The novel mTOR inhibitor RAD001 (Everolimus) induces antiproliferative effects in human pancreatic neuroendocrine tumor cells

Background/Aim: Tumors exhibiting constitutively activated PI(3) K/Akt/mTOR signaling are hypersensitive to mTOR inhibitors such as RAD001 (everolimus) which is presently being investigated in clinical phase II trials in various tumor entities, including neuroendocrine tumors (NETs). However, no preclinical data about the effects of RAD001 on NET cells have been published. In this study, we aimed to evaluate the effects of RAD001 on BON cells, a human pancreatic NET cell line that exhibits constitutively activated PI(3) K/Akt/mTOR signaling. Methods: BON cells were treated with different concentrations of RAD001 to analyze its effect on cell growth using proliferation assays. Apoptosis was examined by Western blot analysis of caspase-3/PARP cleavage and by FACS analysis of DNA fragmentation. Results: RAD001 potently inhibited BON cell growth in a dose-dependent manner which was dependent on the serum concentration in the medium. RAD001-induced growth inhibition involved G0/G1-phase arrest as well as induction of apoptosis. Conclusion: In summary, our data demonstrate antiproliferative and apoptotic effects of RAD001 in NET cells in vitro supporting its clinical use in current phase II trials in NET patients. Copyright (c) 2007 S. Karger AG, Basel

Characterization of TOR complex 2 (TORC2) in "Saccharomyces cerevisiae"

The TOR proteins play a central role in the control of cell growth. TOR proteins are the founding members of the phosphatidylinositol-related kinase (PIKK) family of protein kinases. In S. cerevisiae there are two TOR proteins, TOR1 and TOR2. TOR1 and TOR2 regulate cell growth via a rapamycin-sensitive pathway controlling translation, transcription, nutrient uptake, ribosome biogenesis and autophagy. TOR2 also has a unique, rapamycin-insensitive function, which is the control of the actin cytoskeleton. Recently, it has been found that the TOR proteins exist in two distinct complexes, TOR complex(TORC1) and TOR complex(TORC2). While TORC1 mediates the rapamycin-sensitive pathway, TORC2 is responsible for the control of the actin cytoskeleton. TORC1 comprises three proteins, TOR1 or TOR2, KOG1 and LST8. TORC2 consists of five proteins, TOR2, AVO1, AVO2, AVO3 and LST8. Thus, these structurally and functionally distinct TOR complexes account for the diversity of TOR signaling in yeast. Here we focused on the characterization of TORC2. Our studies suggest that TORC2 exists in an oligomeric state and that AVO1 and likely AVO3 act as scaffold proteins required for the integrity of TORC2. We also found that AVO1 plays a role as an adaptor protein mediating efficient phosphorylation of substrates. LST8 in turn appears to modulate TOR2 kinase activity possibly by binding directly to the TOR2 kinase domain. LST8 is common to both TORC1 and TORC2 and may therefore be important to respond to upstream signaling factors

Quantitative MRI establishes the efficacy of PI3K inhibitor (GDC-0941) multi-treatments in PTEN-deficient mice lymphoma

Aim: To assess the efficacy of multiple treatment of phosphatidylinositol- 3-kinase (PI3K) inhibitor on autochthonous tumours in phosphatase and tensin homologue (Pten)-deficient genetically engineered mouse cancer models using a longitudinal magnetic resonance imaging (MRI) protocol. Materials and Methods: Using 3D MRI, B-cell follicular lymphoma growth was quantified in a Pten +/-Lkb1 +/hypo mouse line, before, during and after repeated treatments with a PI3K inhibitor GDC-0941 (75 mg/kg). Results: Mean pre-treatment linear tumour growth rate was 165±12.8 mm 3/week. Repeated 28-day GDC-0941 administration, with 21 days 'off-treatment', induced average tumour regression of 41±7%. Upon cessation of the second treatment (which was not permanently cytocidal), tumours re-grew with an average linear growth rate of 40.1±15.5 mm 3/week. There was no evidence of chemoresistance. Conclusion: This protocol can accommodate complex dosing schedules, as well as combine different cancer therapies. It reduces biological variability problems and resulted in a 10-fold reduction in mouse numbers compared with terminal assessment methods. It is ideal for preclinical efficacy studies and for phenotyping molecularly characterized mouse models when investigating gene function.</p

How moderate changes in Akt T-loop phosphorylation impact on tumorigenesis and insulin resistance

The Akt signalling pathway plays vital roles in controlling cellular responses to insulin as well as in proliferation and survival. Inhibition of Akt signalling leads to insulin resistance and type 2 diabetes, whereas hyperactivation of Akt promotes tumorigenesis. In this study, we investigate how modest changes in the activity of the Akt signalling pathway, to an extent that might be achieved by drug treatment, would impact on insulin resistance and tumorigenesis. Using insulin-resistant PDK1(K465E/K465E) PH domain knock-in mice, we found that introducing the PTEN(+/−) mutation to slightly stimulate Akt restored normal insulin sensitivity. Introducing the PDK1(K465E/K465E) PH domain knock-in mutation into cancer-prone PTEN(+/−) mice, lowered Akt activity only by about 50%, but led to a delay in tumour onset of ∼4 months in a broad range of tumours. This was also accompanied by slower growth of B cell follicular lymphomas, as monitored by magnetic resonance imaging. Our findings imply that signal transduction inhibitors that lead to a modest reduction in Akt activity would not only delay onset of tumours possessing elevated phosphoinositide 3-kinase pathway activity but would also reduce the growth rate of developed tumours

Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation

Members of the PAR-1/MARK kinase family play critical roles in polarity and cell cycle control and are regulated by 14-3-3 scaffolding proteins, as well as the LKB1 tumour suppressor kinase and atypical protein kinase C (PKC). In this study, we initially investigated the mechanism underlying the interaction of mammalian MARK3 with 14-3-3. We demonstrate that 14-3-3 binding to MARK3 is dependent on phosphorylation, and necessitates the phosphate-binding pocket of 14-3-3. We found that interaction with 14-3-3 was not mediated by the previously characterised MARK3 phosphorylation sites, which led us to identify 15 novel sites of phosphorylation. Single point mutation of these sites, as well as the previously identified LKB1- (T211) and the atypical PKC sites (T564/S619), did not disrupt 14-3-3 binding. However, a mutant in which all 17 phosphorylation sites had been converted to alanine residues ( termed 17A-MARK3), was no longer able to bind 14-3-3. Wild-type MARK3 was present in both the cytoplasm and plasma membrane, whereas the 17A-MARK3 mutant was strikingly localised at the plasma membrane. We provide data indicating that the membrane localisation of MARK3 required a highly conserved C-terminal domain, which has been termed kinase-associated domain-1 (KA-1). We also show that dissociation of 14-3-3 from MARK3 did not affect catalytic activity, and that a MARK3 mutant, which could not interact with 14-3-3, was normally active. Finally, we establish that there are significant differences in the subcellular localisation of MARK isoforms, as well as in the impact that atypical PKC overexpression has on 14-3-3 binding and localisation. Collectively, these results indicate that 14-3-3 binding to MARK isoforms is mediated by multiple phosphorylation sites, and serves to anchor MARK isoforms in the cytoplasm

Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8(+) T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8(+) T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8(+) T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP- E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. (C) 2018 AACR

Identification of protor as a novel rictor-binding component of mTOR complex-2

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR–Raptor (regulatory associated protein of mTOR)–mLST8 (termed mTORC1), and complex 2, comprising mTOR–Rictor (rapamycininsensitive companion of mTOR)–mLST8–Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser&lt;sup&gt;473&lt;/sup&gt;). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role