Research Grants Awarded
Causes and consequences of sub-nuclear re-distribution of telomerase in breast pre-malignancy
Background: In order to achieve immortality, pre-malignant breast cells must stabilize their telomeres. My recent data indicate that nuclear re-distribution of existing levels of telomerase, rather than up-regulation of telomerase as previously supposed, may be sufficient for stabilization of telomeres in cultured human mammary epithelial cells (HMEC). In addition, several telomere-associated proteins (TRF2, Ku80, WRN) change dramatically as HMEC undergo immortalization. Objective/Hypothesis: I propose that in finite lifespan cells, telomerase is sequestered in the nucleolus, away from the telomeres, and is unavailable for telomere stabilization. During the process of immortalization, telomerase gains access to the telomeres through up-regulation and/or re-distribution. In the latter case, the total cellular levels of telomerase do not change significantly, but are instead redirected to the telomeres by changes in expression, localization, or function of telomere- and telomerase-associated proteins. Specific Aims: Using a well-characterized HMEC culture system, I plan to: 1) determine whether endogenous telomerase is sequestered within nucleoli or other sub-nuclear organelles in finite lifespan HMEC; 2) evaluate the differences in the abundance and/or distribution of telomere- and telomerase-associated factors in finite lifespan and immortalized HMEC; 3) directly assess the effects of these factors on telomerase localization and proliferative potential of HMEC; and 4) evaluate the differences in the abundance and/or distribution of telomere- and telomerase-associated factors in breast tissues with different histopathologic features. Study Design: (1) I will perform immunofluorescent co-localization studies of telomerase and selected markers of nucleoli, Cajal bodies, and PML bodies, in finite lifespan and immortalized HMEC. (2) I will determine whether immortalization correlates with changes in the levels of expression, and/or localization, of TRF2, Ku80, DNA-PKcs, WRN, nucleolin, and PinX1. (3) I will perturb the expression of these proteins in finite lifespan HMEC using shRNA and cDNA, to test whether these perturbations change the distribution of endogenous telomerase and/or the proliferative lifespan of these cells. (4) I will perform semi-quantitative immunohistochemical and immunofluorescent analyses of telomere- and telomerase-associated proteins in sections of breast tumor specimens, and determine whether any likely correlations exist with histological features or clinical parameters such as tumor size, grade, lymph node status, or ER presence. Potential Outcomes and Benefits of Research: My hypothesis could explain why as many as 75% of malignant breast lesions have equivalent or lower levels of telomerase than normal breast tissues. If correct, such a finding would indicate that certain telomere- or telomerase-associated proteins are likely to be better markers of breast pre-malignancy than telomerase itself.
Multiple errors in cellular growth control mechanisms contribute to invasive breast cancer. We use human mammary epithelial cells (HMEC) cultured from normal tissue to model the processes that lead to unlimited growth (immortality), a prerequisite for the acquisition of malignant traits. HMEC from normal tissues are limited in the number of cell divisions they can undergo in culture. In contrast, cells from breast tumor tissues are immortal. Immortality is associated with an enzymatic activity called telomerase, which is dependent upon the expression of a protein called hTERT. Telomerase acts to maintain telomeres, the ends of human chromosomes. Telomeres protect the chromosomes from biochemical reactions that can damage genetic material. In normal, non-immortal HMEC, telomeres become shorter with each cell division, eventually resulting in growth arrest or cell death. While this is deleterious for the individual cell, it is beneficial to the whole organism, because it ensures that cells that may have accumulated defects are not propagated indefinitely. It is widely believed that normal breast tissues and HMEC derived from them do not express hTERT and have no associated telomerase enzyme activity, while almost all human breast cancer tissues and immortal cells lines display telomerase activity, and maintain intact telomeres indefinitely. Most researchers accept that hTERT expression and telomerase activity must be increased in order to achieve immortality. However, recent studies employing more sensitive methodology have indicated that normal breast tissues have higher hTERT expression and telomerase activity than 75% of malignant breast lesions. Thus, hTERT levels and telomerase activity by themselves are not necessarily useful markers of pre-malignancy in breast tissues. I propose that in at least some non-immortal cells, telomerase is present at levels that would be sufficient for maintaining intact telomeres, but that this telomerase is sequestered away from the telomeres. During the process of immortalization, telomeres are stabilized through increase or re-distribution of hTERT. In the latter case, the total levels of hTERT do not change, but are instead redirected to the telomeres by changes in other proteins. A better understanding of the mechanisms of immortalization that actually occur during cancer progression is required for studying therapeutic intervention into the immortalization process. Having elucidated this alternative, never-before proposed mechanism of immortalization, I will advance the field by explaining why telomerase levels are not always predictive of breast cancer progression. I will also identify new proteins participating in HMEC immortalization, which have the potential of becoming informative markers of breast pre-malignancy. This could allow for development of new methods of early detection of breast cancer, new prognostic information, and new ways of inhibiting tumor progression.