Isolation of Cancer Precursors from Normal Human Breasts

Institution: University of California, San Francisco
Investigator(s): Bob Liu, Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12FB-0114 Award: $45,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2006)
My mentor’s lab (Dr. Thea Tlsty, UCSF) is focused on understanding the earliest events of breast cancer development, specifically those that can cause normal cells to become malignant. We employ a cell culture model system of growing normal human mammary epithelial cells (HMEC) to identify early transformation. From normal HMEC, we have identified a rare population of cells that have abnormal growth control and malignant characteristics; we refer to these cells as “variant” HMEC (vHMEC). vHMEC do not express a tumor suppressor gene, called p16INK4a, due to hypermethylation of the p16INK4a promoter. The p16INK4 gene, which produces a physiological inhibitor of the cyclin D-CDK4 complex, is considered as an important tumor suppressor gene. Thus, alterations in p16INK4 functions, or gene regulation, are thought to lead to a variety of changes associated with early tumor formation. For example, the loss of p16INK4a is sufficient to induce centrosomal abnormalities leading to genomic instability in the vHMEC. Also, vHMEC overexpress cyclooxygenase-2 (COX-2), which can provide vHMEC with the ability to undergo angiogenesis and acquire invasive properties. Our lab has also shown that there are cells with hypermethylated p16INK4a promoter (i.e., an epigenetic change) and COX-2 overexpression in normal breast tissues from diseasefree women.

Given these observations, we hypothesize that vHMEC exist in normal mammary tissue, and that these cells are likely candidates to be breast cancer precursors in vivo. To test this hypothesis, I have identified cell surface markers (CD73+CD90-) that facilitated the prospective isolation of a subpopulation of cells from normal mammary reduction tissues having extended growth characteristics similar to that of cultured

vHMEC. Therefore, my overall aims are to:
1) test whether the expression of p16INK4a is silenced in the CD73+CD90-subpopulation;
2) determine the cell of origin of the CD73+CD90- subpopulation in the mammarytissue; and
3) assess whether the frequency of CD73+CD90- cells is correlated with humanbreast cancer progression.

These aims will be addressed with combinations of techniques from cell biology (fluorescent-activated cell sorting, immunocytochemistry, confocal microscopy, and tissue culturing of primary mammary cells), molecular biology (immunoblot analysis and methylation specific PCR), and clinical pathology (immunohistochemistry). Since we believe that cells with early malignant characteristics exist in normal breast tissues of health women, we hypothesize that all women have the potential to develop breast cancer due to activation and progression of these pre-malignant cells. We may be able to use markers, CD73 and CD90, to detect these pre-malignant cells in vivo. These markers may also be important therapeutic targets for the elimination of these pre-malignant cells.

Final Report (2007)
The goal of this project was to develop biomarkers that can be used in early detection, prognosis, and prevention. I developed cell surface markers (CD73+CD90-) that facilitated isolation of cells that have a similar growth patterns as our human mammary epithelial cell culture model system. The CD (cluster of differentiation) system was first developed for leukocytes (white blood cells), and these “markers” allow complex cell populations of many types to be “sorted” based on what molecules are present on their surface. Using this approach identified a rare population of mammary epithelial cells, called vHMEC, from disease-free women that have pre-malignant characteristics. We believe that these cells may be precursors to development of human breast cancer. Thus, the markers CD73 and CD90 may have potential to be important in early detection, prognosis, and prevention. Thus, our aims were to characterize the cell populations that are identified and/or sorted by the CD73 and CD90 markers, and to determine whether these cell populations have malignant characteristics.

Significant progress was made in the 1-yr of CBCRP funding. First, we obtained results to suggest that there are sub-populations of cells in the human mammary glands that have different propensity to methylate promoters (i.e. “silence” genes) of tumor suppressors, such as p16. This is very exciting, because we are not aware of others who have been able to use cell surface markers, such as CD73 and CD90, to identify cells with propensity to undergo nuclear DNA promoter methylation. Next, we have obtained clues from microarray “gene profiling” to suggest that CD73”high” and CD90”low” cells overlap with basal-like breast cancers. Basal-like breast cancers have the worst prognosis due to their rapid growth, high metastatic potential, and lack of good chemotherapeutic treatments (basal tumors tend to be both ER and Her-2 negative). If CD73 and CD90 can be used to enhance detection/prognosis of basal-like tumors, then these markers would be a tremendous asset in identifying and perhaps treating this type of aggressive tumor.