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Mutant Stem Cells May Seed Cancer

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Science  05 Sep 2003:
Vol. 301, Issue 5638, pp. 1308-1310
DOI: 10.1126/science.301.5638.1308

Some cancers are sustained by a small minority of cells; their resemblance to normal stem cells might explain why many cancers are so hard to eradicate, and it has researchers rethinking cancer treatments

Stem cells have acquired a golden glow in the past few years as a possible tool for reversing the damage diseases wreak on organs. Many researchers predict that stem cell transplants—whether derived from embryonic tissue or from adult cells that retain the flexibility to develop into various tissues—will someday repair hearts crippled by heart attacks or brains under attack by Alzheimer's or Parkinson's disease. But the very qualities that make these cells so attractive for medicine—especially their capacity to replicate ad infinitum—also hint at a dark side. Recent evidence suggests that they may be the source of the mutant cells that give rise to cancerous tumors and maintain their growth.

Researchers have identified what they call “cancer stem cells” in blood cancers, such as leukemias, and in breast and brain cancers. The finding raises the possibility that the mutations that drive cancer development may have originated in the body's small supply of naturally occurring stem cells. Cancer stem cells resemble those normal cells in several ways. In particular, both types are self-renewing. This means that when they divide, one of the daughter cells differentiates into a particular cell type that eventually stops dividing, but the other retains its stem cell properties, including the ability to divide in the same way again.

The new work shows that cancer stem cells, which form only a small proportion of the total tumor cell population, are the only tumor cells with the capacity to keep tumors growing. “Most people think that cancer is cells that grow too much, but only one in a million [leukemia cells] has the ability to sustain the disease,” says stem cell biologist John Dick of the University of Toronto.

Irving Weissman, a stem cell expert at Stanford University School of Medicine in California, describes the therapeutic implications of the work as “absolutely huge … it has the chance of leading to new ways of thinking about cancer and new kinds of therapies.” To cure cancer completely, Weissman and others say, it may be necessary to design therapies that target cancer stem cells, assuming that can be done without also wiping out the stem cells needed to maintain tissues such as the bone marrow and intestinal lining. In addition, the growing suspicion that stem cells may be the source of some cancers sounds a note of caution for efforts to use stem cells for organ repair.

Bad seeds

The idea of cancer stem cells has been kicking around for 40 years. An early indication that such cells might exist came from the finding that just 1% or less of leukemia cells grow and form colonies in lab dishes. This implied that only those few cells could drive tumor growth in patients, but the work more or less petered out in the mid-1970s because researchers weren't able to assess the carcinogenic potential of the cells in living animals. Only in the early 1990s did that begin to change, aided by a mouse model Dick and his colleagues were using to study the development of human hematopoietic stem cells, which give rise to the various types of blood cells.

A potent few.

Breast cancer cells with stem cell-like markers formed a large tumor (bottom left and mouse's left side), whereas injections of other breast cancer cells didn't grow (top left and mouse's right side).

CREDIT: AL-HAJJ ET AL., PNAS 100 (7), 3983 (2003)

The model starts with an extremely immunodeficient mouse strain called the NOD/SCID mouse. The researchers irradiate the animals to destroy their bone marrow and then introduce human stem cells to see if they will produce a new complement of blood cells. After showing that normal human hematopoietic stem cells can do this, Dick and his team used the approach to study the cancer-causing power of acute myeloid leukemia (AML) cells freshly harvested from human patients. By progressively diluting a known number of leukemia cells, they established that only the very rare AML cell—about one in a million—has the ability to reproduce the disease in the animals.

Because this was a much smaller fraction of cells than could form colonies in culture, the result indicated that the simple ability to grow didn't equate with the ability to develop into leukemias in living animals. Dick speculated that the leukemia-initiating cells had a greater developmental potential than the vast majority of clone-forming cells and might even be stem cell-like.

