Siddhartha Mukherjee - The Emperor of All Maladies: A Biography of Cancer

by Siddhartha Mukherjee

  Of the twenty thousand genes in a cancer cell, Weinberg reasoned the vast majority were likely normal and only a small minority were mutated proto-oncogenes. Now imagine, for a moment, being able to take all twenty thousand genes in the cancer cell, the good, the bad, the ugly, and transferring them into twenty thousand normal cells, such that each cell receives one of the genes. The normal, unmutated genes will have little effect on the cells. But an occasional cell will receive an oncogene, and, goaded by that signal, it will begin to grow and reproduce insatiably. Reproduced ten times, these cells will form a little clump on a petri dish; at twelve cell divisions, that clump will form a visible "focus"--cancer distilled into its primordial, elemental form.

  The snowstorm was Weinberg's catharsis; he had rid himself of retroviruses. If activated oncogenes existed within cancer cells, then transferring these genes into normal cells should induce these normal cells to divide and proliferate. For decades, cancer biologists had relied on Rous sarcoma virus to introduce activated src into cells and thereby incite cell division. But Weinberg would bypass Rous's virus; he would determine if cancer-causing genes could be transferred directly from cancer cells to normal cells. At the end of the bridge, with snow still swirling around him, he found himself at an empty intersection with lights still flashing. He crossed it, heading to the cancer center.

  Weinberg's immediate challenge was technical: how might he transfer DNA from a cancer cell to a population of normal cells? Fortunately, this was one of the technical skills that he had so laboriously perfected in the laboratory during his stagnant decade. His chosen method of DNA transfer began with the purification of DNA from cancer cells, grams of it precipitated out of cell extracts in a dense, flocculent suspension, like curdled milk. This DNA was then sheared into thousands of pieces, each piece carrying one or two genes. To transfer this DNA into cells, he next needed a carrier, a molecule that would slip DNA into the interior of a cell. Here, Weinberg used a trick. DNA binds to the chemical calcium phosphate to form minuscule white particles. These particles are ingested by cells, and as the cells ingest these particles, they also ingest the DNA pieces bound to the calcium phosphate. Sprinkled on top of a layer of normal cells growing in a petri dish, these particles of DNA and calcium phosphate resemble a snowglobe of swirling white flakes, the blizzard of genes that Weinberg had so vividly imagined in his walk in Boston.

  Once that DNA blizzard had been sprinkled on the cells and internalized by them, Weinberg envisioned a simple experiment. The cell that had received the oncogene would embark on unbridled growth, forming the proliferating focus of cells. Weinberg would isolate such foci and then purify the DNA fragment that had induced the proliferation. He would thus capture a real human oncogene.

  In the summer of 1979, Chiaho Shih, a graduate student in Weinberg's lab, began to barrel his way through fifteen different mouse cancer cells, trying to find a fragment of DNA that would produce foci out of normal cells. Shih was laconic and secretive, with a slippery, quicksilver temper, often paranoid about his experiments. He was also stubborn: when he disagreed with Weinberg, colleagues recalled him thickening his accent and pretending not to understand English, a language he spoke with ease and fluency under normal circumstances. But for all his quirks, Shih was also a born perfectionist. He had learned the DNA transfection technique from his predecessors in the lab, but even more important, he had an instinctive feel for his cells, almost a gardener's instinct to discriminate normal versus abnormal growth.

  Shih grew enormous numbers of normal cells in petri dishes and sprinkled them weekly with genes derived from his panel of cancer cells. Plate after plate of transfected cells piled up in the laboratory. As Weinberg had imagined in his walk across the river, Shih soon stumbled upon a crucial early result. He found that transferring DNA from mouse cancer cells invariably produced foci in normal cells, proof that oncogenes could be discovered through such a method.*

  Excited and mystified, Weinberg and Shih performed a bolder variant of the experiment. Thus far they had been using mouse cancer cell lines to obtain their DNA. Changing tactics and species, they moved on to human cancer cells. "If we were going to trap a real oncogene so laboriously," Weinberg recalled, "we thought that we might as well find it in real human cancers." Shih walked over to the Dana-Farber Cancer Institute and carried back a cancer cell line derived from a patient, Earl Jensen, a long-term smoker who had died of bladder cancer. DNA from these cells was sheared into fragments and transfected into the normal human cell line. Shih returned to his microscope, scouring plate after plate for foci.

