What Rb does in normal cells is still an unfolding puzzle. Its name, as it turns out, is quite a misnomer. Rb, retinoblastoma, is not just mutated in rare eye tumors in children. When scientists tested the gene isolated by Dryja, Friend, and Weinberg in other cancers in the early nineties, they found it widely mutated in lung, bone, esophageal, breast, and bladder cancers in adults. Like ras, it is expressed in nearly every dividing cell. And it is inactivated in a whole host of malignancies. Calling it retinoblastoma thus vastly underestimates the influence, depth, and prowess of this gene.
The retinoblastoma gene encodes a protein, also named Rb, with a deep molecular "pocket." Its chief function is to bind to several other proteins and keep them tightly sealed in that pocket, preventing them from activating cell division. When the cell decides to divide, it tags Rb with a phosphate group, a molecular signal that inactivates the gene and thus forces the protein to release its partners. Rb thus acts as a gatekeeper for cell division, opening a series of key molecular floodgates each time cell division is activated and closing them sharply when the cell division is completed. Mutations in Rb inactivate this function. The cancer cell perceives its gates as perpetually open and is unable to stop dividing.
The cloning of ras and retinoblastoma--oncogene and anti-oncogene--was a transformative moment in cancer genetics. In the decade between 1983 and 1993, a horde of other oncogenes and anti-oncogenes (tumor suppressor genes) were swiftly identified in human cancers: myc, neu, fos, ret, akt (all oncogenes), and p53, VHL, APC (all tumor suppressors). Retroviruses, the accidental carriers of oncogenes, faded far into the distance. Varmus and Bishop's theory--that oncogenes were activated cellular genes--was recognized to be widely true for many forms of cancer. And the two-hit hypothesis--that tumor suppressors were genes that needed to be inactivated in both chromosomes--was also found to be widely applicable in cancer. A rather general conceptual framework for carcinogenesis was slowly becoming apparent. The cancer cell was a broken, deranged machine. Oncogenes were its jammed accelerators and inactivated tumor suppressors its missing brakes.*
In the late 1980s, yet another line of research, resurrected from the past, yielded a further bounty of cancer-linked genes. Ever since de Gouvea's report of the Brazilian family with eye tumors in 1872, geneticists had uncovered several other families that appeared to carry cancer in their genes. The stories of these families bore a familiar, tragic trope: cancer haunted them generation upon generation, appearing and reappearing in parents, children, and grandchildren. Two features stood out in these family histories. First, geneticists recognized that the spectrum of cancers in every family was limited and often stereotypical: colon and ovarian cancer threading through one family; breast and ovarian through another; sarcomas, leukemias, and gliomas through a third. And second, similar patterns often reappeared in different families, thereby suggesting a common genetic syndrome. In Lynch syndrome (first described by an astute oncologist, Henry Lynch, in a Nebraskan family), colon, ovarian, stomach, and biliary cancer recurred generation upon generation. In Li-Fraumeni syndrome, there were recurrent bone and visceral sarcomas, leukemias, and brain tumors.
Using powerful molecular genetic techniques, cancer geneticists in the 1980s and 1990s could clone and identify some of these cancer-linked genes. Many of these familial cancer genes, like Rb, were tumor suppressors (although occasional oncogenes were also found). Most such syndromes were fleetingly rare. But occasionally geneticists identified cancer-predisposing gene alterations that were quite frequently represented in the population. Perhaps the most striking among these, first suggested by the geneticist Mary Claire-King and then definitively cloned by Mark Skolnick's team at the pharma company Myriad Genetics, was BRCA-1, a gene that strongly predisposes humans to breast and ovarian cancer. BRCA-1 (to which we will return in later pages) can be found in up to 1 percent of women in selected populations, making it one of the most common cancer-linked genes found in humans.
By the early 1990s, the discoveries of cancer biology had thus traversed the gap between the chicken tumors of Peyton Rous and real human cancers. But purists still complained. The crusty specter of Robert Koch still haunted the genetic theory of cancer. Koch had postulated that for an agent to be identified as the "cause" of a disease, it must (1) be present in the diseased organism, (2) be capable of being isolated from the diseased organism, and (3) re-create the disease in a secondary host when transferred from the diseased organism. Oncogenes had met the first two criteria. They had been found to be present in cancer cells and they had been isolated from cancer cells. But no one had shown that a cancer gene, in and of itself, could create a bona fide tumor in an animal.
