Funding Proposal:

Study Signal Transduction Within Cells

February 5, 2002

Dr. Gerald A. Soff, M.D.; Northwestern University ( g-soff@northwestern.edu )

Holly A. Hanford; Northwestern University ( h-hanford@northwestern.edu )

Specific Aims

           Angiogenesis, the process by which existing blood vessels sprout to create new vessels, is a necessary physiological process for embryonic development as well as reproductive cycles in adult females (1).  Angiogenesis is also recognized as a characteristic for pathological conditions such as wound healing and tumor growth (2).  Growth of solid tumors depends upon induction of angiogenesis in order to provide adequate oxygen and nutrients to proliferating cells and thus avoid necrosis.  In addition, vasculature provides a physical route for metastasis, (spread to distant sites) (3).  More recently, accumulating evidence has linked angiogenesis to the pathology of leukemia.  Investigators have observed increased angiogenesis and angiogenic factors in the bone marrows of patients with acute and chronic leukemia.  And in a series of animal studies of leukemia, angiogenesis inhibitors have proved an effective therapy, free of toxicity (4, 5).  Various angiogenic inhibitors are currently in clinical trial for the treatment of leukemia (4-9).

A balance of angiogenic stimulators, such as basic fibroblastic growth factor (bFGF) and vascular endothelial growth factor (VEGF), and inhibitors, such as angiostatin, prevent unnecessary blood vessel development in healthy organisms.  During tumorigenesis, tumor cells induce angiogenesis by up-regulation of stimulators as well as down-regulation of inhibitors (10).  Angiostatin is a potent human inhibitor of angiogenesis.  Therefore, angiostatin represents a potential pharmacological tool for inhibiting solid tumor growth and metastasis as well hematological malignancies. 

 The mechanism of angiogenic inhibition by angiostatin is not fully characterized.  The process of angiogenesis involves the endothelial cells, which line blood vessels (4, 5).  In the process of new blood vessel formation, the endothelial cells migrate and proliferate (11,12,13). Our laboratory has shown that these endothelial cell processes are targeted by, and suppressed by angiostatin (3, 12).  A proposed explanation for the antiangiogenic effect of angiostatin is the induction of endothelial cell-specific apoptosis.  (Apoptosis is the process of programmed cell death).  Several studies have clearly demonstrated that angiostatin induces apoptosis in endothelial cells, and our laboratory has found angiostatin-induced apoptosis to be a caspase-dependent phenomenon which is triggered within 1 hour of exposure to physiological concentrations of angiostatin (3,11,14).  Furthermore, the apoptotic effect of angiostatin was specific for endothelial cells, and no effect was observed on a series of other cell types.   These findings verify the potential for angiostatin as an effective anti-cancer agent.  This proposal aims to further elucidate antiangiogenic mechanisms by testing the hypothesis that angiostatin induces endothelial cell-specific apoptosis through a signal transduction pathway initiated at the membrane and propagated through intracellular second messengers. 

Understanding the mechanism by which angiostatin acts to inhibit angiogenesis and cancer growth offers several important benefits.  Firstly, it may allow for more precise use of the angiostatin, by helping to tailor the dose and schedules of the treatment.  Most importantly, however, understanding of how angiostatin works may facilitate generation of small molecule drugs which can mimic the effect of angiostatin and allow for cancer treatment with an orally available, inexpensive drug.

AIM1.   Investigate potential cell signaling molecules involved in mediating angiostatin-induced apoptosis.

Several studies within the angiogenesis field allude to candidate effector molecules for angiostatin signaling.  Shichiri and Hirata identified the expression of the immediate early gene c-fos; the cell growth-related genes MAPK2, ET_B, and preproendothelin1; and the apoptosis-related genes p53, bcl-2, bad, and bax as regulated by angiostatin (15).  Additionally, activation of focal adhesion kinase (FAK) in response to angiostatin treatment has been suggested (11).  Finally, Maeshima, et.al. report that the angiogenic inhibitor tumstatin acts as an inhibitor of protein synthesis through an integrin and FAK-mediated pathway to induce endothelial cell apoptosis (16).  All of these genes represent reasonable starting points in the investigation of downstream effector molecules.

 AIM 2.  Identify and characterize the cell surface molecules responsible for initiating angiostatin signal transduction.

 Studies in our laboratory using fluorogenic peptide substrates for various caspases have revealed a role for caspase 8, the primary effector of the cell surface death-receptor pathway in angiostatin-induced apoptosis (14).  Such data suggest that angiostatin may directly or indirectly initiate a caspase cascade stemming from the cell surface, resulting in apoptosis.  Furthermore, in preliminary studies in our laboratory, Hanford and Soff have demonstrated that angiostatin binds specifically to the surface of endothelial cells.  Thus far, the only protein reported to bind AS on the surface of endothelial cells is ATP synthase, but this interaction is hypothesized to render endothelial cells more susceptible to hypoxic stress independently of apoptosis (17, 18).  The second aim is to determine the identity of angiostatin-binding proteins on the cell surface responsible for the induction of apoptosis.

 Experimental Design and Methods

 Specific Aim 1.  Investigate potential cell signaling molecules involved in mediating angiostatin-induced apoptosis.

 Rationale:  Recent studies implicate various cytoplasmic cell signaling molecules in angiostatin-initiated signal transduction.  Using reverse transcriptase:polymerase chain reaction (RT-PCR), Shichiri and Hirata document down-regulation of the survival factors, c-fos and bcl-2 in response to angiostatin treatment.  The authors also note down-regulation of MAPK-2, a growth related gene.  Alternatively, they report up-regulation of the pro-apoptotic genes bad, bax, and p53 (15).  These data clearly support investigation of the c-Jun and MAPK pathways following angiostatin treatment.

