William H. Goodson

ACTIVATION OF THE mTOR PATHWAY BY LOW LEVELS OF XENOESTROGENS IN BREAST EPITHELIAL CELLS FROM HIGH-RISK WOMEN

Breast cancer is an estrogen-driven disease. Consequently, hormone replacement therapy correlates with disease incidence. However, increasing male breast cancer rates over the past three decades implicate additional sources of estrogenic exposure including wide spread estrogen-mimicking chemicals or xenoestrogens (XEs), such as bisphenol-A (BPA). By exposing renewable, human, high-risk donor breast epithelial cells (HRBECs) to BPA at concentrations that are detectable in human blood, placenta and milk, we previously identified gene expression profile changes associated with activation of mammalian target of rapamycin (mTOR) pathway genesets likely to trigger prosurvival changes in human breast cells. We now provide functional validation of mTOR activation using pairwise comparisons of 16 independent HRBEC samples with and without BPA exposure. We demonstrate induction of key genes and proteins in the PI3K-mTOR pathway—AKT1, RPS6 and 4EBP1 and a concurrent reduction in the tumor suppressor, phosphatase and tensin homolog gene protein. Altered regulation of mTOR pathway proteins in BPA-treated HRBECs led to marked resistance to rapamycin, the defining mTOR inhibitor. Moreover, HRBECs pretreated with BPA, or the XE, methylparaben (MP), surmounted antiestrogenic effects of tamoxifen showing dose-dependent apoptosis evasion and induction of cell cycling. Overall, XEs, when tested in benign breast cells from multiple human subjects, consistently initiated specific functional changes of the kind that are attributed to malignant onset in breast tissue. Our observations demonstrate the feasibility of studying renewable human samples as surrogates and reinforce the concern that BPA and MP, at low concentrations detected in humans, can have adverse health consequences.

Bisphenol-A-induced inactivation of the p53 axis underlying deregulation of proliferation kinetics, and cell death in non-malignant human breast epithelial cells

Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway—accompanied by dose-dependent evasion of apoptosis and increased proliferation—in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21WAF1 and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ERα (Estrogen Receptor): ERβ ratio—an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures—another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth—a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.

New models and online calculator for predicting non-sentinel lymph node status in sentinel lymph node positive breast cancer patients

BackgroundCurrent practice is to perform a completion axillary lymph node dissection (ALND) for breast cancer patients with tumor-involved sentinel lymph nodes (SLNs), although fewer than half will have non-sentinel node (NSLN) metastasis. Our goal was to develop new models to quantify the risk of NSLN metastasis in SLN-positive patients and to compare predictive capabilities to another widely used model.MethodsWe constructed three models to predict NSLN status: recursive partitioning with receiver operating characteristic curves (RP-ROC), boosted Classification and Regression Trees (CART), and multivariate logistic regression (MLR) informed by CART. Data were compiled from a multicenter Northern California and Oregon database of 784 patients who prospectively underwent SLN biopsy and completion ALND. We compared the predictive abilities of our best model and the Memorial Sloan-Kettering Breast Cancer Nomogram (Nomogram) in our dataset and an independent dataset from Northwestern University.Results285 patients had positive SLNs, of which 213 had known angiolymphatic invasion status and 171 had complete pathologic data including hormone receptor status. 264 (93%) patients had limited SLN disease (micrometastasis, 70%, or isolated tumor cells, 23%). 101 (35%) of all SLN-positive patients had tumor-involved NSLNs. Three variables (tumor size, angiolymphatic invasion, and SLN metastasis size) predicted risk in all our models. RP-ROC and boosted CART stratified patients into four risk levels. MLR informed by CART was most accurate. Using two composite predictors calculated from three variables, MLR informed by CART was more accurate than the Nomogram computed using eight predictors. In our dataset, area under ROC curve (AUC) was 0.83/0.85 for MLR (n = 213/n = 171) and 0.77 for Nomogram (n = 171). When applied to an independent dataset (n = 77), AUC was 0.74 for our model and 0.62 for Nomogram. The composite predictors in our model were the product of angiolymphatic invasion and size of SLN metastasis, and the product of tumor size and square of SLN metastasis size.ConclusionWe present a new model developed from a community-based SLN database that uses only three rather than eight variables to achieve higher accuracy than the Nomogram for predicting NSLN status in two different datasets.

