Editor's note: This article has been updated with a correction to the image caption. The image depicts a tumor cell cluster, not an individual tumor cell.
NCI Frederick’s Frank Cuttitta, Ph.D.; academic scientists; and an antibody manufacturer have unmasked an enigmatic protein and overturned decades of convention surrounding its relevance to cancer and biology.
They’ve demonstrated that the appropriately named Cripto-3 (CR3), a protein long believed to be a negligible genetic byproduct in humans, is actually produced in multiple physiological settings.
They’ve also identified some tissues in which it can be found and have confirmed its presence in some human cancers, raising it as a potential target for treatment.
Their findings appear in Cancers.
Confirmation that CR3 is a produced protein now opens the door to a multitude of studies into its functions and involvement in health and disease. Many questions about the nature of those functions remain, but the scientific community has the ability to begin answering them.
“The collective Cripto field of study has to rethink what role CR3 may play in normal and malignant physiology,” said Cuttitta, the study’s lead author and a special volunteer in the Tumor Angiogenesis Unit of the Mouse Cancer Genetics Program at NCI Frederick.
Role in Tissues, Role in Cancer, Many Opportunities
CR3 was long an inscrutable subject due to its similarity to a related, more-understood protein, Cripto-1 (CR1).
The two differ by just six of the 188 amino acids spread throughout their structures. They’re the molecular equivalent of identical twins who, in passing, can only be differentiated by a few freckles, a hairstyle, or a slight lilt in voice.
Until now, there were no tools to make that distinction, leading some scientists to believe that CR3 was just the byproduct of a pseudogene, a section of DNA that doesn’t make a protein or other products. Past studies showed the possible existence of CR3 in humans, but unable to see past CR1, couldn’t confirm it had meaningful biological roles.
But the team was able to tell the difference between CR3 and CR1 thanks to a new antibody-based approach. That meant they could begin figuring out CR3’s roles in the human body.
To that end, they ran a series of assays and experiments on biopsies and blood samples obtained from people with and without cancer, trying to pinpoint where CR3 was and what it was doing.
“We had to be extremely critical of our approach since our reported data was going against previously established conventions,” Cuttitta said.
They found that CR1 and CR3 can exist in different tissues, a phenomenon they termed “anatomical separation.” That, in turn, was evidence that CR3 has a genuine function separate from that of CR1. As a rule, cells don’t produce near-identical proteins with overlapping functions, Cuttitta said, so seeing CR3 and CR1 sequestered in separate tissues was a sign that they’re both unique.
The team also found CR3 was prevalent in different parts of tumors than CR1—more evidence. Its relationship with patients’ prognosis and disease severity likewise differed from that of CR1.
The data suggest future research should investigate CR3’s role in cancer in more detail and assess its potential as a cancer target, Cuttitta said. The team has already begun some studies in that area.
New Antibodies Allow Scientists to Pinpoint CR3 in the Human Body
Two new antibodies, NCI 5G1-1 and NCI 5G11-2, designed by GenScript Biotech, were the key to at last distinguishing CR3. In this case, the antibodies acted like molecular signal flares, attaching to a target protein and tagging it so that scientists can detect it via various experimental methods.
Cuttitta and colleagues enlisted GenScript to handle the herculean task of designing the antibodies, with input from the rest of the team. Design efforts zeroed in on a small structural difference between CR1 and CR3 that the team believed might be enough to distinguish one from the other. If they got it right, an antibody designed to attach to and tag CR3 shouldn’t attach to CR1, and vice versa.
The antibodies had to be engineered from scratch, a lengthy and intricate process that involved using mouse antibodies as a base.
Yet it paid off. Across a series of experiments, NCI 5G1-1 proved capable of tagging CR1 without erroneously tagging CR3, and NCI 5G11-2 likewise tagged CR3 without tagging CR1. None of the other commercial antibodies the team tested was able to do that.
With the mysterious CR3 revealed and distinguishable, studies like the team’s are just the beginning.
“Given the fact that selective anti-CR1/CR3 reagents have never been available before, an entire untapped investigative avenue in the Cripto field of study is now open utilizing our NCI reagents,” Cuttitta said.
Samuel Lopez leads the editorial team in Scientific Publications, Graphics & Media (SPGM). He writes for newsletters; informally serves as an institutional historian; and edits scientific manuscripts, corporate documents, and sundry other written media. SPGM is the creative services department and hub for editing, illustration, graphic design, formatting, multimedia, and training in these areas.