A ballsy search for cancer targets

There are no magic bullets in the fight against cancer. But by targeting proteins found almost exclusively in tumor cells and the testes, researchers may have discovered the closest thing yet. Megan Scudellari explores how a handful of young investigators hope to turn magic into reality.

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Staring at the list of proteins on her computer screen, Angelique Whitehurst was baffled. It was 2006, and Whitehurst, then a postdoc at the University of Texas Southwestern Medical Center in Dallas, was harnessing the recently discovered power of genome-wide RNA interference to single out the genes and their corresponding proteins responsible for drug resistance in human cancer cells. Although her screen flagged many of the usual suspects, including dozens of proteins involved in cell division and protein translation, one subset of molecular targets stood out like a sore thumb. “I kept thinking, 'What are all these testis proteins doing in here?'” she recalls.
Whitehurst, an energetic blond with a contagious smile, plopped down in front of her computer and dug into the literature. The interlopers, she found out, were cancer-testis antigens, or CT antigens, members of a group of proteins normally only highly expressed in the germ cells of the testes (and to a lesser extent in the ovaries), yet also found in a wide variety of tumor types.
That fortuitous discovery changed Whitehurst's research trajectory. Four years after publishing the finding¹, Whitehurst has devoted her lab to teasing apart what the elusive CT antigens actually do in cancerous tissues. That knowledge, she now believes, could lead to the development of new, more sophisticated cancer drugs.
CT antigens—so named because they can evoke an immune response in people with cancer—are tantalizing therapeutic targets because of their unique pattern of expression in the body. Since they are not highly expressed outside testes or ovaries, drugs aimed at blocking their function should only affect tumor cells, with no effect on noncancerous tissues outside the gonads. Although such drugs could damage people's reproductive organs, researchers see this as an acceptable risk for cancer patients, the majority of whom are well into their golden years when diagnosed with the disease.
With that in mind, if drugs that target CT antigens pan out, they could be the nearest thing oncology has to a magic bullet. Unlike chemotherapy regimens—which kill healthy cells in addition to tumor cells, leading to common side effects such as bleeding, pain and hair loss—wiping out cells expressing CT antigens should theoretically cause “no side effects, no off-target effects on normal tissues, none at all,” says Steven Rosenberg, head of tumor immunology at the US National Cancer Institute in Bethesda, Maryland.

Dan Sears

Today, Whitehurst and a small but devout band of young investigators, many of whom stumbled on CT antigens through unbiased genetic screens like hers, are rapidly identifying the functions of this unique subset of proteins. Some of these antigens degrade tumor suppressors, they are learning, whereas others protect cancer cells from deadly replication errors. Notably, such findings are having immediate applications in vaccine clinical trials. Even more importantly, the research could lead to the development of small-molecule drugs with the potential to succeed where cancer vaccines have failed.
“It's a really ripe field that we can do a lot in, because not much is known about these antigens,” says Whitehurst, now an associate professor at the University of North Carolina at Chapel Hill School of Medicine. “There's just enough known to know they are important.”

Name calling

Part of the reason that CT antigens have flown under the radar, scientists say, comes down to their peculiar name. Twenty years ago, researchers at the Belgian branch of the Ludwig Institute for Cancer Research discovered the first of these proteins in a human melanoma cell line. Because it evoked an immune reaction by activating human T cells in vitro, they accurately but unfortunately labeled it 'melanoma antigen family A, 1′ (MAGE-A1).

“Targeting CT antigens should theoretically cause no side effects, no off-target effects on normal tissues, none at all.”

Despite the fact that the finding appeared in Science², most people within the cancer field took no notice of the work. “To basic biologists, [an antigen] wasn't very interesting,” says Ryan Potts, a tumor immunologist at UT Southwestern Medical Center.
In addition to the alienating name, there was also an experimental hiccup: with no direct equivalents of many important human CT antigens in mice, cancer researchers couldn't study the proteins in animal models. And as Andrew Simpson, scientific director of the Ludwig Institute's New York branch, puts it: “If you can't study it in the mouse, it might as well not exist.”
Within immunology circles, however, CT antigen research continued, focused primarily on vaccines. In the 1990s, the discovery of MAGE-A1 was followed by the isolation and characterization of similar melanoma-associated antigens (MAGEs). Soon thereafter, scientists identified NY-ESO-1, a different type of antigen expressed in normal testes and ovaries as well as across a wide array of human cancers, including those of the skin, breast, bladder, prostate and kidney³.

UT Southwestern Medical Center

Both MAGE and NY-ESO-1 soon became major targets of experimental therapeutic vaccines. These vaccines delivered some form of the CT antigen—a strand of DNA or a segment of the protein—in the hopes of activating a T cell immune response that would destroy this foreign agent as well as any cancer cells expressing it.
To date, NY-ESO-1 has been the subject of more than 40 phase 1 clinical trials and MAGE the focus of 20. “But none of those have been very successful,” says Rosenberg.
A few products have made it to later-stage testing. For instance, the British drug giant GlaxoSmithKline (GSK) is currently running two phase 3 trials of a MAGE vaccine—one in 1,300 people with stage 3 melanoma, and a second in 2,270 people with non–small cell lung cancer—with a smaller trial ongoing in people with metastatic melanoma. But Lloyd Old, director of the Ludwig's New York branch, is not holding his breath that these will work. A central reason these cancer vaccines have failed in the clinic, he says, is that tumors are skilled at subverting immune attacks. “And without controlling cancer immunosuppression,” he argues, “we will not see the potential of [CT antigen–based] cancer vaccines.”
A more fundamental problem could be simply that researchers don't know what CT antigens actually do in normal or cancerous tissues. Old says that CT antigens might have a critical function during the early development of an embryo, but it may be one that can be dangerous and cause malignancy in adult cells. “It may be that cancer reinitiates a program that is part of gametogenesis, and therefore acquires all the attributes of pregnancy and development,” he says.