 While activation of focal adhesion kinase (FAK) in response to angiostatin treatment has been observed (11), it is not clear if FAK may have a role in angiostatin-mediated inhibition of angiogenesis.  FAK has been reported to promote the processes of cell migration and survival (19), which would be expected to promote angiogenesis.  Therefore, exploration of FAK activation in response to angiostatin treatment, is necessary to clarify the potential involvement of FAK in angiostatin signal transduction.

 Finally, Maeshima et.al. reported that the angiogenic inhibitor tumstatin acts as an inhibitor of protein synthesis through an integrin and FAK-mediated pathway to induce endothelial cell apoptosis (16).  (Integrins are cell-matrix adhesion molecules, capable of relaying both outside-in and inside-out signals.)  Tumstatin was found to inhibit protein synthesis through inactivation of FAK in an a_Vb₃-dependent manner.  Furthermore, several integrins have been implicated in binding and mediating signal for various angiogenic inhibitors (20-22).  All of the genes noted above represent reasonable starting points in the investigation of downstream effector molecules. 

 Experimental Approach:  In order to experimentally address the second messengers required for angiostatin-mediated cell signaling, gene expression will be examined through Northern blotting.  Radiolabeled cDNA probes corresponding to various apoptosis related factors, including c-fos, bad, bax, bcl-2, p53, and others implicated by the Shichiri and Hirata study will be hybridized to mRNA isolated from bovine aortic endothelial cells (BAEC), a well-characterized endothelial cell line known to respond to angiostatin.  Verification of differential gene expression in response to angiostatin treatment will pinpoint cytoplasmic molecules involved in angiostatin-mediated signaling.  Alternatively, microarrays can be used to achieve the same ends, by identifying a series of genes whose expression is regulated directly or indirectly by angiostatin.  Follow-up studies will dissect the pathways identified through differential gene expression.

 Additionally, antibodies specific for activated, tyrosine-phosphorylated FAK will be used to directly monitor activation or inhibition of FAK in response to angiostatin treatment.  Alternatively, FAK activity can be investigated using kinase assays with ³²P-ATP.  If regulation of FAK activity by angiostatin is observed, subsequently FAK regulation by angiostatin will be assessed, in the presence of extracellular matrix proteins known to affect FAK.   Investigation of the downstream effectors of FAK, including phosphoinositol-3-kinase (PI3K) and protein kinase B/Akt (PKB/Akt) will be facilitated through functional and immunoassays.

 Finally, based upon the evidence that tumstatin affects protein synthesis in an integrin-dependent fashion, preliminary experiments will be done to determine protein synthesis and integrin-dependence in angiostatin-initiated apoptosis.   The global effect of angiostatin treatment on protein synthesis can be examined through ³⁵S-Methionine incorporation.   Integrin involvement in angiostatin signal transduction will be addressed through the use of integrin blocking antibodies during angiostatin functional assays.  Integrin blocking antibodies and peptides are commercially available for a variety of integrin subtypes.  Alternately, b3-subunit deficient cells obtained from knockout mice may address the same question.

Specific Aim 2:  Identify and characterize the cell surface molecules responsible for initiating angiostatin signal transduction.

Rationale:  Preliminary studies in our laboratory using a cell-based assay with biotin-labeled angiostatin demonstrated that angiostatin binds specifically to the surface of endothelial cells, but not other cell types.  Further studies are necessary to quantify the amount of angiostatin binding per cell.  The only protein on the surface of endothelial cells reported to bind angiostatin in the literature is the a/b-subunits of ATP synthase (17, 18).  Those authors demonstrated that angiostatin binds to ATP synthase and inactivates it.   They hypothesized that inactivation of the ATP synthase activity renders cells more susceptible to the hypoxic conditions encountered by migrating endothelial cells (17, 18).  This proposal sets forth to determine the identity of other angiostatin-binding proteins on the cell surface responsible for the induction of apoptosis.

 Experimental Approach:  Our laboratory has employed biotin-labeled angiostatin to verify binding to the surface of endothelial cells.   This technique does not facilitate quantification of binding sites per cell.   Flow cytometry has been used to demonstrate binding and quantify various surface receptors (22,17,23).  BAEC-D cells pre-incubated with biotinilated angiostatin will be exposed to streptavidin-conjugated FITC, and fluorescent signal will be detected and quantified by flow cytometry.  Radioligand binding using I¹²⁵-angiostatin serves as an alternative procedure (17,24).

 Biotinylation of angiostatin can also serve as a probe for protein-protein binding.  Cell lysates of BAEC-D will be separated via 1-dimensional SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane by standard Western blotting protocol.   Ligand blotting will be performed as described (24).  Protein bands displaying angiostatin binding will be excised and in-gel digested with trypsin to create peptide fragments.  Matrix assisted laser desorption ionization and time of flight detection (MALDI-TOF) will be used to identify proteins corresponding to the peptide composition (17, 25, 26).  If necessary, protein separation can be optimized through the use of 2-dimensional electrophoresis.  An alternative method for detecting protein-protein interactions is GST pulldown (27).  This technique will depend upon generation of a glutathione s-transferase fusion to AS or AS fragments using the pGEX fusion vector (Pharmacia).  Identification of angiostatin binding partners will be verified and exploration of signaling pathways will follow.

 Summary;

 These studies will serve to advance the understanding of the antiangiogenic/Antitumoral activity of angiostatin.  The results obtained may accelerate the ability to translate the basic science of angiogenesis inhibition to the “bedside” and provide the foundation for more effective and well tolerated cancer therapy. 

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