Retroviruses and the Genetic Origins of Cancer, 1970-1993

In an unusually close collaboration with his colleague J. Michael Bishop between 1970 and 1984, Harold Varmus discovered that the many different forms of cancer all arise from a common genetic mechanism involving specific genes present in the normal cells of many different species. The intractable secrets of cancer lie hidden in the chromosomes of normal cells. When these normal cellular genes undergo mutations or inaccurate expression (the reading out of the genetic code by a cell's molecular machinery), the result can be cancer, the progressive multiplication of cells under conditions that impose constraints on the growth of normal cells. Varmus and Bishop's genetic theory of cancer explained several facts that had long puzzled cancer researchers: that the disease befalls some while it spares others even under similar environmental conditions, that it can be inherited but most often is triggered by environmental factors, that its risk increases with age (because an accumulation of gene mutations is required).

Their discovery deepened our understanding of the genetic basis of cancer, of cell growth and differentiation, of the control of gene expression in higher organisms, and of the molecular processes of evolution. "More than any other single experiment," the prominent cancer virologist Robert Weinberg has written, "[Varmus and Bishop's] work defined a milestone in twentieth-century cancer research, because it refocused thinking on the ultimate origins of cancer, directing attention to a site deep inside the cell." Until Varmus and Bishop's pathbreaking research, scientists had focused on cancer-causing viruses and the multitude of environmental carcinogens. Since then, genomes of different species have been the center of cancer research.

Varmus and Bishop's discovery resulted from detailed studies of the biochemistry, genetics, and life cycle of retroviruses, viruses that can incorporate their genes into the chromosomes of infected cells, thereby reengineering the cell's chemical processes to produce new virus particles. One of the genetic changes induced by retroviruses is the insertion of oncogenes, genetic sequences that are capable of transforming normal cells into cancer cells. Since most viruses have fewer than a dozen genes, compared to ca. 30,000 in the DNA of human cells, studying viruses greatly simplified the search for cancer-causing genes.

Varmus and Bishop began their joint research in 1970 trying to uncover one of the great contemporary challenges in biomedical research: to understand how retroviruses reproduced and caused cancer. During the 1960s, Howard Temin had proposed that retroviruses, whose genetic material consists of double-stranded RNA (ribonucleic acid), made double-stranded DNA (deoxyribonucleic acid) copies of themselves, which Temin called a provirus. The provirus could then insert itself into the DNA of infected cells, from where it directed the synthesis of new RNA virus particles. Temin suggested that proviruses implanted oncogenes in the DNA of host cells, resulting in cancer.

Temin's provirus hypothesis was greeted with much skepticism during the 1960s because it implied that the direction of genetic information could be reversed, in violation of what Francis Crick had called the "central dogma" of biology: that genetic information always flowed forward in the cell through a unidirectional molecular circuitry made up of DNA, messenger RNA, and protein-producing structures called ribosomes. However, Temin was vindicated in 1970 when he and David Baltimore found, independently of each other, a novel enzyme, reverse transcriptase, which directed just such a synthesis of retroviral RNA into a DNA provirus.

The second conceptual framework that guided Varmus and other cancer virologists during the early 1970s was the "virogene-oncogene" hypothesis, put forth by Robert Huebner and George Todaro in 1969. They suggested that all forms of cancer arise from the activation of oncogenes that had been embedded in normal cells through infection of germ cells (cells that are part of the germline, and are involved in the reproduction of organisms) by RNA tumor viruses in an early stage of evolution. These endogenous viral oncogenes lie silent until activated by carcinogenic substances, upon which they triggered neoplastic growth.

Varmus and Bishop set out to test and add experimental detail to the provirus and oncogene-virogene hypotheses: to elucidate more fully the life cycle of retroviruses, to detect the provirus in infected cells (not just to prove its existence indirectly by following the action of reverse transcription), to track the provirus in time and in vivo (in living organisms), not just in a laboratory dish. To do so, they chose a well-established experimental organism, Rous sarcoma virus (RSV, isolated by Peyton Rous in 1909), a retrovirus that triggers cancer in chicken and the microorganism in which scientists first detected an oncogene. Yet, research which began as an outgrowth of recent advances in virology unexpectedly led them in a new direction, towards uncovering the genetic origin of cancer.

In 1970, the oncogene of Rous sarcoma virus, SRC (for sarcoma, and pronounced "sark," denoting the type of connective tissue cancer it causes), was identified and shown to be the first clear example of a gene that can transform a cell from normal to perpetual cancerous growth. To test the virogene-oncogene hypothesis and to prove the existence of proviruses directly in infected cells, Varmus and Bishop had to demonstrate the presence in animal cells of DNA sequences that derived from retroviral RNA, namely the SRC oncogene. In other words, they had to compare a single viral gene, SRC, with the DNA of an animal infected by the virus, a difficult experimental challenge before the advent of restriction mapping and gene cloning.

In 1971, Varmus devised a molecular probe using radioactive tagging that could identify SRC genes amidst the multitude of other genes in vertebrate cells. In 1976, Varmus, Bishop, and French postdoctoral researcher Dominique Stehelin reported in Nature that they had made the surprising discovery that SRC was nearly identical to a sequence in the normal cellular DNA of several different species of birds. Subsequent research in the Varmus-Bishop lab by Deborah Spector found a SRC proto-oncogene in fish as well as in several mammals, including mice, cows, and humans, which made their research of immediate interest to a wide range of scientists. Moreover, Varmus and his collaborators were able to detect the provirus of RSV directly by means of molecular hybridization, to measure new copies of RSV DNA following infection and track DNA synthesis in time, to determine that reverse transcription occurred in the cytoplasm of the cell, and to distinguish between linear, circular, and integrated forms of the provirus.

