Xenoestrogen-specific perturbations in the human breast

Institution: California Pacific Medical Center Research Institute
Investigator(s): Shanaz  Dairkee , Ph.D. -
Award Cycle: 2011 (Cycle 17) Grant #: 17UB-8702 Award: $899,961
Award Type: SRI Request for Proposal (RFP)
Research Priorities
Etiology and Prevention>Prevention and Risk Reduction: ending the danger of breast cancer

Initial Award Abstract (2011)

Introduction: To prevent breast cancer, it is critical to acquire the knowledge of events that occur in benign (non-malignant) cells before they become cancerous (malignant). To this end, we have developed live interactive processes to evaluate the effects of potentially carcinogenic chemicals on non-malignant human breast cells that can be studied experimentally. We have already demonstrated that environmental chemicals cause dramatic changes in cell programming, which are similar to the effects of estrogens. Since estrogens are involved in the origins of breast cancer, it is important to regulate the use of chemicals that mimic this hormone, also known as xenoestrogens (XEs). For example, the well-known carcinogen, stilbestrol or DES, is an estrogen mimic. Our major goal is to employ non-malignant cells in assays to screen chemicals for estrogen-like effects.

Central hypothesis: We hypothesize that continued exposure of non-malignant breast cells to XEs - at levels already reported in mother’s blood, cord blood, placenta, mother’s milk and human tissue - alters central functions of cell metabolism, and redirects exposed cells on the path to cancer.

General methodology: Using bisphenol A (BPA) as a test chemical, we have previously identified six sets of genes that are significantly affected in human breast cells. These changes, which allow damaged cells to survive and acquire additional defects, predict specific functional deficiencies also found within aggressive breast tumors. Our goal is to build on these findings in 3 steps: (1) Define consistent changes caused by a wide panel of known XEs in non-malignant breast cells using established cell biology assays. This will establish a basic battery of tests to screen unknown chemicals, which might behave as XEs. (2) Apply novel technologies and computational approaches to enhance the understanding of XE-induced functional changes thereby facilitating the development of additional tests. This will allow an expanded choice of reliable screens from a comprehensive battery of assays for XE effects. (3) Apply our ability to obtain significant data from small numbers of cells within clinical samples to develop assays utilizing readily renewable and repeatedly available sources of non-malignant breast cells such as human milk.

Innovative elements: In the public debate on the safety of chemicals, providing a recommendation is often a dilemma for scientists who are skeptical of arbitrary end points without a clear understanding of why or how biological effects of XEs, such as the feminization of animals, or enhanced growth activity in breast cancer cell lines, come about. We address scientific skepticism by identifying a series of simple metabolic functions that can define pertinent cancer related effects - or lack of effects -- of a chemical on human cells. We have already shown that four inter-related attributes of breast cancer (oxidative stress, metabolic pathway signaling, apoptosis, and proliferation) consistently occur after exposure of living cells to BPA, and to another possible XE - methylparaben. Our innovative approach to ascertain these and other functional changes at the cellular level provides the needed mechanistic framework for whole animal studies and advances standardized XE screening, as well as breast cancer prevention research. To extend the study of tiny samples from human breast tissue to younger women who are closer to the earliest steps of carcinogenesis, and to establish other renewable sources of breast epithelium for carcinogenicity research, we will optimize techniques we have already used to grow stem cells present in human milk.

Community involvement: Our central aim is to develop unequivocal criteria to differentiate between chemicals that exert estrogenic effects and those that do not. To make research data widely available, we have already actively disseminated our results to the lay press, public forums, and the broadcast media. To fulfill the overall goal of disease prevention, we will work closely with MORE GREEN MOMS, an advocacy group that is focused on environmental exposure of the most vulnerable members of society – our children. We have also actively supported others in the field, such as the collaborative between the Natural Resources Defense Council and UC Berkeley School of Public Health in a CBCRP-sponsored effort to define the current status of chemical tests for breast cancer. Our present proposal is a focused response to the recommendations of that initiative. Finally, to engage a wide representation of community leadership in this cause, we have proactively sought the support of business and professional leaders outside of usual advocacy groups.

