A Novel Biological Framework for the Role of Xenoestrogens

Institution: California Pacific Medical Center Research Institute
Investigator(s): Shanaz  Dairkee , Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12IB-0115 Award: $279,242
Award Type: IDEA
Research Priorities
Etiology>Environment and gene/environment interactions: nature vs. nurture

Initial Award Abstract (2006)
Xenoestrogens (XEs) are known to elicit estrogen like effects. Long banned in the United States, the pesticide DDT is a well-known example of this class of compounds. Like many XEs, it exerts physiological effects, whereby genetic males grow to look like females externally. Since excessive exposure to estrogen is believed to increase the risk of breast cancer, it seems likely that agents, such as XEs that mimic the effects of this hormone may confer similar risks in the human population. In recent studies it has been demonstrated that cells in laboratory dishes when exposed to bisphenol A (BPA), a chemical used to make the type of plastics commonly used in disposable beverage bottles, display a pattern of gene activation similar to that induced by estrogens. A major limitation of previous data relevant to tumor initiation is that it is based on the XE response of cancer cell lines. These cells are already malignant and have undergone decades of selection for survival and rapid growth in the laboratory. They are thus biologically far removed from the normal target tissue of individuals who may be at an increased risk for breast cancer development due to inadvertent exposure to excessive XE levels in their environment. The key aspect of our project is to study the molecular changes induced by XEs directly in live cells within nonmalignant breast tissue of high-risk individuals.

To circumvent the disadvantages of an incremental learning process requiring translation from cancer cell line and/or animal work to human disease induced by environmental agents, such as XEs, here we propose two innovative approaches:
1) Simulating the exchange of molecular signals that occur within breast tissue by using a co culture set up of normal human breast epithelial cells and stromal fibroblasts,
2) Employing live, normal breast cells collected from non-cancerous contralateral tissue of breast cancer patients by random periareolar fine needle aspiration (RPFNA).

In conjunction with gene chip technology optimized by our collaborators at Stanford University, these novel cellular systems will be used to identify a gene ‘signature’ depicting response to the widely pervasive XE, BPA. We will then ask whether the changes in gene expression induced by BPA can be reversed with the estrogen antagonist, tamoxifen.

This proposal is aimed at addressing the widespread concern among individuals, families, and society at large regarding regions of California where the incidence of breast cancer is significantly higher, potentially due to environmental factors. It is anticipated that assays based on target human cells will be significantly more reliable in screening chemicals that pose a specific threat to human health. We hope to delineate a small well-defined set of genetic markers, such that exposure to various XEs might be readily identified. Ultimately, the analysis of these markers in clinical tumor tissue could reliably distinguish individuals exposed to excessive XE levels. Such data will be invaluable for capturing new and effective molecular targets to assist in cancer prevention.

Final Report (2008)
Given the suspected carcinogenic influence of estrogens in hormone-sensitive tissues, such as the human breast, exposure to estrogen mimicking compounds or xenoestrogens (XEs) is a likely risk factor for cancer development and, therefore, important to investigate for the induction of causal changes at the genome-wide level. While strategies for evaluating XEs have previously relied on endpoints often questionably related to human breast biology, we have developed and implemented a novel in vitro (cell –based) model system to study the role of the ubiquitous XE, bisphenol A (BPA) in malignant breast disease. BPA directly enters human consumption by leaching out from polycarbonate plastic containers of food and beverages, and from epoxy resins used as dental sealants.

Comprising our in vitro model system are non-malignant cells derived by “random periareolar fine needle aspiration” (RPFNA) of the contralateral breast of consented patients undergoing surgical excision of benign or malignant breast lesions. Due to the dearth of live populations of non-cancerous target cells in the past, investigators filled the gap with readily available advanced breast cancer cell lines. The feasibility of direct laboratory experimentation with high-risk, non-cancerous RPFNA clinical material meets the critical need to recapitulate early effects of the cellular microenvironment (i.e., including stromal cells with breast epithelial cells), which promote subsequent tumor development and progression. Our studies employing cells from high-risk women have identified gene 'signatures' (collective increases or decreases in the expression level of specific human genes) reflecting distinctive patterns of response to estrogen, progesterone, and BPA. These data demonstrate that BPA-induced genomic changes were closely similar to the effects of estrogen exposure. Most significantly, these changes promote survival by activating specific cellular pathways without measurable proliferative changes. In principle, altered cell survival pathways contribute to the accumulation of a significant degree of genetic damage, thus facilitating malignant progression. It is likely that tumors arising from such precursors might be more aggressive as they would be relatively resistant to common clinical treatments, such as radiation and chemotherapy. Indeed, in striking agreement with this reasoning, we observed that the gene expression patterns reflecting BPA response detected in our model were significantly associated with aggressive, high-grade primary breast tumors within two independent patient populations. In other words, tumors, which displayed a ' genetic signature' of BPA exposure, were more likely to confer poor prognosis.

Our findings validate the assumption that high-risk RPFNA cultures provide a clear window to glimpse the past profile of cells, which harbor the potential to progress to full-blown cancer. Together with the use of gene expression profiling, our efforts have been remarkably successful in providing a biologically meaningful evaluation of chemicals, which might pose a long-term threat to human health. In addition to reliable model systems to serve as a substitute for direct experimentation on humans, this research has led to a new paradigm, which suggests that the ultimate severity of breast cancer outcome is determined in the earliest response of susceptible, non-malignant cells to a potential carcinogen, possibly BPA, encountered long before the manifestation of clinical cancer.

Symposium Abstract (2007)
Objective: An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. The goals of this study were (1) to expand the representation of clinical breast cancer in basic research by isolating and immortalizing tumor cells derived from a wide spectrum of pathological specimens (2) to compare gene expression profiles before and after immortalization of such tumor cell populations, with those of currently used breast cancer cell lines, and (3) to evaluate the significance of immortalization associated changes identified here in determining tumor aggressiveness and patient outcome.

Description of work: Expression profiling analyses of primary breast tumor cultures before and after induction with the immortalizing gene, hTERT, as well as widely used spontaneously immortalized breast cancer cell lines, identified a common signature characteristic of tumor cell immortalization. An important and unique feature of this “Immortalization Signature” (ImmSig) was the significant upregulation of oxidoreductase genes. Silencing the hTERT gene by RNA interference reversed ImmSig expression, increased cellular reactive oxygen species (ROS) levels, altered mitochondrial membrane potential, and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, ImmSig expression was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of ER positive tumors.

Clinical relevance: Our data support the notion that ImmSig expression assists in surmounting normal barriers related to oxidative and replicative stress response in tumor cells. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.

Bisphenol A induces a profile of tumor aggressiveness in high-risk cells from breast cancer patients.
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Dairkee SH, Seok J, Champion S, Sayeed A, Mindrinos M, Xiao W, Davis RW, Goodson WH
Yr: 2008 Vol: 68 Nbr: 7 Abs: Pg:2076-80