Research activities focus on sarcomas and encompass diverse areas, ranging from biochemical studies and translational research aimed at developing new therapies for treating sarcomas, to biochemical predictors of disease. Our laboratories investigate the relationship between biological parameters and clinical parameters for sarcomas, including:

1. correlations of disease outcome with various treatment and lifestyle parameters using an extensive patient database
2. predictors of metastasis in malignant bone and soft tissue tumors by flow cytometric analyses of DNA ploidy and cell cycle parameters
3. mechanisms and potential markers related to metastasis in skeletal tumors by molecular and biochemical studies.

We have recorded data regarding our patients with bone and soft tissue tumors in a FOXPRO system for the past 30 years. To date, we have information on 15,000 patients that includes demographic data, diagnosis, stage, anatomical site, operative procedures, use of adjuvant therapy, laboratory DNA ploidic data, and outcome in terms of local recurrence, metastasis, and death.

At the molecular level, we are focusing on chemotherapeutic drug resistance and mechanisms of antiangiogenesis agents. The sensitivity of sarcoma cells to the novel marine chemotherapeutic, Ecteinascidin-743 (ET- 743), has led us and others to consider this compound as an important component of future treatment options for sarcomas such as chondrosarcoma. However, as with other chemotherapeutics, the propensity of tumor cells to develop resistance to ET-743 poses a significant challenge for employing this drug over an extended period of time as a cancer treatment. A human chondrosarcoma cell line developed in our laboratories, CS-1, was treated successively with increasing concentrations of ET 743, yielding a variant cell line displaying a significant degree of resistance to the cytotoxic action of this drug. Various experiments were performed to identify molecular aberrations between the parent and resistant cell line. Although no significant differences in the activity of membrane transporters such as P-glycoprotein or Multi drug Resistance Protein were detected, the cell migratory ability of the ET-743-resistant cell variant was reduced, as was its attachment capability to gelatin-coated cell culture dishes. Staining of the actin containing cytoskeleton with fluorescent-labeled phalloidin revealed significant differences in the cytoskeleton architecture between the parent and ET-743-resistant CS-1 cell lines. Comparison of serum-free conditioned medium from both cell lines showed conspicuous differences in the levels of several proteins, including a quartet of high molecular weight proteins ( ¡Ý 140 kDa). The protein sequences of two of these high molecular weight proteins, present at significantly higher concentrations in conditioned medium obtained from the parent cell line, corresponded to subunits of type I and type IV collagen. Analysis of type I collagen _1 chain mRNA revealed a significantly lower level in the ET-743-resistant CS-1 cell line. Thus, prolonged exposure to ET-743 may cause distinct changes in cell function through cytoskeleton rearrangement and/or modulation of collagen levels. Our goal is to further dissect the molecular mechanisms underlying the development of chemoresistance, in order to formulate strategies to minimize or prevent this phenomenon, as well as develop diagnostic tests for indicating the onset of chemoresistance.

We have also been studying the properties of a novel, protein designated plasminogen-related protein B (PRP-B), encoded for by plasminogen-related gene B (PRG-B), with the goal of extending our understanding of the antiangiogenic process as it relates to tumor growth as well as gaining insights into biological processes that are insensitive to the action of antiangiogenesis inhibitors. We have been accumulating evidence that PRP-B may function as part of a host defense mechanism to thwart tumor growth, most likely by inhibiting angiogenesis. To understand the function of this 9kDa protein, we prepared and characterized a recombinant form of the protein (rPRP-B), which we have employed in mouse tumor studies. These in vivo experiments have clearly demonstrated a marked antitumor activity. When administered systemically, rPRP-B significantly inhibited growth of different tumors when grown in mice, although the corresponding homologous region in plasminogen (i.e., the plasminogen activation peptide) was much less effective at curtailing tumor growth. Interestingly, when rPRP-B was administered in combination with a chemotherapeutic, tumor necrosis of a human chondrosarcoma in an athymic mouse xenograft model was significantly enhanced relative to individual chemotherapeutic or antiangiogenic treatment, suggesting that rPRP-B may have utility as an adjunctive cancer therapeutic, similar to Avastin (the anti-VEGF antibody developed by Genentech). Noteworthy was our finding that tumor vascularization was severely depressed only in the combination therapy, suggesting that a partial block in angiogenesis, which was observed in the individual therapies, is capable of slowing tumor growth substantially but is inadequate in terms of producing tumor necrosis.

We have also initiated laboratory studies on cyclooxygenase-2 (COX-2) expression in musculoskeletal tumors, following the observation that a patient originally treated in the Orthopaedic Oncology Service showed remarkable improvement when subsequently treated with Celebrex. A clinical trial to test Celebrex in conjunction with standard treatment for skeletal tumors is in the advanced planning stage. In recent years it has become evident that COX-2 may play a role in carcinogenesis, and there is now a mounting body of evidence that COX-2 inhibitors may be of some value in cancer management.

Faculty
Francis Hornicek, MD, PhD
Co-Director

David Harmon, MD
Henry Mankin, MD
Christine Towle, PhD
Lawrence Weissbach, PhD, MBA

Selected Publications
Sutton K, Wright M, Fondren G, Towle CA, Mankin HJ. Cyclooxygenase-2 expression in chondrosarcoma. Oncology 2003 (in press).

Rodriguez EK, Hornicek FJ, Gebhardt MC, Mankin HJ. Metachronous osteosarcoma: A report of five cases. Clin Orthop 2003; (411):227-35.

Mankin HJ. A computerized system for orthopaedic oncology. Clin Orthop 2002; 398:252-61.

DeLaney TF, Chen GT, Mauceri TC, Munro JJ, Hornicek FJ, Pedlow FX, et al. Intraoperative dural irradiation by customized 192iridium and 90yttrium brachytherapy plaques. Int J Radiat Oncol Biol Phys 2003; 57(1):239-45.

Rosenthal DI, Hornicek FJ, Torriani M, Gebhardt MC, Mankin HJ. Osteoid osteoma: Percutaneous treatment with radiofrequency energy. Radiology 2003; 229(1):171-5.

Rosenthal DI, Marota JJ, Hornicek FJ. Osteoid osteoma: Elevation of cardiac and respiratory rates at biopsy needle entry into tumor in 10 patients. Radiology 2003; 226(1):125-8.

Morioka H, Morii T, Vogel T, Hornicek FJ, Weissbach L. Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro . Exp Cell Res 2003; 287(1):166-77.

Morioka H, Weissbach L, Vogel T, Nielsen GP, Faircloth GT, Shao L, Faircloth GT, Hornicek FJ. Antiangiogenesis treatment combined with chemotherapy produces chondrosarcoma necrosis. Clin Cancer Res 2003; 9(3):1211-7.

Morii T, Weissbach L. Sphingosine 1-phosphate and cell migration: resistance to angiogenesis inhibitors. Biochem Biophys Res Commun 2003; 310:884-888.

Shao L, Kasanov J, Hornicek FJ, Morii T, Vogel T, Weissbach L. Ecteinascidin-743 drug resistance in chondrosarcoma: Transcriptional and cellular alterations. Biochem Pharmacol 2003; 66:2381-2395.