Try this multiple-choice quiz: Which malignancy takes the lives of more American women over age 75? (a) breast cancer or (b) colon cancer?
If you picked (b), you won. In fact, the epidemiology of colorectal carcinoma (CRC) is surprisingly grim. CRC is the second leading cancer killer in the U.S., and the third most common malignancy overall. This year, more than 50,000 Americans will die from CRC, and an estimated 131,600 new cases will be diagnosed.
Diagnosis and prognosis are the names of CRC’s therapeutic game. If detected early enough, the patient’s chance of survival even outright cure beats 90 percent. So in years to come, individuals suspected of having colon cancer may have to thank a colony of knockout mice for playing a role in saving their lives from colon cancer.
A paper in last week’s Science, dated March 1, 2002, tells the story of these unique rodents: Its title: “Colorectal cancer in mice genetically deficient in the mucin Muc2.” The article’s first author is molecular biologist Anna Velcich at the Albert Einstein Cancer Center in Bronx, N.Y.
“Our overall finding,” Velcich told BioWorld Today, “is that by inactivating the mucin gene, the knockout mice spontaneously developed colon cancer. Young mice had the majority of these tumors in their small intestine. As the animals got older, tumors developed in their colon the large intestine which is quite rare in mouse models. What seems to be especially important in our Muc2 protein-lacking mouse model,” she continued, “is that they developed rectal cancer, which is a different pathological and clinical entity from colon cancer.
“The gastrointestinal tract of mice, as well as of men and women,” Velcich explained, “is ordinarily lined along its lumen with a layer of mucus, consisting mostly of proteins called mucins. The most abundant of these is mucin2 Muc2. As we report in Science, we knocked out the Muc2 gene from mice we generated. They eventually developed both gut and rectal tumors.”
“The human genome’s Muc2 gene resides on the short arm of chromosome 11,” she went on. “It’s actually part of a small stretch of the chromosome in which four mucin genes occur packed together. The Muc2 gene encodes the mucin2 protein, which is a building block of the intestinal mucus.”
Does Muc2 Fend Off Cancer-Causing Wastes?
“Many of these Muc2 molecules polymerize,” Velcich pointed out. “They trap water and solutes everything that can stay in there. The main purpose of this elastic substance, which is mucus, is thought to be just to lubricate and protect the inner surface of the intestinal mucosa. It’s like having a shield on top of the cells. If you remove that, you may incur some sort of exposure of the cells to the outside environment. In the intestinal lumen we have a lot of junk noxious agents. And one of the functions of the mucus is to protect the underlying cells from these agents. That is one of the hypotheses in this field.
“We debated a lot whether we can call Muc2 a tumor suppressor gene. Strictly speaking, yes. We knocked it out and we got tumors. So, yes, it behaves as a tumor suppressor. However,” she hedged, “tumor suppressors to me are involved in a more subtle concept. Say you introduce a normal gene into tumor cells, you should be able to suppress the tumor phenotype, and this type of experiment is not feasible for us because the gene is quite difficult to handle.
“Besides that,” Velcich surmised, “I don’t think it would work, because we have tumors that express very high levels of this Muc2 protein, which is actually contributing to a very poor prognosis in that particular subset of tumor cells. I would call this a very dangerous protein. If you don’t have it in the beginning, you may develop colon cancer. But if you have too much in full-blown tumors, you have an increased probability of metastasis. It’s like a Janus double-faced protein.
“Our mice don’t have colon cancer metastases,” she pointed out. “But they do sustain invasion. The intestine has a unit structure, the crypt, which lies on some layers of tissue. When these are destroyed, the tumor cells tend to grow inward. That is called invasion.
“We knew already that there are alterations in mucin in colon cancer,” Velcich went on. “Some full-blown tumors express very high levels of this Muc2 protein; others don’t express it at all. But this was in tumors. What was not known was whether alteration of mucin expression occurred in the early stages of tumor development. And that’s what we have shown in our mice.
“We cloned the human gene first,” she recounted, “and then we cloned the mouse gene for our study on the differentiation of the gut’s goblet cells. But once we had the mouse gene, we were in the position of doing this type of experiment, and testing the function and significance of the Muc2 protein.”
Blow-By-Blow Muc2 Genes Knockout Protocol
“In our vivo experiments,” Velcich continued, “first, we cloned the mouse gene. Next, we designed a vector a delivery system. It was a piece of DNA that allowed replication in bacteria, so we could produce a large amount of this plasmid DNA. Then we introduced this piece of DNA to mouse stem cells, through transfection. The cell that picked up our vector became resistant to the neomycin antibiotic.
“Then we looked for homologous recombination, which means that the piece of DNA had to integrate into the Muc2 gene. It couldn’t be introduced just any place in the cell’s genome. That inactivation had to be targeted. The neomycin-resistant gene was flanked by sequences that were homologous to a portion of the Muc2 genes. It’s not a very frequent event, but it occurs, so we took advantage of it.
“The result,” Velcich summed up, “is that the first-generation progeny were born heterozygous. Only one of their two Muc2 alleles was inactivated. Knocking out both alleles was obtained only upon mating. Both alleles were then inactivated, and they didn’t produce any Muc2 protein. Then spontaneously we didn’t have to do anything with time they developed colorectal tumors.” Q.E.D.