Overexpression of aromatase leads to hyperplasia and changes in the expression of genes involved in apoptosis, cell cycle, growth, and tumor suppressor functions in the mammary glands of transgenic mice.

Our previous studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in aromatase transgenic mammary glands. In this study, we have examined the effects of aromatase overexpression on biochemical changes in the aromatase transgenic mice. Our results show an increase in the expression of both estrogen and progesterone receptors, and their expression is maintained in the transgenic mammary tissue even without circulating ovarian estrogens. Our results also show an increase in the expression of several growth factors and cell cycle genes in the aromatase transgenic mammary glands, which is consistent with the observed increase in proliferating cell nuclear antigen levels and cellular proliferation. Interestingly, we have also observed a decrease in the expression of epidermal growth factor receptor and its ligands, epidermal growth factor and transforming growth factor alpha, as well as several tumor suppressor genes such as p53 and retinoblastoma. This study presents novel and interesting findings that are consistent with the current models of aromatase influence and the complex interactions of biochemical pathways leading to mammary tumorigenesis.

Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities.

Our recent studies have shown that the cellular gene at the mouse mammary tumor virus integration site in the int-5 locus is aromatase. To study the role of int-5/aromatase in normal mammary development and mammary neoplasia, we have generated transgenic mice that overexpress int-5/aromatase under the control of mouse mammary tumor virus enhancer/promoter. All the transgenic virgin (n = 10) and postlactational (n = 15) females that overexpress int-5/aromatase show various histological abnormalities. Overexpression of int-5/aromatase in mammary glands of virgin females leads to the enlargement of 40% of ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of int-5/aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75-80% of ducts and glands that exhibit a range of morphological abnormalities, including formation of hyperplastic alveolar nodule (10%), ductal and glandular hyperplasia (70-80%), ductal and lobular dysplasia (15%), and nuclear abnormalities (2-5%) such as multinucleation and karyomegaly, which are all indicative of preneoplastic changes. Our results show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen as a result of overexpression of int-5/aromatase appears to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. These findings support clinical and experimental data that suggest that early estrogen exposure increases breast cancer risk.

Structure of the int-5, a novel MMTV integration genomic locus containing mouse early transposon LTR homology region.

Previous studies have reported the cloning and identification of the int-5, a novel mouse mammary tumor virus (MMTV) integration locus involved in mammary tumorigenesis. Here we report the characterization of the 5.5 kb region from this novel MMTV integration site. Our results show that the region after the MMTV integration site, a 258 bp sequence is homologous (100%) to the mouse early transposon (mETn) long-terminal repeat and other sequences of the this transposon are not present in the 5.5 kb region. Our results also show for the first time the tumor specific expression of mETn and expression of the region downstream of the MMTV integration site that is homologous to mETn-LTR in D2 mammary tumors.

Aromatase overexpression and breast hyperplasia, an in vivo model--continued overexpression of aromatase is sufficient to maintain hyperplasia without circulating estrogens, and aromatase inhibitors abrogate these preneoplastic changes in mammary glands.

To test directly the role of breast-tissue estrogen in initiation of breast cancer, we have developed the aromatase-transgenic mouse model and demonstrated for the first time that increased mammary estrogens resulting from the overexpression of aromatase in mammary glands lead to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Continued overexpression of aromatase that leads to increased breast-tissue estrogen contributes to a number of epigenetic changes in mammary tissue such as alteration in the regulation of genes involved in apoptosis, activation of genes involved in cell cycle and cell proliferation, and activation of a number of growth factors. Our current studies show aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Preneoplastic and neoplastic changes induced in mammary glands as a result of aromatase overexpression can be completely abrogated with the administration of the aromatase inhibitor, letrozole. Consistent with complete reduction in hyperplasia, we have also seen downregulation of estrogen receptor and a decrease in cell proliferation markers, suggesting aromatase-induced hyperplasia can be treated with aromatase inhibitors. Our studies demonstrate that aromatase overexpression alone, without circulating estrogen, is responsible for the induction of breast hyperplasia and these changes can be abrogated using aromatase inhibitors.

