What Can Be the Why?
Research on retroviruses has revolutionized our understanding of cancer and remains a rich source of insights into how cancer cells work.
Few situations are more exasperating to the inquirer than to watch a tiny nodule form on a rabbit’s skin at a spot from which the chemical agent inducing it has long since been gone, and to follow the nodule as it grows, and only too often becomes a destructive epidermal cancer. What can be the why for these happenings? — Payton Rous, Nobel Prizelecture, December 13, 1966
BY DARRELL E. WARD
In the early 1990s, Kathleen Boris-Lawrie, PhD, was working in the laboratory of Nobel Prize laureate Howard Temin at the University of Wisconsin. She’d constructed a simpler form of bovine leukemia virus (BLV) as a step toward developing a retrovirus vaccine against infectious cancers and AIDS.
Boris-Lawrie, who today is a professor of veterinary biosciences and a researcher with The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James), had modified the BLV genome in several ways.
To prevent replication in host cells, she stripped out two critical regulatory genes, tax and rex. She also removed the tax and rex response elements, which are sequences of bases in the viral RNA. The Tax and Rex proteins bind to these response elements and induce efficient transcription, transport and translation of viral RNA which are required for viral replication. Boris-Lawrie replaced the deleted BLV material with sequences from the genetically simpler spleen necrosis virus.
She put her model to the test and published the 1995 study in the Journal of Virology. The findings noted that, rather than being unable to replicate, the hybrid BLV remained remarkably fecund. The question was, Why?
Peyton Rous initiated the use of tumor-causing viruses in cancer research100 years ago when, in 1911, he discovered that a “filterable element” extracted from tumors in one chicken quickly produced tumors in another chicken. “It was the first example of a cancer-causing infectious agent,” Boris-Lawrie says. That agent was later named the Rous sarcoma virus (RSV), and ultimately its study led to the anticancer drug, Gleevec.
Renato Dulbecco at the California Institute of Technology, along with students Harry Rubin and Howard Temin, gave new life to RSV research in the 1950s. Rubin added RSV particles to normal chicken fibroblasts and found that they induced morphologic and behavioral changes characteristic of cancer cells. Suddenly, scientists realized they could study cancer-cell transformation in a Petri dish.
Temin wanted to know why the virus’s RNA genome could persist in tumor cells. He arrived at a radical explanation: RSV and related viruses copied their RNA genome into DNA after entering host cells.
Until then, genetic information was thought to move from DNA to RNA and never the reverse. But Temin, working with Dulbecco, discovered reverse transcriptase, a viral enzyme that enables RSV and similar viruses to copy their RNA genome into DNA. This “backward” flow of information earned these infectious agents the name “retroviruses.”
David Baltimore at the Massachusetts Institute of Technology, working independently of Temin and Dulbecco, also discovered reverse transcriptase. The three researchers shared the 1975 Nobel Prize in Physiology or Medicine.
J. Michael Bishop and Harold E. Varmus, now director of the National Cancer Institute and former director of the National Institutes of Health, quickly put RSV reverse transcriptase to work. They discovered the first oncogene, SRC (pronounced “sarc”), and explained how RSV transformed cells, dramatically advancing the understanding of cancer.
Using reverse transcriptase, Bishop and Varmus made a single-stranded DNA copy of the wild-type RSV RNA genome.
They fragmented the DNA copy, then hybridized the pieces to the RNA genome of tumor-causing RSV. Some fragments remained unbound, however, and they used these leftovers to probe the host genome. From this they learned that the viral src was a shortened version of a gene found naturally in the host genome.
Bishop and Varmus won the 1989 Nobel Prize in Physiology or Medicine for discovering the cellular origin of retroviral oncogenes.
“Cells with the mutant src, which is found in the virus, make a truncated tyrosine kinase receptor that is constantly active,” Boris-Lawrie says. “In addition, src can have another mutation that makes it even more active, producing strong growth signals that push cells to proliferate.”
Boris-Lawrie was finishing the BLV study when Temin, a lifelong nonsmoker, died of lung cancer in 1994. She moved to Ohio State, set up her lab and began investigating how spleen necrosis virus endows the castrated BLV with the ability to replicate. First, she and her collaborators discovered that unspliced spleen necrosis virus RNA contains an element that boosts protein production 20,000-fold. They described this “post-transcriptional control element” in a 1999 Journal of Virology paper.