In the mid-1980s, a series of remarkable experiments allowed cancer geneticists to meet Koch's final criteria. In 1984, biologists working on stem cells had invented a new technology that allowed them to introduce exogenous genes into early mouse embryos, then create a living mouse out of those modified embryos. This allowed them to produce "transgenic mice," mice in which one or more genes were artificially and permanently modified. Cancer geneticists seized this opportunity. Among the first such genes to be engineered into a mouse was c-myc, an oncogene discovered in lymphoma cells.
Using transgenic mouse technology, Philip Leder's team at Harvard altered the c-myc gene in mice, but with a twist: cleverly, they ensured that only breast tissue in the mouse would overexpress the gene. (Myc could not be activated in all cells. If myc was permanently activated in the embryo, the embryo turned into a ball of overproliferating cells, then involuted and died through unknown mechanisms. The only way to activate myc in a living mouse was to restrict the activation to only a subset of cells. Since Leder's lab was studying breast cancer, he chose breast cells.) Colloquially, Leder called his mouse the OncoMouse. In 1988, he successfully applied for a patent on the OncoMouse, making it the first animal patented in history.
Leder expected his transgenic mice to explode with cancer, but to his surprise, the oncomice sprouted rather mousy cancers. Even though an aggressive oncogene had been stitched into their chromosomes, the mice developed small, unilateral breast cancers, and not until late in life. Even more surprisingly, Leder's mice typically developed cancers only after pregnancy, suggesting that environmental influences, such as hormones, were strictly required to achieve full transformation of breast cells. "The active myc gene does not appear to be sufficient for the development of these tumors," Leder wrote. "If that were the case, we would have expected the uniform development of tumor masses involving the entire bilateral [breast] glands of all five tumor-bearing animals. Rather, our results suggest at least two additional requirements. One of these is likely to be a further transforming event. . . . The other seems to be a hormonal environment related to pregnancy that is only suggested by these initial studies."
To test the roles of other oncogenes and environmental stimuli, Leder created a second OncoMouse, in which two activated proto-oncogenes, ras and myc, were engineered into the chromosome and expressed in breast cells. Multiple tumors sprouted up in the breast glands of these mice in months. The requirement for the hormonal milieu of pregnancy was partially ameliorated. Still, only a few distinct clones of cancer sprouted out of the ras-myc mice. Millions of breast cells in each mouse possessed activated ras and myc. Yet, of those millions of cells, each endowed with the most potent oncogenes, only a few dozen turned into real, living tumors.
Even so, this was a landmark experiment: cancer had artificially been created in an animal. "Cancer genetics," as the geneticist Cliff Tabin recalls, "had crossed a new frontier. It was not dealing with just genes and pathways and artificial lumps in the lab, but a real growing tumor in an animal." Peyton Rous's long squabble with the discipline--that cancer had never been produced in a living organism by altering a defined set of cellular genes--was finally laid to its long-overdue rest.
* In fact, the "normal" cells that Weinberg had used were not exactly normal. They were already growth-adapted, such that a single activated
oncogene could tip them into transformed growth. Truly "normal" cells, Weinberg would later discover, require several genes to become transformed.
+In fact, ras, like src, had also been discovered earlier in a cancer-causing virus--again underscoring the striking capacity of these viruses to reveal the mechanisms of endogenous oncogenes.
* The Laskerites had largely been disbanded in the aftermath of the 1971 National Cancer Act. Mary Lasker was still involved in science policy, although with nowhere near the force and visceral energy that she had summoned in the sixties.
* Although cancer is not universally caused by viruses, certain viruses cause particular cancers, such as the human papilloma virus (HPV), which causes cervical cancer. When the mechanism driving this cancer was deciphered in the 1990s, HPV turned out to inactivate Rb's and p53's signal--underscoring the importance of endogenous genes in even virally induced cancers.
The Hallmarks of Cancer
I do not wish to achieve immortality through my works. I wish to achieve immortality by not dying.