  The experiment worked yet again. As with the mouse cancer cell lines, prominent, disinhibited foci appeared in the dishes. Weinberg pushed Shih to find the precise gene that could convert a normal cell to a cancer cell. Weinberg's laboratory was now racing to isolate and identify the first native human oncogene.

  He soon realized the race had other contenders. At the Farber, across town, Geoff Cooper, a former student of Temin's, had also shown that DNA from cancer cells could induce transformation in cells. So had Michael Wigler at the Cold Spring Harbor Lab in New York. And Weinberg, Cooper, and Wigler had yet other competitors. At the NCI, a little-known Spanish researcher named Mariano Barbacid had also found a fragment of DNA from yet another cancer cell line that would transform normal cells. In the late winter of 1981, all four laboratories rushed to the finish line. By the early spring, each lab had found its sought-after gene.

  In 1982, Weinberg, Barbacid, and Wigler independently published their discoveries and compared their results. It was a powerful, unexpected convergence: all three labs had isolated the same fragment of DNA, containing a gene called ras, from their respective cancer cells.+ Like src, ras was also a gene present in all cells. But like src again, the ras gene in normal cells was functionally different from the ras present in cancer cells. In normal cells, the ras gene encoded a tightly regulated protein that turned "on" and "off" like a carefully modulated switch. In cancer cells, the gene was mutated, just as Varmus and Bishop had predicted. Mutated ras encoded a berserk, perpetually hyperactive protein permanently locked "on." This mutant protein produced an unquenchable signal for a cell to divide--and to keep dividing. It was the long-sought "native" human oncogene, captured in flesh and blood out of a cancer cell. "Once we had cloned a cancer gene," Weinberg wrote, "the world would be at our feet." New insights into carcinogenesis, and new therapeutic inroads would instantly follow. "It was," as Weinberg would later write, all "a wonderful pipe dream."

  In 1983, a few months after Weinberg had purified mutant ras out of cancer cells, Ray Erikson traveled to Washington to receive the prestigious General Motors prize for his research on src activity and function. The other awardee that evening was Tom Frei, being honored for his advancement of the cure for leukemia.

  It was a resplendent evening. There was an elegant candlelit dinner in a Washington banquet hall, followed by congratulatory speeches and toasts. Scientists, physicians, and policymakers, including many of the former Laskerites,* gathered around linen-covered tables. Talk turned frequently to the discovery of oncogenes and the invention of curative chemotherapy. But the two conversations seemed to be occurring in sealed and separate universes, much as they had at Temin's conference in Houston more than a decade earlier. Frei's award, for curing leukemia, and Erikson's award, for identifying the function of a critical oncogene, might almost have been given to two unconnected pursuits. "I don't remember any enthusiasm among the clinicians to reach out to the cancer biologists to synthesize the two poles of knowledge about cancer," Erikson recalled. The two halves of cancer, cause and cure, having feasted and been feted together, sped off in separate taxis into the night.

  The discovery of ras brought one challenge to a close for cancer geneticists: they had purified a mutated oncogene from a cancer cell. But it threw open another challenge. Knudson's two-hit hypothesis had also generated a risky prediction: that retinoblastoma cancer cells contained two inactivated copies of the Rb g
ene. Weinberg, Wigler, and Barbacid had proved Varmus and Bishop right. Now someone had to prove Knudson's prediction by isolating his fabled tumor suppressor gene and demonstrating that both its copies were inactivated in retinoblastoma.

  This challenge, though, came with an odd conceptual twist. Tumor suppressor genes, by their very nature, are asserted in their absence. An oncogene, when mutated, provides an "on" signal for the cells to grow. A tumor suppressor gene when mutated, in contrast, removes an "off" signal for growth. Weinberg and Chiaho Shih's transfection assay had worked because oncogenes can cause the normal cells to divide uncontrollably, thus forming a focus of cells. But an anti-oncogene, transfected into a cell, cannot be expected to create an "anti-focus." "How can one capture genes that behave like ghosts," Weinberg wrote, "influencing cells from behind some dark curtain?"

