Clinical Trial Inspired by Breast Cancer Cells, Cows, and Collaboration

NCI CAM Annual Report-FY10

With more than 13 years of experience running early phase clinical trials, Raymond Perez, M.D., currently the Medical Director of the Clinical Research Center at the University of Kansas, knows that in early drug development “you can’t sustain a clinical program with just one line of research. You have to look all over for opportunities for treatments that are ready to test in people, and help investigators make that happen.”

Dr. Perez and his former colleague Wil­liam Kinlaw, M.D., Professor of Medicine at Dartmouth Medical School (where Dr. Perez led the Phase I clinical trials program at the Norris Cotton Cancer Center from 1998 until 2011 before heading to Kansas) have pursued one such opportunity down a winding path from basic lipid metabolism studies to test­ing in dairy cows, and now to an early phase, NCI-funded clinical trial* testing conjugated linoleic acid (CLA) as a targeted anti-cancer agent.

About 10 years ago, Dr. Perez heard Dr. Kinlaw give a presentation on a protein called Spot 14, which helps cells produce necessary fatty acids from glucose. When the researchers blocked Spot 14 with an antisense nucleotide (a single strand of DNA or RNA, produced in the lab, which will bind to a correspond­ing section of DNA or RNA in the cell and interfere with production of proteins), they saw breast cancer cells die in laboratory cell cultures. “After Dr. Kinlaw’s presentation, I approached him and started talking about Spot 14 and he’d never really thought about it therapeutically,” recounted Dr. Perez.

Over the next several years, Dr. Kinlaw, an endocrinologist by training, moved his labora­tory over to the Norris Cancer Center and his team engaged in a frustrating search for a drug to potentially target Spot 14 in people. No pharmaceutical company would com­mit to producing antisense against Spot 14, he said. Some drugs that he and Dr. Kinlaw tested in animals – such as Cerulenin (an antifungal agent) – were too toxic. Other drugs – such as Orlistat (a weight-loss drug) – were not absorbed into the bloodstream after oral administration.

The researchers put the idea of an anti-Spot 14 therapy aside for a while and concentrated on understanding its biology, showing that the protein is a marker for aggressive primary breast cancers.** Then, serendipitously, Dr. Kinlaw received a call from Dale Bauman, Ph.D., a professor of animal science at Cornell University. Dr. Bauman had been looking at a condition in dairy cows called milk-fat depres­sion. The condition happens when cows eat plants in the wild that contain linoleic acid. Bacteria in their stomachs convert linoleic acid into another compound called conjugated linoleic acid (CLA), which is absorbed into the blood stream and suppresses milk-fat production.

In an experiment, he had fed some cows linoleic acid-containing plants and gave other cows injections of pure CLA, and then performed microarray analysis to analyze gene expression changes. “The biggest hit he got from both was that Spot 14 was turned off,” Dr. Kinlaw recalled. “Dr Bauman had never heard of Spot 14 before so he called me out of the blue.” Interestingly, CLA also interfered with an enzyme called lipoprotein lipase, which cancer cells can use to extract fatty ac­ids from the local circulatory bed, Dr. Kinlaw added.

“We had a series of discussions about this and the obvious thing that came up was that if CLA could shut down lipid synthesis in the cows’ mammary epithelial cells, maybe it could do the same thing in malignant human epithelial cells,” Dr. Kinlaw commented. “We got some CLA and tested it in breast cancer and liposarcoma cells, and it inhibited their growth quite promptly.” ***

CLA is used as a dietary supplement and had shown no severe side effects in a clinical trial testing it for weight loss, Dr. Kinlaw contin­ued. He and other investigators performed a small proof-of-concept pharmacology trial in 23 women diagnosed with breast cancer. Between the 10 to 12 days from diagnosis to mastectomy, the women received 7.5 grams of CLA per day and then tissue taken during their surgeries was examined for Spot 14. “CLA significantly turned down Spot 14 in the tumors, so it did seem to actually work in humans,” Dr. Kinlaw noted.

“We realized that CLA might be the compound that could hit this target; now we have to prove it,” Dr. Perez said. In the ongoing Phase I trial, they will first determine the recommended therapeutic dose of CLA. The study dose will be escalated in patients until side effects are observed or the larger doses no longer increase the effect of CLA on Spot 14, he added. In this first part of the trial, samples of fat tissue from patients will be used to measure the activity of CLA.

Once the recommended dose has been deter­mined, a second group of patients will be given CLA and tumor biopsies will be taken to make sure CLA is hitting its target in the tumors, Dr. Perez continued. The researchers will also look at the effect of CLA on proteins involved in the Spot 14 signaling pathway that also play a role in fat metabolism and examine the com­pound’s effects on cell proliferation, apoptosis (programmed cell death), and other measure­ments of tumor growth.

Dr. Kinlaw is currently writing a grant appli­cation to study the mechanisms behind CLA’s effects on Spot 14 in more detail, and also to explore how the compound might be most effectively delivered to tumors, using mice genetically engineered to lack either Spot 14 or lipoprotein lipase. “These are pretty good models to help us figure out how CLA might be working in humans,” he said.