The tumor cell in Kaposi's sarcoma (KS), the spindle-shaped cell, responds to a variety of growth factors, some of which are produced in an autocrine fashion such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and lL-6, while others are produced by HlV-infected mononuclear cells such as oncostatin M (OSM) and HlV-Tat protein. Our long-term goal is to characterize the signaling pathways by which the above mentioned growth factors sustain KS growth with a view to defining molecular targets which can lead to rational therapy. The principal hypothesis is that bFGF, PDGF, lL-6 and OSM utilize the ras/MAP kinase cascade in an interactive and complementary fashion. Moreover, we propose that KS development in the skin and mucosal membrane results from Synergistic interaction of a signaling pathway associated with Tat-binding integrins (beta1 and beta2 sub-classes) with the ras/MAP kinase cascade.
In Aim 1, we will delineate the intermediary components between receptor tyrosine protein kinases (PTK's) or receptor-associated PTK's (including src, JAK1, JAK2, and focal adhesion kinase or FAK) and Ras protein, with specific emphasis on the role of SH2 domain proteins (Grb2, Shc) and guanine nucleotide exchange factors such as hSOS. This will be accomplished by stimulating KS-derived cell lines from AIDS patients with bFGF, PDGF, IL-6, OSM and Tat protein under optimal proliferative conditions and conducting immune complex kinase assays (ICK's), immune precipitation, Western blotting, affinity chromatography with fusion proteins, antiphosphotyrosine immunoblotting and GTP uptake studies. Control experiments will be conducted with small and large vessel endothelial cells (EC's). We will also investigate the role of serine/threonine kinases which act downstream of Ras, including Raf-1, MEK-1 and MEKK. Relating to Tat protein, we will determine whether integrins using the intermediary PTK, FAK, intersect with the above pathways.
Under Aim 2, we will attempt to disrupt KS cell growth in vitro by targeting select steps in the aforementioned signaling cascade. We will utilize different kinds of PTK inhibitors to interfere with cellular activation by RTK's and receptor-associated PTK's. We will disrupt cellular p2lRas function by transfecting a dominant negative ras allele or use farnesyl transferase inhibitors to gain understanding of the overall importance of the ras/MAP kinase cascade in KS growth. We will determine the importance of autologous bFGF production by transfecting KS cells with a kinase-defective bFGF receptor. We will study the effect of a biological response modifier already in use for KS treatment, lFNalpha, to determine how the use of kinases of the JAK family and a novel class of transcription factors affect KS cell growth in parallel with MAP kinase. We will use RGD-containing peptides which compete with binding of Tat or extracellular matrix proteins to integrin receptors to determine their effect on growth pathways.
| Barillari, G; Sgadari, C; Palladino, C et al. (1999) Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin. J Immunol 163:1929-35 |
| Faris, M; Ensoli, B; Kokot, N et al. (1998) Inflammatory cytokines induce the expression of basic fibroblast growth factor (bFGF) isoforms required for the growth of Kaposi's sarcoma and endothelial cells through the activation of AP-1 response elements in the bFGF promoter. AIDS 12:19-27 |
| Faris, M; Ensoli, B; Stahl, N et al. (1996) Differential activation of the extracellular signal-regulated kinase, Jun kinase and Janus kinase-Stat pathways by oncostatin M and basic fibroblast growth factor in AIDS-derived Kaposi's sarcoma cells. AIDS 10:369-78 |