Role of MMTV integration locus cellular genes in breast cancer.

Mouse mammary tumorigenesis as a result of mouse mammary tumor virus (MMTV) integrations has helped to identify a wide variety of interesting genes that play a role in mammary development and tumorigenesis. Several such genes int1/wnt1, wnt3, wnt 10B, int2/fgf3, fgf4, int3/notch and int6 have been shown to be genetically altered in naturally formed mammary tumors as a consequence of MMTV integration. Some of these genes have been well characterised and examined in in vivo breast cancer transgenic models for their potential for tumorigenesis. Overexpression of one or more of these genes have resulted in a striking proliferation of mammary gland epithelium of both female and male transgenic mice. Our own studies have demonstrated overexpression of int5/aromatase in mammary glands of virgin and postlactational females leads to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer, that may, in turn, increase the risks for developing breast cancer. Therefore, further understanding of these genes should provide new insights to their involvement and mechanism of action in breast cancer.

The overexpression of int-5/Aromatase, a novel MMTV integration locus gene, is responsible for D2 mammary tumor cell proliferation.

Our recent studies have shown that the cellular gene at the mouse mammary tumor virus (MMTV) integration site in the int-5 locus in BALB/c D2 precancerous hyperplastic alveolar nodules is identical to the gene encoding aromatase (CYP19), a member of the cytochrome P450 gene superfamily. MMTV integrated within the 3' untranslated region of the aromatase gene is responsible for the overexpression of this gene (int-5/aromatase) in mammary tumors. This paper describes the biological significance of overexpression of int-5/aromatase in D2 tumor cells. Using a cell line derived from the D2 tumor, we have demonstrated the effect of the aromatase substrate, androstenedione, on the proliferation of tumor cells. Proliferative effects of androstenedione were blocked by an aromatase inhibitor, providing evidence for the role of int-5/aromatase in this process. Furthermore, the androstenedione-mediated proliferation was inhibited by the addition of anti-estrogen ICI 164,384, suggesting that the estrogen formed from the conversion of androstenedione by int-5/aromatase acts like a mitogen to stimulate the growth of D2 tumor cells. This model with its known mechanism of aromatase activation should prove useful for studying the role of intra-tumoral estrogen in mammary cancer, for evaluating the effects of aromatase inhibitors, and for comparing breast cancer treatments.

The nature and expression of int-5, a novel MMTV integration locus gene in carcinogen-induced mammary tumors.

Our previous studies have resulted in the identification and cloning of int-5, a novel site of mouse mammary tumor virus (MMTV) integration, from BALB/c D2 precancerous hyperplastic alveolar nodules (HAN). This paper presents a detailed characterization of the int-5 locus from both D2 HAN and normal genome and expression of the unique gene from the MMTV integration site. Our results show that the cellular gene at the MMTV integration site in the int-5 locus is identical to the gene encoding aromatase (CYP19), a member of the cytochrome P-450 gene superfamily. MMTV is integrated within exon 10 in the 3' untranslated region of the aromatase gene. This gene (int-5/aromatase) is expressed in normal mammary gland and overexpressed in mammary tumors. These results suggest that the overexpression of this gene may be responsible for mammary tumorigenesis. This is the first demonstration of integration of MMTV in a cellular gene that plays a role in hormone-dependent breast cancers.

A novel in vitro and in vivo breast cancer model for testing inhibitors of estrogen biosynthesis and its action using mammary tumor cells with an activated int-5/aromatase gene.

