Genes Involved in Immortalization of Human Mammary Cells

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Martha Stampfer, Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4JB-0119 Award: $251,476
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1998)
Normal human mammary cells have a limited lifespan. After a certain number of cell divisions, they are no longer capable of further proliferation. Most scientists now believe that this limited lifespan, referred to as cellular senescence, arose as a cancer prevention mechanism in long-lived organisms such as humans. This hypothesis is based on two lines of evidence. First, invasive human breast carcinomas (and most adult cancers) are thought to result from accumulation of multiple genetic errors - around 9 errors have been estimated for breast cancer development. The limited proliferative capacity of normal breast cells makes it highly unlikely that so many errors could accumulate in the absence of some means of overcoming cellular senescence. Second, the vast majority of human breast cancers (and most human cancers) express an enzymatic activity, called telomerase, which is associated with an unlimited proliferative capacity - or immortality. This activity is not found in non-tumor breast tissues. Cells derived from normal human breast tissues exhibit cellular senescence when cultured under laboratory conditions, whereas some cells from tumor tissues can express an unlimited proliferative potential in culture.

Since it is thought that nearly all breast cancers have undergone changes that allow them to overcome normal cellular senescence, uncovering the underlying mechanisms that allow these cells to gain an extended or unlimited potential may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention in breast carcinogenesis. This proposal is concerned with uncovering those mechanisms involved in loss of normal cellular senescence and thus potentially responsible for the progression of early stages of breast cancer.

The vast majority of breast cancers originate from a type of cell called an epithelial cell. Our laboratory has pioneered the development and use of cultured human mammary epithelial cells (HMEC) in order to study the changes in growth control processes that occur during breast cancer progression. Using chemical carcinogens, we have been able to transform normal finite lifespan HMEC to immortality. However, we still do not know what molecular changes have permitted these transformed cells to overcome cellular senescence. The goal of this proposal is to identify the individual genes that normally prevent immortalization, based on the assumption that these genes are mutated in immortally transformed cells.

To identify such genes, we will use a relatively new methodology that employs gene fragments that may act as genetic suppressor elements. Finite lifespan HMEC which are likely to need just one more mutation to become immortal will be exposed to gene fragments (packaged in viral vectors) that may be able to disrupt the normal functions of the immortalization suppressor genes. Since immortally transformed cells will maintain growth after the finite lifespan cells have stopped growing, immortalized cells can be easily detected. The virally inserted gene fragments can then be identified by molecular markers attached to the viral vectors. The significance of the isolated and sequenced gene fragments will then be gauged by comparing their expression in normal vs. transformed HMEC, as well as by functional tests in which the full-length gene is introduced into immortal HMEC. The discovery of genes that normally suppress immortalization may aid in the design of drugs that specifically target the immortalization growth defect, provide diagnostic information, and greatly facilitate further studies on HMEC immortalization

Final Report (2001)
Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions-a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). When specific oncogenes are inappropriately turned on in a key cellular signaling pathway (Ras, Raf) they can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to abnormally high levels of oncogenic RAF changes from being tumor suppressive to tumor enhancing, and what are the molecular underpinnings of this response. Our data indicate:

1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of mortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited.

2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response.

Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means if enforcing a Raf induced growth arrest and that this tumor suppressive function is lost at a specific stage n malignant transformation. Further studies to elucidate the ways by which immortal, converted HMEC escape this arrest may provide a more complete model of breast carcinogenesis as well as ways to intervene in that process.