Breast Cancer Research at Berkeley Lab
Part 2: The Dark Side of Immortality

Second in a series on research at Berkeley Lab that aims to eradicate the scourge of breast cancer.

Addressing some 2,0000 researchers, physicians, policy-makers and breast cancer survivors gathered in Philadelphia for the 2005 Era of Hope conference, Berkeley Lab Distinguished Scientist and breast-cancer research pioneer Mina Bissell explained how, under certain conditions, the phenotype of a cell — its physical characteristics — can play an even bigger role in breast cancer development than its genotype, or genetic characteristics. (See Part 1 of this series.)

Martha Stampfer and Paul Yaswen

Bissell's findings on the importance of cell phenotypes have added a new dimension in the study of breast cancer development, but they do not diminish the important role played by genetics. In another pair of presentations at the Era of Hope conference, two long-time Berkeley Lab collaborators, cell biologists Martha Stampfer and Paul Yaswen, discussed the complex interplay of genetics that enables human mammary epithelial cells (HMECs) to become "immortalized" — able to grow and divide indefinitely. Cell immortalization is considered a critical step down the road to malignancy and invasive cancer.

When immortality is a threat

"Once HMECs have been immortally transformed, the introduction of one or two oncogenes can further transform these cells towards malignancy," says Stampfer. "Cells that have not been immortally transformed cannot be rendered malignant by those same oncogenes."

Since 1976, Stampfer has worked to develop cultures of HMECs that can be used by cancer researchers to study the normal mechanisms by which proliferation and differentiation in human cells are controlled, and to understand how these normal processes become altered as a result of immortal and malignant transformation.

Normal HMECs, like most normal human cells, have a limited capacity for replication. After about 50 to 80 divisions, an HMEC either stops dividing or dies. This stringently enforced finite lifespan, often referred to as "replicative senescence," is thought to have arisen in long-lived organisms, such as humans, as a means of preventing cancer.

	Since 1976, Berkeley Lab's Martha Stampfer has been developing cultures of human mammary epithelial cells for the study of breast cancer.

"Development of most adult onset cancers requires multiple genetic changes that accumulate with successive cell divisions," Stampfer says. "The limited cell division capacity of normal cells makes it highly unlikely that a single cell could accumulate so many genetic errors."

Immortalized cells undergo alterations that enable them to overcome the natural barrier of replicative senescence so that their growth and division continues unchecked. This is often accompanied by genomic instability, which allows more genetic errors to arise with each cell division, opening the door to malignancy.

Says Stampfer, "The long-term objective of my program has been to develop and characterize an HMEC system for use in a wide variety of studies on human cell biology and carcinogenesis. The goal is to understand the normal growth control processes in HMECs and to determine how these processes may be altered as a result of immortal and malignant transformation."

The p53 protein

Just how powerful a research tool these cultured HMEC lines can be was demonstrated when Stampfer and Yaswen used them to reveal important new information about the relationship between a tumor-suppressing protein called p53 and telomeres, the structures that protect the ends of chromosomes and enable tumor cells to grow and divide indefinitely. The p53 protein, which is retained in most breast tumors but is lost more frequently in other cancers, was shown to act as a sort of emergency brake to slow or even stop the development of cancer in its early stages.

"We found that p53 plays a role in cancer suppression both before and after cells become immortalized," says Stampfer. "The role of p53 after immortalization had not been discovered in previous studies because the p53 gene had been inactivated before the transformation of HMECs to immortality."

Telomeric DNA is shown in these breast cells as pink dots. In the normal breast cells, on the left, the telomeric DNA is in far more ample supply than in the cancerous cells on the right. (Images courtesy of Alan Meeker)

Stampfer and Yaswen led a research effort showing that the presence of p53 in newly immortalized cells prevents the cells from expressing high levels of telomerase, the enzyme that maintains the telomeres. Without sufficient levels of telomerase, cell replication slows to a crawl, even though the cells are characterized as immortal. The presence of p53 in most breast cancer cells, acting to slow the cell division process, may explain why breast cancer is less aggressive than many other forms of cancer.

