Early Transitions in Breast Cancer

Institution: University of California, San Francisco
Investigator(s): Thea Tlsty, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9WB-0107 Award: $199,424
Award Type: STEP Award
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2003)
One of the major problems with treating breast cancer is that it is detected too late. Our best approach to treating or preventing cancer is early detection and an understanding of the earliest steps in the transition of normal cells to malignant ones. A laboratory-based model that mimics these transitions would provide molecular markers to allow us to detect cancer earlier and provide targets for preventive therapy. We believe we have identified such a model that has allowed us to identify a subpopulation of variant human mammary epithelial cells (vHMEC) that exhibit remarkable characteristics similar to those seen in pre-malignant lesions of breast cancer. These cells are present in healthy women with no known predisposition to breast cancer. By silencing the p16 gene, the vHMEC have acquired the ability to continue dividing until loss of the DNA integrity at the end of their chromosomes (telomeric sequences) causes chromosomal abnormalities. We propose to study how these putative precursor cells transition to malignancy.

We hypothesize that the variant mammary epithelial cells are early precursors to breast cancer. These cells have many of the characteristics of early stages of cancer such as the same types of chromosomal damage, abnormalities in the structures organizing cell division (centrosomes), short telomeric sequences and gene activation patterns commonly observed in cancer cells. One would predict that if variant HMEC are precursors to cancer they should not only be found in the body (as we have already shown) but they should be present in at least a fraction of pre-malignant lesions. To test our hypothesis, we will look for markers of variant HMEC in these lesions.

The methodology we will use to examine pre-malignant lesions for markers of variant HMEC is at the same time both old and new. We will use a powerful new microscope (dual photon confocal microscopy) and new labeling techniques to view the structures within a cell that we believe are critical to cancer transition. While the staining techniques to view whole chromosomes, chromosome ends (telomeres) and centrosomes are commonly used, the ability to see and count these structures in intact human tissue is new.

We are studying cells that have never before been examined in the body (vHMEC). By identifying new markers that are unique to these putative precursor cells, we are able to see patches of these cells in healthy women, before cancer is formed. By studying the cells in plastic dishes we have learned about novel markers that can now be seen in these cells in the human body.

Final Report (2006)
Our studies have identified a rare population of cells, variant human mammary epithelial cells (vHMEC) from disease-free women that, by loss of certain cell cycle checkpoint controls, have the ability to accumulate chromosomal abnormalities and express phenotypes that are critical to malignant progression. The loss of p16 expression, a distinguishing feature of vHMEC permits the population to proliferate in culture and additional 30-50 population doublings beyond the time the majority of the population activates a proliferative growth arrest. These cells continue to erode telomeric sequences until they reach a critically short length and generate telomeric dysfunction. We believe that molecular characterization of these cells provides valuable insight into genesis of telomeric dysfunction and genomic instability that prime cells for transformation.

Through microarray analysis and subsequent validation, we determined that silencing of p16 in vitro was also associated with the upregulation of the stress activated protein, COX-2. To determine the significance of our in vitro findings, we demonstrate that overexpression of COX-2 characterized pre-malignant lesions of the breast and are statistically correlated with dysfunctional p16/Rb signaling and short telomeres. To understand how COX-2 expression is modulated in p16 silenced cells, we probed for relevant regulatory pathways. We demonstrate that the stress-activated protein kinase, p38 and p53 are necessary for COX-2 overeexpression in our in vitro model of premalignancy. We further illustrate that COX-2 overexpression diminishes the cell's response to DNA damage, through which may facilitate genomic instability. We next determined if these regulatory molecules are recapitulated in vivo, how early during breast carcinogenesis is this stress activated pathway initiated and what is the relationship to the onset of proliferation, a major variable in the risk of cancer progression. We demonstrate that stress activation, COX-2 and activated p38, is robustly upregulated during atypical ductal hyperplasia and DCIS compared to normal tissue, before the observed diminution in invasive carcinoma. The onset of proliferation, measured by Ki67, and the accumulation of p53 often a surrogate for mutant p53 was significantly elevated in high grade DCIS and remained elevated in invasive breast tumors. These findings demonstrate that stress activation precedes the onset of proliferation. The implication is that these two events, stress activation and onset of proliferation mark two important transitions during premalignancy.

Although breast cancer is reflected by the aberrant proliferation of epithelial cells, considerable data has shown that the surrounding stroma contributes to cancer progression. We demonstrate that carcinoma-associated fibroblasts (CAF) co-cultured with metastatic breast tumor cells in cleared murine fat pads increase tumor incidence and weight, while decreasing tumor latency. Although CAF co-cultured with non-tumorigenic, vHMEC, do not generate tumors in mice, they do activate malignant phenotypes such as elevated COX-2 expression, epithelial-to-mesenchymal transition and increased proliferation in novel in vitro assays. These data indicate that mammary CAF can stimulate the growth, as well alter the morphology and motility/invasion of non-tumorigenic mammary epithelial cells.

Histologically normal human mammary epithelia with silenced p16ink4A overexpress COX-2, promoting a malignant program
Periodical:Cancer Cell
Index Medicus:
Authors: Crawford, Y., Gauthier, M.L., Joubel, A., Mantel, K., Afshari, C.A., Tlsty, T.
Yr: 2004 Vol: 5 Nbr: Abs: Pg:263-73

Genetic and epigenetic changes in mammary epithelial cells may mimic early events in carcinogenesis.
Periodical:Journal of Mammary Gland Biology and Neoplasia
Index Medicus: J Mammary Gland Biol Neoplasia
Authors: Tlsty, T.D., Crawford, Y.G., Holst, C.R., Fordyce, C.A., Zhang, J., McDermott, K., et al.
Yr: 2004 Vol: 9 Nbr: Abs: Pg:263-74

p38 regulates COX-2 in human mammary epithelial cells and is activated in pre-malignant tissue.
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Gauthier, M.L., Pickering, C.R., Miller, C. J., Chew, K.L., Fordyce, C.A., et al.
Yr: 2005 Vol: 65 Nbr: Abs: Pg:1793-9

Abrogated Response to Cellular Stress Identifies DCIS Associated with SubsequentTumorEvents and Defines Basal-like BreastTumors
Periodical:Cancer Cell
Index Medicus:
Authors: Gauthier, M.L., Berman, H.K., Miller, C., Kozakeiwicz, K., Chew, K. et al.
Yr: 2007 Vol: 12 Nbr: Abs: Pg:479-491