  In the mid-1980s, cancer geneticists had begun to glimpse shadowy outlines behind retinoblastoma's "dark curtain." By analyzing chromosomes from retinoblastoma cancer cells using the technique pioneered by Janet Rowley, geneticists had demonstrated that the Rb gene "lived" on chromosome thirteen. But a chromosome contains thousands of genes. Isolating a single gene from that vast set--particularly one whose functional presence was revealed only when inactive--seemed like an impossible task. Large laboratories professionally equipped to hunt for cancer genes--Webster Cavenee's lab in Cincinnati, Brenda Gallie's in Toronto, and Weinberg's in Boston--were frantically hunting for a strategy to isolate Rb. But these efforts had reached a standstill. "We knew where Rb lived," Weinberg recalled, "but we had no idea what Rb was."

  Across the Charles River from Weinberg's lab, Thad Dryja, an ophthalmologist-turned-geneticist, had also joined the hunt for Rb. Dryja's laboratory was perched on the sixth floor of the Massachusetts Eye and Ear Infirmary--the Eyeball, as it was known colloquially among the medical residents. The ophthalmological infirmary was well-known for its clinical research on eye diseases, but was barely recognized for laboratory-based research. Weinberg's Whitehead Institute boasted the power of the latest technologies, an army of machines that could sequence thousands of DNA samples and powerful fluorescent microscopes that could look down into the very heart of the cell. In contrast, the Eyeball, with its proud display of nineteenth-century eyeglasses and lenses in lacquered wooden vitrines, was almost self-indulgently anachronistic.

  Dryja, too, was an unlikely cancer geneticist. In the mid-1980s, having completed his clinical fellowship in ophthalmology at the infirmary in Boston, he had crossed town to the science laboratories at Children's Hospital to study the genetics of eye diseases. As an ophthalmologist interested in cancer, Dryja had an obvious target: retinoblastoma. But even Dryja, an inveterate optimist, was hesitant about taking on the search for Rb. "Brenda [Gallie] and Web [Cavenee] had both stalled in their attempts [to clone Rb]. It was a slow, frustrating time."

  Dryja began his hunt for Rb with a few key assumptions. Normal human cells, he knew, have two copies of every chromosome (except the sex chromosomes), one from each parent, twenty-three pairs of chromosomes in all, a total of forty-six. Every normal cell thus has two copies of the Rb gene, one in each copy of chromosome thirteen.

  Assuming Knudson was right in his two-hit hypothesis, every eye tumor should possess two independent inactivating mutations in the Rb gene, one in each chromosome. Mutations, Dryja knew, come in many forms. They can be small changes in DNA that can activate a gene. Or they can be large structural deletions in a gene, stretching over a large piece of the chromosome. Since the Rb gene had to be inactivated to unleash retinoblastoma, Dryja reasoned that the mutation responsible was likely a deletion of the gene. Deleting a sizable piece of a gene, after all, is perhaps the quickest, crudest way to paralyze and inactivate it.

  In most retinoblastoma tumors, Dryja suspected, the two deletions in the two copies of the Rb gene would lie in different parts of the gene. Since mutations occur randomly, the chance of both mutations lying in precisely the same region of the gene is a little akin to rolling double sixes in dice that have one hundred faces. Typically, one of the deletions would "hit" the front end of the gene, while the other deletion might hit the back end (in both cases, the functional consequences would be the same--inactivating Rb). The two "hits" in most tumors would thus be asymmetric--affecting two different parts of the gene on the two chromosomes.

  But even hundred-headed dice, rolled many times, can yield double sixes. Rarely, Dryja knew, one might encounter a tumor in which both hits had deleted exactly the same part of the gene on the two sister chromosomes. In that case, that piece of chromosome would be completely missing from the cell. And if Dryja could find a method to identify a completely missing piece of chromosome thirteen in a retinoblastoma tumor cell, he would instantly land on the Rb gene. It was the simplest of strategies: to hunt the gene with absent function, Dryja would look for absence in structure.