NYU Langone Medical Center

Old's hypothesis is echoed in the history of cancer research. In 1902, the Scottish embryologist John Beard proposed the 'trophoblast theory of cancer', the idea that tumor cells are virtually indistinguishable from trophoblasts, rapidly-growing cells that surround an embryo during development. The concept is also similar to the cancer stem cell theory, which posits that cancer is caused and maintained by a small group of hardy, self-renewing cells. Nowadays, mounting evidence has linked germ cell development and cancer progression: cancer cells and early embryonic cells produce similar hormones, grow rapidly, form blood vessels to support themselves, and locally suppress the immune system.
Today, Whitehurst and others, including Hearn Cho of the New York University Cancer Institute, are poised to prove or disprove the cancer-development link by determining the role of CT antigens in tumor cells. “It's surprising that it's taken this long for people to get interested in function,” says Cho. “From a 10,000-foot view, understanding the relationship between [CT antigens'] normal function and their pathological function in cancer is going to be very enlightening.”

Standing up to antigens

In Whitehurst's sunlit office, extensive lists of CT antigens fill a whiteboard on the wall: some boxed into categories, others punctuated by question marks, with last-minute additions squeezed into corners. “We want to see what's in there that's fundamentally targetable,” says Whitehurst. “We'll only know after we do it.”
Earlier this year Whitehurst received a three-year, $750,000 grant from Stand Up To Cancer, a California-based charity, to study 115 CT antigens found in solid tumors. With the award, she plans to compartmentalize the antigens into functional groups based on their roles in tumor cells, then narrow in on which might be the best therapeutic targets.
To this end, Whitehurst will do what she does best—high-throughput RNA interference screens. By depleting the expression of each CT antigen in tumor cells derived from a variety of cancers, she hopes to reveal how CT antigens are involved in well-known tumor-associated pathways, such as the p53 and Wnt pathways. Small-molecule drugs targeting these cancer-specific pathways could open up a whole new therapeutic arm of drug treatment, and prove more successful than the lackluster CT antigen vaccine trials, she says.
Whitehurst has already validated the utility of this approach. Reporting last year, she showed that acrosin binding protein (ACRBP)—one of the four CT antigens reported in her original 2007 paper—helps cancer cells survive by allowing tumor cells to divide even in the presence of significant mitotic abnormalities. She also studied patient tumor samples and found that high expression of ACRBP correlated with reduced survival time and faster relapse rates among people with ovarian cancer⁴.

“Understanding the relationship between CT antigens' normal function and their pathological function in cancer is going to be very enlightening.”

The discovery suggests that the ACRBP pathway could be an ideal therapeutic target, especially in ovarian cancer. If the antigen expression is reduced, errors in cell division might hamper cancer's ability to thrive. But ACRBP is not the only CT antigen whose function is now known.
Around the same time that Whitehurst was tackling ACRBP, Ryan Potts, then a doctoral student who also happened to be at UT Southwestern, stumbled across a MAGE protein in an independent yeast study. To figure out its function, he did protein purification and mass spectrometry experiments involving it and eight other MAGE proteins.
In the end, Potts showed that the various MAGE proteins he studied fuel tumor growth by binding to and enhancing the activity of ubiquitination enzymes that help prime p53, the potent tumor suppressor, and other proteins for degradation by the cell's machinery. “That potentially gives us a mechanism of how these MAGE proteins are functioning as oncogenes,” says Potts, who published the results last year⁵.
Independent validation for these findings came earlier this year when NYU's Cho reported similar data showing how MAGE proteins can disrupt p53's antitumor activity⁶. “There has been speculation for decades [over] whether MAGE or other CT antigens are functionally important,” he says. “Our data show they are.”
Similarly to Whitehurst, Potts is now beginning a systematic examination of the tumor-causing potential of all 50 MAGE proteins. He plans to study which of the members of the MAGE family cause normal human cell lines to turn cancerous and which promote tumor formation in mouse models.
Cho, meanwhile, is moving his research into the clinic. On the basis of some of his work linking MAGE proteins to multiple myeloma, he convinced GSK to launch another clinical trial with its MAGE vaccine product. The 16-person study, conducted in collaboration with the Ludwig Institute, opened on 15 June.
Cho is optimistic that functional knowledge will improve current vaccine trials. But the real value, he notes, may be in the development of small-molecule drugs targeting the newly discovered functional pathways. “Whether it's specifically targeting [CT antigen] activity or hitting important downstream targets...this is a very exciting development,” he says. “I think we're just scratching the surface here. It's a new frontier, undeveloped in cancer research.”