Parkash S. Gill - US grants

The goal of this proposal is to elucidate the pathogenesis of Kaposi sarcoma (KS) in the presence or absence of IEV infection, based on the hypothesis that glucocorticoids and certain cytokines/monokines (IL-l, TNF, IL-6, novel growth factors) act directly and indirectly to induce and enhance the growth of KS. A case-control study will be employed for HIV-related KS among homosexual men with a relatively intact immune system (CD4),200/dl, no AMS-defining opportunistic infections). Cases will be accrued from the KS clinics at the LAC/USC Medical Center (N=160). Three age-matched controls will be identified per case. Group I controls will be EIV-positive clinically asymptomatic homosexual friend controls; Group 2 controls will be HIV-negative homosexual friend controls; Group 3 controls will be HIV-negative heterosexual neighborhood controls. Classic KS cases will be identified from the Los Angeles County population based cancer registry (CSP) (n=272). Controls for these cases will be age-matched neighborhood controls identified using a predetermined algorithm. An epidemiologic questionnaire, blood and urine specimens will be obtained on all participants. In addition KS tissue will be obtained on all A.MS-KS cases and on a sample of clinic KS cases. We will determine (1) if plasma ACTH, serum cortisol and urinary free cortisol are elevated in KS cases compared to controls; (2) if serum and short term blood monocyte/macrophage culture supernatant cytokine/monokine levels (TNF, EL-1, EL-6) and urinary IL-6 levels are present at higher levels in cases compared to controls; (3) if certain epidemiologic characteristics are KS risk factors, such as use of steroids, history of certain sexually transmitted diseases, indices of altered immune function, etc. A secondary objective of the proposal is to establish a serum and tissue bank for use in future studies.

The goal of this proposal is to elucidate the pathogenesis of Kaposi sarcoma (KS) in the presence or absence of IEV infection, based on the hypothesis that glucocorticoids and certain cytokines/monokines (IL-l, TNF, IL-6, novel growth factors) act directly and indirectly to induce and enhance the growth of KS. A case-control study will be employed for HIV-related KS among homosexual men with a relatively intact immune system (CD4),200/dl, no AMS-defining opportunistic infections). Cases will be accrued from the KS clinics at the LAC/USC Medical Center (N=160). Three age-matched controls will be identified per case. Group I controls will be EIV-positive clinically asymptomatic homosexual friend controls; Group 2 controls will be HIV-negative homosexual friend controls; Group 3 controls will be HIV-negative heterosexual neighborhood controls. Classic KS cases will be identified from the Los Angeles County population based cancer registry (CSP) (n=272). Controls for these cases will be age-matched neighborhood controls identified using a predetermined algorithm. An epidemiologic questionnaire, blood and urine specimens will be obtained on all participants. In addition KS tissue will be obtained on all A.MS-KS cases and on a sample of clinic KS cases. We will determine (1) if plasma ACTH, serum cortisol and urinary free cortisol are elevated in KS cases compared to controls; (2) if serum and short term blood monocyte/macrophage culture supernatant cytokine/monokine levels (TNF, EL-1, EL-6) and urinary IL-6 levels are present at higher levels in cases compared to controls; (3) if certain epidemiologic characteristics are KS risk factors, such as use of steroids, history of certain sexually transmitted diseases, indices of altered immune function, etc. A secondary objective of the proposal is to establish a serum and tissue bank for use in future studies.

