EWS-FLI1 and RHA Interaction Gives New understanding of Ewing's sarcoma

Thu, 01 Jun 2006 12:57:37 PST

( from http://www.rxpgnews.com ) Using molecular and cell-based models, researchers at Georgetown University Medical Center have refined the picture of how a cancer-promoting protein associated with Ewing's sarcoma functions. And in the process, they have hit upon a possible strategy for treatment of the cancer, which is a rare and highly malignant cancer that most often strikes teens and young adults.

In the June 1st issue of the journal Cancer Research, published by the American Association for Cancer Research, the researchers report that the oncoprotein, EWS-FLI1, teams up with a helicase protein that bends the shape of RNA, and together they work to promote or repress transcription of various other proteins, leading to cancer development.

But because it is not possible to directly shut down helicase proteins, given their vital general role in protein transcription, and given that no one has figured out how to clinically inactivate EWS-FLI1 alone, the researchers propose driving a wedge-like drug between the two proteins that would eliminate their interaction.

"Proteins are three-dimensional structures, and the space between EWS-FLI1 and the helicase might be targetable by a small molecule that keeps the proteins apart," says the study's lead author Jeffrey Toretsky, M.D., an associate professor in the departments of Oncology and Pediatrics at the Lombardi Comprehensive Cancer Center. "It could render EWS-FLI1 harmless while not affecting its partnering helicase protein."

Toretsky adds that the findings may also help explain the workings of a number of difficult-to-treat sarcomas that are caused by the same sort of genetic rearrangements seen in Ewing's sarcoma. "Understanding the molecules EWS-FLI1 interacts with may provide insights into similar diseases," he said. "Very little work has been done to study the functional partners of these translocation proteins, and this study may offer a new research approach."

Ewing's sarcoma results in solid tumors in bone or soft tissue and is caused by a translocation between two chromosomes which fuses the EWS gene from chromosome 22 to the FLI1 gene of chromosome 11. This fusion produces the EWS-FLI1 fusion protein which is only found in tumors. This translocation is present in 95 percent of tumors, according to Toretsky, a recognized expert on Ewing's sarcoma. Chromosomal translocations are the cause of many sarcomas and leukemias, and the resulting fusion proteins represent targets for new therapies.

The potential for a drug that would target the fusion proteins in tumor cells was first put forward by scientist Paul Ehrlich more than 100 years ago. In Ewing's sarcoma, the proteins that are created from chromosomal translocations are unique and only occur in the tumor cells.

Therefore, fusion protein produced by translocation in Ewing's Sarcoma represents "an ideal target" for therapy, according to Aykut Üren, MD, PhD, assistant professor of oncology and a co-author on the paper. Previously published animal studies using "antisense" molecules have indeed shown that when the EWS-FLI1 is inactivated, Ewing's Sarcoma does not develop. But antisense, which modifies gene expression, is technologically inappropriate for human therapy, and to date, no one has been able to design a drug that would shut down EWS-FLI1 in humans. "The fusion protein is known to be oncogenic, but we suspected that it must work with other molecules, and these molecular interactions could offer us opportunities for treatment," Toretsky said.

To find out if their hypothesis was true, the research team utilized a virus that displayed random, but identifiable, protein sequences and mixed the viruses with EWS-FLI1. "We wanted to see what viruses stuck to the EWS-FLI1 protein," Toretsky said. They found a certain peptide sequence attached to EWS-FLI1 in every experiment they conducted, and a search of the Human Genome database showed these sequences belonged to RNA helicase A (RHA), a common helicase not known to be oncogenic.

They then conducted a number of rigorous studies to prove that EWS-FLI1 and RHA did indeed bind to each other in a complex. Furthermore, when EWS-FLI1was expressed along with RHA, the cells demonstrated increased hallmarks of cancer development.

"The two proteins appear to work together in order for EWS-FLI1 to have maximal oncogenic potential," Toretsky said. "Since EWS-FLI1 needs RHA, our goal is to keep them apart."

Nanoparticles Shown to Inhibit Ewings Sarcoma

Thu, 21 Apr 2005 17:48:38 PST

( from http://www.rxpgnews.com ) A novel delivery system that transports gene silencing nanoparticles into tumor cells has been shown to inhibit Ewings sarcoma in an animal model of the disease.

