some conditions loss of p53) was targeted in basal cells using K5CREERT2 or in luminal cells using K8CREERT2 were FACS isolated and RNA was extracted before being hybridized Affymetrix microarrays.

Dataset Information

High-dose oncogenic PIK3CA drives constitutive cellular stemness through increased NODAL/TGFb signaling

Publications

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

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