“We learned to our surprise that a cellular protein—not a viral protein—was modulating this effect,” Boris-Lawrie says. They speculated that host cells probably also use this protein to enhance translation. “We believed that if we identified the protein, we could use the retrovirus as a model system to learn something important about RNA translation,” she says.
Next, the researchers discovered that the post-transcriptional control element was a specialized structure on viral RNA consisting of two stem-like configurations topped with a loop—like golf tees each with a ball in profile. They described this 2003 finding in the Journal of Virology.
The breakthrough came three years later. The elusive protein was RNA helicase A (RHA), an enzyme involved in transporting mRNA from the nucleus to the cytoplasm and in initiating translation. When RHA was knocked out, the retrovirus no longer made several vital proteins.
In addition, Boris-Lawrie and her colleagues showed that an important cell growth-control gene that is lost in many cancers, JunD, produces an unspliced mRNA and interacts with RHA.
RHA binds to the post-transcriptional control element on junD mRNA, allowing translation. If RHA is missing, the protein isn’t made.
“Retroviruses seem to use RHA to enhance production of their own proteins, and cells use it to control the amount of particular proteins they make, many of which are involved in growth control,” says Boris-Lawrie. Nature Structural and Molecular Biology featured the study on the cover of the June 2006 issue.
“These findings provided important insights into how cells regulate certain growth proteins, many of which play an important role in cancer, along with showing how viruses use cell mechanisms to establish an infection,” Boris-Lawrie says.
That the retrovirus needs RHA to make these essential proteins suggests that the enzyme is a potential target for antiretroviral therapy and for blocking cancer-cell growth, Boris-Lawrie says.
When Jeffrey Parvin, MD, PhD, professor of biomedical informatics, came to Ohio State from Harvard, he’d already discovered that RHA interacts with BRCA1, a tumor-suppressor gene involved in DNA repair. Research by others has shown that mutations in RHA can prevent its proper interaction with BRCA1, causing a failure of BRCA1-associated DNA repair.
“Studies of breast-cancer patients show that RHA mutations can occur independently of BRCA1 mutations,” Boris-Lawrie says. “Other studies show that patients with the BRCA1 phenotype but without BRCA1 mutations had mutations in RHA. This could explain why patients without BRCA1 mutations can have the BRCA1 cancer phenotype,” Boris-Lawrie says.
“We are now assessing breast-cancer cohorts to see if RHA is deregulated in breast cancer,” she continues.
Rex, Tax and HBZ
Twenty years ago, Patrick Green, PhD, professor of Veterinary Biosciences and of Molecular Virology, Immunology, and Medical Genetics, and co-leader of the OSUCCC – James Viral Oncology Program, experienced a “why” moment that guided his research for a decade.
Green was drawn to the retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) and its close relative HTLV-2 early on. HTLV-1 causes adult T-cell leukemia/lymphoma and the demyelinating disease, tropical spastic paraparesis. HTLV-2 is not clearly associated with any disease.
Using gel electrophoresis to separate HTLV-2 proteins into bands for identification, he noticed that a key regulatory protein called Rex formed two bands on the gel, whereas the HTLV-1 Rex protein formed only one. (Improvements in technology would later show that HTLV-1 Rex also segregates into two close bands.)
“I spent about ten years working to understand why Rex from HTLV-2 showed two bands—we thought it might be an important difference between the two viruses,” Green says.
He learned that one of the bands represents the active, phosphorylated form of the Rex protein while the other is the inactive, dephosphorylated form.
Phosphorylation, which adds an energy-laden phosphate group to certain molecules, had changed the shape of the activated Rex, resulting in a second band.
Rex helps transport unspliced viral RNA from the nucleus to the cytoplasm, Green notes. “Interestingly, we found the active form in the nucleus and the inactive form in the cytoplasm. We proposed that active, phosphorylated Rex binds with the target RNA in the nucleus and exports it to the cytoplasm. There, Rex is dephosphorylated, drops its RNA payload and relocates to the nucleus.”
This mechanism enables the virus to stockpile inactive Rex in the cytoplasm that would be immediately available for viral replication, he says. “In short order the protein could be activated, move to the nucleus and start exporting viral RNA. This could happen really quickly because the protein doesn’t have to be made from scratch.”
