Obesity-Mediated Regulation of HGF/c-Met Is Associated with Reduced Basal-Like Breast Cancer Latency in Parous Mice

It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-T_Ag mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-T_Ag mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-T_Ag mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential microenvironmental alterations in c-Met signaling may play a role in etiology.     

Persistent changes in gene expression induced by estrogen and progesterone in the rat mammary gland.

Epidemiological studies have consistently shown that an early full-term pregnancy is protective against breast cancer. We hypothesize that the hormonal milieu that is present during pregnancy results in persistent changes in the pattern of gene expression in the mammary gland, leading to permanent changes in cell fate that determine the subsequent proliferative response of the gland. To investigate this hypothesis, we have used suppression subtractive hybridization to identify genes that are persistently up-regulated in the glands of E- and progesterone (P)-treated Wistar-Furth rats 28 d after steroid hormone treatment compared with age-matched virgins. Using this approach, a number of genes displaying persistent altered expression in response to previous treatment with E and P were identified. Two markers have been characterized in greater detail: RbAp46 and a novel gene that specifies a noncoding RNA (designated G.B7). Both were persistently up-regulated in the lobules of the regressed gland and required previous treatment with both E and P for maximal persistent expression. RbAp46 has been implicated in a number of complexes involving chromatin remodeling, suggesting a mechanism whereby epigenetic factors responsible for persistent changes in gene expression may be related to the determination of cell fate. These results provide the first support at the molecular level for the hypothesis that hormone-induced persistent changes in gene expression are present in the involuted mammary gland.     

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2-dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.     

Effects of Age and Parity on Mammary Gland Lesions and Progenitor Cells in the FVB/N-RC Mice

The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3-4 pregnancies) females were euthanized at 16-22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer.     

Relationship between histology,development and tumorigenesis of mammary gland in female rat

The mammary gland is a dynamic organ that undergoes structural and functional changes associated with growth, reproduction, and post-menopausal regression. The postnatal transformations of the epithelium and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The increased cancer incidence in mammary glands of humans and similarly of rodents in association with their development is believed to be partly explained by proliferative activity together with lesser degree of differentiation, but it is not completely understood how the virgin gland retains its higher susceptibility to carcinogenesis. During its developmental cycle, the mammary gland displays many of the properties associated with breast cancer. An early first full-term pregnancy may have a protective effect. Rodent models are useful for investigating potential breast carcinogens. The purpose of this review is to help recognizing histological appearance of the epithelium and the stroma of the normal mammary gland in rats, and throughout its development in relation to tumorigenic potential.     

Parity and breast cancer: evidence of a dual effect.

Epidemiological studies have shown that early first pregnancy reduces the risk of developing breast cancer, which indicates that initiation of the disease occurs at an early age. Thus the subclinical lesion of breast cancer might already be present in the breast before childbearing begins and the growth of any such focus might be modified by the endocrine changes of pregnancy. To test this hypothesis the relation between parity and age at presentation was studied in 341 unselected patients with breast cancer presenting to a single clinic. The mean age at presentation was 5.2 years lower in parous than nulliparous women (p < 0.001) and fell with increasing parity. It is concluded that reproductive history influences not only the risk of breast cancer but also the latent interval of a proportion of breast carcinomas.     

Developmental stage determines the effects of MYC in the mammary epithelium

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.     

Activation of Stat5 and induction of a pregnancy-like mammary gland differentiation by eicosapentaenoic and docosapentaenoic omega-3 fatty acids

The protective effect of early pregnancy against breast cancer can be attributed to the transition from undifferentiated cells in the nulliparous to the differentiated mature cells during pregnancy. Considerable evidence suggests strongly that the n-3 polyunsaturated fatty acid (PUFA) content of adipose breast tissue is inversely associated with an increased risk of breast cancer. Here, we report that there was a decrease in the n-6/n-3 PUFA ratio and a significant increase in concentration of n-3 PUFA docosapentaenoic acid and eicosapentaenoic acid in the pregnant gland. The functional role of n-3 PUFAs on differentiation was supported by the studies in the fat-1 transgenic mouse, which converts endogenous n-6 to n-3 PUFAs. Alternation of the n-6/n-3 ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid, in the mammary gland of fat-1 mouse resulted in development of lobulo-alveolar-like structure and milk protein beta-casein expression, mimicking the differentiated state of the pregnant gland. Docosapentaenoic acid and eicosapentaenoic acid activated the Jak2/Stat5 signaling pathway and induced a functional differentiation with production of beta-casein. Expression of brain type fatty acid binding protein brain type fatty acid binding protein in virgin transgenic mice also resulted in a reduced ratio of n-6/n-3 PUFA, a robust increase in docosapentaenoic acid accumulation, and mammary differentiation. These data indicate a role of mammary derived growth inhibitor related gene for preferential accumulation of n-3 docosapentaenoic acid and eicosapentaenoic acid in the differentiated gland during pregnancy. Thus, alternation of n-6/n-3 fatty acid compositional ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid and eicosapentaenoic acid, is one of the underlying mechanisms of pregnancy-induced mammary differentiation.     