Genetic alterations in primary breast cancers and their metastases: direct comparison using modified comparative genomic hybridization.

Breast tumor development and progression are thought to be driven by an accumulation of genetic alterations, but little is known about the specific changes that occur during the metastatic process. We analyzed pairs of primary breast cancers and their matched lymph node metastases from 11 patients, pairs of primaries and distant metastases from three patients, and pairs of primaries, and local recurrences from two patients by using comparative genomic hybridization (CGH). Simultaneous hybridization analysis of primary versus matched lesion DNAs from 11 patients was also performed (modified CGH). This modified approach was useful not only for confirming CGH results but also for demonstrating quantitative differences between aberrations present at both sites. Frequent chromosomal changes present at both sites (>35% of 16 cases) were 1q, 8q, and 17q gains and 6q, 8p, 9q, 13q, 16q, 17p, and Xp losses. The total number of aberrations detected exclusively in the lymph nodes or distant metastases was higher than that in the primary tumors (2.5 vs. 0.7, P< 0.05). We found high‐level amplifications in four metastases (two lymph nodes and two distant metastases), but none in any primary tumor. These findings suggest that progression from primary breast cancer to metastasis may be associated with the acquisition of further genetic changes. Although further investigations are required, it was of interest that 3 of 11 patients (27%) showed 18q loss solely in their lymph node metastases. Genes Chromosom. Cancer 19:267–272, 1997.

Wound Collagen Accumulation in Obese Hyperglycemic Mice

We used C57-BL ob/ob mice as a model to study wound healing in type II (adult-onset) diabetes. Planimetry was used to assess rate of closure in standard open skin wounds. In agreement with previous subcutaneous wound collagen-accumulation studies, closure was slower in the ob/ob mice. Subcutaneous implants were used to evaluate wound collagen accumulation. Weanling mice have collagen accumulation similar to lean littermates (mean 3.43 μg/ cm vs. 3.46 μg/cm), but the same ob/ob animals had decreased wound collagen (mean 2.39 μg/cm vs. 3.02 μg/cm, P < 0.04) when mature. Other ob/ob animals fed a restricted diet (and thus not obese) had normal collagen accumulation at the same age. Neither insulin nor diet restriction restored wound collagen accumulation in phenotypically obese mice. Because collagen accumulation is not improved by measures that control hyperglycemia (insulin and diet restriction) and the defect was seen only in phenotypically obese ob/ob mice, the decreased wound collagen accumulation may be due in part to structural changes in adipose tissue.

Stimulation of wound blood vessel growth by wound macrophages.

Healthy wound healing is an ordered sequence of events beginning with an inflammatory response and culminating in the synthesis of collagen by a population of new fibroblasts. In virtually all wounds, the blood supply for these new fibroblasts is the result of an intense ingrowth of new vessels. The cause of this “neovascularization” is not yet well understood. Traditionally, it has been held that dead tissue and blood clot provide the stimulus for repair. It is correct that these elements will provoke an inflammatory response, but in the clinical situation, open and dead space wounds will continue to heal long after clot and dead tissue have been removed. Clearly, a broader explanation is needed for the continued growth and neovascularization of wound tissue in these situations. A newer hypothesis is that a cellular component of the wound-presumably a cell involved in the inflammatory response -responds to the multiple stimuli of injury. This cell (or cells) then translates these stimuli into chemical signals which trigger regeneration of cells, collagen formation, and fibrosis. Such signals would include a stimulus for blood vessel ingrowth. A likely cell to perform this “translator” function is the macrophage. This cell has been shown to be present in wounds and also to be necessary for repair. Silver [9], while measuring oxygen tension in healing wounds, observed that macro-

Development of a new miniature method for the study of wound healing in human subjects

