Inspired by a fairy tale called “The Three Princes of Serendip,“ English writer Horace Walpole invented a word that still holds special relevance for many researchers.

Walpole summarized the plot of the fairy tale in a letter: “... as their three highnesses traveled, they were always making discoveries by accidents and sagacity, of things which they were not in quest of ... .“

Serendip was an old name for the island country of Sri Lanka. From it Walpole created the word serendipity.

The progress of science owes much to serendipity and to the sagacity of researchers resourceful enough to follow unexpected clues.

A recent example appears in the June issue of Nature Genetics. Frank Tufaro, an associate professor of microbiology and immunology at the University of British Columbia, in Vancouver, and his co-authors went looking for genes involved in the biosynthesis of glycoaminoglycans (GAGs). These cell-surface molecules have diverse functions, including the recognition of growth factors that induce cell growth and development.

The researchers found what they were looking for and something unexpected. They uncovered the function of a cancer gene. As a result, they have established a basis for understanding the role of a family of tumor suppressors.

Their paper, “The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate,“ described for the first time the function of an EXT gene, part of a family of genes responsible for skeletal abnormalities and bone cancer.

Study Links GAG Synthesis To Bone Cancer

EXT genes are responsible for hereditary multiple exostoses (HME), an autosomal-dominant trait affecting two out of every 100,000 people. It is the cause 15 percent of all bone tumors and half of all benign bone tumors.

HME-associated genes encode proteins called EXTs. EXTs may be involved in other disorders as well. Although mutations in EXT genes are believed to be responsible for most cases of HME, the function of the genes was unknown until Tufaro's group began its search for genes involved in GAG synthesis.

The Canadian team used a cleverly designed assay system consisting of mouse cells and herpes simplex virus type I (HSV-1). The virus can only infect cells that express normal heparan sulfate GAGs on their surface. Tufaro et al. had mouse cells that lacked the ability to synthesize heparan sulfate GAGs. This was indicated by the inability of the herpes virus to infect the cells.

Starting with 10 million complementary DNAs (cDNAs) from HeLa cells, the researchers divided them up into pools of 100,000. They added the cDNAs to the GAG-negative mouse cells.

After several cycles of screening and subdividing, the researchers isolated a line that had become susceptible to HSV-1 infection after being transfected by one of the cDNAs. This cell line had recovered the ability to synthesize GAGS. Upon analysis, the cDNA responsible for this change was identified as EXT1, one of the known EXT genes. Now the researchers had an enzyme involved in GAG synthesis (which they were seeking) and a function for a cancer gene (which they were pleased to find). They established a link between GAG synthesis and bone cancer.

“The key is we have a functional assay for EXT1. That was really the breakthrough. Now we can look at the function of these other genes [in the EXT family],“ Tufaro told BioWorld International.

Further testing demonstrated the ability of the EXT1 gene to alter the expression of GAGs in other types of cultured cells. Mutated versions of the gene, however, did not reverse the infection-resistance of cells.

The EXT1 protein was identified as a glycoprotein present in the endoplasmic reticulum (ER), a large network of internal membranes containing enzymes that synthesize a large variety of biomolecules. The protein's likely configuration in the ER suggests that it may be a glycosyl transferase, a type of enzyme involved in GAG synthesis.

“We are working with a group of carbohydrate chemists to sort out exactly what the changes are [in the GAG affected by the mutation],“ Tufaro said.

Other experiments in progress are designed to see if the lack of EXT causes tumors in tissues.

“And we are looking now at patient material and trying to characterize changes in the cell surface,“ Tufaro said.

The significance of the work is increased by suggestions in the literature that EXT genes may be linked to other types of cancers, including breast cancer. It is possible the EXT genes are a class of tumor suppressors that can influence the development of cancers by changing cell surface proteins, heparan sulfate GAGs.

“There are many, many heparan and GAG binding proteins that are crucial in regulating not only development but also the growth of cells in the mature adult. It seems obvious to me and to others that that is a good place to start. It is a testable model,“ Tufaro said.

Neurovir Holds EXT Patent

One of the major growth factors that may interact with GAGs affected by the EXT1 mutation is fibroblast growth factor.

Tufaro also noted HME has been linked to mutations in three different EXT genes. The EXT2 mutation, for example, does not appear to have the same activity as the EXT1 mutation.

“I think that is important because it says we are not just dealing with overlapping activities,“ Tufaro said. “We have a whole pathway within the EXT gene family. That is what we are working hard to sort out.“

In addition to his academic career, Tufaro is a founder and the chief scientific officer of Neurovir Inc., in Vancouver. This company is developing the use of herpes viruses as vectors for gene therapies to treat cancers. It is now considering expanding its interests in light of the results described in the Nature Genetics paper.

“We have a patent on the use of EXT based on what we now know about it. Neurovir is interested in possibly developing it and licensing it to another company specifically interested in tumor suppressors,“ Tufaro said. *

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