* The term oncogene had been coined earlier by two NCI scientists, Robert Huebner and George Todaro, in 1969, although on scant evidence.
+Art Levinson, in Mike Bishop's lab at UCSF, also discovered this phosphorylating activity; we will return to Levinson's discovery in later pages.
The Wind in the Trees
The fine, fine wind that takes its course through the chaos of the world
Like a fine, an exquisite chisel, a wedge-blade inserted . . .
--D. H. Lawrence
The developments of the summer of 1976 drastically reorganized the universe of cancer biology, returning genes, again, to its center. Harold Varmus and Michael Bishop's proto-oncogene theory provided the first cogent and comprehensive theory of carcinogenesis. The theory explained how radiation, soot, and cigarette smoke, diverse and seemingly unrelated insults, could all initiate cancer--by mutating and thus activating precursor oncogenes within the cell. The theory made sense of Bruce Ames's peculiar correlation between carcinogens and mutagens: chemicals that cause mutations in DNA produce cancers because they alter cellular proto-oncogenes. The theory clarified why the same kind of cancer might arise in smokers and nonsmokers, albeit at different rates: both smokers and nonsmokers have the same proto-oncogenes in their cells, but smokers develop cancer at a higher rate because carcinogens in tobacco increase the mutation rate of these genes.
But what did human cancer genes look like? Tumor virologists had found src in viruses and then in cells, but surely other endogenous proto-oncogenes were strewn about in the human cellular genome.
Genetics has two distinct ways to "see" genes. The first is structural: genes can be envisioned as physical structures--pieces of DNA lined up along chromosomes, just as Morgan and Flemming had first envisioned them. The second is functional: genes can be imagined, a la Mendel, as the inheritance of traits that move from one generation to the next. In the decade between 1970 and 1980, cancer genetics would begin to "see" cancer-causing genes in these two lights. Each distinct vision would enhance the mechanistic understanding of carcinogenesis, bringing the field closer and closer to an understanding of the core molecular aberration in human cancers.
Structure--anatomy--came first. In 1973, as Varmus and Bishop were launching their initial studies on src, a hematologist in Chicago, Janet Rowley, saw a human cancer gene in a physical form. Rowley's specialty was studying the staining patterns of chromosomes in cells in order to locate chromosomal abnormalities in cancer cells. Chromosome staining, the technique she had perfected, is as much an art as a science. It is also an oddly anachronistic art, like painting with tempera in an age of digital prints. At a time when cancer genetics was zooming off to explore the world of RNA, tumor viruses, and oncogenes, Rowley was intent on dragging the discipline back to its roots--to Boveri's and Flemming's chromosomes dyed in blue. Piling anachronism upon anachronism, the cancer she had chosen to study was chronic myelogenous leukemia (CML)--Bennett's infamous "suppuration of blood."
Rowley's study was built on prior work by a duo of pathologists from Philadelphia who had also studied CML. In the late 1950s, Peter Nowell and David Hungerford had found an unusual chromosomal pattern in this form of leukemia: the cancer cells bore one consistently shortened chromosome. Human cells have forty-six chromosomes--twenty-three matched pairs--one inherited from each parent. In CML cells, Nowell found that one copy of the twenty-second chromosome had its head lopped off. Nowell called the abnormality the Philadelphia chromosome after the place of its discovery. But Nowell and Hungerford could not understand where the decapitated chromosome had come from, or where its missing "head" had gone.
Rowley, following this study, began to trace the headless chromosome in her CML cells. By laying out exquisitely stained photographs of CML chromosomes enlarged thousands of times--she typically spread them on her dining table and then leaned into the pictures, hunting for the missing pieces of the infamous Philadelphia chromosome--Rowley found a pattern. The missing head of chromosome twenty-two had attached itself elsewhere--to the tip of chromosome nine. And a piece of chromosome nine had conversely attached itself to chromosome twenty-two. This genetic event was termed a translocation--the flip-flop transposition of two pieces of chromosomes.
Rowley examined case after case of CML patients. In every single case, she found this same translocation in the cells. Chromosomal abnormalities in cancer cells had been known since the days of von Hansemann and Boveri. But Rowley's results argued a much more profound point. Cancer was not disorganized chromosomal chaos. It was organized chromosomal chaos: specific and identical mutations existed in particular forms of cancer.
