Ribonucleases May Hold Clues to Killing Cancer

University of Wisconsin biochemistry professor Ronald T. Raines nominates pancreatic ribonuclease A (RNase-A) as "probably the most-studied enzyme of the 20th century." But in the next breath, he admits, "There are ribonucleases floating through your bloodstream right now, and we don't really know [their function]." Raines and Richard Youle, director of the biochemistry section in the National Institute of Neurological Disorders and Stroke at Bethesda, Md., led two research groups that are friendly rivals--sharing reagents and corresponding, but not collaborating--in a competition to discover how ribonucleases can best be used to treat cancer. Both groups published papers recently that bore remarkable resemblances in their methods and results, giving both men added hope that they're headed in the right direction through a field still clouded by enigma. Among what is known about the RNase A superfamily is that the enzymes are potent catalysts of RNA cleavage. However, human RNase (hRNase-A) is not cytotoxic. Produced by the pancreas and secreted into the cell's endoplasmic reticulum (ER), from there entering the gut, it is thought to aid in digestion. The Northern leopard frog (Rana pipiens) carries a homologue of hRNase-A, called onconase, that exhibits potent anticancer activity in vitro. Publicly held biotechnology company Alfacell Corp. of Bloomfield, N.J., currently has Phase III clinical trials under way to treat malignant mesothelioma, the asbestos-related cancer, with a drug based on onconase. Work by Youle's and Raines' groups has helped to clarify how onconase and a few other nonhuman ribonucleases, including that found in the bovine pancreas and semen, manage to be cytotoxic. Last fall, Raines and colleagues showed that the ability of a ribonuclease to resist binding to a protein aptly called Kibonuclease Inhibitor (RI) determines its cytotoxicity.1 This month, Youle's team published findings that hRNase altered by addition of the human iron-storage protein, transferrin, not only becomes 5,000 times more toxic than wild-type hRNase but will target cancer cells in vitro.2 An objective of both researchers was to enhance RNase cytotoxicity by blocking its binding with RI. The method both chose was steric hindrance, creating a dimer or molecular compound that they then placed at the RI receptor site of the RNase. Youle calls steric hindrance a crude but effective tactic, "like putting a boulder in the doorway." Raines thinks of it as "protein terrorism" rather than engineering, because the goal is to destroy RI. His dimer included an extra amino acid instead of transferrin. Both ways worked, but transferrin dramatically increased uptake of RNase specifically in cancer cells, which express many iron receptors on their surfaces. Both groups are trying to discover how RNase gets into the cytosol--that part of the cell excluding the mitochondria and the ER--where RI and most RNA are found. "It's known that ribonucleases, to be cytotoxic, need to be active enzymes," Raines says. "They need to be able to cleave RNA." Youle explains, "Although it's very logical that RI is binding to ribonucleases, nobody has been able to point out a place in the cell where both happen to be together." Other questions concern safety and efficacy in vivo. Youle, who has begun working with mice, says an advantage of RNase is, "It's very much like self. The toxins are completely different; there's nothing remotely like them in your body. The more foreign a substance is, the more rapidly it builds antibodies." Raines reveals that his team is characterizing other ribonucleases that are "more toxic than onconase." He adds, "It's not clear yet how broadly useful ribonucleases will be. I think everyone's waiting to see what happens with onconase." Stanislaw M. Mikulski, executive vice president and medical director of Alfacell, says accrual has been completed for the Phase III trial. Funded entirely by Alfacell, it involves 152 patients in 33 centers nationwide who primarily suffer from unresectable mesothelioma. In the randomized design, 91 people will receive onconase as a sole agent and 61 will be given the chemotherapeutic drug doxorubicin (Adriamycin). Side effects of onconase, including some renal function problems, lassitude, and flu-like symptoms, have been relatively mild to date. Hair loss or major organ toxicities have not been observed. There is no reliable treatment for malignant mesothelioma and once the disease manifests, average survival is one to two years. Mikulski expects results to be available in about six months. "Tumor cells develop resistance to single-point compounds because they are extremely resilient and smart, if you will, about how to beat the assault," he comments. However, there is evidence that onconase does not merely degrade RNA--it can attack several points in the regulatory mechanisms of cell life and tumor development. For example, it increases expression of other inhibitor proteins, downregulates a cyclin important to the cell cycle, and activates apoptosis.3 Despite remaining uncertainties about the protein's mechanism, Mikulski is sure of one thing: "There is a fabulous lesson of nature in what we are observing." P.A. Leland et al., "Ribonuclease A variants with potent cytotoxic activity," Proceedings of the National Academy of Sciences, 95:10407-12, Sept. 1, 1998. M. Suzuki et al., "Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction," Nature Biotechnology, 17:265-70, March 1999. G. Juan et al., "G1 arrest of U937 cells by onconase is associated with suppression of cyclin D3 expression, induction of p16INK4A, p21WAF1/CIP1 and p27KIP and decreased pRb phosphorylation," Leukemia, 12:1241-8, August 1998.

Ribonucleases May Hold Clues to Killing Cancer

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