Association of TGF-ß2 levels in breast milk with severity of breast biopsy diagnosis.

PURPOSE: TGF-ß plays a dual role in breast carcinogenesis, acting at early stages as tumor-suppressors and later as tumor-promoters. TGF-ß isoforms are expressed in breast tissues and secreted in milk, suggesting that analysis of levels in milk might be informative for breast cancer risk. Accordingly, we assessed TGF-ß2 levels in milk from women who had undergone a breast biopsy and related the concentrations to diagnosis. METHODS: Milk donated by women who had undergone or were scheduled for a breast biopsy was shipped on ice for processing and testing. Breast cancer risk factors were obtained through a self-administered questionnaire, and biopsy diagnoses were extracted from pathology reports. TGF-ß2 levels in milk, assessed as absolute levels and in relation to total protein, were analyzed in bilateral samples donated by 182 women. Linear regression was used to estimate relationships of log-transformed TGF-ß2 levels and TGF-ß2/ total protein ratios to biopsy category. RESULTS: Milk TGF-ß2 levels from biopsied and non-biopsied breasts within women were highly correlated (r (2) = 0.77). Higher mean TGF-ß2 milk levels (based on average of bilateral samples) were marginally associated with more severe breast pathological diagnosis, after adjusting for duration of nursing current child (adjusted p trend = 0.07). CONCLUSIONS: Our exploratory analysis suggests a borderline significant association between higher mean TGF-ß2 levels in breast milk and more severe pathologic diagnoses. Further analysis of TGF-ß signaling in milk may increase understanding of postpartum remodeling and advance efforts to analyze milk as a means of assessing risk of breast pathology.

SKP2 overexpression is associated with increased serine 10 phosphorylation of p27 (pSer10p27) in triple-negative breast cancer.

S-phase kinase-associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin-dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1 /S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)-negative breast cancers and demonstrated that triple-negative tumors were more likely to have high expression of SKP2 than were non-triple negative, ERα-negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. Use of the triple-negative TMX2-28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple-negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers.

Parity-related molecular signatures and breast cancer subtypes by estrogen receptor status.

INTRODUCTION: Relationships of parity with breast cancer risk are complex. Parity is associated with decreased risk of postmenopausal hormone receptor-positive breast tumors, but may increase risk for basal-like breast cancers and early-onset tumors. Characterizing parity-related gene expression patterns in normal breast and breast tumor tissues may improve understanding of the biological mechanisms underlying this complex pattern of risk. METHODS: We developed a parity signature by analyzing microRNA microarray data from 130 reduction mammoplasty (RM) patients (54 nulliparous and 76 parous). This parity signature, together with published parity signatures, was evaluated in gene expression data from 150 paired tumors and adjacent benign breast tissues from the Polish Breast Cancer Study, both overall and by tumor estrogen receptor (ER) status. RESULTS: We identified 251 genes significantly upregulated by parity status in RM patients (parous versus nulliparous; false discovery rate = 0.008), including genes in immune, inflammation and wound response pathways. This parity signature was significantly enriched in normal and tumor tissues of parous breast cancer patients, specifically in ER-positive tumors. CONCLUSIONS: Our data corroborate epidemiologic data, suggesting that the etiology and pathogenesis of breast cancers vary by ER status, which may have implications for developing prevention strategies for these tumors.

The RhoA pathway mediates MMP-2 and MMP-9-independent invasive behavior in a triple-negative breast cancer cell line.

Breast cancer is a heterogeneous disease that varies in its biology and response to therapy. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk among patients diagnosed with triple-negative and/or basal-like breast cancers. A greater understanding of the molecular mechanisms underlying cancer cell spreading is needed as 90% of cancer-associated deaths result from metastasis. We previously demonstrated that the Tamoxifen-selected, MCF-7 derivative, TMX2-28, lacks expression of estrogen receptor α (ERα) and is highly invasive, yet maintains an epithelial morphology. The present study was designed to further characterize TMX2-28 cells and elucidate their invasion mechanism. We found that TMX2-28 cells do not express human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR), in addition to lacking ERα, making the cells triple-negative. We then determined that TMX2-28 cells lack expression of active matrix metalloproteinases (MMPs)-1, MMP-2, MMP-9, and other genes involved in epithelial-mesenchymal transition (EMT) suggesting that TMX2-28 may not utilize mesenchymal invasion. In contrast, TMX2-28 cells have high expression of Ras Homolog Gene Family Member, A (RhoA), a protein known to play a critical role in amoeboid invasion. Blocking RhoA activity with the RhoA pathway specific inhibitor H-1152, or a RhoA specific siRNA, resulted in inhibition of invasive behavior. Collectively, these results suggest that TMX2-28 breast cancer cells exploit a RhoA-dependent, proteolytic-independent invasion mechanism. Targeting the RhoA pathway in triple-negative, basal-like breast cancers that have a proteolytic-independent invasion mechanism may provide therapeutic strategies for the treatment of patients with increased risk of metastasis.