Green then focused on Tax, an HTLV regulatory protein that increases the transcription of viral proteins. Using a complex recombinant virus model, he and his colleagues showed that Tax is critical for malignant transformation of host cells. “Without Tax, cell transformation doesn’t happen and the virus is essentially dead,” Green says.
They also learned how Tax transforms host cells: The protein activates NFκB, a master regulator of gene transcription. “Without NFkB activation, transformation doesn’t occur,” Green says.
But this presented a quandary. Many ATL cells stop making Tax, yet NFκB is constitutively active. “Something else was picking up what Tax was no longer there to do,” Green says. He believes the culprit is a viral protein called Hbz, which is present in almost all ATL tumor cells and also plays a role in NFκB signaling.
Hbz was discovered relatively recently, and interest in it has grown over the last decade, Green says. “We hypothesize that Tax, along with Hbz, is ultimately required for malignant transformation, and that the two may work in concert, or that Hbz substitutes for various Tax functions,” he says.
“Our lab focuses on how viral proteins interact with host cells. We try to address questions using natural target cells and physiological levels of gene expression, and we have a variety of infectious proviral clones in which we can manipulate RNA and protein expression.”
This enabled his lab to quickly answer important questions about Hbz. “We provided the first evidence that this protein is directly involved in proliferation of both infected cells and tumor cells,” Green says. “It is likely expressed early in replication, suggesting that infected cells require it to survive—perhaps to suppress innate immunity—or to stimulate growth. We’re looking at that now.
“Once we understand these virus-host interactions, we might be able to use small molecular inhibitors to disrupt them.”
Green has collaborated with colleagues in Japan to produce transgenic mice that express Hbz and develop tumors. “This boosts our original hypothesis that Hbz is a transforming factor, along with Tax,” he says.
“Overall, our evidence suggests that HTLV-1 tumorigenesis involves multiple oncogenes, with Tax as the initiator and Hbz providing infected cell persistence and tumor maintenance,” he says. “There is no published data yet to determine whether targeting Hbz will benefit patients, but that is forthcoming. It’s the holy grail for us at this point.”
Laboratory research lays the groundwork for new rational therapies for cancer, Green says. “You first have to identify the pathways involved and learn how they work and what they lead to. Then you can define how to inhibit, regulate or disrupt what the viral protein is doing to that pathway.
“The basic-research side feeds into the preclinical-research side, which feeds the development of clinical trials that evaluate novel agents in patients,” he says. Peyton Rous’ filterable elements continue to contribute to science’s understanding of cancer beyond what might be expected. That and the determination to answer, “What can be the why?”
Program Project Grant: Using Retrovirus Models to Understand Cancer
Retrovirus research at Ohio State is supported in part by a five-year, $10.9 million Program Project Grant (PPG) from the National Cancer Institute. Patrick Green, PhD, professor of Veterinary Biosciences at Ohio State’s College of Veterinary Medicine, and co-leader of the OSUCCC – James Viral Oncology Program, assumed leadership of the PPG after the original principal investigator, Michael Lairmore, DVM, PhD, professor of Veterinary Biosciences, transitioned from Ohio State to become dean of the University of California Davis School of Veterinary Medicine in October 2011. The goal of the PPG is to identify new therapeutic targets against retroviral-induced lymphoma and associated syndromes, such as hypercalcemia.
The PPG has five interrelated projects, each with a principal investigator:
Project 1: Stefan Niewiesk, DVM, PhD, associate professor of Veterinary Biosciences, and Mamuka Kvatstahelia, PhD, associate professor of Pharmaceutics, are investigating the role of accessory proteins in the human T-lymphotropic virus type 1 (HTLV-1), which causes an aggressive lymphoma and leukemia and a number of immune system disorders.
Project 2: Patrick Green, PhD, is defining novel post-transcriptional mechanismsof HTLV, particularly the p28 protein and its contribution to viral replication and cellular transformation.
Project 3: Kathleen Boris-Lawrie, PhD, is investigating translational control mechanisms in retroviruses and host-cell growth-control genes that involve RNA helicase A.
Project 4: Katherine Weilbaecher, MD, of Washington University, St. Louis, and Thomas Rosol, PhD, of Ohio State, are studying the bone microenvironment in osteolytic and osteoblastic tumor models.
Project 5: Lee Ratner, MD, of Washington University, St. Louis, is examining the role of inflammation in tax-mediated carcinogenesis using transgenic mouse models that express HTLV-1 tax.