Yale researchers have identified an unusual molecular process in normal tissues that causes RNA molecules produced from separate genes to be clipped and stitched together. The discovery that these rearranged products exist in normal as well as cancerous cells potentially complicates the diagnosis of some cancers and raises the possibility that anti-cancer drugs like Gleevec could have predictable side effects.The work is reported in the journal Science.

“Our findings are surprising because we identified in normal cells certain types of gene products— so called chimeric RNAs and proteins—thought to be found only in cancerous cells or in cells on their way to becoming cancerous,” said Jeffrey Sklar, professor of pathology and laboratory medicine at Yale School of Medicine, and senior researcher on the study.

Chimeric proteins are considered to be key factors that drive many forms of cancer. They arise from chromosome abnormalities in which segments of the chromosomes are rearranged. At the sites where chromosome segments reattach, genes fuse giving rise to chimeric RNA, which in turn is used to construct the chimeric protein. Gleevec, a highly successful new anti-cancer drug, was developed to target the chimeric protein product of one such gene fusion.

Sklar’s group earlier discovered that a particular gene fusion, with its associated chimeric RNA and protein, is the probable cause of certain endometrial cancers. Unexpectedly, they also found the same chimeric RNA and protein in healthy uterine tissue — where the chromosomes and genes showed no abnormalities.

“Extensive experiments on the normal tissues and cultured cells from those tissues indicated to us that a previously little-known process, the direct splicing together of two RNAs from separate genes—or trans-splicing—is responsible for producing the chimeras,” said Sklar.

They also found that level of the chimeric molecules in normal cells was decreased by elevated estrogen and increased by reduced oxygen — conditions that control the synchronized cyclic behavior of normal cells that line the inside of the uterus.

These observations suggest that trans-splicing between the RNAs might be common in other normal tissues, because gene fusions have been identified in cancers that arise in many tissues.

“These findings may bring new insights into how cancers operate. It seems that rather than scrambling chromosomes to invent new genes, cancers mimic normal cellular processes, but in an exaggerated and unregulated fashion. You might say that cancers are clever but not very original,” said Yale Research scientist Hui Li, lead author of the paper.

According to the researchers, these results indicate that caution should be exercised in using chimeric gene products as markers for cancer, as is widely done now in cancer diagnosis. Additionally, cancer drugs that target products of chromosomal abnormalities may have varying degrees of toxicity because those same targets may be present in normal cells due to the trans-splicing of RNA.

A  group of small, non-coding RNA molecules may serve as a future marker to improve cancer staging and may also be able to convert some advanced tumors to more treatable stages, report a University of Chicago-based research team in the April 1, 2008, issue of the journal Genes & Development.Carcinomas are cancers that develop from epithelial tissue, which lines internal and external body surfaces. When normal cells are transformed into cancer cells, this epithelial tissue can take on the characteristics of embryonic tissue, known as mesenchymal tissue, which is comprised of unspecialized cells that will develop, as the embryo matures, into more specialized tissues.

That process also goes in reverse. Epithelial to mesenchymal transition (EMT) occurs, for example, during wound healing. In cancer, however, this process can produce invasive and mobile cells that can pass through membranes and travel to distant sites, where they seed new tumors.

“There are a bewildering numbers of pathways or stimuli that can either trigger EMT or reverse that process,” said study author Marcus E. Peter, PhD, professor in the Ben May Department for Cancer Research at the University of Chicago. “What we have identified is a master regulator of EMT that is probably controlled by many of these stimuli.”

Peter and colleagues showed that this master regulator consists of a specific group of microRNAs, a family called miR-200. MicroRNAs are tiny RNA molecules that have very important roles in gene regulation. They have multiple targets and act mainly by attaching themselves to specific sites in messenger RNA to prevent the production of proteins.

The authors studied a standard panel of 60 established human tumor cell lines representing nine different human cancers, as well as several specimens of human primary ovarian cancer. They showed that miR-200 was always present in epithelial (less invasive) and not in mesenchymal (more invasive) types of tumors.

“The importance of this finding is, first, that miR-200 may represent a good marker to stage cancer,” Peter said, and “second, that reintroducing miR-200 into late cancer cells could provide a new form of treatment, preventing these cells from going through EMT and becoming more invasive.”

Physicians already have a set of fairly reliable markers for carcinoma. Tumors with high levels of E-cadherin tend to be tightly tethered to nearby cells and less likely to break free and travel to other sites. Those with high Vimentin levels represent mesenchymal cells able to pass though other tissues.

Peter and colleagues found that miR-200 added mechanistic depth to those markers. Every tumor cell line the researchers tested that had the epithelial marker E-cadherin and not the mesenchymal marker Vimentin, had high amounts of miR-200. Every cell line with high Vimentin and no E-cadherin had no detectable miR-200.

“So we were able to show a complete correlation between miR-200 and E-cadherin/Vimentin expression,” Peter added.

The authors found that miR-200 microRNAs helped regulate EMT transition. They bind directly to non-coding regions in the RNA of ZEB1 and ZEB2, known blockers of E-cadherin transcription. Both ZEB proteins have previously been implicated in human malignancies, ZEB1 in aggressive colorectal and uterine cancers, and ZEB2 in advanced stages of ovarian, gastric and pancreatic tumors.

By inhibiting miR-200, Peter and his coworkers could induce EMT. More important, by introducing miR-200, they managed to activate production of E-cadherin protein and reverse tumors from a more-invasive mesenchymal into a less-invasive epithelial form.

“In a previous paper we found that another micro RNA, let-7, drives tumor progression at an earlier stage,” Peter said. “Let-7 appears to be a key player in preventing a cancer from becoming more aggressive. Now we want to figure out how these two micro RNAs work together to regulate carcinogenesis.”

Once they understand this process, they want to use these microRNAs to treat cancer. Both microRNA families have the connection to drug resistance as well as to cancer stem cells, sub-population of cancer cells that have self-renewal properties and the ability to give rise to new tumors that are more resistant to current therapy.

“Our aim is not only to make tumors less invasive by reintroducing let-7 and miR-200,” explained Peter. “We hope that we’ll make tumors more sensitive to drugs and be able to target the stem cell population, which gives tumors their renewal capacity.”

“The idea is a two-hit strategy,” Peter said, “hit them first with the microRNA and make those drug-resistant cells sensitive again, then hit them again with low levels of conventional chemotherapy.”