The Toronto team provided direct evidence for that supposition. They characterized the leukemia-initiating cells according to surface proteins called markers that distinguish the various cell types of the hematopoietic system. The leukemia-initiating cells turned out to belong to an exclusive group. They were positive for the CD34 marker and negative for CD38—the same as human hematopoietic stem cells—and did not carry markers of more mature cells. “They're very similar to normal stem cells,” Dick says.

The cancer cells' resemblance to normal stem cells held up even though AML is a heterogeneous disease, with several different subtypes depending on which genetic abnormalities the patients' cells carry. Dick and his colleagues characterized the leukemia-initiating cells from the various AML subtypes and found that all belonged to that same CD34+/CD38 class. When put into NOD/SCID mice, however, each cell type produced a leukemia identical to that in the patient from which it had originally been isolated. All in all, Dick concludes, it's likely that the initial mutations that gave rise to the leukemias arose in normal stem cells, causing them to take the wrong developmental pathway.

Stanford's Weissman has come to a similar conclusion by tracing a common genetic abnormality leading to AML. This is the so-called 8;21 chromosomal translocation, in which the AML gene on chromosome 21 is fused to the ETO gene on chromosome 8, thus leading to production of an abnormal hybrid protein that alters gene expression. Weissman found the translocation both in stem cells and in more mature blood cells of several different types in AML patients. That suggests that the translocation originated in a stem cell that then gave rise to the different mature cells as well as replicas of itself.

But even though the translocation is necessary for this type of AML to develop, Weissman says it is not sufficient. Stem cells that were isolated from patients in remission and carried the abnormality, he and his colleagues found, differentiated normally in culture. This indicates that additional mutations, possibly occurring at a later developmental stage, are necessary to produce leukemia. Unlike Dick, who sees the original hematopoietic stem cell as potentially leukemic, Weissman thinks that the leukemia stem cell may be one or more steps down the differentiation pathway.

The cells of solid tumors are harder to isolate and study than those of the hematopoietic system, but recent evidence suggests that solid tumors also contain stem-like cells. Some of it comes from research on breast cancer by Michael Clarke and his colleagues at the University of Michigan Medical School in Ann Arbor. They, too, used the NOD/SCID mouse to assay for cells able to initiate cancer, in this case injecting human breast cancer cells into mouse mammary glands.

Necessary for growth.

Without a gene called Tcf-4, fewer projections grow in the lining of the mouse intestine (right) compared to a control (left). The abnormality is apparently due to a stem cell deficit.

CREDIT: KORINEK ET AL., NATURE GENETICS 19, 379 (1998)

The Michigan team found that, as with AML, only a small fraction of breast cancer cells form tumors in the NOD/SCID mouse, as reported in the 1 April issue of the Proceedings of the National Academy of Sciences. These cells could be distinguished from non-tumor-initiating cells based on their surface markers. (They were CD44 positive, CD24 negative or low.) “We can prospectively identify cells that are capable of driving tumor growth and metastasis,” Clarke says.

At present, Clarke and his colleagues can't be positive that the cell population they identified is derived from normal stem cells. But the cancer-initiating cells possess the key stem cell characteristic of self-renewal. The tumors growing in the mouse mammary pads contained their own small populations of CD44+/CD24−/low cells, which could be isolated and used to initiate tumor growth in other mice. It's still possible, however, that more mature cells regained the ability to self-renew, Clarke says.

Mutant stem cells may seed brain tumors as well. In work due to appear in the 15 September issue of Cancer Research, a team led by Peter Dirks at the Hospital for Sick Children in Toronto isolated an apparent stem cell for brain cancer. The researchers found that a variety of brain cancers, from relatively slow-growing astrocytomas to highly aggressive medulloblastomas and glioblastomas, contain small populations of cells with very similar, stem cell-like properties. They carry markers previously associated with normal neural stem cells and do not have markers characteristic of more differentiated cells. The cells also had the ability to self-renew and to differentiate, at least in lab cultures. Together, these qualities suggest that the different types of tumors derive from a common stem cell population, Dirks says.

