DALLAS--A team of developmental biologists examining how tumor growth factor (TGF)-beta sends messages into cells has come up with the first animal model for colon cancer that closely mimics the human disease.
The "knock-out" mouse lacks the gene for the intracellular protein Smad3, which transduces signals from the TGF-beta receptor to the cell nucleus and alters gene expression. Mice without Smad3 cannot respond to TGF-beta regulatory signals. They also develop colorectal cancers remarkably like those in humans. Such tumors are deeply invasive, occur at both ends of the bowel, and metastasize to lymph nodes.
The new mouse model "should provide a powerful tool for identifying the genetic and molecular alterations that lead to malignancy" as well as providing a useful model for trials of drugs to prevent or treat human colorectal cancers, the researchers said in their report in the September 18th issue of Cell.
The research group is led by Luis F. Parada, PhD, and Jonathan M. Graff, MD, PhD, and includes James A. Richardson, DMV, and graduate student Yuan Zhu, who made some of the key observations and is first author on the Cell paper. All are at the University of Texas Southwestern Medical Center.
"We have a particular interest in how external signals are transmitted to the cell nucleus, resulting in modification of gene expression and leading to organized embryogenesis," Dr. Parada told Oncol-ogy News International. "Jon Graff had discovered and studied Smad genes in the frog, and we decided to investigate these genes further in the mouse."
The investigators expected that homozygous Smad3-negative mutations would be lethal during embryonic development and that heterozygotes (having one normal Smad3 gene) might eventually develop some form of cancer.
Mr. Zhu mutated the mouse Smad3 gene to test this idea (see figure). The gene was inactivated in mice by homologous recombination.
"To our surprise (and initial chagrin), the homozygous mice were born and appeared normal," Dr. Parada said. Smad3 was clearly not required for normal embryonic development. However, Mr. Zhu kept the mice and examined them regularly to see what would happen as they aged. "To our delight, colon cancer developed in these mice after several months," Dr. Parada said.
The final surprise was that colon cancer developed in all 129 of the inbred mice who were homozygous for the Smad3 mutation. Heterozygous mice are viable and fertile, and have been observed for up to 9 months with no apparent signs of disease.
The current model for development of colorectal adenocarcinoma suggests that the disease progresses from normal to hyperplastic colonic epithelium, then to benign adenomatous polyps and to invasive and then metastatic carcinoma.
"It would appear that this Smad3 knock-out mouse resembles the histo-pathology of the human condition with considerable fidelity," Dr. Parada said. "If this is the case, given the 100% incidence of cancer within a defined window of time, these mice may be an ideal substrate for therapeutic and pharmacological studies." Among the similarities to human disease: The mouse tumors are invasive and metastatic, limited to the colorectum, found mostly at the end of the lower intestine and at the boundary with the small intestine, and exhibit most of the stages of colon cancer progression.
To produce the homozygotes, the chimeric mice (lacking a Smad3 allele) were bred both with the mouse strain from which the blastocyst came, to produce an inbred strain of Smad3-negative heterozygotes (see figure), and with a different strain, to produce hybrid heterozygotes.
Mating heterozygotes from the inbred strain to each other resulted in the Smad3 homozygous mice who all developed colon cancer. However, homozygotes produced by mating heterozygotes from the hybrid strain were less likely to develop colon cancer, and, when they did, the tumors developed much later.
"This raises the new question of what are the ‘modifier’ genes present in the other mouse strain that so dramatically reduce the incidence of cancer," Dr. Parada said. Finding out why these homozygous mice are less susceptible to colon cancer could open the door to new preventive approaches in humans.
The findings confirm a central role for the loss of TGF-beta signaling in at least some forms of colon cancer. TGF-beta has a variety of functions, including inhibiting the proliferation of cells such as colonic epithelium. A TGF-beta receptor mutation has also been reported in a patient with colorectal cancer.
Three receptors are involved in transducing TGF-beta signals: Smad2, Smad3, and Smad4. Previous attempts to produce homozygous Smad2 or Smad4 knock-out mice were unsuccessful because the mice all died in utero or shortly after birth, long before tumors could form. Successful production of Smad3 knock-outs that develop colon cancer constitutes the "proof of principal" that loss of the ability to respond to TGF-beta sets up the colon for neoplastic change.