Final Report (2015)

The long-term goal of our research is to develop reliable methods to identify chemicals present in the environment that play a role in breast carcinogenesis. In this project, we focused on chemicals with estrogenic properties, known as xenoestrogens (XEs), to ask whether laboratory exposure to such chemicals of healthy breast samples, isolated by fine needle aspiration of human volunteers, could initiate a spectrum of cellular perturbations commonly observed in cancerous breast tissue. Moreover, we asked whether the type of perturbation and its intensity would vary from chemical to chemical. To address these questions, it is essential that the tests are performed on healthy cells, not cancer cells, and that such test cells used in laboratory assays continue to be estrogen responsive and maintain estrogen receptor (ER)-positive features, characteristic of human breast epithelial cells. As we have optimized the conditions for the growth of ER-positive healthy breast cells over the past 7 years, here we successfully employed such cells as potential targets of chemical carcinogenesis. Each of our published studies regarding the effects of XEs has used independent sets of healthy breast specimens, whereby XE-induced perturbations observed in one set of samples are confirmed with usual or additional experimental assays in another set of human donor samples. Although this work has not reached a scale typical of population-based epidemiological studies, our approach of continued data validation with additional cases is novel in experimental studies with live human samples.

In our aim to determine the carcinogenic potential of low dose Xes that enter the human body via ingestion, inhalation, or contact with common consumer products, we have selected chemicals at the highest volumes of production and distribution. These chemicals are: bisphenol A (BPA), methyl paraben (MP), perfluorooctanoic acid (PFOA), and terephthalic acid (TPA). To measure molecular and functional perturbations after XE exposure of healthy breast cells, we evaluated several endpoints and pathways that are characteristic of breast cancer. Major highlights from our data regarding the impact of Xes on healthy breast cells collected over the project period include: (1) persistent shifts in the Estrogen Receptor Alpha (Era): Estrogen Receptor Beta (Erß) ratio, whereby levels of Era are increased while those of Erß are reduced; (2) phosphorylation of Era, which results in its activation, and whereby signaling pathways tightly regulated by natural estrogens during the menstrual cycle or in pregnancy become hyperactive; (3) increased expression of the estrogen regulated genes cyclin D and cathepsin D, involved in regulating cellular functions such as proliferation, angiogenesis, and cell death; (4) activation of molecular signaling related to cell survival by the PI3K/AKT/mTOR pathway; (5) increase in number of proliferating cells; (6) induction of DNA-repair proteins, suggesting the presence of genetic damage; and (7) evasion of programmed cell death signals, allowing genetically damaged cells to persist for future growth, instead of being eliminated. Notably, in an attempt to reverse deleterious changes in healthy cells, we observed that adverse effects of Xes were effectively surmounted by dietary components, such as curcumin (Dairkee et al., Carcinogenesis 34: 703, 2013).

In our studies aimed at comparing different estrogenic chemicals, our data revealed diversity in the biological modes of action of various Xes on healthy breast cells. For example, we found that TPA, a major chemical precursor of polyethylene terephthalate (PET) containers used for the storage of food and beverages, was similar to other Xes in causing some perturbations mentioned above, but dissimilar in that it inhibits cell proliferation (Luciani-Torres et al., Carcinogenesis 36: 168, 2015). Such a result in the setting of a common carcinogen testing scenario would lead to the assumption that TPA is ‘safe’ at low doses. However, while cell proliferation is halted in the presence of TPA due to DNA damage detected by the cell’s surveillance mechanism, TPA exposure also compromises the elimination of damaged cells by programmed cell death. Consequently, it is possible that damaged cells could progress to cancerous precursors at a future time. Our research in this project has also revealed the impact of Xes as ‘mixtures’ circulating in the blood of humans at any given time. In this regard, our data demonstrate that the degree of cellular and molecular perturbation detected in healthy cells exposed to mixtures of BPA, MP, and PFOA is not merely an additive effect of three vs. one chemical. Instead, it reflects a synergistic impact of each of the three chemicals, which is several times greater than statistically expected of an additional increase in dose contributed by each, thus questioning the safety standards commonly based on single XE tests of chemical carcinogenicity. Overall, the breadth of findings in this project pertaining to the direct effects of chemicals on healthy human breast cells provides strong evidence that experimental application of population-based healthy tissue samples in robust in vitro chemical tests could generate much-needed rationale for improved regulation of exposure to agents potentially carcinogenic to humans.

Bisphenol-A-induced inactivation of the p53 axis underlying deregulation of proliferation kinetics, and cell death in non-malignant human breast epithelial cells.
Index Medicus: Carcinogenesis
Authors: Dairkee SH, Luciani-Torres MG, Moor DH, Goodson WH 3rd.
Yr: 2013 Vol: 34 Nbr: 3 Abs: Pg:703-12

Exposure to the polyester PET precursor terephthalic acid induces and perpetuates DNA damage-harboring non-malignant human breast cells
Index Medicus: Carcinogenesis
Authors: Maria Gloria Luciani-Torres, Dan H.Moore, William H.Goodson III and Shanaz H.Dairkee
Yr: 2015 Vol: 36 Nbr: 1 Abs: Pg:168-176