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Predicting metastasis in osteosarcoma

By Jay D. Lenn

OREF-funded research seeks to spare patients unnecessary pain

Osteosarcoma, a primary bone cancer, appears most often in long bones—such as the femur, tibia, and humerus—during periods of rapid growth. Consequently, it usually affects children and young adults between the ages of 10 and 20 years old.

At the time of diagnosis, about 80 percent of patients with osteosarcoma have metastatic disease, usually involving the lungs. Metastatic disease is clinically apparent, however, in only about 15 percent of these patients.

Hue H. Luu, MD

As a result, systemic chemotherapy treatments are generally prescribed for all osteosarcoma patients. If clinicians could identify whether the metastatic disease will develop, however, some patients could be spared the chemotherapy and its side effects. That’s the challenge taken on by Hue H. Luu, MD. A recipient of a 2007 Orthopaedic Research and Education Foundation (OREF) Research Grant, Dr. Luu is investigating a biological marker that may help identify patients whose osteosarcoma is not likely to spread.

Osteosarcoma genetics
Dr. Luu’s 2007 grant is his second OREF award related to osteosarcoma. In 2002, he received an OREF Resident Research Award to study genetic factors regulating metastasis in osteosarcoma. “We are now focusing on a particular gene identified in our initial investigations,” he explains.

The gene is S100A6, which encodes the S100 calcium binding protein A6. In previous studies of tissue samples from osteosarcoma patients, Dr. Luu and his colleagues found that S100A6 was often expressed in osteosarcoma. Significant overexpression of S100A6 correlated with a decreased likelihood of metastatic disease or its later development.

Dr. Luu and his colleagues are trying to determine how S100A6 exerts its influence. Their preliminary studies show that overexpression of S100A6 may enhance cell adhesion and inhibit cell migration—factors that would suppress osteosarcoma metastasis (Fig. 1).

For metastasis to occur, a cancer cell must override mechanisms controlling the normal interaction of cells with each other and with components of the mesh-like substance within the extracellular space. Metastasis also requires that cancer cells migrate through the blood vessels to a secondary site, usually the lungs.

Dr. Luu is currently testing the following hypotheses:

  • Overexpression of S100A6 promotes cell adhesion and inhibits cell migration in cancer cells with a high affinity for metastasis.
  • Overexpression of S100A6 inhibits pulmonary metastasis in a mouse model of human osteosarcoma.
  • Knockdown of S100A6—genetically engineered disabling of S100A6—inhibits cell adhesion and promotes cell migration in highly metastatic cancer cells.
  • Knockdown of S100A6 promotes pulmonary metastasis in the mouse model.

Preliminary efforts
First, Dr. Luu and his team had to establish a highly metastatic cell line. They began with an established line of human osteosarcoma cells that didn’t have a particularly strong or predictable likelihood for metastasis. These cells were injected into the tibias of mice. When pulmonary metastasis occurred, the metastasized cells were harvested and the cycle was repeated using the harvested cells.

“By the end we developed a line of cells that grew every time we injected it into the animal,” says Dr. Luu. “Six weeks later the lungs were full of metastases.”

This line of cells is being used in both in vitro and in vivo experiments to test the role of S100A6. The original cell line serves as the experimental control.

Dr. Luu’s group also needed to engineer a mechanism to “switch off” S100A6 in the cancer cells. They developed a system to identify vulnerable sites in the gene’s chemical code and then designed a genetic switch—specially tailored small interfering RNA—that could be introduced to disable the S100A6 gene.

With his in vitro experiments completed, Dr. Luu has begun testing his hypotheses in the mouse model. His preliminary data to date are consistent with his original findings about the role of S100A6 in promoting cell adhesion and inhibiting cell migration (Fig. 1).

Patients’ interest
Dr. Luu and his patients are excited about the potential predictive value of S100A6 overexpression. “Patients do Internet searches and know that most of them have metastatic disease. Some of them have asked me what we’re doing to understand this,” says Dr. Luu. He adds that his patients are inspired by the work he and other scientists are doing in this field and are often interested in contributing to the research group’s osteosarcoma tissue bank.

As a clinician and scientist, Dr. Luu appreciates the value of research. “Science is critical to our clinical practice. We need to invest in biomedical research that helps find new avenues for treating patients and understanding basic processes in disease.”

According to Dr. Luu, OREF not only funds research, it also promotes and facilitates scientific mentoring. “Mentorship is critical,” he says, “because it helps you to develop scientific expertise and navigate the challenges of establishing a career and securing research funds.”

Value of mentorship
Dr. Luu learned the benefits of a mentor long before his medical residency. As a high school student, he had the opportunity to work in the lab of Kathleen Green, PhD, at Northwestern University. “I was expecting to wash dishes or do very basic tasks,” he recalls, “but she got me involved in cloning. She made an impact on my life and stimulated my interest in research.”

Developing a research career is a long, arduous undertaking; those who have established research careers provide valuable insight into the process. “OREF gives us opportunities to seek out mentors and get advice from these individuals,” says Dr. Luu.

AAOS Now
August 2009 Issue
http://www.aaos.org/news/aaosnow/aug09/research3.asp