PTEN and the pan phosphoinositide 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002) exert significant control over tumor-induced angiogenesis and tumor growth in vivo. The LY294002 compound is not a viable drug candidate due to poor pharmacologic variables of insolubility and short half-life. Herein, we describe the development and antitumor activity of a novel RGDS-conjugated LY294002 prodrug, termed SF1126, which is designed to exhibit increased solubility and bind to specific integrins within the tumor compartment, resulting in enhanced delivery of the active compound to the tumor vasculature and tumor. SF1126 is water soluble, has favorable pharmacokinetics, and is well tolerated in murine systems. The capacity of SF1126 to inhibit U87MG and PC3 tumor growth was enhanced by the RGDS integrin (AvB3/A5B1) binding component, exhibiting increased activity compared with a false RADS-targeted prodrug, SF1326. Antitumor activity of SF1126 was associated with the pharmacokinetic accumulation of SF1126 in tumor tissue and the pharmacodynamic knockdown of phosphorylated AKT in vivo. Furthermore, SF1126 seems to exhibit both antitumor and antiangiogenic activity. The results support SF1126 as a viable pan PI3K inhibitor for phase I clinical trials in cancer and provide support for a new paradigm, the application of pan PI3K inhibitory prodrugs for the treatment of cancer. [Cancer Res 2008;68(1):206-15]

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is mutated or lost in 60% to 70% of advanced gliomas and is associated with malignant phenotypic changes such as migration, which contribute to the morbidity and mortality of this disease. Most of the tumor suppressor function of PTEN has been attributed to its ability to dephosphorylate the second messenger, phosphatidylinositol 3,4,5-triphosphate, resulting in the biological control of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Despite recent work suggesting that the protein phosphatase activity of PTEN controls glioma cell migration, the mechanisms by which this occurs are unclear. Herein, we show using glioma cell lines (U87MG and U373MG) stably transfected with wildtype PTEN or catalytically altered mutants of PTEN that PTEN controls integrin-directed migration in a lipid phosphatase, PI3K/AKT-independent manner. Confirming this observation, we show that the stable overexpression of COOH-terminal Src kinase, the physiologic negative regulator of SRC family kinases (SFK), or treatment with the SFK inhibitor PP1 abrogates glioma migration. The results provide direct evidence that the downstream effect of the protein phosphatase activity of PTEN is to suppress SFK and FYN, and to regulate RAC-GTPase activity after A v integrin stimulation. Furthermore, studying vitronectin-directed migration using (a) Fyn small interfering RNA and (b) astrocytes from Fyn heterozygous (+/À) mice, Pten heterozygous (+/À) mice, Pten and Fyn double heterozygous (+/À) mice, or Fyn knockout (À/À) mice confirmed a role of FYN in A v integrin-mediated haptotaxis in glial cells. Our combined results provide direct biochemical and genetic evidence that PTEN's protein phosphatase activity controls FYN kinase function in glioma cells and regulates migration in a PI3K/AKT-independent manner.

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by Fc␥RIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.Syk is a 72-kDa protein-tyrosine kinase that plays a central role in coupling immune recognition receptors to multiple downstream signaling pathways (1,2). This function is a property of both its catalytic activity and its ability to participate in interactions with effector proteins containing SH2 1 domains.For example, after the engagement of antigen receptors on B cells, Syk is phosphorylated on three tyrosines that lie within the linker B region, which separates the N-terminal tandem pair of SH2 domains from the catalytic domain (3). Phosphorylation of the first, at Tyr-317 (numbering based on the murine Syk sequence), is catalyzed primarily by Lyn, a Src family kinase. This creates a docking site for c-Cbl, a potential negative regulator of Syk-dependent signaling (4, 5). Indeed, mutant forms of Syk containing substitutions of Phe for Tyr at position 317 exhibit enhanced activity in B cells and mast cells (3,5,6). To date, c-Cbl is the only protein identified that is capable of binding to Syk at pTyr-317. Phosphorylation of Tyr-342 and 346 forms a docking site for multiple SH2 domaincontaining proteins including phospholipase C-␥, Vav, and Fgr (7-10). Mutant forms of Syk containing substitutions of Phe for Tyr-342 or both Tyr-342 and 346 exhibit a reduced ability to couple immune recognition receptors to the activation of downstream effectors such as phospholipase C-␥2 in B cells and mast cells (10, 11).Syk also is required for the activation of phosphoinositide 3-kinase (PI3K) in response to a variety of signals (12-18) including engagement of the B cell antigen receptor (BCR) (12) and macrophage or neutrophil Fc␥ receptors (13,14). Furthermore, the expression of a constitutively active ...

Herein, we report the first evidence that c-SRC is required for retinoic acid (RA) receptor (RAR) signaling, an observation that suggests a new paradigm for this family of nuclear hormone receptors. We observed that CSK negatively regulates RAR functions required for neuritogenic differentiation. CSK overexpression inhib-

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a−/− embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a −/− mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a−/− mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.

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The aim of this study was to investigate whether luteal phase ovarian oestrogen is required for blastocyst implantation and pregnancy maintenance in the rhesus monkey. Preimplantation embryos were retrieved from naturally ovulated, mated embryo donor monkeys. In group I, developmentally normal, age- and stage-matched embryos were transferred to recipient monkeys showing naturally synchronized ovulatory cycles. Immediately prior to embryo transfer, recipients were subjected to bilateral ovariectomy, and following transfer they were treated with i.m. injections of either progesterone (group Ia, n = 4), or oestradiol + progesterone (group Ib, n = 2). Recipient monkeys of group Ic (n = 4) were subjected to sham ovariectomy and vehicle injection. In group Ia, progesterone supplementation alone led to three pregnancies and live births. In group Ib, there was one live birth. In the control group Ic, four transfers resulted in two live births and one abortion on cycle day 58. Analysis of serum progesterone and oestradiol profiles showed that oestradiol had declined to undetectable levels within 3-5 days after ovariectomy in group Ia recipients, and the area under the curve of serum oestrogen concentrations during the peri-implantation period (days 10-20 after ovulation) were less (P < 0.001) in group Ia compared with group Ic. There were no changes in the area under the curve among serum progesterone concentrations in all the subgroups. In group II, long-term ovariectomized embryo recipients (n = 4) were primed with oestradiol till cycle day 11 of simulated transfer cycle, and received progesterone treatment from cycle day 10 till the end of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)