Direct positive regulation of PTEN by the p85 subunit of phosphatidylinositol 3-kinase.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is deregulated in many human diseases including cancer, diabetes, obesity, and autoimmunity. PI3K consists of a p110 catalytic protein and a p85alpha regulatory protein, required for the stabilization and localization of p110-PI3K activity. The p110-PI3K enzyme generates the key signaling lipid phosphatidylinositol 3,4,5-trisphosphate, which is dephosphorylated by the PI3-phosphatase PTEN. Here we show another function for the p85alpha regulatory protein: it binds directly to and enhances PTEN lipid phosphatase activity. We demonstrate that ectopically expressed FLAG-tagged p85 coimmunoprecipitates endogenous PTEN in an epidermal growth factor dependent manner. We also show epidermal growth factor dependent coimmunoprecipitation of endogenous p85 and PTEN proteins in HeLa cells. Thus p85 regulates both p110-PI3K and PTEN-phosphatase enzymes through direct interaction. This finding underscores the need for caution in analyzing PI3K activity because anti-p85 immunoprecipitations may contain both p85:p110-PI3K and p85:PTEN-phosphatase enzymes and thus measure net PI3K activity. We identify the N-terminal SH3-BH region of p85alpha, absent in the smaller p55alpha and p50alpha isoforms, as the region that mediates PTEN binding and regulation. Cellular expression of p85DeltaSH3-BH results in substantially increased magnitude and duration of pAkt levels in response to growth factor stimulation. The ability of p85 to bind and directly regulate both p110-PI3K and PTEN-PI3-phosphatase allows us to explain the paradoxical insulin signaling phenotypes observed in mice with reduced PI3K or PTEN proteins. This discovery will impact ongoing studies using therapeutics targeting the PI3K/PTEN/Akt pathway.

Comparing safety and adequacy between surgical biopsy versus core needle biopsy in diagnosing neuroblastoma.

BACKGROUND: Adequate tissue biopsy is essential for diagnosis and risk stratification of neuroblastoma (NB). Historically, NB diagnosis has relied on tissue obtained via surgical biopsy. However, core needle biopsy may provide a safe and adequate method of obtaining tissue in pediatric patients. AIM: The aim of this study is to compare the adequacy and safety between core needle biopsy and surgical biopsy for the diagnosis of NB in children at our institution. METHODS: Institutional approval was obtained. Medical records of patients diagnosed with NB from 2004 - 2019 were retrospectively reviewed. Patients had either core needle biopsy (CNB) or surgical biopsy (SB) including open/minimally invasive biopsy. Data included patient demographics, tumor location and size, sample adequacy for diagnosis and risk stratification, post-biopsy complications, length of hospital stay, and need for repeat biopsy. Statistical analysis was conducted using the Mann-Whitney U test or Student's t-test. RESULTS: Thirty-eight patients were included; 53 biopsies were performed including 41 SB and 12 CNB. Patient and tumor characteristics were similar in both groups, as well as the biopsy adequacy for diagnosis and risk stratification. In all cases, there was no need for repeat biopsy. The CNB group demonstrated reduced length of stay (2 ± 0.4 days vs 5 ± 0.5 days; P < 0.0001) and fewer complications (8%) than the SB group (44%) (P = 0.038). CONCLUSION: Core needle biopsy is an acceptable modality for diagnosis and risk stratification in the pediatric population. Advantages include decreased length of stay and fewer post-procedure complications.

The p85alpha subunit of phosphatidylinositol 3'-kinase binds to and stimulates the GTPase activity of Rab proteins.

Rab5 and Rab4 are small monomeric GTPases localized on early endosomes and function in vesicle fusion events. These Rab proteins regulate the endocytosis and recycling or degradation of activated receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGFR). The p85alpha subunit of phosphatidylinositol 3'-kinase contains a BH domain with sequence homology to GTPase activating proteins (GAPs), but has not previously been shown to possess GAP activity. In this report, we demonstrate that p85alpha has GAP activity toward Rab5, Rab4, Cdc42, Rac1 and to a lesser extent Rab6, with little GAP activity toward Rab11. Purified recombinant Rab5 and p85alpha can bind directly to each other and not surprisingly, the p85alpha-encoded GAP activity is present in the BH domain. Because p85alpha stays bound to the PDGFR during receptor endocytosis, p85alpha will also be localized to the same early endosomal compartment as Rab5 and Rab4. Taken together, the physical co-localization and the ability of p85alpha to preferentially stimulate the down-regulation of Rab5 and Rab4 GTPases suggests that p85alpha regulates how long Rab5 and Rab4 remain in their GTP-bound active state. Cells expressing BH domain mutants of p85 show a reduced rate of PDGFR degradation as compared with wild type p85 expressing cells. These cells also show sustained activation of the mitogen-activated protein kinase and Akt pathways. Thus, the p85alpha protein may play a role in the down-regulation of activated receptors through its temporal control of the GTPase cycles of Rab5 and Rab4.