Timing of dietary fat exposure and mammary tumorigenesis: Role of estrogen receptor and protein kinase C activity

The possible association between a high fat diet and increased breast cancer risk has remained controversial. This largely reflects the conflicting data obtained from migrant, case control and animal studies, which generally support this association, and cohort studies which often fail to show a link between fat and breast cancer. The mammary gland is particularly sensitive to estrogens during the fetal development, leading us to hypothesize that dietary fat levels during this period may significantly influence breast cancer risk. Using chemically-induced mammary tumors in rats as our experimental model, we have demonstrated the ability of a maternal diet, high in the polyunsaturated fatty acid (PUFA) linoleic acid, to alter mammary gland differentiation, accelerate the onset of sexual maturation, and increase breast cancer risk. The mammary glands of female rats exposed to a highfat diet in utero have more of the undifferentiated structures (terminal end buds) and fewer of the differentiated structures (alveolar buds) than the glands of rats exposed to a low-fat diet in utero. Furthermore, these mammary glands contain lower levels of total estrogen receptors and have reduced total protein kinase C activity. These effects appear to be mediated by an increase in tne serum estradiol levels of pregnancy, which are elevated at least 30% in pregnant dams fed a high fat diet. Furthermore, the administration of estradiol to pregnant dams produce effects on mammary gland development, onset of puberty and sensitivity to chemical carcinogenesis comparable to those seen in the offspring of rats fed a high fat diet during pregnancy. Our results, thus, support the hypothesis based on epidemiological data that high maternal estrogen levels increase daughters' breast cancer risk. The results also suggest that a high-fat diet may be an important factor in increasing pregnancy estrogenic activity.     

Can modulation of mammary gland development by dietary factors support breast cancer prevention?

Breast cancer continues to be a major challenge for public health, since it is the most common cancer of women in the Western world, and its prevalence is still increasing. In order to achieve better results in the prevention and treatment of breast cancer it is crucial to identify the mechanisms behind its initiation, i.e. the changes and deviations that have occurred in the mammary gland growth. It has long been known that a woman's reproductive history is the strongest breast cancer risk factor if genetic background and age are excluded. The reproductive hormones, and the timing of events leading to changes in these hormones, and consequently, in the mammary gland, are the most important players. However, it has become obvious that dietary components may also contribute to breast cancer risk through their effects on the mammary gland. The past few years have added important information to our knowledge of the mechanisms behind breast cancer initiation at the level of target cells (mammary stem cells) and gene expression (genetic 'fingerprint' associated with persistent pregnancy-induced protection against breast cancer), as well as of the effects of certain dietary factors (steroid action modulators). These results and their links to breast cancer initiation and progression will be discussed.     

Prospective breast cancer risk factors prediction in Saudi women

Women's health is affected by breast cancer worldwide and Saudi Arabia (SA) is no exception. Malignancy has enormous consequences for social, psychological and public health. The aim of this study was to examine the risk factors for Saudi women from breast cancer using logistic regression models. In 135 patient cases for different stages of breast cancer was used to study case management, 270 healthy women from King Abd Alla Medical City, Mecca, SA were taken to predict the probability of women developing breast cancer, logistic regression was analyzed taking factors such as age, marital status, family history, parity, age at first full-term pregnancy, menopausal status, body mass index (BMI) and breast feeding. The logistic regression model showed that there are important risk factors (age, marital status, family history, parity, age at first full-term pregnancy, menopausal status, body mass index, and breast feeding) in development of breast cancer. Fewer cases were observed in unmarried women, age ≤30, BMI ≤20. In contrast, more cases were found with women age 41–50 married, BMI > 30, a smaller number of children, not breast feeding, age of first pregnancy ≥30, menopausal status age at 46–50. Based on our data there is role of risk factors in developing breast cancer, less BMI, the increase number of children, breast feeding, which are playing as protective factor for breast cancer.     

The role of CSF-1 in normal physiology of mammary gland and breast cancer: an update

Colony stimulating factor (CSF-1) and its receptor (CSF-1R, product of c-fms proto-oncogene) were initially implicated as essential for normal monocyte development as well as for trophoblastic implantation. However, studies have demonstrated that CSF-1 and CSF-1R have additional roles in mammary gland development during pregnancy and lactation. This apparent role for CSF-1/CSF-1R in normal mammary gland development is very intriguing because this receptor/ligand pair has also been found to be important in the biology of breast cancer in which abnormal expression of CSF-1 and its receptor correlates with tumor cell invasiveness and adverse clinical prognosis. Recent findings also implicate tumor-produced CSF-1 in promotion of bone metastasis in breast cancer, and a certain membrane-associated form of CSF-1 appears to induce immunity against tumors. This review aims to summarize recent findings on the role of CSF-1 and its receptor in normal and neoplastic mammary development that may elucidate potential relationships of growth factor-induced biological changes in the breast during pregnancy and tumor progression.     