The fact that a homologue, or corresponding genetic sequence, of SRC is found in a wide range of species and has been preserved for more than a billion years indicated to Varmus and Bishop that it originated in normal cells, not in the retrovirus that carries it. They concluded that the SRC proto-oncogene, as they called the cellular homologue of the SRC oncogene, was captured from the genome of a host cell by an invading retrovirus far in the evolutionary past in a chance event known as viral transduction. As retroviruses insert themselves into the DNA of host cells and from there direct the synthesis of new virus particles, they can on rare occasions make copies not just of their own viral genes, but capture adjacent cellular genes--including cellular proto-oncogenes--which they then carry, or transduct, into other organisms they subsequently infect. The process by which retroviruses capture proto-oncogenes damages these genes in ways that can turn them into full-fledged oncogenes, which induce malignant growth when the virus infects another cell.

Through an accident of nature, retroviruses had singled out proto-oncogenes, extracted them from cells, and brought them to the attention of scientists, well before scientists could have found these genes among the convolutions of the immensely long chain of human DNA. (This accident, which was of such benefit to science, brought no evolutionary benefit to the viruses themselves--they can reproduce and live even when they lose their oncogene through mutation.) Retroviruses remain vital tools for the isolation of oncogenes and for elucidating fundamental processes within human cells.

The fact that the cellular version of SRC and other proto-oncogenes survived through many stages of evolution also indicated that they must perform a vital function, most likely in directing the growth and development of cells, although their precise role has not been elucidated. However, while the SRC gene is expressed at a low rate in normal tissue cells, the version of the gene carried by RSV is expressed at a much higher rate. Cells infected by the virus are thus transformed into ever-growing and dividing cancer cells. Varmus and Bishop hypothesized that the cellular version of the gene may cause malignancy not just as a result of viral transduction (as a retroviral oncogene), but also if it undergoes mutation under the influence of environmental carcinogens like radiation, toxins, or tobacco smoke, or if the regulatory mechanism for its expression is damaged during cell division.

Varmus and Bishop showed the virogene-oncogene hypothesis to be faulty on two counts: first, proto-oncogenes were not noxious cancer genes lying in wait until stirred into action by a carcinogen, but were part of the genetic machinery of normal cells and carried out a function vital for the developing cell; and secondly, they were found in cells that showed no sign of viral infection, meaning that they had not been deposited in the germ line by a virus at some distant point in the past, but rather originated in healthy cells and were later introduced in modified, oncogenic form into the genome of an evolving retrovirus.

From their discovery of proto-oncogenes in normal cells Varmus and Bishop made important inferences, the accuracy of which has been borne out by subsequent research. They reasoned that SRC was the archetype of an array of genes that give rise to cancer, and that the oncogenes of other retroviruses likely also stemmed from cellular proto-oncogenes. More than a hundred proto-oncogenes have since been found by looking for cellular homologues of retroviral oncogenes.

Varmus and Bishop further hypothesized that all cancers, including those not caused by oncogenic viruses, are the result of damage to cellular proto-oncogenes. Damage that turns proto-oncogenes into oncogenes can occur not just through transduction by retroviruses, but when genes move from one chromosome to another in a process called chromosomal translocation. More commonly damage stems from a wide range of mutagenic and carcinogenic agents, including chemicals and radiation, as well as from retroviruses that do not carry oncogenes, such as the mouse mammary tumor virus which Varmus began to use as a model for studies of carcinogenesis in the mid-1970s. Retroviruses without oncogenes can trigger cancer by causing so-called insertion mutations in proto-oncogenes adjacent to the insertion site. This discovery opened the way for the identification of many mutant proto-oncogenes in human tumors that have no viral homologue.

Finally, Varmus and Bishop proposed that cancer can ensue when so-called tumor suppressor genes, genes that control the expression of proto-oncogenes, themselves undergo mutation or are deleted, leaving the latter free to divide unhindered. It is thus the behavior of both proto-oncogenes and tumor suppressor genes that drives the malignant growth of cancer cells.

To the extent that his administrative duties since 1999 as President and Chief Executive Officer of Memorial Sloan-Kettering Cancer Center, and as Director of the National Cancer Institute have allowed, Varmus has directed a small laboratory that carries on his genetic studies of retroviral oncogenesis and replication. He and his coworkers devise mouse models of human cancer in order to untangle the relationship between normal patterns of cell growth and development, and oncogenic events that disturb these patterns. His research aims at uncovering the molecular mechanisms that enable cancer cells to maintain their ability to divide indefinitely, and that make it possible for certain cells, called cancer stem cells, to give rise to cancer repeatedly. Varmus's laboratory is focusing on mutations of the RAS proto-oncogene, which, like SRC, first came to the attention of molecular biologists as a retroviral oncogene. Many human tumors, in particular cancers of the lung, colon, and pancreas, involve mutations in the three closely related versions of RAS found in the human genome. The mutation involves the alteration of a single letter in the genetic code, yet the consequence is a highly potent cancer gene. Besides this, Varmus is continuing his investigation, begun in the early 1990s, of how a protein called Wnt establishes cell signaling pathways that are instrumental in normal cell development and gene expression, but also in neoplasia of the mouse mammary gland and several human tissues, including the colon and liver. Since about 2002, Varmus and his colleagues have also been studying mutations of the EGF (epidermal growth factor) receptor gene, mutations that make certain lung cancers susceptible to treatment with anti-cancer agents called tyrosine kinase inhibitors.