Karen A. Dunphy

Radiation Acts on the Microenvironment to Affect Breast Carcinogenesis by Distinct Mechanisms that Decrease Cancer Latency and Affect Tumor Type

Tissue microenvironment is an important determinant of carcinogenesis. We demonstrate that ionizing radiation, a known carcinogen, affects cancer frequency and characteristics by acting on the microenvironment. Using a mammary chimera model in which an irradiated host is transplanted with oncogenic Trp53 null epithelium, we show accelerated development of aggressive tumors whose molecular signatures were distinct from tumors arising in nonirradiated hosts. Molecular and genetic approaches show that TGFβ mediated tumor acceleration. Tumor molecular signatures implicated TGFβ, and genetically reducing TGFβ abrogated the effect on latency. Surprisingly, tumors from irradiated hosts were predominantly estrogen receptor negative. This effect was TGFβ independent and linked to mammary stem cell activity. Thus, the irradiated microenvironment affects latency and clinically relevant features of cancer through distinct and unexpected mechanisms.

Repression of mammary stem/progenitor cells by p53 is mediated by notch and separable from apoptotic activity

Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li‐Fraumeni syndrome). The tumors represent the basal‐like subtype, which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere‐forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3‐fold increase in the frequency of long‐term regenerative mammary stem cells in Trp53−/− mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere‐forming assays and the DsRed‐let7c‐sensor. The frequency of long‐term label‐retaining epithelial cells was decreased in Trp53−/− mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ‐secretase (N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester) reduced the number of Trp53−/− mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells through Notch and that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells. STEM CELLS 2011;29:119–127

Estrogen and progesterone regulate radiation-induced p53 activity in mammary epithelium through TGF-beta-dependent pathways

DNA damage normally induces p53 activity, but responses to ionizing radiation in the mammary epithelium vary among developmental stages. The following studies examined the hormones and growth factors that regulate radiation-responsiveness of p53 in mouse mammary epithelium. Immunoreactive p21/WAF1 and TUNEL staining were used as indicators of p53 activity following exposure to ionizing radiation. In ovariectomized mice, radiation-induced accumulation of p21/WAF1 was minimal in the mammary epithelial cells (<1%). Systemic injections of estrogen and progesterone (E+P) for 72 h were necessary to recover maximal expression of p21/WAF1 following ionizing radiation (55%). The effects of E+P on radiation-induced p21/WAF1 were p53-dependent as responses were absent in Trp53−/− mice. Though hormonal treatments stimulated increases in the proportion of cycling cells (PCNA-positive), this was not directly correlated with p53 activity. Whole organ cultures were used to determine whether E+P act directly upon the mammary gland. Treatment with E+P was sufficient to render p53 responsive to radiation, but TGF-β-neutralizing antibodies blocked responsiveness. In the absence of E+P, TGF-β1 alone did not alter p53 activity. These results demonstrate that estrogen and progesterone together with TGF-β signaling are necessary for maintenance of p53 activity in the mammary epithelium.

MiR203 mediates subversion of stem cell properties during mammary epithelial differentiation via repression of ΔNP63α and promotes mesenchymal-to-epithelial transition.

During reproductive life, the mammary epithelium undergoes consecutive cycles of proliferation, differentiation and apoptosis. Doing so relies on the retained proliferative capacity, prolonged lifespan and developmental potency of mammary stem cells (MaSCs). ΔNp63α, the predominant TP63 isoform in mammary epithelia, is robustly expressed in MaSCs and is required for preservation of self-renewing capacity in diverse epithelial structures. However, the mechanism(s) underlying subversion of this activity during forfeiture of self-renewing capacity are poorly understood. MicroRNAs (miRNAs) govern critical cellular functions including stem cell maintenance, development, cell cycle regulation and differentiation by disrupting translation of target mRNAs. Data presented here indicate that expression of miR203, a miRNA that targets ΔNp63α and ΔNp63β is activated during luminal epithelial differentiation and that this pattern is observed in the murine mammary hierarchy. In addition, we present evidence that the transcription factor Zeb1 represses miR203 expression, thus enhancing ΔNp63α protein levels. Furthermore, ectopic miR203 suppresses ΔNp63α expression, proliferation and colony formation. The anti-clonogenic effects mediated by miR203 require suppression of ΔNp63α. In addition, ectopic miR203 promotes mesenchymal-to-epithelial transition and disrupts activities associated with epithelial stem cells. These studies support a model in which induction of miR203 mediates forfeiture of self-renewing capacity via suppression of ΔNp63α and may also have anti-tumorigenic activity through its reduction of EMT and cancer stem cell populations.

Estrogen and progesterone induce persistent increases in p53-dependent apoptosis and suppress mammary tumors in BALB/c-Trp53+/- mice

IntroductionTreatment with estrogen and progesterone (E+P) mimics the protective effect of parity on mammary tumors in rodents and depends upon the activity of p53. The following experiments tested whether exogenous E+P primes p53 to be more responsive to DNA damage and whether these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome.MethodsMice that differ in p53 status (Trp53+/+, Trp53+/-, Trp53-/-) were treated with E+P for 14 days and then were tested for p53-dependent responses to ionizing radiation. Responses were also examined in parous and age-matched virgins. The effects of hormonal exposures on tumor incidence were examined in BALB/c-Trp53+/- mammary tissues.ResultsNuclear accumulation of p53 and apoptotic responses were increased similarly in the mammary epithelium from E+P-treated and parous mice compared with placebo and age-matched virgins. This effect was sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c-Trp53+/- mice but these responses were intermediate compared with Trp53+/+ and Trp-/- tissues, indicating haploinsufficiency. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. The majority of tumors lacked estrogen receptor (ER), but ER+ tumors were observed in both nulliparous and parous mice. However, apoptotic responses to ionizing radiation and tumor incidence did not differ among outgrowths of epithelial transplants from E+P-treated donors and nulliparous donors.ConclusionTherefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppress mammary tumorigenesis, but this effect was not retained in epithelial outgrowths.