--Woody Allen
Scurrying about in its cage in the vivarium atop Harvard Medical School, Philip Leder's OncoMouse bore large implications on small haunches. The mouse embodied the maturity of cancer genetics: scientists had created real, living tumors (not just abstract, etiolated foci in petri dishes) by artificially manipulating two genes, ras and myc, in an animal. Yet Leder's experiment raised further questions about the genesis of cancer. Cancer is not merely a lump in the body; it is a disease that migrates, evolves, invades organs, destroys tissues, and resists drugs. Activating even two potent proto-oncogenes had not recapitulated the full syndrome of cancer in every cell of the mouse. Cancer genetics had illuminated much about the genesis of cancer, but much, evidently, remained to be understood.
If two oncogenes were insufficient to create cancers, then how many activated proto-oncogenes and inactivated tumor suppressors were required? What were the genetic steps needed to convert a normal cell into a cancer cell? For human cancers, these questions could not be answered experimentally. One could not, after all, proactively "create" a human cancer and follow the activation and inactivation of genes. But the questions could be answered retrospectively. In 1988, using human specimens, a physician-scientist named Bert Vogelstein at Johns Hopkins Medical School in Baltimore set out to describe the number of genetic changes required to initiate cancer. The query, in various incarnations, would preoccupy Vogelstein for nearly two decades.
Vogelstein was inspired by the observations made by George Papanicolaou and Oscar Auerbach in the 1950s. Both Papanicolaou and Auerbach, working on different cancers, had noted that cancer did not arise directly out of a normal cell. Instead, cancer often slouched toward its birth, undergoing discrete, transitional stages between the fully normal and the frankly malignant cell. Decades before cervical cancer evolved into its fiercely invasive incarnation, whorls of noninvasive premalignant cells could be observed in the tissue, beginning their first steps in the grisly march toward cancer. (Identifying and eradicating this premalignant stage before the cancer spreads is the basis for the Pap smear.) Similarly, Auerbach had noted, premalignant cells were seen in smokers' lungs long before lung cancer appeared. Colon cancer in humans also underwent graded and discrete changes in its progression, from a noninvasive premalignant lesion called an adenoma to the highly invasive terminal stage called an invasive carcinoma.
Vogelstein chose to study this progression in colon cancer. He collected samples from patients representing each of the stages of colon cancer. He then assembled a series of four human cancer genes--oncogenes and tumor suppressors--and assessed each stage of cancer in his samples for activations and inactivations of these four genes.*
Knowing the heterogeneity of every cancer, one might naively have presumed that every patient's cancer possessed its own sequence of gene mutations and its unique set of mutated genes. But Vogelstein found a strikingly consistent pattern in his colon cancer samples: across many samples and many patients, the transitions in the stages of cancer were paralleled by the same transitions in genetic changes. Cancer cells did not activate or inactivate genes at random. Instead, the shift from a premalignant state to an invasive cancer could precisely be correlated with the activation and inactivation of genes in a strict and stereotypical sequence.
In 1988, in the New England Journal of Medicine, Vogelstein wrote: "The four molecular alterations accumulated in a fashion that paralleled the clinical progression of tumors." He proposed, "Early in the neoplastic process one colonic cell appears to outgrow its companions to form a small, benign neoplasm. During the growth of [these] cells, a mutation in the ras gene . . . often occurs. Finally, a loss of tumor suppressor genes . . . may be associated with the progression of adenoma to frank carcinoma."
Since Vogelstein had preselected his list of four genes, he could not enumerate the total number of genes required for the march of cancer. (The technology available in 1988 would not permit such an analysis; he would need to wait two decades before that technology would become available.) But he had proved an important point, that such a discrete genetic march existed. Papanicolaou and Auerbach had described the pathological transition of cancer as a multistep process, starting with premalignancy and marching inexorably toward invasive cancer. Vogelstein showed that the genetic progression of cancer was also a multistep process.
This was a relief. In the decade between 1980 and 1990, proto-oncogenes and tumor suppressor genes had been discovered in such astonishing numbers in the human genome--at last count, about one hundred such genes--that their abundance raised a disturbing question: if the genome was so densely littered with such intemperate genes--genes waiting to push a cell toward cancer as if at the flick of a switch--then why was the human body not exploding with cancer every minute?