Kaposi's sarcoma is a tumor in which the lesion predominantly consists of aberrant vascular proliferation. The tumor tissue, or KS-derived spindle cells, when placed subcutaneously in the nude mouse, induces a vascular lesion resembling Kaposi's sarcoma and is of mouse tissue origin. Further, the lesion demonstrates a delayed (12 hr) vascular hyperpermeability response. Thus, this tumor provides an excellent model system to not only understand more about the tumor itself, but also the signals involved in angiogenesis, and to study inhibitors that could later be used for therapy of a variety of diseases, including vascular diseases, malignancy and inflammatory disease. Primary mediators of angiogenesis include: (i) fibroblast growth factors, basic and acidic, both of which lack signal peptide and thus are not secreted, and (ii) vascular endothelial cell growth factor or vascular permeability factor (VDGF/VPF) which has signal peptide and is secreted. VEGF/VPF and FGFs are produced by Kaposi's sarcoma derived spindle cells. Secondary mediators of angiogenesis (such as TNF, TFG-alpha and beta, angiogenin etc) induce angiogenic lesions only in vivo. Thus, these factors must exert their response through the induction of primary mediators. KS derived spindle cells produces many of these cytokines (TGF-beta, IL-1 and IL-6). Further, steroid hormones regulate cytokine expression which in turn regulates angiogenic factors. Alternatively steroid hormones may directly regulate angiogenic factors. We have also shown that glucocorticoids pardoxically enhance IL-6 expression, which in turn upregulates VEGF/VPF. Based on the above, we propose to study the following using vascular endothelial cells and vascular smooth muscle cells as controls: (1) The expression of known primary mediators of angiogenesis in KS-derived spindle cells and their regulation by steroid hormones and cytokines; (ii) Define further the presence or absence of a novel angiogenic factor expressed by KS-derived spindle cells; (iii) Study the transduction pathway of IL-6 and VEGF/VPF; (iv) Define the mechanism of the paradoxical upregulation of IL-6 with glucocorticoids in KS derived spindle cells and compare to other cell types.

The purpose of this study is to determine the effectiveness and toxicity of TNP-470, an inhibitor of angiogenesis, in the treatment of subjects with HIV associated with Kaposi's sarcoma.

human therapy evaluation; neoplasm /cancer chemotherapy; paclitaxel; Kaposi's sarcoma; drug metabolism; clinical research; human subject;

DESCRIPTION: (Adapted from Applicant's Abstract) Kaposi's Sarcoma (KS) is the most common tumor associated with HIV infection. KS represents a vascular proliferation characterized by multiple lesions at initial presentation. an identical tumor develops in other settings without HIV infection, including the classic form seen predominantly in older Eastern European men, an African form, renal transplant recipients, and iatrogenically induced by glucocorticoids disease. Clinically, KS is a heterogeneous course that appears to be independent of the severity of immunodeficiency. KS in highly vascular angiogenic tumor that appears to be of endothelial cell origin. Gill's group has demonstrated in the past that angiogenic factors bFGF, VEGF and IL8 and inducers of angiogenesis IL6, TGFbeta, IL1beta and TNFalpha are all expressed in KS cells and more recently that VEGF is an autocrine factor, a permeability factor and a survival factor for KS cells in culture. He has also found that all known members of the VEGF family and VEGF-R family are expressed in both KS cell lines and KSHV infected EC cultures. He proposes that VEGF is the critical growth factor for KS and that VEGF/VEGF-R proteins represent the convergence points for all KS growth factor cascades. Therefore, he proposes to both demonstrate this and define the roles of individual VEGF/VEGF- R family members in both the KS Y-1 spindle cell line and in the recently developed KSHV infected BMEC system of Flore et al, as well as confirm expression of all of these proteins in primary KS tumor tissue. A comprehensive set of experiments focused on the following three Specific Aims are proposed. Specific Aim 1 is designed to use specific reagents to define and compare the expression and roles of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF, including the use of antisense phosphorothioate oligonucleotide inhibitors and VEGF-R antibodies to examine effects on cell proliferation, migration and survival. The new Specific Aim 2 involves an investigation of the importance of the greatly increased constitutive levels of ERK activation (an apparent downstream VEGF effect) found in KS cells compared to normal HUVEC or skin cells. Whether this is needed for cell survival and whether KSHV infection also induces ERK activation as a appropriate critical event will be examined as well as pharmacological intervention at other stages of MAPK signal transduction. New Specific Aim 3 represents an attempt to establish the hierarchy and possible convergence of IL1beta and IL6 autocrine growth factor action in relation to VEGF survival signaling by individually blocking all three loops and studying the impact on cell growth, migration, apoptosis and second messenger activity.