A new miniaturized method for evaluation of healing potential in human subjects has been developed. A small, standardized, subcutaneous wound is made in a standard location. Expanded PTFE tubing is placed in the wound and removed at specific intervals. The ingrowth of connective tissue is evaluated by histology and by measurement of hydroxyproline accumulation. Beginning on the fifth day, hydroxyproline accumulation correlates with visual accumulation of tissue in the tubing. Preliminary results show decreased hydroxyproline accumulation in the wounds of patients with preoperative debility. This is to be expected on the basis of previous clinical experience and laboratory studies. This method has the further advantage that the catheters may be placed with local anesthesia in outpatients and therefore may be applied systematically to populations, as well as for observation in patients requiring operation.

Continuous direct tissue oxygen tension measurement by a new method using an implantable silastic tonometer and oxygen polarography

A new method of tissue oxygen tension measurement that is suitable for human use has been described. An implanted Silastic tube tonometer is combined with polarographic oxygen electrodes. The techniques of preparation, insertion, and measurement have been described. The advantages and disadvantages of this method have been considered in relation to established methods of tissue oxygen tension measurement. The method is suitable for clinical use and can be used to aid clinical decision-making.

Correlation of bromodeoxyuridine (BRDU) labeling of breast carcinoma cells with mitotic figure content and tumor grade.

Tumor proliferation is inversely associated with survival in patients with breast carcinoma. Labeling of tumor cells with bromodeoxyuridine (BRDU) correlates highly with that seen with [3H]thymidine, the current “gold standard” for measuring tumor S-phase. However, the relationship of BRDU labeling to mitotic figure content and tumor grade remains incompletely defined. To determine this, we labeled 55 breast carcinomas with BRDU in vivo and correlated the results with mitotic figure content. The BRDU labeling index was the number of BRDU-positive cells/2, 000 tumor cells, the mitotic figure index was the number of mitotic figures per 1, 000 tumor cells, and the mitotic figure count was the number of mitotic figures per 10 high-powered fields. BRDU labeling was also correlated with tumor grade (ScarffBloomRichardson). The BRDU labeling index correlated highly with the mitotic figure index (r = 0.814, p = 7.0 ± 10−14), mitotic figure count (r = 0.725, p = 6.0 ± 10−10), and tumor grade (r = 0.68, p = 1.1 ± 10−8). The correlation of BRDU labeling with mitotic figure content was strong enough to suggest that a very carefully measured mitotic figure index provides an estimate of tumor growth fraction equivalent to the BRDU labeling index. Also, analysis of variance showed that the mitotic figure index was twice as precise as the mitotic figure count in estimating BRDU labeling, and thus was a more accurate measure of tumor proliferation. Moreover, measurements made by the mitotic figure index were as precise as those made by BRDU labeling. However, which method is optimal for estimating tumor proliferation rate remains unclear. Further studies are indicated.

Augmentation of some aspects of wound healing by a “Skin Respiratory Factor”

Skin Respiratory Factor (SRF), a water soluble extract of Brewer’s Yeast, Saccharomyces cerevisiae, was brought to the attention of our laboratory by the Review Panel on Over-The-Counter Hemorrhoidal Preparations, an advisory committee of the Food and Drug Administration. The Panel, which is assessing SRF as an ingredient in a proprietary hemorrhoidal product, Preparation H, requested us to evaluate SRF as a wound-healing agent. Since there was evidence from the manufacturer that SRF influences oxygen consumption by fibroblasts [2], and since some aspects of wound healing, including collagen formation, epithelization, and wound strength, can be influenced by agents altering oxygen consumption, this laboratory considered undertaking an evaluation of SRF to be a matter of scientific interest. Our evaluation of SRF utilized a number of established methods of studying wound healing, and the results were of sufficient interest to warrant publication. The commercial material is standardized as units with 1 unit (U) of SRF increasing the uptake of oxygen by minced rat abdominal skin (1 mg dry weight) by 1% in a I-hr measurement by Warburg manometry. Yeast extracts contained 8000-12,000 U/g, and the material supplied by the manufacturer for this study contained 8100 U/g.2