Cancer geneticists already knew two answers to this question. First, proto-oncogenes need to be activated through mutations, and mutations are rare events. Second, tumor suppressor genes need to be inactivated, but typically two copies exist of each tumor suppressor gene, and thus two independent mutations are needed to inactivate a tumor suppressor, an even rarer event. Vogelstein provided the third answer. Activating or inactivating any single gene, he postulated, produced only the first steps toward carcinogenesis. Cancer's march was long and slow and proceeded though many mutations in many genes over many iterations. In genetic terms, our cells were not sitting on the edge of the abyss of cancer. They were dragged toward that abyss in graded, discrete steps.
While Bert Vogelstein was describing the slow march of cancer from one gene mutation to the next, cancer biologists were investigating the functions of these mutations. Cancer gene mutations, they knew, could succinctly be described in two categories: either activations of proto-oncogenes or inactivations of tumor suppressor genes. But although dysregulated cell division is the pathological hallmark of cancer, cancer cells do not merely divide; they migrate through the body, destroy other tissues, invade organs, and colonize distant sites. To understand the full syndrome of cancer, biologists would need to link gene mutations in cancer cells to the complex and multifaceted abnormal behavior of these cells.
Genes encode proteins, and proteins often work like minuscule molecular switches, activating yet other proteins and inactivating others, turning molecular switches "on" and "off" inside a cell. Thus, a conceptual diagram can be drawn for any such protein: protein A turns B on, which turns C on and D off, which turns E on, and so forth. This molecular cascade is termed the signaling pathway for a protein. Such pathways are constantly active in cells, bringing signals in and signals out, thereby allowing a cell to function in its environment.
Proto-oncogenes and tumor suppressor genes, cancer biologists discovered, sit at the hubs of such signaling pathways. Ras, for instance, activates a protein called Mek. Mek in turn activates Erk, which, through several intermediary steps, ultimately accelerates cell division. This cascade of steps, called the Ras-Mek-Erk pathway--is tightly
regulated in normal cells, thereby ensuring tightly regulated cell division. In cancer cells, activated "Ras" chronically and permanently activates Mek, which permanently activates Erk, resulting in uncontrolled cell division--pathological mitosis.
But the activated ras pathway (Ras- Mek - Erk) does not merely cause accelerated cell division; the pathway also intersects with other pathways to enable several other "behaviors" of cancer cells. At Children's Hospital in Boston in the 1990s, the surgeon-scientist Judah Folkman demonstrated that certain activated signaling pathways within cancer cells, ras among them, could also induce neighboring blood vessels to grow. A tumor could thus "acquire" its own blood supply by insidiously inciting a network of blood vessels around itself and then growing, in grapelike clusters, around those vessels, a phenomenon that Folkman called tumor angiogenesis.
Folkman's Harvard colleague Stan Korsmeyer found other activated pathways in cancer cells, originating in mutated genes, that also blocked cell death, thus imbuing cancer cells with the capacity to resist death signals. Other pathways allowed cancer cells to acquire motility, the capacity to move from one tissue to another--initiating metastasis. Yet other gene cascades increased cell survival in hostile environments, such that cancer cells traveling through the bloodstream could invade other organs and not be rejected or destroyed in environments not designed for their survival.
Cancer, in short, was not merely genetic in its origin; it was genetic in its entirety. Abnormal genes governed all aspects of cancer's behavior. Cascades of aberrant signals, originating in mutant genes, fanned out within the cancer cell, promoting survival, accelerating growth, enabling mobility, recruiting blood vessels, enhancing nourishment, drawing oxygen--sustaining cancer's life.
These gene cascades, notably, were perversions of signaling pathways used by the body under normal circumstances. The "motility genes" activated by cancer cells, for instance, are the very genes that normal cells use when they require movement through the body, such as when immunological cells need to move toward sites of infection. Tumor angiogenesis exploits the same pathways that are used when blood vessels are created to heal wounds. Nothing is invented; nothing is extraneous. Cancer's life is a recapitulation of the body's life, its existence a pathological mirror of our own. Susan Sontag warned against overburdening an illness with metaphors. But this is not a metaphor. Down to their innate molecular core, cancer cells are hyperactive, survival-endowed, scrappy, fecund, inventive copies of ourselves.
Siddhartha Mukherjee - The Emperor of All Maladies: A Biography of Cancer Page 46