"Our findings have suggested that even though newly immortal cells have gained the potential to express telomerase, their very short telomeres and low level of telomerase activity may render them extremely sensitive to telomerase inhibitors, especially when p53 is still present," Stampfer says. "Mutation or loss of p53 can propel precancerous cells from an indolent, genetically unstable, slow-growing state to a rapidly growing and evolving state that leads to a full-blown cancer."

Telomerase and immortal transformation

In order for an HMEC to undergo immortal transformation, it must overcome two barriers. The first of these barriers, easily overcome in cultured HMECs, is mediated by a tumor-suppressing protein called retinoblastoma.

The second barrier, much more difficult to overcome, is mediated by the length of the DNA in the telomeres. Once the telomeric DNA become too short the ends of affected chromosomes may become damaged, and the host HMECs can lose the ability to make accurate copies of their genomes. If the shortening of the telomeric DNA continues — in the absence of sufficient telomerase, a cell loses some of its telomeric DNA with each successive division — most cells stop dividing altogether or die. This process, known as the "telomere crisis," is a natural defense mechanism aimed at preventing the spread of genetically damaged cells.

In the march to malignancy, there is a progressive shortening of telomere lengths that is accompanied by an increase in genomic instability. In some cases, this genomic instability can give rise to genetic aberrations that lead to invasive cancer.

At the Era of Hope conference Paul Yaswen reported that in some cases the genomic instability associated with dysfunctional telomeres can give rise to genetic errors that actually lead to immortalization and, ultimately, malignancy. According to Yaswen, genetic analyses have shown that the expressions of more than 1,500 genes are deregulated by genome aberrations in breast cancer. The observations that he, Stampfer, and their collaborators have recorded are consistent with clinical observations that, during the early "pre-invasive" stages of cancer, the telomeres of cells in a troubled area are very short. Furthermore, the loss or absence of the p53 protein is associated with patients facing the worst prognosis.

In her presentation, Stampfer said that one of her goals is to test the hypothesis that small amounts of telomerase synthesized in newly immortalized HMECs may be able to protect the telomeric ends, thus suppressing genomic instability.

The Role of ZNF217

Yaswen has collaborated with Joe Gray, Berkeley Lab's Associate Laboratory Director for Life and Environmental Sciences, and Colin Collins of the University of California at San Francisco in a search for oncogenes in a specific area of chromosome 20 known to be amplified in a large number of human breast cancers. The researchers, led by Gray, identified ZNF217 as a gene in this region whose level of expression consistently matched the levels of amplification found in breast tumors and cancerous cell lines.

"ZNF217 was relatively inactive in normal breast cells, but highly active in a number of breast cancer cell lines," Yaswen says. "The next logical step was to try to determine the role of the ZNF217 gene, if any, on breast cancer progression, by putting it into normal breast cells."

Introducing extra copies of ZNF217 into cultures of normal human mammary epithelial cells caused some of those cells to become immortal. The cells took on other characteristics as well, similar to changes observed in Stampfer's cultures of immortalized breast cells when they were exposed to chemical carcinogens. And ZNF217-treated cells displayed a new resistance to TGF b (Transforming Growth Factor beta), a substance that normally stops the growth of many different types of cells.

Yaswen and collaborators, including Stampfer, are now studying possible cooperative interactions of ZNF217 and c-myc, another oncogene linked to breast cancer, during HMEC immortalization and the progression towards malignancy.

"We hypothesize that most spontaneous human solid tumors arise from telomerase deficient cells which acquire malignant changes during the period of genomic instability associated with telomere dysfunction," says Yaswen. "Our research suggests that augmenting the molecular mechanisms and processes that monitor and prevent the growth of cells with dysfunctional telomeres may be useful for preventing immortalization and malignancy."

Additional information

• More about Martha Stampfer's work with HMEC cultures
• More about Paul Yaswen's research
• More about the 2005 Era of Hope meeting of breast cancer researchers
• Read Part 1 of Breast Cancer Research at Berkeley Lab