Oncogenic transformation of mammary epithelial cells by transforming growth factor beta independent of mammary stem cell regulation.

BACKGROUND: Transforming growth factor beta (TGFß) is transiently increased in the mammary gland during involution and by radiation. While TGFß normally has a tumour suppressor role, prolonged exposure to TGFß can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFß during involution to determine the persistent effects on premalignant mammary epithelium. METHOD: CDßGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFß (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFß and then assessed for markers of EMT and transformation. RESULTS: The 14-day exposure to TGFß induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFß. TGFß-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFß-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFß-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFß2 and changes in extra cellular matrix. CONCLUSION: This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

Loss of SFRP1 promotes ductal branching in the murine mammary gland.

BACKGROUND: Secreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of SFRP1 expression is found in breast cancer along with a multitude of other human cancers. Activated Wnt signaling leads to inappropriate mammary gland development and mammary tumorigenesis in mice. When SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer stem-like cells. However, the effects of SFRP1 loss on mammary gland development in vivo are yet to be elucidated. The work described here was initiated to investigate the role of SFRP1 in mammary gland development and whether SFRP1-/- mice exhibit changes in mammary gland morphology and cell signaling pathways shown to be associated with SFRP1 loss in vitro. RESULTS: 10 week old nulliparous SFRP1-/- mammary glands exhibited branching with clear lobulo-alveolar development, which normally only occurs in hormonally stimulated mid-pregnant wt mammary glands. Explant cultures of SFRP1-/- mammary glands display increased levels of a well known Wnt signaling target gene, Axin2. Histomorphologic evaluation of virgin glands revealed that by 10 weeks of age, the duct profile is markedly altered in SFRP1-/- mice showing a significantly higher density of ducts with distinct alveoli present throughout the mammary gland, and with focal ductal epithelial hyperplasia. These findings persist as the mice age and are evident at 23 weeks of age. Changes in gene expression, including c-Myc, TGFß-2, Wnt4, RANKL, and Rspo2 early in mammary gland development are consistent with the excessive hyper branching phenotype. Finally, we found that loss of SFRP1 significantly increases the number of mammary epithelial cells capable of mammosphere formation. CONCLUSIONS: Our study indicates that SFRP1 gene is critical for maintaining proper mammary gland development, and that reduced levels of SFRP1 results in hyperplastic lesions and its loss may be a critical event in cancer initiation.

Down-regulation of sfrp1 in a mammary epithelial cell line promotes the development of a cd44high/cd24low population which is invasive and resistant to anoikis.

BACKGROUND: The Wnt family of secreted proteins is implicated in the regulation of cell fate during development, as well as in cell proliferation, morphology, and migration. Aberrant activation of the Wnt/beta-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway by competing with the Frizzled receptor for Wnt ligands resulting in an attenuation of the signal transduction cascade. Loss of SFRP1 expression is observed in breast cancer, along with several other cancers, and is associated with poor patient prognosis. However, it is not clear whether the loss of SFRP1 expression predisposes the mammary gland to tumorigenesis. RESULTS: When SFRP1 is knocked down in a non-malignant immortalized mammary epithelial cell line (76 N TERT), nuclear levels of beta-catenin rise and the Wnt pathway is stimulated. The SFRP1 knockdown cells exhibit increased expression of the pro-proliferative Cyclin D1 gene and increased cellular proliferation, undergo a partial epithelial-mesenchymal transition (EMT), are resistant to anchorage-independent cell death, exhibit increased migration, are significantly more invasive, and exhibit a CD24low/CD44high cell surface marker expression pattern. CONCLUSION: Our study suggests that loss of SFRP1 allows non-malignant cells to acquire characteristics associated with breast cancer cells.

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-beta-neutralizing antibodies blocked responsiveness. In the absence of E+P, TGF-beta1 alone did not alter p53 activity. These results demonstrate that estrogen and progesterone together with TGF-beta signaling are necessary for maintenance of p53 activity in the mammary epithelium.

DNA methylation in paired breast epithelial and white blood cells from women undergoing reduction mammoplasty.

BACKGROUND: The extent to which white blood cell (WBC) DNA methylation provides information on the status of breast epithelial cell DNA is unknown. PATIENTS AND METHODS: We examined the correlation between methylation in Ras-association domain family-1 gene (RASSF1), a tumor-suppressor gene, and methylation in repetitive elements in paired sets of DNA from WBC and breast epithelial cells collected from 32 women undergoing reduction mammoplasty. RESULTS: We observed no evidence of correlation in methylation levels for ALU, long interspersed nuclear element-1 (LINE1) or juxtacentromeric satellite-2 (SAT2) (r=0.02 for LINE1, p=0.98; r=0.28 for ALU, p=0.12; r=0.26 for SAT2, p=0.17) for matched sets of DNA from WBC and breast epithelial cells. Variability in these markers across individuals and in the same tissue was low. Five women had an average methylation level above 5% for RASSF1 in breast epithelial cell DNA; however, average methylation levels in WBC DNA for these women were all below 1%. CONCLUSION: Methylation patterns in WBC DNA did not reflect methylation patterns in the breast.

