ESPE Abstracts

Background and Aim: Pediatric patients with germline mutations in the tumor suppressor gene PTEN (PTEN hamartoma tumor syndrome, PHTS) frequently develop lipomas. PTEN antagonizes phosphoinositide 3-kinase (PI3K) signaling, which can induce adipogenesis upon activation through insulin. The PI3K downstream target AKT can deactivate FOXO1 via phosphorylation, initiating the expression of the lipogenesis activating transcription factor SREBP1. To study the underlying mechanisms of lipoma formation in patients with PHTS, we asked whether there is a link between reduced PTEN in adipose progenitor cells (APCs) and aberrant growth of adipose tissue.

Methods: We downregulated PTEN via siRNA (KD) or CRISPR (CR) in APCs and compared proliferation, in vitro differentiation and gene expression (RNA sequencing, RT-qPCR, Western blots) in control and knockdown cells. To investigate whether the effects observed in cells with PTEN downregulation could be reversed, we overexpressed constitutively active FOXO1 in PTEN CR cells.

Results: PTEN downregulation led to an increase in proliferation (1.4±0.2 fold in KD, 1.5±0.1 fold in CR P < 0.05) and differentiation (1.77±0.07 fold in KD, 5.6±1.9 fold in CR, P < 0.01) of APCs. Protein levels of the senescence associated protein p21 were reduced (0.33±0.07 fold, P < 0,001) in PTEN KD APCs explaining the accelerated growth. RNA sequencing and qPCRs revealed a downregulation of FOXO1 (0.75±0.03 fold, P = 0,018) on mRNA level while inactivation through phosphorylation increased (6.6±3.5 fold, P = 0.046). SREBP1 protein levels (1.5±0.1 fold, P = 0.047) were higher after PTEN KD and may account for the enhanced adipogenesis. To validate this we overexpressed constitutively active FOXO1 in PTEN CR cells and observed that adipogenesis was attenuated (0.73±0.07 fold, P = 0,065) while SREBP1 protein levels were reduced (0.49±0.14 fold, P = 0,024).

Conclusion: We found enhanced proliferation through cell cycle activation in APCs with PTEN downregulation. Additionally, adipogenesis increased mediated through inhibition of FOXO1 signaling. These effects may account for the adipose tissue overgrowth observed in patients with PTEN mutations.