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Corticorelin acetate (CrA) is a synthetic form of corticotropin-releasing factor undergoing clinical trials in the treatment of peritumoral brain edema (PBE). We sought to investigate preclinically its potential as an antitumor agent against human solid tumors and to assess its ability to enhance the therapeutic activity of bevacizumab (BEV) in these same models. The in vivo efficacy of CrA as a single agent and in combination with the antiangiogenic agent, BEV, was examined in two preclinical human tumor models, the MX-1 breast and Colo-205 colon carcinomas. These models were selected based on their known sensitivity to BEV and were tumor types in which BEV has been approved for clinical use. The corneal micropocket assay was also performed to assess the antiangiogenic activity of CrA relative to BEV. The exposure level of CrA in the mouse using a typical preclinical regimen was measured so as to compare it to reported clinical exposure levels. CrA was active as a single agent in the MX-1 breast carcinoma, but did not exhibit statistically significant activity as a single agent in the Colo-205 colon carcinoma under the doses and schedules used in the study. When BEV, which was active or near active in both the MX-1 and Colo-205 models, was administered concomitantly with CrA, therapeutic outcomes were observed that were significantly better than those obtained using either monotherapy. These therapeutic potentiations using CrA plus BEV were obtained in the absence of any observable increase in toxicities. CrA was active in the corneal micropocket assay, producing a substantial (>70%) inhibition of neovascularization. A representative CrA regimen in mice produced an exposure within eightfold of human exposure determined at one-half the current clinical dose. The application of CrA for the treatment of PBE likely involves its activity as an antiangiogenic agent, which may be one possible mechanism to explain its observed preclinical antitumor activity. That activity, as well as its ability to provide an enhanced therapeutic outcome when given in conjunction with BEV in the absence of increased toxicity, supports the use of CrA clinically as other than a replacement therapy for dexamethasone in PBE.

Phospholipase D (PLD) is a PtdCho-hydrolyzing enzyme that plays central signaling functions in eukaryotic cells. We previously demonstrated that action of a set of four nonclassical and membrane-associated Sec14p-like phosphatidylinositol transfer proteins (PITPs) is required for optimal activation of yeast PLD in vegetative cells. Herein, we focus on mechanisms of Sfh2p and Sfh5p function in this regulatory circuit. We describe several independent lines of in vivo evidence to indicate these SFH PITPs regulate PLD by stimulating PtdIns-4,5-P2 synthesis and that this stimulated PtdIns-4,5-P2 synthesis couples to action of the Stt4p PtdIns 4-kinase. Furthermore, we provide genetic evidence to suggest that specific subunits of the yeast exocyst complex (i.e. a component of the plasma membrane vesicle docking machinery) and the Sec9p plasma membrane t-SNARE are regulated by PtdIns(4,5)P2 and that Sfh5p helps regulate this interface in vivo. The collective in vivo and biochemical data suggest SFH-mediated stimulation of Stt4p activity is indirect, most likely via a substrate delivery mechanism.

All eukaryotic cells examined to date exhibit an array of cytosolic polypeptides that have the ability to catalyze the energy-independent transfer of lipids and/or phospholipids between membrane bilayers in vitro (Wirtz, 1991; Bankaitis et al., 1996). These phospholipid transfer proteins (PLTPs) are classified as a function of their lipid headgroup specificities in the in vitro transfer reaction, and phosphatidylinositol transfer proteins (PITPs) represent a class of oligospecific PLTPs. PITPs effect efficient transfer of PI or the zwitterionic phospholipid phosphatidylcholine (PC), and a distinct preference is exhibited towards PI as transfer substrate. While the physiological functions of PITPs, and PLTPs in general, have presented a long-standing mystery for reasons reviewed elsewhere (Wirtz, 1991; Bankaitis et al., 1996), much progress has been made in the past several years on this problem. The discovery that the Saccharomyces cerevisiae SEC14 gene product (Secl4p) is the yeast PITP has provided the first opportunity to dissect the in vivo function of a PITP (Bankaitis et al., 1990). Immediate insights were forthcoming on this issue as genetic and biochemical analyses had identified Secl4p as a cytosolic factor that is essential for viability of the yeast cell because it is required for the biogenesis of secretory vesicles dedicated to the transport of secretory glycoproteins from a late Golgi compartment (Novick et al., 1980; Bankaitis et al., 1989; Cleves et al., 1991). The outstanding questions that remain include the following: (i) what is the mechanism of Secl4p function in the stimulation of protein export from the yeast Golgi complex?, and (ii) how does Secl4p harness its PI and PC transfer activities to biological function? In this manuscript, we will discuss the recent progress we have made on these issues.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20+is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that thespo20+gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

Phospholipase D (PLD) is a PtdCho-hydrolyzing enzyme that plays central signaling functions in eukaryotic cells. We previously demonstrated that action of a set of four nonclassical and membrane-associated Sec14p-like phosphatidylinositol transfer proteins (PITPs) is required for optimal activation of yeast PLD in vegetative cells. Herein, we focus on mechanisms of Sfh2p and Sfh5p function in this regulatory circuit. We describe several independent lines of in vivo evidence to indicate these SFH PITPs regulate PLD by stimulating PtdIns-4,5-P2 synthesis and that this stimulated PtdIns-4,5-P2 synthesis couples to action of the Stt4p PtdIns 4-kinase. Furthermore, we provide genetic evidence to suggest that specific subunits of the yeast exocyst complex (i.e. a component of the plasma membrane vesicle docking machinery) and the Sec9p plasma membrane t-SNARE are regulated by PtdIns(4,5)P2 and that Sfh5p helps regulate this interface in vivo. The collective in vivo and biochemical data suggest SFH-mediated stimulation of Stt4p activity is indirect, most likely via a substrate delivery mechanism.

Phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs) are ubiquitous and highly conserved proteins that are believed to regulate lipid-mediated signaling events. Their ubiquity and conservation notwithstanding, PITPs remain remarkably uninvestigated. Little is known about the coupling of specific PITPs to explicit cellular functions or the mechanisms by which PITPs interface with apppropriate cellular functions. The available information indicates a role for these proteins in regulating the interface between lipid metabolism and membrane trafficking in yeast, signaling in plant development, the trafficking of specialized luminal cargo in mammalian enterocytes, and neurological function in mammals. Herein, we review recent advances in PITP biology and discuss as yet unresolved issues in this field.Key words: phosphatidylinositol transfer protein, secretion, lipid signaling, phosphoinositide.

Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes. Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner. Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L. japonicus late nodulin Nlj16. Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules. The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene. This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene. We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24–65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet,SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate thatSFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with “bypass-Sec14p” mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

A new yeast strain, designated pstB2, that is defective in the conversion of nascent phosphatidylserine (PtdSer) to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase 2, has been isolated. The pstB2 strain requires ethanolamine for growth. Incubation of cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrates a 50% increase in the labeling of PtdSer and a 72% decrease in PtdEtn formation in the mutant relative to the parental strain. The PSTB2 gene was isolated by complementation, and it restores ethanolamine prototrophy and corrects the defective lipid metabolism of the pstB2 strain. The PSTB2 gene is allelic to the pleiotropic drug resistance gene, PDR17, and is homologous to SEC14, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein. The protein, PstB2p, displays phosphatidylinositol but not PtdSer transfer activity, and its overexpression causes suppression of sec14 mutants. However, overexpression of the SEC14 gene fails to suppress the conditional lethality of pstB2 strains. The transport-dependent metabolism of PtdSer to PtdEtn occurs in permeabilized wild type yeast but is dramatically reduced in permeabilized pstB2 strains. Fractionation of permeabilized cells demonstrates that the pstB2 strain accumulates nascent PtdSer in the Golgi apparatus and a novel light membrane fraction, consistent with a defect in lipid transport processes that control substrate access to PtdSer decarboxylase 2.

Corticorelin acetate (CrA) is a synthetic form of corticotropin-releasing factor undergoing clinical trials in the treatment of peritumoral brain edema (PBE). A corneal micropocket angiogenesis assay (CMA) was performed to assess the activity of CrA relative to bevacizumab (BEV). We also investigated the in vivo anti-tumor efficacy of CrA as a single agent and in combination with BEV, in two preclinical human tumor models, the MX-1 breast and Colo-205 colon carcinomas. These models were selected based on their sensitivity to BEV. In the CMA, six groups of female mice were implanted with pellets containing VEGF or no VEGF, and treated for 7 days. The vehicle control group received phosphate buffered saline, sc, bid. The positive reference group received BEV, ip, qd, at 5 mg/kg/inj. The two remaining groups received CrA sc, bid, at 0.1 mg/kg/inj or 0.2 mg/kg/inj. Treatment outcome was determined from semi-quantitative characterization of neovascularization. CrA produced a substantial (>70%) inhibition of neovascularization (p In the xenograft studies, mice were implanted sc in the flank with approximately 1 mm3 tumor fragments obtained from donor passage mice (MX-1), or with 1 × 106 tissue culture-derived Colo-205 cells implanted in 50% matrigel. When tumors were generally between 80-240 mg (mm3), mice were distributed to control and treatment sets. CrA was administered at two dose levels, 0.1 and 0.2 mg/kg/injection, sc, twice daily (bid). BEV was administered at a dose of 5 mg/kg/inj, ip, twice weekly. All treatments were continued until each group achieved its tumor target size, 1 gm in the Colo-205 experiment, and 1.5 gm in the MX-1 experiment. A log cell kill (LCK) of ≥1.0, equivalent to a delay in tumor growth of ≥ 3.32 x tumor volume doubling time, accompanied by statistical significance (p CrA was active as a single agent in the MX-1 breast carcinoma (1.0 LCK; p Conclusion: CrA has demonstrated anti-angiogenic activity in vivo, which may be one possible mechanism to explain its observed preclinical antitumor activity. Importantly it enhances the antitumour activity of BEV in both MX-1 and Colo-205 models. Given the good safety profile of CrA and its lack of hypertensive side effects in clinical trials, CrA should be evaluated clinically in combination with BEV. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1554.

In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.