DESCRIPTION (provided by applicant): Kaposi's sarcoma (KS) develops as a vascular proliferate process in response to infection of endothelial cells or their precursors with HHV-8. KS is a highly vascular tumor with aberrant vascular structures and extravasated red blood cells. KS tumor cells express cell surface receptors unique and restricted to endothelial cells such as VEGFR-2 and VEGFR-3. The VEGF family of proteins is overexpressed in KS and provides strong growth and survival signals for this tumor. It is now known that arterial and venous endothelial cells are phenotypically distinct, even at the level of the capillary beds. The most significant cell surface proteins which mark this distinction are ephrin B2, expressed on arterial endothelial cells, and its receptor, EphB4, which is expressed on venous endothelial cells. The absence of either protein interferes with proper vessel maturation. Ephrin B2 induction leads to increased sprouting of vessels, whereas the opposite is true for EphB4 induction. Due to the highly vascular nature of KS we wished to determine if KS revealed markers for artery or vein, and if there was an imbalance in their expression. Remarkably, we found expression of ephrin B2, but not EphB4. In order to understand how HHV-8 could participate in this phenotype, we determined that ephrin B2 was induced by HHV-8 infection of endothelial cells. Further, HHV- 8 vGPCR alone could induce ephrin B2 and VEGF. VEGF is known to itself induce ephrin B2 in favor of EphB4 during development. The possibility that HHV-8 directly or indirectly induces the arterial phenotype of KS will be investigated in the current proposal. We have also determined that VEGF-C can induce ephrin B2, while other KS growth factors do not. We hypothesize that absence or reduction of EphB4 may be responsible for the aberrant nature of the KS vasculature. We propose that HHV-8 induces arterial marker expression by the direct effect of viral proteins or by the regulation of cellular autocrine or paracrine molecules. The precise mechanism of ephrin B2 induction through VEGF and VEGF-C will also be studied. In the current proposal we plan to profile KS lesions and cells for arterial and venous specific markers, and study how HHV-8, VEGF and VEGF-C induce ephrin B2. This work is anticipated to enhance our understanding of KS pathogenesis, provide opportunities for novel therapies for KS, and contribute to the understanding of vascular biology.

The Translational and Clinical Sciences Program enables USC Norris discoveries to be translated to the clinic by conducting innovative trials relevant to our patient population. Members have diverse expertise from basic to clinical investigation and are highly engaged. Leadership is enriched by an inter-programmatic Steering Committee, which leverages expertise in genomics, biomarkers, bio-imaging and drug development, and regular meetings with disease and thematic teams to ensure that translational and clinical research occurs in an interdisciplinary and coordinated manner. New targets are selected from basic science Research Programs, with translation supported and accelerated by collaborative teams focused on developing novel therapeutics, diagnostics and biomarkers and on executing clinical trials. Members actively participate in intra- and interprogrammatic research using the expertise of USC Norris Shared Resources and clinical resources. Priority themes are novel targets, enhanced efficacy of antibodies using drug conjugates, immunotherapeutics, and cell therapies, and epigenetic targets. We have identified and validated tumor-associated targets, developed agents that have been taken to first in human studies, developed companion imaging agents, and initiated multiple high priority investigator initiated trials. Several novel targets have been chosen and moved through different stages of translation that are already or soon to be in the clinic. Accompanying biomarkers and imaging probes have also been developed for several targets and integrated into trials. Multiple high-impact trials have been conducted, including first in human novel agents discovered and developed at USC Norris and positive Phase II trials that have moved to Phase III. USC Norris PIs have served as lead investigators for several multicenter Phase III NCTN trials. The Program?s 61 members come from six schools and 21 departments. They have $16M in total funding (direct costs) of which 31% is from NCI, 25% is from other NIH sources, and 13% from other peer-reviewed funding sources. The Program has been highly productive with 1,030 publications during the project period, of which 27% were intra-programmatic, 31% were interprogrammatic and 42% were inter-institutional.