7 October 1997 UROT97\CAVEOLIN.MS-- 830 words
Second PCa metastasis gene found
Caveolin protein is the difference
Lake Tahoe, NV--One cell in a human prostate tumor is metastatic, while its sister cell, in the same cell line, is not. What's the difference between the two?
In this case, the only difference is that the metastatic cell expresses the protein caveolin, Timothy C. Thompson, PhD, of the Baylor College of Medicine, Houston, TX, discovered. Caveolin is not found in normal prostate epithelial cells, or in carcinoma in situ, but it's "very prominently" expressed in metastatic cells, he said. So now two genes have now been identified as promoting prostate cancer metastasis. (The other is thymosin beta-15.)
The tool that found caveolin's role in metastases was a mouse system for comparing cancer cells, Dr. Thompson explained at the Fourth Annual Scientific Retreat of The Association for the Cure of Cancer of the Prostate (CaP CURE) here this September.
Dr. Thompson's group at Baylor used differential display of messenger RNA (mRNA) to find the difference between the 2 cell lines. This technique can pinpoint the genes that mutate from one generation to the next as a cell line becomes more aggressive.
The mRNA from the non-metastatic cancer line was converted to complimentary DNA (cDNA) and amplified with polymerase chain reaction (PCR). The cDNA was sorted out by molecular weight on a gel. Then the mRNA from the metastatic line was treated the same way. The gels looked identical except where the mRNA was unique to one line--and differential mRNAs were examined more closely as candidate oncogenes. Dr. Thompson identified one mRNA as a sequence that produced caveolin.
"Caveolin is normally expressed in endothelial cells, in some types of smooth muscle cells," said Dr. Thompson. It is a structural protein of cavaolae, an organelle in the trans-Golgi network. It is involved in the localization of signal transduction molecules, which may behave abnormally in cancer.
"In normal prostate epithelial cells, caveolin is expressed at low or undetectable levels," said Dr. Thompson. "In metastases it's very prominently expressed, in the mouse system and in humans. It seems to be involved in the way that prostate cancer metastasizes, and breast cancer as well." In 51 radical prostatectomy specimens, 43 stained for caveolin, he previously reported.
Dr. Thompson also demonstrated that caveolin (with other factors) actually caused metastases. When he turned the caveolin expression back down, the cells weren't as metastatic. "We took 3 metastatic cell lines, and made anti-sense clones," by inserting anti-sense sequences into the cell lines, he said. That reversal lowered the cells' caveolin protein production "substantially," and also lowered their metastatic activity, when they were returned to mouse prostates.
In order to develop prostate cancer in mice, Dr. Thompson first knocked out a DNA error-correcting mechanism, and then infected them with a virus that turned two important cell cycle growth switches permanently on. He started with cancer-susceptible mice that had the p53 gene knocked out. "p53 is the classic tumor suppression gene," he said. It is now becoming accepted that loss of p53 is associated with metastases. The p53 gene, on chromosome 17, normally stops cell division in the G1 stage when DNA is damaged, and makes the cell wait until the DNA is repaired.
Dr. Thompson then used a retrovirus to transform the ras and myc oncogenes. The normal ras and myc genes promote cell growth at appropriate times in the cell cycle, but the ras and myc oncogenes just keep on promoting cell growth. As an added benefit, the retrovirus marks the clones for identification.
"From the p53 knockout mouse, we use the urogenital sinus tissue that derives prostate cancer, we infect it with this virus, and it makes primary prostate cancer," said Dr. Thompson. Then they transfer the prostate cancer cells into a normal male mouse.
"These tumors are polyclonal, highly heterogeneous, similar to human prostate cancer," said Dr. Thompson. "But only a few of those cells will metastasize."
Dr. Thompson's group found metastatic cells, and then went back to get clones of their original primary tumors, he said. "They are clonally marked so we can grow cell lines out of them and know that the metastasis was derived from the same clone of the primary tumor."
"Then we do differential display," said Dr. Thompson. There is a long process of screening, and using fragments as probes on Northern blots. "Ultimately we confirm or refute that a particular sequence is overexpressed or underexpressed in the metastases relative to the primary tumor."
Other genes that have been implicated in prostate cancer are thymosin beta-15 and kai-1. The thymosin beta-15 gene was similarly discovered by comparing a metastatic and non-metastatic prostate and then breast cancer cell lines in rats and humans. Thymosin beta-15 binds to actin, and regulates cell motility, but beyond that the mechanism is murky.
The kai-1 gene, as its name implies, is a tumor suppressor gene that suppresses cell motility, and declines when prostate cancer becomes metastatic. "kai" stands for kang ai I, Chinese for "anticancer".