Chromosomal translocations can create new genes called chimeras by fusing two genes formerly located on two different chromosomes--the "head" of chromosome nine, say, fused with the "tail" of a gene in chromosome thirteen. The CML translocation, Rowley postulated, had created such a chimera. Rowley did not know the identity or function of this new chimeric monster. But she had demonstrated that a novel, unique genetic alteration--later found to be an oncogene--could exist in a human cancer cell, revealing itself purely by virtue of an aberrant chromosome structure.
In Houston, Alfred Knudson, a Caltech-trained geneticist, also "saw" a human cancer-causing gene in the early 1970s, although in yet another distinct sense.
Rowley had visualized cancer-causing genes by studying the physical structure of the cancer cell's chromosomes. Knudson concentrated monastically on the function of a gene. Genes are units of inheritance: they shuttle properties--traits--from one generation to the next. If genes cause cancer, Knudson reasoned, then he might capture a pattern in the inheritance of cancer, much as Mendel had captured the idea of a gene by studying the inheritance of flower color or plant height in peas.
In 1969, Knudson moved to the MD Anderson Cancer Center in Texas, where Freireich had set up a booming clinical center for childhood cancers. Knudson needed a "model" cancer, a hereditary malignancy whose underlying pattern of inheritance would reveal how cancer-causing genes worked. The natural choice was retinoblastoma, the odd, rare variant of eye cancer that de Gouvea had identified in Brazil with its striking tendency to erupt in the same family across generations.
Retinoblastoma is a particularly tragic form of cancer, not just because it assaults children but because it assaults the quintessential organ of childhood: the tumor grows in the eye. Afflicted children are sometimes diagnosed when the world around them begins to blur and fade. But occasionally the cancer is incidentally found in a child's photograph when the eye, lit by a camera flash, glows eerily like a cat's eyes in lamplight, revealing the tumor buried behind the lens. Left untreated, the tumor will crawl backward from the eye socket into the optic nerve, and then climb into the brain. The primary methods of treatment are to sear the tumor with high doses of gamma radiation or to enucleate the eye surgically, leaving behind an empty socket.
Retinoblastoma has two distinct variants, an inherited "familial" form and a sporadic form. De Gouvea had identified the familial form. Children who suffer from this familial or inherited form may carry strong family histories of the disease--fathers, mothers, cousins, siblings, and kindred affected--and they typically develop tumors in both eyes, as in de Gouvea's case from Rio. But the tumor also arises in children with no family history of the disease. Children with this sporadic form never carry a history in the family and always have a tumor in only one eye.
This pattern of inheritance intrigued Knudson. He wondered whether he could discern a subtle difference in the development of cancer between the sporadic and the inherited versions using mathematical analyses. He performed the simplest of experiments: he grouped children with the sporadic form into one cohort and children with the familial form in a second. And sifting through old hospital records, Knudson tabulated the ages in which the disease struck the two groups, then plotted them as two curves. Intriguingly, he found that the two cohorts developed the cancers at different "velocities." In inherited retinoblastoma, cance
r onset was rapid, with diagnosis typically two to six months after birth. Sporadic retinoblastoma typically appeared two to four years after birth.
But why did the same disease move with different velocities in different children? Knudson used the numbers and simple equations borrowed from physics and probability theory to model the development of the cancer in the two cohorts. He found that the data fit a simple model. In children with the inherited form of retinoblastoma, only one genetic change was required to develop the cancer. Children with the sporadic form required two genetic changes.
This raised another puzzling question: why was only one genetic change needed to unleash cancer in the familial case, while two changes were needed in the sporadic form? Knudson perceived a simple, beautiful explanation. "The number two," he recalled, "is the geneticist's favorite number." Every normal human cell has two copies of each chromosome and thus two copies of every gene. Every normal cell must have two normal copies of the retinoblastoma gene--Rb. To develop sporadic retinoblastoma, Knudson postulated, both copies of the gene needed to be inactivated through a mutation in each copy of the Rb gene. Hence, sporadic retinoblastoma develops at later ages because two independent mutations have to accumulate in the same cell.