Further support for the idea that brain tumors originate in stem cells comes from Luis Parada of the University of Texas Southwestern Medical Center in Dallas and his colleagues. They created a mouse model of brain cancer by mutating an oncogene called neurofibromatosis 1 along with the p53 tumor-suppressor gene in an early precursor of brain neurons and other cells. The mice develop numerous brain tumors, Parada reported in February at a meeting of the American Association for Cancer Research. Imaging studies showed that although the tumors can end up anywhere in the brain, they all originate in two areas, the lateral ventricles and hippocampus—both places where brain stem cells are located.

Preliminary evidence suggests that the proportion of stem cells in a tumor may determine how deadly it is—a finding that could have prognostic implications. Dirks found that fast-growing medulloblastomas and glioblastomas had many more putative tumor stem cells than astrocytomas had. And Clarke notes that an extremely aggressive breast cancer that he studied contained about 25% stem cells. He cautions, however, that so far researchers have studied tumors from only a small number of patients, and more work will be needed to confirm these findings.

A common path

Another line of evidence suggesting that cancers originate from stem cells comes from studies of the biological machinery underlying self-renewal. Normal and cancer stem cells show some striking similarities. Recently, for example, researchers have shown that the genes Bmi-1 and Wnt, both of which can cause cancer when mutated, are needed for self-renewal in normal and cancer stem cells.

The Bmi-1 gene participates in normal hematopoietic development, and its malfunction has been linked to AML. In work reported in the 15 May issue of Nature, two teams, one led by Clarke and Weissman and the other including Guy Sauvageau of the University of Montreal and Julie Lessard of the Clinical Research Institute of Montreal, link the gene to self-renewal.

To test whether cells missing Bmi-1 can self-renew, the researchers transplanted stem cells from Bmi-1 knockout mice into normal mice that had been irradiated to destroy their bone marrow. The stem cells produced a normal complement of blood cells—but only briefly. By 8 weeks, blood cells derived from the transplanted cells had almost disappeared, and when bone marrow taken from the animals was put into a second series of mice, no Bmi-1-deficient blood cells could be detected.

Bmi-1 is also needed for the self-renewal of leukemia cells, Sauvageau and Lessard showed. About 5 years ago, Sauvageau and his colleagues discovered that they could cause an AML-like disease in mice by introducing two oncogenes, Meis1a and Hoxa9, into the bone marrow cells of the animals. In the current work, Sauvageau and Lessard introduced these genes into fetal liver cells, which contain hematopoietic stem cells, from normal mice and animals in which Bmi-1 had been inactivated.

Altered cells from both types of mice transiently produced leukemias when transplanted into irradiated mice, but the cells lacking Bmi-1 could not be transmitted to a second set of recipients. This result shows that without Bmi-1, leukemia stem cells die out, just as normal stem cells do.

The Wnt gene is likewise the focus of a great deal of research by both cancer researchers and developmental biologists. The protein encoded by the gene normally controls cell fate decisions during the development of many of the body's tissues. It exerts its effects by binding to, and thus activating, a receptor on the cell surface membrane. This in turn sets off a series of changes inside the cell, culminating in the activation of genes governing cell division and differentiation.

Evidence suggests that operation of this pathway, sometimes called the “Wnt cascade,” is needed for stem cell maintenance. For example, about 5 years ago, Hans Clevers of the University of Utrecht in the Netherlands and his colleagues created a strain of mice in which the gene encoding a protein called Tcf-4 had been knocked out. Tcf-4 operates at the end of the Wnt cascade, working with certain other proteins to activate gene expression in response to Wnt signals. Animals lacking the gene died shortly after birth and displayed a single defect: The stem cells that produce the lining of the intestines appeared to be missing. The Wnt cascade is also needed for self-renewal of hematopoietic stem cells, Weissman's team in collaboration with Roel Nusse, also at Stanford, and his colleagues reported a few months ago.

Abnormal activation of the Wnt pathway, and presumably of self-renewal, can lead to cancer. For example, the APC gene, which is mutated early in the development of 90% of colon cancers, is part of the Wnt cascade. As a result of this mutation, Clevers says, “you have an active Wnt cascade without [an incoming] Wnt signal.”