In this classic Trojan horse approach, a protein called transferrin that normally delivers iron into cells is modified to also smuggle into tumor cells siRNA (short interfering RNA) encased in nano-sized sugar polymers. The siRNA was designed to target a specific growth-promoting gene called EWS-FLI1 thats active only in Ewings sarcoma tumors.

Once inside these cells, the genetic machinery of the tumor cells are effectively silenced or shut down, preventing further growth.

This is the first study to show that systemic administration of siRNA can inhibit disseminated tumor growth, said Siwen Hu, a postdoctoral fellow at Childrens Hospital of Los Angeles and the University of Southern California, and one of the studys lead investigators.

We conclude that this novel delivery system is a powerful and simple method to induce gene silencing, with the potential to move to clinical trials, said Hu, who presented the results at the 96th Annual Meeting of the American Association for Cancer Research.

In recent years, scientists have been intrigued by the potential of siRNA to block the activity of genes that promote the growth of tumors. Harnessing the power of this new technology, however, has proved daunting for a variety of reasons, including the ability to deliver these bits of genetic material in high concentrations to specific tumor sites, while avoiding degradation.

To overcome these hurdles, the scientists employed a sugar-containing polymer invented by chemical engineers at the California Institute of Technology. For this experiment, the polymer binds to and condenses the engineered siRNA into nanoparticles that, in effect, form a protective shield around their precious genetic cargo. These nanoparticles, in turn, are attached to transferrin, a protein that typically carries iron molecules through the bloodstream until it meets up with a transferrin receptor on the surface of another cell. The transferrin binds tightly to a receptor on the cells surface, where it is drawn inside and surrounded by a small vesicle. The vessels are acidified, causing the nanoparticles to release its contents the siRNA.

Since transferrin receptors are upregulated in tumor cells, this delivery system will home in on tumor cells, leaving normal cells in tact, Hu said.

To test their new delivery system, the scientists targeted tumor cells from the patients of Ewings sarcoma, a rare and often deadly bone cancer that generally strikes young adults. Despite aggressive therapy, about 40 percent of patients with Ewings family tumors and 95 percent with metastases die as a result of their disease.

Scientists now recognize that Ewings sarcoma results when two chromosomes break and trade their genetic content in whats technically called a translocation, activating the oncogene EWS-FLI1 which triggers the tumor growth characteristic for this cancer.

In their experiment, siRNA was delivered to this growth-promoting region of the tumor cell, effectively reducing cell replication by 80 percent.

The scientists then tried their novel technology in laboratory mice grafted with human Ewings sarcoma tumors. Following three consecutive days of treatment, the scientists observed strong, but transient, inhibition of tumor growth.

However, when used over longer durations (twice-weekly injections up to four weeks), the results were striking.

Long-term treatments with this delivery system markedly inhibited tumor growth, with little or no tumor growth in many animals, said Hu.

Future experiments will combine the novel delivery system with small molecular anti-tumor agents, with hopes of creating a new and effective way to treat Ewings sarcoma and other tumors in the clinic.

Clinically, Ewings patients are treated with combination of chemotherapeutic agents, but despite aggressive treatments, the patient outcomes are poor, said Hu.

The delivery system were developing can shield the drugs from degradation before reaching the target sites, while delivering siRNA for more specificity and potency so as to lower the required dose for efficacy.

The study was a collaborative effort between the laboratory of Timothy J. Triche, at Childrens Hospital of Los Angeles; and the laboratory of Mark E. Davis, at Caltech. Also participating in the study were Jeremy D. Heidel and Derek W. Barlett, both at Caltech.

Support for this research came from the Las Madrinas endowment in Molecular Genetics and Molecular Pathology at the Childrens Hospital of Los Angeles, the Whitaker Foundation, with the National Science Foundation funding part of the work at Caltech.

RxPG News : Bone Cancer

EWS-FLI1 and RHA Interaction Gives New understanding of Ewing's sarcoma

Nanoparticles Shown to Inhibit Ewings Sarcoma

Nanoparticles Shown to Inhibit Ewings Sarcoma