A new human adipocyte model with PTEN haploinsufficiency.

Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology.

A tiered high-throughput screening approach for evaluation of estrogen and androgen receptor modulation by environmentally relevant bisphenol A substitutes.

Bisphenol A (BPA) is a high production volume chemical with a broad application spectrum. As an endocrine disrupting chemical, mainly by modulation of nuclear receptors (NRs), BPA has an adverse impact on organisms and is identified as a substance of very high concern under the European REACH regulation. Various BPA substitution candidates have been developed in recent years, however, information concerning the endocrine disrupting potential of these substances is still incomplete or missing. In this study, we intended to investigate the endocrine potential of BPA substitution candidates used in environmentally relevant applications such as thermal paper or epoxy resins. Based on an extensive literature and patent search, 33 environmentally relevant BPA substitution candidates were identified. In order to evaluate the endocrine potential of the BPA replacements, a screening cascade consisting of biochemical and cell-based assays was employed to investigate substance binding to the NRs estrogen receptor α and ß, as well as androgen receptor, co-activator recruitment and NR-mediated reporter gene activation. In addition, a computational docking approach for retrospective prediction of receptor binding was carried out. Our results show that some BPA substitution candidates, for which so far no or only very few data were available, possess a substantial endocrine disrupting potential (TDP, BPZ), while several substances (BPS, D-8, DD70, DMP-OH, TBSA, D4, CBDO, ISO, VITC, DPA, and DOPO) did not reveal any NR binding.

Simvastatin induces apoptosis in PTEN­haploinsufficient lipoma cells.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E­binding protein (4E­BP)­1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator­activated receptor (PPAR)γ expression. The small interfering RNA­mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Resveratrol Potentiates Growth Inhibitory Effects of Rapamycin in PTEN-deficient Lipoma Cells by Suppressing p70S6 Kinase Activity.

Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations in PTEN frequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygous PTEN deficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizes PTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation in PTEN-deficient lipoma cells. Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis. PTEN expression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced in PTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primary PTEN wild-type preadipocytes were less affected compared to PTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes.

Phosphatidylinositol 3-kinase (PI3K) signalling regulates insulin-like-growth factor binding protein-2 (IGFBP-2) production in human adipocytes.

OBJECTIVE: Insulin-like-growth factor binding protein 2 (IGFBP-2) is thought to be a marker for the phosphatase and tensin homolog (PTEN) status and activity of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. We aimed to evaluate whether or not lipoma cells of a patient with a heterozygous deletion in the PTEN gene would produce more IGFBP-2 than PTEN non deficient control cells. Moreover, we analysed the influence of pharmacological inhibitors of the PI3K/AKT/mTOR pathway on IGFBP-2 production. DESIGN: PTEN deficient preadipocytes and control PTEN non deficient preadipocytes were differentiated in vitro and treated with the respective inhibitors. PTEN was transiently down regulated by siRNA in human preadipocytes. IGFBP-2 mRNA and protein expression and IGFBP-2 in culture supernatant were measured. RESULTS: PTEN deficient lipoma cells were found to produce IGFBP-2 during in vitro differentiation in comparable amounts to PTEN non deficient cells. In contrast, acute down regulation of PTEN in preadipocytes resulted in enhanced production of IGFBP-2. Incubation with the PI3K inhibitors LY294002 and wortmannin decreased IGFBP-2 mRNA and protein. Neither the mTOR complex 1 inhibitor rapamycin nor PD98059, an inhibitor of MEK (mitogen-activated protein kinase kinase), showed a significant effect on IGFBP-2 production. CONCLUSION: IGFBP-2 production in PTEN deficient preadipocytes was not influenced by PTEN deficiency or by inhibition of mTORC1 and MAPK. In contrast, inhibition of PI3K decreased IGFBP-2 expression and secretion.

Sirolimus treatment of severe PTEN hamartoma tumor syndrome: case report and in vitro studies.

BACKGROUND: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer predisposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient. METHODS: The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide-3-kinase (PI3K)/AKT/mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro. RESULTS: Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly. CONCLUSION: Sirolimus treatment led to an improvement of the patient's clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.