Children with the inherited form of retinoblastoma, in contrast, are born with a defective copy of Rb. In their cells, one gene copy is already defective, and only a single additional genetic mutation is needed before the cell senses the change and begins to divide. These children are thus predisposed to the cancer, and they develop cancer faster, producing the "rapid velocity" tumors that Knudson saw in his statistical charts. Knudson called this the two-hit hypothesis of cancer. For certain cancer-causing genes, two mutational "hits" were needed to provoke cell division and thus produce cancer.
Knudson's two-hit theory was a powerful explanation for the inheritance pattern of retinoblastoma, but at first glance it seemed at odds with the initial molecular understanding of cancer. The src gene, recall, requires a single activated copy to provoke uncontrolled cell division. Knudson's gene required two. Why was a single mutation in src sufficient to provoke cell division, while two were required for Rb?
The answer lies in the function of the two genes. Src activates a function in cell division. The mutation in src, as Ray Erikson and Hidesaburo Hanafusa had discovered, creates a cellular protein that is unable to extinguish its function--an insatiable, hyperactive kinase on overdrive that provokes perpetual cell division. Knudson's gene, Rb, performs the opposite function. It suppresses cell proliferation, and it is the inactivation of such a gene (by virtue of two hits) that unleashes cell division. Rb, then, is a cancer suppressor gene--the functional opposite of src--an "anti-oncogene," as Knudson called it.
"Two classes of genes are apparently critical in the origin of the cancers of children," he wrote. "One class, that of oncogenes, acts by virtue of abnormal or elevated activity. . . . The other class, that of anti-oncogenes [or tumor suppressors], is recessive in oncogenesis; cancer results when both normal copies have been mutated or deleted. Some persons carry one such mutation in the germline and are highly susceptible to tumor because only one somatic event is necessary. Some children, even though carrying no such mutation in the germline, can acquire tumor as a result of two somatic events."
It was an exquisitely astute hypothesis spun, remarkably, out of statistical reasoning alone. Knudson did not know the molecular identity of his phantasmic anti-oncogenes. He had never looked at a cancer cell to "see" these genes; he had never performed a biological experiment to pin down Rb. Like Mendel, Knudson knew his genes only in a statistical sense. He had inferred them, as he put it, "as one might infer the wind from the movement of the trees."
By the late 1970s, Varmus, Bishop, and Knudson could begin to describe the core molecular aberration of the cancer cell, stitching together the coordinated actions of oncogenes and anti-oncogenes. Cancer genes, Knudson proposed, came in two flavors. "Positive" genes, such as src, are mutant activated versions of normal cellular genes. In normal cells, these genes accelerate cell division, but only when the cell receives an appropriate growth signal. In their mutant form, these genes are driven into perpetual hyperactivity, unleashing cell division beyond control. An activated proto-oncogene, to use Bishop's analogy, is "a jammed accelerator" in a car. A cell with such a jammed accelerator careens down the path of cell division, unable to cease mitosis, dividing and dividing again relentlessly.
"Negative" genes, such as Rb, suppress cell division. In normal cells, these anti-oncogenes, or tumor suppressor genes, provide the "brakes" to cellular proliferation, shutting down cell division when the cell receives appropriate signals. In cancer cells, these brakes have been inactivated by mutations. In cells with missing brakes, to use Bishop's analogy again, the "stop" signals for mitosis can no longer be registered. Again, the cell divides and keeps dividing, defying all signals to stop.
Both abnormalities, activated proto-oncogenes and inactivated tumor suppressors ("jammed accelerators" and "missing brakes"), represent the core molecular defects in the cancer cell. Bishop, Knudson, and Varmus did not know how many such defects were ultimately needed to cause human cancers. But a confluence of them, they postulated, causes cancer.
A Risky Prediction
They see only their own shadows or the shadows of one another, which the fire throws on the opposite wall of the cave.
