Sarcoma research within the Surgical Oncology Research Laboratories is performed in the laboratory of Dr. Sam Yoon. Dr. Yoon's laboratory studies the process of tumor angiogenesis, or the formation of new tumor blood vessels. This research is conducted in a variety of approaches including:

1. basic science studies which look into the mechanisms of tumor blood vessel formation
2. translational research studies which apply new basic science knowledge to human disease
3. clinical trials which investigate new anti-angiogenic strategies in sarcoma patients

The following is an overview of current research projects:

Knockdown of angiogenic factors by RNA interference inhibits sarcoma angiogenesis

Sam S. Yoon, MD
Principal Investigator

Group Members:
Namali Fernando
Kara Detwiller

RNA interference is a recently described phenomenon in mammalian cells. Introduction into cells of short interfering RNAs (siRNAs), which are double-stranded and 20-25 nucleotide in length, lead to their incorporation into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs). The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA. By generating siRNAs to specific gene sequences, gene expression can be effectively "knocked-down" or "silenced."

Our laboratory has generated siRNAs to three angiogenic factors -- vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiopoietin 2 (Ang2). Studies with several different VEGF siRNAs in human sarcoma cells revealed two VEGF siRNAs that led to near complete inhibition of VEGF expression. This VEGF inhibition was persistent even in hypoxic conditions, during which VEGF gene expression is normally upregulated. When sarcoma cells that stably express these VEGF siRNAs are implanted into mice, tumor growth is severely inhibited and tumor blood vessel density is reduced. We are currently studying the effect of siRNAs targeting bFGF and Ang2 along to determine if these siRNAs combined with VEGF siRNAs will have a synergistic inhibitory effect on tumor angiogenesis.

A phase II study of neoadjuvant bevacizumab and radiation therapy for resectable soft tissue sarcomas

Sam S. Yoon, MD
Principal Investigator

Co-Investigators:
Thomas DeLaney, MD
David Harmon, MD
Dushyant Sahani, MD
Andrew Rosenberg, MD
Peter Park, PhD

The long term objective of this project is to determine if anti-angiogenic agents can increase the efficacy of radiation therapy for solid tumors. Human cancers such as soft tissue sarcomas (STS) often have areas of hypoxia secondary to irregular and porous tumor blood vessels, and hypoxia significantly reduces the efficacy of radiation therapy. Inhibition of vascular endothelial growth factor (VEGF) leads to normalization of tumor blood vessels and improvement in tumor oxygenation. Several pre-clinical studies have demonstrated potentiation of radiation therapy with anti-VEGF agents, but there is little clinical data in humans examining the role of anti-VEGF agents in this setting.

Bevacizumab is a humanized anti-VEGF monoclonal antibody that binds VEGF and inhibits its activity, and clinical trials of bevacizumab have demonstrated efficacy against metastatic colorectal cancer and renal cancer. We hypothesize that bevacizumab can alter the vasculature in STS, improve tumor oxygenation, and increase the efficacy of radiation therapy. To address this hypothesis, we have designed a phase II study to examine the use of neoadjuvant bevacizumab combined with radiation therapy for patients with primary STS.

The specific aims are

1. To determine the response rate of neoadjuvant bevacizumab combined with radiation therapy for intermediate and high-risk STS.
2. To analyze the biologic effects of this regimen on tumor vasculature, blood flow, and oxygenation.

To accomplish these specific aims, primary tumors will be assessed for response to this regimen using RECIST criteria. Serial blood samples will be collected to measure levels of circulating angiogenic factors, and tumor biopsies before and after treatment will be analyzed for changes in VEGF and hypxia inducible factor 1a (HIF-1a) levels, blood vessel density, and expression of hypoxia-responsive and angiogenesis-related genes. Perfusion CT scans will be used to assess tumor blood flow and vascular permeability.

Role of Down syndrome critical region (DSCR) proteins, endogenous inhibitors of calcineurin, in tumor angiogenesis

Sam S. Yoon, MD
Sandra Ryeom, PhD
Principal Investigators

Group Members:
Lila Gollogly
Namali Fernando

Vascular endothelial growth factor (VEGF) is critical for endothelial cell function. Ligation of VEGF to the VEGFR-2 (KDR/Flk-1) receptor causes a rapid increase in intracellular calcium and the activation of calcineurin. Calcineurin is a calcium-activated ser/thr phosphatase that couples intracellular calcium to transcriptional programs in the immune, nervous and cardiac systems. Activated calcineurin triggers the nuclear import of NF-AT transcription factors to drive expression of cell-specific genes. Recently, a family of three endogenous, negative regulators of calcineurin termed the Down syndrome candidate region (DSCR) proteins have been discovered. DSCR1 is the mostly widely studied of the three, and interestingly gene expression profiling of endothelial cells has identified DSCR1 as the most highly upregulated gene following VEGF stimulation, suggesting DSCR1 forms a negative feedback mechanism for calcineurin signaling.

In collaboration with Dr. Sandra Ryeom (Harvard Medical School), our laboratory has generated retroviral and lentiviral vectors carrying the DSCR genes and transduced endothelial cells with these vectors. Overexpression of DSCR1 in endothelial cells results in decreased endothelial cell proliferation and capillary tube formation in vitro. Tumors implanted into transgenic mice that overexpress Csp1 grow dramatically more slowly than tumors implanted into normal mice, and analysis of tumors reveals decreased blood vessel density. We have also recently discovered that DSCR1 regulation of calcineurin is disrupted in endothelial cell-derived tumors (i.e. angiosarcomas). The restoration of DSCR1 expression in these tumor cells decreased proliferation and tumor formation.

Current work in the lab is exploring alterations in DSCR1 regulation of calcineurin in other types of cancers. In addition, we are examining the role of the other DSCR genes in endothelial cells and angiogenesis.

Tumor angiogenesis in different organ environments

Sam S. Yoon, MD
Principal Investigator

Group Members:
Sung Hwan Kim, MD, PhD
Namali Fernando

The human body is composed of over 10 12 endothelial cells lining the inside of blood vessels and covering a surface area of over 1000 square meters. Cancers must induce these normally quiescent endothelial cells to proliferate and form new blood vessels in order to support tumor growth. In fact, endothelial cells comprise about 5% of the total population of cells in a tumor. The endothelial cells that form new blood tumor blood vessels originate from the host organ or tissue in which the tumor is growing. These endothelial cells have markedly different histologic and physiologic characteristics based on their site of origin.

The process of new blood vessel formation, or angiogenesis, is regulated by a balance between pro-angiogenesis and anti-angiogenesis factors. Given the prominent differences in endothelial cells in various organs, we hypothesized that the efficacy of specific angiogenic factors would vary according to the location in which the cancer was growing. To test this hypothesis, we first engineered stable transfectants from RenCa mouse renal carcinoma cells that constitutively secrete the anti-angiogenic agent endostatin. Following inoculation into the subcutaneous flank region of mice, endostatin-producing cells are dramatically growth-inhibited compared to flank tumors from control cells after 3 weeks. Similar results were obtained when we examined a mouse model of experimental lung metastases, but surprisingly, endostatin had little or no effect in preventing the formation of experimental liver metastases. These results suggest that endostatin may be less efficacious against liver sinusoidal endothelial cells.

We are currently examining other pro-angiogenic (VEGF, PDGF) and anti-angiogenic factors (tumstatin, thrombospondin 1) to determine further differences in the efficacy of these factors in different organs or tissues. In addition, we are isolating endothelial cells from different sites and examining their response to various angiogenic factors in vitro .