  To identify such a missing piece, Dryja needed structural mileposts along chromosome thirteen--small pieces of DNA called probes, which were aligned along the length of the chromosome. He could use these DNA probes in a variant of the same "sticking" reaction that Varmus and Bishop had used in the 1970s: if the piece of DNA existed in the tumor cell, it would stick; if the piece did not exist, the probe would not stick, identifying the missing piece in the cell. Dryja had assembled a series of such probes. But more than probes, he needed a resource that he uniquely possessed: an enormous bank of frozen tumors. The chances of finding a shared deletion in the Rb gene in both chromosomes were slim, so he would need to test a vast sample set to find one.

  This, then, was his crucial advantage over the vast professional labs in Toronto and Houston. Laboratory scientists rarely venture outside the lab to find human samples. Dryja, a clinician, had a freezer full of them. "I stored the tumors obsessively," he said with the childlike delight of a collector. "I put news out among patients and doctors that I was looking for retinoblastoma cases. Every time someone saw a case, they would say, 'Get that guy Dryja.' I would then drive or fly or even walk to pick up the samples and bring them here. I even got to know the patients by name. Since the disease ran in families, I would call them at home to see if there was a brother or sister or cousin with retinoblastoma. Sometimes, I would know [about a tumor] even before doctors knew."

  Week after week, Dryja extracted the chromosomes from tumors and ran his probe set against the chromosomes. If the probes bound, they usually made a signal on a gel; if a probe was fully missing, the signal was blank. One morning, having run another dozen tumors, Dryja came to the lab and held up the blot against the window and ran his eyes left to right, lane after lane automatically, like a pianist reading a score. In one tumor, he saw a blank space. One of his probes--H3-8, he had called it--was deleted in both chromosomes in that tumor. He felt the brief hot rush of ecstasy, which then tipped into queasiness. "It was at that moment that I had the feeling that we had a gene in our hands. I had landed on retinoblastoma."

  Dryja had found a piece of DNA missing in tumor cells. Now he needed to find the corresponding piece present in normal cells, thus isolating the Rb gene. Perilously close to the end, Dryja was like an acrobat at the final stretch of his rope. His one-room lab was taut with tension, stretched to its limit. He had inadequate skills in isolating genes and limited resources. To isolate the gene, he would need help, so he took another lunge. He had heard that researchers in the Weinberg lab were also hunting for the retinoblastoma gene. Dryja's choices were stark: he could either team up with Weinberg, or he could try to isolate the gene alone and lose the race altogether.

  The scientist in Weinberg's lab trying to isolate Rb was Steve Friend. A jovial, medically trained molecular geneticist with a quick wit and an easy manner, Friend had casually mentioned his interest in Rb to Dryja at a meeting. Unlike Dryja, working with his growing stash of tumor samples, Friend had been building a collection of normal cells--cells in which the Rb gene was completely intact. Friend's approach had been to find genes that were present in normal
retinal cells, then to try to identify ones that were abnormal in retinoblastoma tumors--working backward toward Dryja.

  For Dryja, the complementarity of the two approaches was obvious. He had identified a missing piece of DNA in tumors. Could Friend and Weinberg now pull the intact, full-length gene out of normal cells? They outlined a potential collaboration between the two labs. One morning in 1985, Dryja took his probe, H3-8, and virtually ran across the Longfellow Bridge (by now, the central highway of oncogenesis), carrying it by hand to Friend's bench at the Whitehead.

  It took Friend a quick experiment to test Dryja's probe. Using the DNA "sticking" reaction again, Friend trapped and isolated the normal cellular gene that stuck to the H3-8 probe. The isolated gene "lived" on chromosome thirteen, as predicted. When Dryja further tested the candidate gene through his bank of tumor samples, he found precisely what Knudson had hypothesized more than a decade earlier: all retinoblastoma cells contained inactivations in both copies of the gene--two hits--while normal cells contained two normal copies of the gene. The candidate gene that Friend had isolated was indisputably Rb.

  In October 1986, Friend, Weinberg, and Dryja published their findings in Nature. The article marked the perfect complement to Weinberg's ras paper, the yin to its yang--the isolation of an activated proto-oncogene (ras) and the identification of the anti-oncogene (Rb). "Fifteen years ago," Weinberg wrote, "Knudson provided a theoretical basis for retinoblastoma tumorigenesis by suggesting that minimally two genetic events are required to trigger tumor development." Weinberg noted, "We have isolated [a human gene] apparently representing one of this class of genes"--a tumor suppressor.