ESPE Abstracts

Introduction: PTEN hamartoma tumor syndrome (PHTS) is a rare genetic disorder caused by germline mutations in the tumor suppressor gene Phosphatase and tensin homologue (PTEN), a negative regulator of the phosphoinositide-3 kinase (PI3K)/AKT/ mechanistic target of rapamycin (mTOR) pathway. Children with PHTS frequently develop adipose tissue overgrowth, so called lipomas that can lead to loss of organ function due to displacing lipoma growth. Currently, except for surgical resection, no systemic therapy exists. We aimed to establish an ex vivo, Pten deficient lipoma slice culture model as a platform to study and compare the efficiency of different PI3K inhibitors to counteract proliferation and lipid accumulation.

Methods: Lipoma tissue and control epigonadal white adipose tissue (WAT) from mice with conditional Pten and retinoblastoma (Rb) knockout was cultivated on air-liquid interface membranes. After 72 hours of treatment slices were fixed, H&E stained and analyzed using automatic image analysis or frozen for measurement of gene expression and determination of PI3K pathway activation via Western blotting.

Results: We found excellent tissue preservation and no impairment of cell viability after 72h of slice culture. After treatment with the pan-PI3K inhibitor wortmannin we detected a decrease of adipocyte size in both lipoma and control WAT slices. Interestingly, the PIK3CA-specific inhibitor alpelisib showed opposite effects in lipoma and control tissue, with bigger adipocytes in lipoma, and smaller ones in epigonadal WAT. The proliferation marker Pcna was significantly downregulated both in wortmannin- and alpelisib-treated lipoma tissue (wortmannin: by 0.23fold, P=0.02, n=8, alpelisib: by 0.51fold, P=0.0001, n=8). In contrast to wortmannin, alpelisib incubation lead to a significant downregulation of fibrosis marker Tgfβ (by 0.59fold, P=0.0004, n=6). The adipogenic transcription factor Pparγ was downregulated in lipoma, but not in control adipose tissue after alpelisib incubation, but not after incubation with wortmannin, indicating a differential effect on adipocyte differentiation. Interestingly, we observed a significant upregulation of glucose transporter Glut1 after treatment with alpelisib (5.39fold, P= 0.0043, n=7), and wortmannin (0.72fold, P=0.046, n=7). On the protein level, we detected a decrease of AKT phosphorylation after wortmannin and alpelisib treatment. We did not see consistent differences between lipoma and control slices regarding the effect of inhibitors on AKT pathway activation.

Conclusion: We established an ex vivo model for the investigation of inhibitor efficacy against lipoma development. Incubation with PI3K pathway inhibitors did affect adipocyte size, gene expression and PI3K pathway activation both in lipoma and control tissue.