Cancer Cell Discrimination Using Host–Guest “Doubled” Arrays

We report a nanosensor that uses cell lysates to rapidly profile the tumorigenicity of cancer cells. This sensing platform uses host-guest interactions between cucurbit[7]uril and the cationic headgroup of a gold nanoparticle to non-covalently modify the binding of three fluorescent proteins of a multi-channel sensor in situ. This approach doubles the number of output channels to six, providing single-well identification of cell lysates with 100% accuracy. Significantly, this classification could be extended beyond the training set, determining the invasiveness of novel cell lines. The unique fingerprint of these cell lysates required minimal sample quantity (200 ng, ∼1000 cells), making the methodology compatible with microbiopsy technology.

The role of lipolysis stimulated lipoprotein receptor in breast cancer and directing breast cancer cell behavior.

The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.

The Role of Activin in Mammary Gland Development and Oncogenesis

TGFβ contributes to mammary gland development and has paradoxical roles in breast cancer because it has both tumor suppressor and tumor promoter activity. Another member of the TGFβ superfamily, activin, also has roles in the developing mammary gland, but these functions, and the role of activin in breast cancer, are not well characterized. TGFβ and activin share the same intracellular signaling pathways, but divergence in their signaling pathways are suggested. The purpose of this review is to compare the spatial and temporal expression of TGFβ and activin during mammary gland development, with consideration given to their functions during each developmental period. We also review the contributions of TGFβ and activin to breast cancer resistance and susceptibility. Finally, we consider the systemic contributions of activin in regulating obesity and diabetes; and the impact this regulation has on breast cancer. Elevated levels of activin in serum during pregnancy and its influence on pregnancy associated breast cancer are also considered. We conclude that evidence demonstrates that activin has tumor suppressing potential, without definitive indication of tumor promoting activity in the mammary gland, making it a good target for development of therapeutics.

Genetic variation in sensitivity to estrogens and breast cancer risk

Breast cancer risk is intimately intertwined with exposure to estrogens. While more than 160 breast cancer risk loci have been identified in humans, genetic interactions with estrogen exposure remain to be established. Strains of rodents exhibit striking differences in their responses to endogenous ovarian estrogens (primarily 17β-estradiol). Similar genetic variation has been observed for synthetic estrogen agonists (ethinyl estradiol) and environmental chemicals that mimic the actions of estrogens (xenoestrogens). This review of literature highlights the extent of variation in responses to estrogens among strains of rodents and compiles the genetic loci underlying pathogenic effects of excessive estrogen signaling. Genetic linkage studies have identified a total of the 35 quantitative trait loci (QTL) affecting responses to 17β-estradiol or diethylstilbestrol in five different tissues. However, the QTL appear to act in a tissue-specific manner with 9 QTL affecting the incidence or latency of mammary tumors induced by 17β-estradiol or diethylstilbestrol. Mammary gland development during puberty is also exquisitely sensitive to the actions of endogenous estrogens. Analysis of mammary ductal growth and branching in 43 strains of inbred mice identified 20 QTL. Regions in the human genome orthologous to the mammary development QTL harbor loci associated with breast cancer risk or mammographic density. The data demonstrate extensive genetic variation in regulation of estrogen signaling in rodent mammary tissues that alters susceptibility to tumors. Genetic variants in these pathways may identify a subset of women who are especially sensitive to either endogenous estrogens or environmental xenoestrogens and render them at increased risk of breast cancer.

Oxybenzone Alters Mammary Gland Morphology in Mice Exposed During Pregnancy and Lactation

Abstract Hormones and endocrine-disrupting chemicals are generally thought to have permanent “organizational” effects when exposures occur during development but not adulthood. Yet, an increasing number of studies have shown that pregnant females are disrupted by endocrine-disrupting chemical exposures, with some effects that are permanent. Here, we examined the long-term effects of exposure to oxybenzone, an estrogenic chemical found in sunscreen and personal care products, on the morphology of the mammary gland in mice exposed during pregnancy and lactation. Female mice were exposed to vehicle or 30, 212, or 3000 µg oxybenzone/kg/d, from pregnancy day 0 until weaning. A nulliparous group, receiving vehicle treatment, was also evaluated. Mammary glands were collected 5 weeks after involution for whole-mount, histological, immunohistochemical, and molecular analyses. Exposure to 3000 µg oxybenzone/kg/d induced permanent changes to ductal density that was significantly different from both the nulliparous and vehicle groups. The two highest doses of oxybenzone similarly induced an intermediate phenotype for expression of progesterone receptor. A monotonic, dose-dependent increase in cell proliferation was also observed in the oxybenzone-treated females, becoming statistically significant at the highest dose. Finally, oxybenzone exposure induced an intermediate phenotype for Esr1 expression in all oxybenzone-treated groups. These data suggest that oxybenzone, at doses relevant to human exposures, produces long-lasting alterations to mammary gland morphology and function. Further studies are needed to determine if exposure to this chemical during pregnancy and lactation will interfere with the known protection that pregnancy provides against breast cancer.