Studies of Telomere Capping Dysfunction in Breast Cancer

Institution: Lawrence Berkeley National Laboratory
Investigator(s): David Gilley, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8KB-0119 Award: $117,712
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2002)
One of the earliest changes during the development of breast cancers is the disruption or rearrangement of the genome within a small subset of normal (i.e., non-cancerous) breast cells. Disruption of the genome or loss of genome integrity can cause normal cellular growth to become deregulated. Cells that lose growth control then divide abnormally and can develope eventually into a cancerous tumor. Recently, there has been an accumulation of evidence that proper maintenance of the very ends of chromosomes (called "telomeres") may play an important role in the loss of genome integrity during breast cancer development. The enzyme responsible for synthesizing the specific DNA at the ends of chromosomes, telomerase, is "activated" in almost all breast tumors that have been examined. In addition, mice, which normally do not develop breast cancer, develop breast adenocarcinomas by the artificial creation of a dysfunctional, or non-capped, telomere. Finally, telomere associations or telomere fusions (which cause genome disruptions) have been shown to occur in a model cellular breast cancer system that is widely used and accepted.

The main specific aims are, first, to use a model cell system for breast cancer to study early stages of cancer progression using microscopic inspection of chromosomes (fluorescence in situ hybridization or FISH). Previous work has suggested a 4-stage progression of breast cancer in this cellular model system that involves senescence (i.e. cell aging), agonescence (escape from cell aging), conditionally immortal, and fully immortal. Our goal is to relate these stages to points at which chromosomal alterations and telomere capping dysfunction occur. The second aim is to follow-up by relating these FISH-detected events with molecular alterations in key temomere-associated proteins, such as Ku, PKCs, Tin2, TRF1 and 2, and Tankyrase. These studies will use antibodies and immunoprecipitation experiments to develop more detailed picture of the molecular associations between these individual telomere proteins. The goal is to develop both a chronological and physical model of how telomere protein complexes are related to stages in breast cell immortalization- the first stage of breast cancer progression.

Finally, in combination with these aims we will also explore the potential of using a PCR-based approach to detect telomere-associated chromosomal fusions. Since tumors are heterogeneous, it is a challenging problem in clinical pathology to detect advanced and potential malignant disease within a background of mostly normal and pre-neoplastic cells. We are hoping that success in a PCR detection test might lead to a clinical application using small needle biopsies of DCIS or other early stage tumors.


Final Report (2003)
Note: Dr. Gilley resigned his CBCRP New Investigator award after one year to accept a faculty position at the Indiana University School of Medicine, Molecular Genetics and Gene Therapy Division.

Background: One of the earliest changes that takes place in the development of breast cancers is that the genetic material or genome is disturbed or rearranged within a small subset of normal breast cells. This rearrangement of the genome has very negative effects, causing normal cell growth to go out of control. Cells that lose growth control then divide abnormally, developing into a cancerous tumor. Recently, there has been an accumulation of evidence that the proper maintenance of the very ends of chromosomes (telomeres) might play an important role in loss of genome integrity during breast cancer development. The enzyme responsible for synthesizing the specific DNA at the ends of chromosomes, telomerase, is activated in almost all breast tumors that have been examined in this way. In addition, mice, which normally do not develop breast cancer, develop breast adenocarcinomas by the artificial creation of a dysfunctional or non-capped telomere. We have recently discovered the first possible evidence of telomere dysfunction in human breast cancer.

Topic addressed: We are studying how breast cancer begins. Since its known that loss of genome stability occurs very early, we are studying what starts these adverse genomic rearrangements. Specifically, we are testing whether loss of chromosome end (telomere) capping takes place in culture models for breast cancer, tumor derived breast cancer lines and actual breast tumor tissue. Our hypothesis is that the ends of chromosomes become uncapped or unprotected and then fuse with other uncapped chromosome ends. Once chromosome ends fuse together, during cell division, chromosomes break or the number of chromosome is disturbed in the daughter cells produced. Genomic instability ultimately leads to the development of breast cancer.

Progress toward specific aims: During the year of CBCRP funding, we discovered that an important telomere protein called TRF2 is dramatically altered in amount during immortalization of human mammary epithelial cells, in breast tumor derived cell lines and in actual breast tumor sections. This is the first evidence that telomere dysfunction occurs in human breast cancers. High levels of endogenous TRF2 in immortalized HMEC and breast tumor-derived cell lines may contribute to the cells' ability to survive gross genomic instability due to critically short telomeres and may help facilitate immortalization. These results suggest that TRF2 overexpression might be a critical step in immortalization and tumorigenesis - indicative of a possible compensatory mechanism to overcome an early event of telomere dysfunction.

Future directions:
1) Determine whether altered TRF2 abundance/localization correlates with changes in related proteins/functions. We will take advantage of the easily manipulated HMEC immortalization system to undertake detailed mechanistic studies of the specific alteration(s) required for TRF2 up-regulation and its consequences.
2) Determine whether TRF2 up-regulation in immortalized HMEC correlates with alterations in protein-protein interactions or phosphorylation states.
3) Determine the prevalence of altered TRF2 abundance among breast tissues. This objective will establish the generality of altered TRF2 expression and start to examine its relevance to particular stages of breast cancer progression.

Impact: The altered regulation of TRF2 in immortally transformed and tumor derived breast cells is a novel finding with potentially important implications both for better understanding of the etiology of the early stages of breast cancer and possible clinical applications. Our proposed studies may permit defining the causes and consequences of HMEC immortalization as it actually occurs during human breast carcinogenesis.