This report is the first to analyze differences in the normal breast microenvironment of premenopausal women, and to show fundamental differences in the ability of breast ECM to influence the aggressiveness and tumorigenicity of breast cancer cells. The comprehensive LC-MS/MS identification of whole tissue hormone metabolites, as well as unique ECM proteins between the AA and CAU women, offers a novel insight into the intricacy of the breast microenvironment.
One limitation of this study, which must be addressed, is the lack of descriptive clinical data on the breast tissue isolated from the reduction mammoplasty patients. The tissue collected for fibroblast and whole breast tissue ECM isolation were considered pathological medical waste; therefore, informative clinical data including parity, body mass index, breast density, oral contraceptive use, phase of menstrual cycle were not available. Whether these important factors potentially had confounding effects on the observed results is regrettably unknown. In attempts to limit these effects, each experiment contained multiple replicates, and was repeated using as many different pools of patient samples feasible. A total of 50 CAU and 53 AA samples were used in the different analyses. However, the possibility remains that inherent factors from the tissue source could remain. Furthermore, we obtained samples from southern, eastern, and midwestern US, which may help eliminate the effects of socioeconomic factors if the samples had been obtained from one small geographical region. Determining whether clinical factors, or genetics, or a combination of the two, systematically relate as to why AAs develop a more aggressive cancer is not the purpose of this study. The objective of this study was not to determine how these discrepancies develop, but rather to use the information obtained to study their influence on breast cancer behavior. In addition, patient samples were pooled in order to obtain sufficient amount of ECM to perform these experiments. Although pooled samples are not ideal, a consistent pattern was observed with all results obtained even in this potentially confounding situation. Since no patient sample could be used twice, this suggests that our conclusions were not skewed by any single sample. Future studies to determine specific components in ECM responsible for these effects will require examination of individual tissues, if enough material becomes available from a single patient. Overall, the results presented provide valuable data for further investigation into the role of the microenvironment in cancer disparities, and potentially as a basis for future studies investigating factors such as parity and phase of menstrual cycle on breast cancer cell behavior.
Collectively, the data presented in this report suggest that the AA breast microenvironment is less permissive of tumor growth compared to the CAU breast microenvironment. Therefore, it is not surprising that only the more aggressive cells are able to survive and proliferate unrestrictedly in the suppressive microenvironment of AA breast tissue. The comparatively suppressive effects of the AA ECM may arise from both a physical restriction due to the types of structural material present in the ECM, and chemically from the signals present, or absent, in the microenvironment. Numerous reports have indicated that the spatial organization and composition of the ECM influence mammary cell behavior, and that alterations in ECM receptor expression facilitate malignant transformation .
The premenopausal stroma is not a static compartment; proliferation in the breast varies with the menstrual cycle, which requires the expansion and deposition of new ECM . Increased deposition of molecules such as collagen can alter the ECM biophysical properties and increase extracellular cellular tension. ECM composition and rigidity modulate cell-ECM interactions and have a significant impact on cell functions. Indeed, mammary epithelial cells cultured on matrices with increased stiffness have disrupted cell-cell junctions, increased proliferation, perturbed endogenous basement membrane assembly, and a dedifferentiated phenotype [44, 45]. The development of breast cancer is characterized by the loss of tissue organization and an increase in tissue rigidity, suggesting that aberrant tension may facilitate the acquisition of a malignant phenotype . For example, primary mammary epithelial cells cultured on floating collagen gels were shown to differentiate in response to lactogenic hormones only when plated on collagen gels with reduced tensional forces. When plated on gels with increased tension, the extracellular forces promoted cell spreading, increased MMP activity, and inhibited acini formation and cellular differentiation . Interestingly, triple-negative tumors (ER-/PR- and lacking HER2 amplification) are composed of undifferentiated cells, potentially resulting from a small, localized area of matrix stiffness and high tension. Paszek et al. demonstrated that matrix stiffness promotes tumor-like behavior in mammary cells, and blocking integrin-dependent cell contractility reverted the malignant phenotype in culture . Thus, if the premenopausal AA microenvironment is comparatively more restrictive in its composition/organization, as our data suggests, this may predispose AAs to triple-negative breast cancer. Further studies on this topic are warranted.