Activation of host wound responses in breast cancer microenvironment.

PURPOSE: Cancer progression is mediated by processes that are also important in wound repair. As a result, cancers have been conceptualized as overhealing wounds or "wounds that do not heal," and gene expression signatures reflective of wound repair have shown value as predictors of breast cancer survival. Despite the widespread acknowledgment of commonalities between host responses to wounds and host responses to cancer, the gene expression responses of normal tissue adjacent to cancers have not been well characterized. EXPERIMENTAL DESIGN: Using RNA extracted from histologically normal breast tissue from 107 patients, including 60 reduction mammoplasty patients and 47 cancer patients, we measured whole genome expression profiles and identified a gene expression signature that is induced in response to breast cancer. RESULTS: This signature represents an in vivo "wound response" signature that is differentially expressed in the normal tissue of breast cancer patients compared with those without disease and is highly accurate (at least 92% sensitivity and 98% specificity) in distinguishing diseased and nondiseased. The in vivo wound response signature is highly prognostic of breast cancer survival, and there is a strong association between the groups identified by this signature and those identified using serum-treated fibroblasts and other microenvironment-derived or microenvironment-related signatures. CONCLUSIONS: The prevalence of the wound response signature in histologically normal tissue adjacent to breast cancer suggests that microenvironment response is an important variable in breast cancer progression and may be an important target for clinical interventions.

Insulin-like growth factor-I inhibits growth regulatory responses engaged by estrogen and progesterone in the mouse mammary gland.

The pathways and key players that regulate parity-induced protection against breast cancer, conferred by estrogen (E) and progesterone (P), have not fully been explained. Interestingly, in rodents, high levels of circulating insulin-like growth factor-I (IGF-I) appear to block this parity-induced protection. Using an in-vitro mouse mammary gland whole organ culture system, we investigated the mechanisms by which IGF-I affects the protective effects of E+P. Earlier studies have demonstrated that important changes induced by parity include both the enhanced sensitivity to p53 activation, and the cell death that occurs in response to DNA damage. Remarkably, when IGF-I was added to cultures containing E+P and irradiated, both cell death and expression of the tumor suppressor protein p53 were inhibited (P<0.03 and <0.006, respectively). In addition, quantitative real-time PCR analysis of numerous genes identified by microarray as being upregulated in response to prolonged exposure to pregnancy levels of E+P suggested that a subset was affected by the addition of IGF-I. IGF-I suppressed the hormone-induced expression of insulin-like growth factor-binding protein-3, transforming growth factor beta-1, and cellular retinol-binding protein-1 (P<0.01 for all genes); all of which are genes that are important in growth suppression and the regulation of survival of cells. Overall, the observed reductions in p53 sensitivity and hormone-induced gene expression in response to IGF-I in vitro suggest a mechanism for the blockade in parity-induced protection previously seen in response to IGF-I in vivo.

Rhodiola crenulata induces death and inhibits growth of breast cancer cell lines.

Diverse compounds from many different chemical classes are currently targeted in preclinical analyses for their ability to act as both chemopreventive and chemotherapeutic agents. Phenolic phytochemicals from Rhodiola crenulata has such potential. This Rhodiola species is a perennial plant that grows in the Tundra, Siberia, and high-elevation regions of Tibet. The phenolic secondary metabolites isolated from R. crenulata were recently analyzed in a preclinical setting for their ability to treat lymphosarcomas and superficial bladder cancers. However, the effects of R. crenulata have yet to be examined for its implications in breast cancer prevention or for its chemotherapeutic abilities. Therefore this study investigated the effects of R. crenulata on breast cancer both in vivo and in vitro. Experiments using aggressive human-derived MDA-MB-231 and mouse-derived V14 breast cancer cell lines demonstrated that phenolic-enriched R. crenulata extract was capable of inhibiting the proliferation, motility, and invasion of these cells. In addition, the extracts induced autophagic-like vesicles in all cell lines, eventually leading to death of the tumor cell lines but not the immortal or normal human mammary epithelial cells. Finally, an in vivo experiment showed that phenolic-enriched dietary R. crenulata is effective in preventing the initiation of tumors and slowing down the tumor growth in mice bearing tumor grafts, thereby further demonstrating its possible potential for treatment of breast cancer progression and metastasis.