Molecular mechanisms of viral Carcinogenesis and angiogenesis activation by the Kaposiís sarcoma Herpesvirus (KSHV)
Kaposiís sarcoma (KS) is the most common cancer associated with AIDS (AIDS-KS). It presents as lesions on the skin, GI and lungs that are characterized by proliferation of spindle cells and intense angiogenesis. Angiogenesis, the formation of new blood microvessels from existing vasculature, is a critical step in tumor formation and metastasis. It is mediated by tumor-secreted growth factors in endothelial cells. Treatment of advanced AIDS-KS with chemotherapy in HIV infected patients receiving anti-retroviral therapies has many complications. Thus it is important to seek pathogenesis-based low-toxicity therapies. In 1994 Chang and Moore identified a new herpesvirus implicated in KS, the Kaposiís sarcoma associated herpesvirus (KSHV/ HHV-8). Our laboratory was the first to isolate infectious KSHV virions and to show that they specifically infect B cells. Thus, identifying KSHV or Human Herpesvirus-8 as a new B-cell tropic virus (Mesri, J. Exp. Med. 1996). We hypothesize that KSHV infection leads to cell transformation and an angiogenic conversion of infected cells (angiogenic switch). Our goal is to identify KSHV genes and host mechanisms able to trigger the KS angio-proliferative response in an attempt to achieve a better understanding of the mechanisms of pathogenesis and to identify new molecular targets for treating KS.
Identification of the viral G protein-coupled receptor as an angiogenic oncogene of KSHV
KSHV encodes a constitutively active G protein coupled receptor (vGPCR) homologous to the human receptors for IL-8 and Gro-alpha, two angiogenic chemokines. We demonstrated that vGPCR can transform cells and induce tumorigenesis. More importantly vGPCR expression is sufficient to trigger an angiogenic switch evidenced by the ability of cell supernatants to induce endothelial cell proliferation and mediated by increased secretion of the angiogenic growth factor VEGF (Bais. Nature, 1998 ). These results showed that KSHV encoded genes are able to cause the KS angiogenic and transformed phenotype, and identified vGPCR as a candidate angiogenic oncogene of KSHV. Since KSHV is an endothelial-tropic virus, and endothelial cells express VEGF-receptor we hypothesized that vGPCR expression in these cells naturally infected by KSHV could lead to autocrine transformation. We found that retroviral transduction of vGPCR to HUVEC led to cell immortalization with constitutive expression and activation of the VEGF receptor-2/ KDR (Bais, Cancer Cell, 2003). We demonstrated that the mechanism of immortalization was a combination of constitutive vGPCR and autocrine KDR signaling leading to activation of the PI3K /AKT survival pathway mediated by increased VEGF secretion (Bais, Cancer Cell 2003). These results proved the oncogenicity of vGPCR in endothelial cells. In combination with our previous studies and new studies from other groups showing that vGPCR is the only KSHV gene able to cause KS lesions in mice identify vGPCR as "the major KSHV angiogenic oncogene" and thus a target for KS therapy.
Identification of Cyclooxygenase-2 as a mediator of vGPCR angiogenesis and tumorigenesis and of the COX-2 inhibitory drug Celecoxib as a potential anti-vGPCR oncogenesis agent.
Cyclooxygenase-2 (COX-2) is an inflammatory mediator involved in tumor angiogenesis that is inhibited by FDA approved drugs such as NSAIDs. We demonstrated that vGPCR up-regulation of COX-2 expression was critical for vGPCR pathogenicity in vivo. We found that the COX-2 inhibition impaired vGPCR-driven angiogenesis in mice skin and that the COX-2-selective inhibitory drug celecoxib (Celebrex) inhibited vGPCR tumorigenesis by inhibiting tumor angiogenesis and VEGF secretion. Moreover, we also found that COX-2 is expressed in the KSHV infected spindle cells of the lesions. Thus, our results identify Cox-2 as a surrogate target for vGPCR angiogenicity expressed in KSHV infected cells that can be inhibited by prescription drugs for the prevention and treatment of Kaposiís sarcoma.
A cell and animal model of KSHV-mediated carcinogenesis.
One of the problems with KS and other viral cancers is the lack of cell and animal models that reproduce the human disease. By using the KSHV genome cloned in a Bacterial Artificial Chromosome (Bac36) we were able to generate a mouse cell line of the endothelial progenitor lineage (mECK36) that contains and express the full KSHV genome and possess many characteristics of transformed angiogenic cells. When injected subcutaneously into nude mice the cells induce KS-like spindle cell sarcomas that contain the viral genome and display phenotypic markers of human KS lesions. We showed that the tumor phenotype was linked to viral gene expression by a siRNA vector that specifically silenced the expression of the oncogene vGPCR of KSHV resulting in inhibition of VEGF secretion and tumorigenicity. Thus, the mECK36 define a cell and animal model in which viral gene function is linked to a KS-like molecular and cellular pathogenic phenotype that can be employed to study viral gene function and for preclinical testing of anti-viral and anti-angiogenic targeted therapies.
Future and current research plans:
- Use of gene arrays to identify critical host pathways dys-regulated by KSHV infection in vitro and in mice tumors.
- Use the mECK36 cell and animal model for genetic analysis of pathogenic KSHV gene function and for drug testing.
- Identifying small molecule drugs that can inhibit vGPCR constitutive signaling (reverse agonist)