Telomere Clustering is Lost in Mammary Epithelial Tumors

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Patrick Kaminker, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7FB-0018 Award: $79,999
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2001)
Factors involved in the normal aging process appear to contribute greatly to an individualís risk of getting cancer. Telomeres are composed of tandem repeats of DNA and associated binding proteins located at the ends of human chromosomes and have been linked to both aging and cancer. There exists a very strong relationship between maintaining telomeres and tumor progression. In fact, all tumors must find a means to maintain telomeres or lose their ability to continue dividing indefinitely. A great deal of research has focused on attempts to inhibit telomere maintenance in tumors, however, the number of cell divisions that can occur even without regulating telomeres may limit the potential therapeutic benefit. Our lab has uncovered a novel method of telomere organization that may influence the normal development of mammary cells and protect these cells from the forming tumors.

Because of the importance of telomeres in cancer, major efforts have been focused on understanding the function of telomeres. During our analysis of telomeres in cell culture models we have uncovered a unique structural property of telomeres that is directly related to the normal development of breast cells. If we disrupt these structures, which we call telomere clusters, we see characteristic changes in these cells that resemble the changes seen in breast cancer cells. Our question is what role do these telomere clusters play in the normal development of breast tissue and do they play a role in the formation of breast tumors. We will use standard microscopy techniques to determine the composition of these telomere clusters. Using antibodies to known telomere-binding and DNA-repair proteins we will determine if these proteins can be specifically found in telomere clusters. Once the composition of the clusters is known we will disrupt the clusters by producing abnormal amounts of normal and mutant forms of the cluster components and then determine which components are necessary for maintaining these structures. Using standard cell culture assays and animal models we will determine if disruption of telomere clusters results in the formation of breast cell tumors.

Initial results have uncovered a previously unknown mechanism in which cells can control their normal development and protect against events that may facilitate tumor progression. Using a 3D cell-culture model we can now study how specific genomic structures allow mammary cells to interact with each other and the surrounding environment. This will allow us to follow the pattern of cellular and tissue organization as cells progress into tumors and how the genome directly affects this organization.

Final Report (2004)
Immortality is primary requirement and a preliminary event for malignant conversion. In order for cells to bypass their "mortality' constraints they must overcome one major proliferative barrier; telomere erosion. Telomeres are structures at the ends of linear chromosomes that consist of repetitive DNA sequences and regulatory proteins. They function to maintain the integrity of the genome by protecting the ends of the chromosomes from being recognized by DNA damage repair proteins and undergoing illegitimate recombination. Because of the inherent problem of replicating linear chromosomes, telomeres erode during each round of division and have been proposed by some to be a "mitotic" clock. When cells are grown in the laboratory, telomere length determines their replicative lifespan and activation of telomerase, an enzyme normally absent in most cells, is sufficient to confer immortality. Activation of telomerase is also the primary mechanism tumor cells use to overcome the replicative constraints imposed by telomere shortening. Prior to telomere-dependent growth arrest epithelial cells have an additional proliferative barrier that involves the p16 tumor suppressor. Although this additional barrier is expected to provide greater protection from tumor formation, the majority of human tumors occur in epithelial tissue such as mammary and prostate. These differences indicate that both growth arrest and telomere regulation differ significantly between cells of mesenchymal (structural cells such as bone, cartilage, or blood vessels) and epithelial origin. Our studies have focused on the specific differences in telomere regulation between both cell types.

We initially observed a distinct difference in subnuclear localization of a telomere-associated protein, TIN2 that resembled the telomere "clustering" phenotype observed in yeast. In fibroblast cells, TIN2 is seen only as multiple, distinct foci at telomeres and we predicted that telomeres underwent a distinct organization in human mammary epithelial cells (HMECs) during morphogenesis and differentiation. Moreover, disruption of these clusters by introducing a mutant form of TIN2 into these cells disrupted morphogenesis, prevented "telomere clustering", and disrupted growth arrest. The phenotype of non-malignant HMECs, grown in three-dimensional cultures, resembled the malignant variant of these cells, prompting our study to determine what the contribution of telomeres and TIN2 had to tumor progression.

We made several key discoveries towards identifying the role of telomeres and TIN2 towards regulation of growth arrest during the period of this funding. Principally, we have found that TIN2 clusters are independent of telomeres and form at heterochromatin rich domains in the nucleus as a function of growth arrest. Furthermore, disruption of TIN2 clusters disrupts the ability of HMECs from maintaining growth arrest and the absence of distinct TIN2 clusters in malignant HMECs and tissue samples indicates that migration of TIN2 to heterochromatin domains is a checkpoint that is overcome during tumor progression. TIN2 appears unique with respect to clustering as other telomere-regulatory and DNA damage repair proteins normally found at telomeres do not change their nuclear distribution during different phases of the cell cycle. Our recent findings also indicate that TIN2 clusters form as a function of telomere length. Activation of telomerase in HMECs is sufficient to lengthen telomeres whereas in fibroblasts activation of telomerase only maintains telomeres at their current length. By introducing telomerase into HMECs we eliminate TIN2 cluster formation during initiation of growth arrest, indicating that as telomeres erode cells alter the nuclear distribution of TIN2 in order to induce proper growth arrest. Cells undergoing permanent, telomere -dependent growth arrest (agonescence) also displays TIN2 clusters.

Characterizing the effects of TIN2 on growth arrest yielded very useful data on the mechanism by which cells bypass this checkpoint. Although initially proposed in the grant we were unable to determine the effects of TIN2 disruption on tumor formation. We plan to complete this in the future as well as identify other components of the clusters and determine what signaling pathways trigger TIN2 redistribution.

Telomeres, aging and cancer: In search of a happy ending
Index Medicus: Oncogene
Authors: Kim HS, Kaminker P, Campisi J
Yr: 2002 Vol: 21 Nbr: 4 Abs: Pg:503-11