--Plato
The philosopher of science Karl Popper coined the term risky prediction to describe the process by which scientists verify untested theories. Good theories, Popper proposed, generate risky predictions. They presage an unanticipated fact or event that runs a real risk of not occurring or being proven incorrect. When this unanticipated fact proves true or the event does occur, the theory gains credibility and robustness. Newton's understanding of gravitation was most spectacularly validated when it accurately presaged the return of Halley's comet in 1758. Einstein's theory of relativity was vindicated in 1919 by the demonstration that light from distant stars is "bent" by the mass of the sun, just as predicted by the theory's equations.
By the late 1970s, the theory of carcinogenesis proposed by Varmus and Bishop had also generated at least one such risky prediction. Varmus and Bishop had demonstrated that precursors of oncogenes--proto-oncogenes--existed in all normal cells. They had found activated versions of the src proto-oncogene in Rous sarcoma virus. They had suggested that mutations in such internal genes caused cancer--but a crucial piece of evidence was still missing. If Varmus and Bishop were right, then mutated versions of such proto-oncogenes must exist inside cancer cells. But thus far, although other scientists had isolated an assortment of oncogenes from retroviruses, no one had isolated an activated, mutated oncogene out of a cancer cell.
"Isolating such a gene," as the cancer biologist Robert Weinberg put it, "would be like walking out of a cave of shadows. . . . Where scientists had previously only seen oncogenes indirectly, they might see these genes, in flesh and blood, living inside the cancer cell."
Robert Weinberg was particularly concerned with getting out of shadows. Trained as a virologist in an era of great virologists, he had worked in Dulbecco's lab at the Salk Institute in the sixties isolating DNA from monkey viruses to study their genes. In 1970, when Temin and Baltimore had discovered reverse transcriptase, Weinberg was still at the bench, laboriously purifying genes out of monkey viruses. Six years later, when Varmus and Bishop had announced the discovery of cellular src, Weinberg was still purifying DNA from viruses. Weinberg felt as if he was stuck in a perpetual penumbra, surrounded by fame but never famous himself. The retrovirus revolution, with all its mysteries and rewards, had quietly passed him by.
In 1972, Weinberg moved to MIT, to a small laboratory a few doors down from Baltimore's lab to study cancer-causing viruses. "The chair of the department," he said, "considered me quite a fool. A good fool. A hardworking fool, but still a fool." Weinberg's lab occupied a sterile, uninspiring space at MIT, in a sixties-style brut
alist building served by a single creaking elevator. The Charles River was just far enough to be invisible from the windows, but just near enough to send freezing puffs of wind through the quadrangle in the winter. The building's basement connected to a warren of tunnels with airless rooms where keys were cut and machines repaired for other labs.
Labs, too, can become machines. In science, it is more often a pejorative description than a complimentary one: an efficient, thrumming, technically accomplished laboratory is like a robot orchestra that produces perfectly pitched tunes but no music. By the mid-1970s, Weinberg had acquired a reputation among his colleagues as a careful, technically competent scientist, but one who lacked direction. Weinberg felt his work was stagnating. What he needed was a simple, clear question.
Clarity came to him one morning in the midst of one of Boston's infamously blinding blizzards. On a February day in 1978, walking to work, Weinberg was caught in an epic snowstorm. Public transportation had ground to a halt, and Weinberg, in a rubber hat and galoshes, had chosen to plod across the blustering Longfellow Bridge from his home to his lab, slowly planting his feet through the slush. The snow blotted out the landscape and absorbed all sounds, creating a silent, hypnotic interior. And as Weinberg crossed the frozen river, he thought about retroviruses, cancer, and human cancer genes.
Src had been so easy to isolate and identify as a cancer-causing gene, Weinberg knew, because Rous sarcoma virus possesses a measly four genes. One could scarcely turn around in the retroviral genome without bumping into an oncogene. A cancer cell, in contrast, has about twenty thousand genes. Searching for a cancer-causing gene in that blizzard of genes was virtually hopeless.
But an oncogene, by definition, has a special property: it provokes unbridled cellular proliferation in a normal cell. Temin had used this property in his cancer-in-a-dish experiment to induce cells to form "foci." And as Weinberg thought about oncogenes, he kept returning to this essential property.
Siddhartha Mukherjee - The Emperor of All Maladies: A Biography of Cancer Page 44