Stem cell sustainer.

In the absence of a Wnt signal (left), any β-catenin not involved in forming adherins junctions (AJs) between cells is tagged for destruction by a complex of proteins including APC. But binding of Wnt (right) to its receptor (Fz) prevents β-catenin breakdown and allows it to join with other proteins to activate gene expression.

CREDIT: ADAPTED FROM L. ALONSO AND E. FUCHS/GENES & DEVELOPMENT; ILLUSTRATION: PRESTON HUEY

Consistent with that, Clevers and his colleagues have found that gene expression patterns in colon cancer cells look a lot like those in colon stem cells. Using DNA microarrays, the researchers analyzed the changes produced by Wnt signaling in colorectal cells. Wnt up-regulates 120 genes and down-regulates 115, they reported in the 18 October 2002 issue of Cell.

When the researchers then looked at normal cells of the gut, they found a similar gene expression pattern in the areas where colon stem cells are located. In contrast, in the areas where differentiated cells are located and Wnt signals are presumably not present, the pattern was just the opposite: The 120 genes up-regulated in the cancer cells were turned down, and those that were down-regulated were turned up. Mutations in the pathway “activate a situation where cells can't shut off the stem cell program,” Clevers concludes. That locks cells with the mutations into a proliferative state during which they can accumulate additional mutations that ultimately produce full-fledged cancers.

Other researchers, including Elaine Fuchs of Rockefeller University in New York City and Fiona Watt of Cancer Research UK's London Research Institute, have shown that the Wnt pathway controls cell fate decisions in the epidermis of the skin. Here, too, malfunctions in the pathway can lead to tumor development. The Fuchs team found evidence that mutations that abnormally activate β-catenin, a key component of the Wnt pathway, cause pilomatricomas, a benign tumor of the hair shaft in humans.

Forewarned

Despite the growing evidence that some cancers arise in stem cells, the case isn't airtight. But those favoring the idea offer two additional arguments. First, stem cells already have the ability to self-renew, whereas more mature cells would somehow have to regain it if they were to turn cancerous.

Second, especially in organs such as the skin and colon lining, where older cells are constantly dying and sloughing off, stem cells may be the only cells that hang around long enough to accumulate the several mutations needed to produce full-fledged cancers. “Cancer [development] is long-term,” Sauvageau says. “You can't have a cancer develop in a [cell] that is short-lived.”

But even if cancer doesn't originate in normal stem cells, tumor growth apparently depends on a small population of stemlike cells that may differ from the bulk of the tumor population in ways that make them resistant to therapy. Current cancer treatments “may not be hitting the cells that can make the tumor come back,” Dick says.

In particular, many cancer therapies are designed to kill dividing cells. Somewhat surprisingly, stem cells are mostly quiescent. They apparently wake up to divide only occasionally—a lifestyle that means they will likely escape the cell-killing effects of standard therapies. Researchers may therefore need to find ways to wipe out cancer stem cells.

And beyond implications for cancer therapy, the findings raise concerns about efforts to use stem cells to treat other diseases. Dick and others working on cancer stem cells note that many potential applications require that stem cells be forced to divide to produce enough cells to replace damaged tissue, and that might facilitate the accumulation of potentially cancer-causing mutations. “We need to think carefully about that,” Dick says.

Weissman points out, however, that even if a stem cell had an initiating mutation, such as the 8;21 translocation of AML, accumulation of the additional mutations needed to produce a cancer would still be relatively unlikely. He cites his team's finding that stem cells bearing the translocation obtained from patients in remission differentiated normally in culture. Many of those patients had been in remission for 15 years or longer, and during that time their stem cells hadn't acquired the additional mutations that would have rekindled leukemia.

Defensive measures may also be possible based on the growing understanding of how stem cells can turn bad. Modern methods of marker analysis and cell-sorting may make it possible to identify and then eliminate cells bearing dangerous mutations. As Cervantes put it: “Forewarned, forearmed; to be prepared is half the victory.”

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