The use of a selective pressure to isolate a more tumorigenic cell is often used in studies seeking to identify the progenitor tumor-initiating cells (cancer stem cell) via culturing the cells in non-adherent conditions . The rationale driving this culture system is that only the progenitor tumor-initiating cells are able to survive and self-renew when contact with the ECM is disrupted, whereas differentiated, non-tumor initiating cells experience anoikis and die . Potentially a similar mechanism of selective pressure is actively selecting for the more aggressive cancer cell in the restrictive premenopausal AA microenvironment.
It is noteworthy that the invasiveness, tumorigenicity, and metastases of the ER+/PR+ cells were enhanced in the presence of the CAU ECM. It has been similarly shown that breast cancer cell proliferation, in response to androgens, was dependent upon both the ER status of the cell and signals from the ECM. Specifically, ER+ MCF7 cells proliferated in the presence of dihydrotestosterone (DHT) by an ERα-dependent mechanism; however, MDA-MB-231 cells responded to DHT by an ER-independent, αvβ3 integrin pathway . Additional estrogen and ECM/integrin interactions related to tumorigenesis have been reported. Hypoxia-inducible factor 1α (HIF-1α), a transcription factor which is overexpressed in the majority of human carcinomas and controls central metastasis-associated pathways, was shown to increase anchorage-independent growth by downregulation of the α5 integrin . Anchorage independent growth and decreased α5 integrin levels were reverted by treatment with the estrogen metabolite, 2-methoxyestradiol, a known pharmacological inhibitor of HIF-1α.
This is the first report to simultaneously analyze the biologically-active estrogens and androgens from each patient in whole breast tissue via LC-MS/MS; previous studies measured blood and urine levels. This method offers a more intimate analysis of the local hormone milieu of the breast microenvironment, compared to measuring circulating levels of hormones. Indeed, it is now well known that the local synthesis from the stromal cells dramatically contributes to the growth, function, and tumorigenesis of ER/PR positive and negative breast cells [51–53]. BRCA1 tumors, the majority of which are ER-, have been effectively prevented by ovariectomy . Furthermore, it is proposed that the increased risk of breast cancer following pregnancy is due to high levels of estrogen and other pregnancy associated hormones that promote the growth of already initiated target cell populations . Interestingly, the majority of breast cancers that develop during this time are ER-/PR- suggesting that hormones affect the local microenvironment.
Different estrogen metabolites have been reported to act as either carcinogens or to protect from tumorigenesis, although their precise mechanisms are yet to be fully defined. The production of 16α-hydroxyestrone has been hypothesized to initiate breast cell transformation by acting as an estrogen agonist, increasing cellular proliferation and generating reactive oxygen species thereby causing DNA damage . Conversely, 2-hydroxyestradiol has been shown to possess estrogen antagonist properties in vivo . In this report, the primary estradiol/estrone metabolites detected were estriol, a product of the 16α-hydroxylation pathway, and 2-methoxyestrone, a product of the 2-hydroxylation pathway. It is of note that these two metabolites appear to be equally balanced in the tissue, as it has been shown that alterations in the ratio of C2/C16 estradiol/estrone hydroxylation can lead to anchorage-independent growth and tumorigenesis . Analysis of these hormone metabolites in breast tissue, as opposed to circulating levels, could potentially be used for early detection of breast cancer in high-risk patients.
Our understanding of the interactions between the numerous cell types within breast, especially during tumorigenesis, still remains vague owing to the complexity of physical and chemical communication, and inherent differences between patients and their resultant types of cancer. However, apart from individual patient differences, there is indisputable evidence that breast carcinomas in premenopausal AA women tend to be triple negative and highly metastatic compared to breast carcinomas in CAU women. Identifying the initiating factors in the development of triple-negative breast cancer in premenopausal AA women will fill a gap of knowledge in breast cancer research. Why these women should have an increased incidence of this disease compared to other racial groups remains elusive.