From milk to malignancy: the role of mammary stem cells in development, pregnancy and breast cancer

Adult stem cells of the mammary gland (MaSCs) are a highly dynamic population of cells that are responsible for the generation of the gland during puberty and its expansion during pregnancy. In recent years significant advances have been made in understanding how these cells are regulated during these developmentally important processes both in humans and in mice. Understanding how MaSCs are regulated is becoming a particularly important area of research, given that they may be particularly susceptible targets for transformation in breast cancer. Here, we summarize the identification of MaSCs, how they are regulated and the evidence for their serving as the origins of breast cancer. In particular, we focus on how changes in MaSC populations may explain both the increased risk of developing aggressive ER/PR(-) breast cancer shortly after pregnancy and the long-term decreased risk of developing ER/PR(+) tumors.     

Protein kinase expression during murine mammary development

The susceptibility of the mammary gland to carcinogenesis is influenced by its normal development, particularly during developmental stages such as puberty and pregnancy that are characterized by marked changes in proliferation and differentiation. Protein kinases are important regulators of proliferation and differentiation, as well as of neoplastic transformation, in a wide array of tissues, including the breast. Using a RT-PCR-based cloning strategy, we have identified 41 protein kinases that are expressed in breast cancer cell lines and in the murine mammary gland during development. The expression of each of these kinases was analyzed throughout postnatal mammary gland development as well as in a panel of mammary epithelial cell lines derived from distinct transgenic models of breast cancer. Although the majority of protein kinases isolated in this screen have no currently recognized role in mammary development, most kinases examined were found to exhibit developmental regulation. After kinases were clustered on the basis of similarities in their temporal expression profiles during mammary development, multiple distinct patterns of expression were observed. Analysis of these patterns revealed an ordered set of expression profiles in which successive waves of kinase expression occur during development. Interestingly, several protein kinases whose expression has previously been reported to be restricted to tissues other than the mammary gland were isolated in this screen and found to be expressed in the mammary gland. In aggregate, these findings suggest that the array of kinases participating in the regulation of normal mammary development is considerably broader than currently appreciated.     

Cold-inducible RNA binding protein in mouse mammary gland development

RNA binding proteins (RBPs) regulate gene expression by controlling mRNA export, translation, and stability. When altered, some RBPs allow cancer cells to grow, survive, and metastasize. Cold-inducible RNA binding protein (CIRP) is overexpressed in a subset of breast cancers, induces proliferation in breast cancer cell lines, and inhibits apoptosis. Although studies have begun to examine the role of CIRP in breast and other cancers, its role in normal breast development has not been assessed. We generated a transgenic mouse model overexpressing human CIRP in the mammary epithelium to ask if it plays a role in mammary gland development. Effects of CIRP overexpression on mammary gland morphology, cell proliferation, and apoptosis were studied from puberty through pregnancy, lactation and weaning. There were no gross effects on mammary gland morphology as shown by whole mounts. Immunohistochemistry for the proliferation marker Ki67 showed decreased proliferation during the lactational switch (the transition from pregnancy to lactation) in mammary glands from CIRP transgenic mice. Two markers of apoptosis showed that the transgene did not affect apoptosis during mammary gland involution. These results suggest a potential in vivo function in suppressing proliferation during a specific developmental transition.     

Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.     

Characterization of the <Emphasis Type="Italic">Six1</Emphasis> homeobox gene in normal mammary gland morphogenesis

Background  

The Six1 homeobox gene is highly expressed in the embryonic mammary gland, continues to be expressed in early postnatal mammary development, but is lost when the mammary gland differentiates during pregnancy. However, Six1 is re-expressed in breast cancers, suggesting that its re-instatement in the adult mammary gland may contribute to breast tumorigenesis via initiating a developmental process out of context. Indeed, recent studies demonstrate that Six1 overexpression in the adult mouse mammary gland is sufficient for initiating invasive carcinomas, and that its overexpression in xenograft models of mammary cancer leads to metastasis. These data demonstrate that Six1 is causally involved in both breast tumorigenesis and metastasis, thus raising the possibility that it may be a viable therapeutic target. However, because Six1 is highly expressed in the developing mammary gland, and because it has been implicated in the expansion of mammary stem cells, targeting Six1 as an anti-cancer therapy may have unwanted side effects in the breast.