We recently showed that the cellular gene int-5/aromatase in BALB/c mammary alveolar hyperplastic nodule (D2 HAN/D2 tumor cells) is activated as a result of mouse mammary tumor virus integration within the 3' untranslated region of the aromatase gene. In the present study, we evaluated the effect of various aromatase inhibitors on androstenedione-mediated tumor cell growth. Also, we compared the effect of the non-steroidal aromatase inhibitor (CGS 16949A) on the inhibition of tumor growth. Our results show that D2 tumor cells respond well to various aromatase inhibitors and antiestrogens. We examined the usefulness of this model by using D2 tumor cells to simulate postmenopausal breast cancer employing both in vitro cell culture and in vivo ovariectomized (OVX) nude mouse. Unlike DMBA-induced tumors or other models, D2 tumor cells form very rapid tumors within a few days in intact mice or OVX nude mice with androstenedione supplementation and respond well to an aromatase inhibitor. This model with its known mechanism of aromatase activation should be useful for studying the role of intra-tumoral estrogen in mammary cancer, for evaluating the effects of aromatase inhibitors and antiestrogens, and for comparing breast cancer treatments.

Acceleration of mammary neoplasia in aromatase transgenic mice by 7,12-dimethylbenz[a]anthracene.

Our studies using the aromatase transgenic mice model have shown that early exposure of mammary epithelium to in situ estrogen as a result of overexpression of aromatase predispose mammary tissue to preneoplastic changes. Here, we hypothesize that the preneoplastic changes induced by mammary estrogen in aromatase transgenic females may be susceptible to environmental carcinogens like 7,12-dimethylbenz[a]anthracene (DMBA), and may result in the acceleration and/or increase in the incidence of breast cancer. Results presented in this study show that tumors appeared in 25% of the mice that were treated with DMBA and all treated transgenic animals had microscopic evidence of neoplastic progression. Control non-transgenic females did not have significant changes even after treatment with DMBA. Consistent with increased neoplastic changes in DMBA-treated aromatase mice, we have seen an increase in the expression of genes involved in cell proliferation and cell cycle. We have also seen changes in the expression of oxidative stress markers and changes in estrogen-mediated growth factors. These studies indicate that more than one event is required for tumor formation, and that early estrogen exposure leading to preneoplastic changes in the mammary epithelial cells increases susceptibility to environmental carcinogens that may result in acceleration and/or an increase in the incidence of breast cancer.

Aromatase overexpression transgenic mice model: cell type specific expression and use of letrozole to abrogate mammary hyperplasia without affecting normal physiology.

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in female mammary glands and gynecomastia and testicular cancer in male aromatase transgenic mice. Both aromatase mRNA and protein are overexpressed in transgenic mammary glands and its expression is not limited to epithelial cells. However, it is more in epithelial than in stromal cells. Our results also indicate aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of the aromatase inhibitor, letrozole. Low concentration of letrozole had no effect on normal physiology as indicated by no significant change in the circulating levels of estradiol and follicle stimulating hormone as well as no change in estrogen responsive genes such as the progesterone receptor and lactoferrin in the uterine tissue. These observations indicate that the expression of aromatase in both epithelial and stromal cells can influence the complex interactions of biochemical pathways leading to mammary carcinogenesis and that the aromatase inhibitor, letrozole can be used as chemopreventive agents without affecting normal physiology.

Overexpression of aromatase in transgenic male mice results in the induction of gynecomastia and other biochemical changes in mammary glands.

Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice.

The transforming growth factor alpha (TGFalpha) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGFalpha and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGFalpha is evident in the transgenic mice overexpressing TGFalpha in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGFalpha transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGFalpha double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGFalpha, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.

Anti-aromatase chemicals in red wine.

Estrogen synthesized in situ plays a more important role in breast cancer cell proliferation than does circulating estrogen. Aromatase is the enzyme that converts androgen to estrogen and is expressed at a higher level in breast cancer tissue than in surrounding noncancer tissue. A promising route of chemoprevention against breast cancer may be through the suppression of in situ estrogen formation using aromatase inhibitors. A diet high in fruits and vegetables may reduce the incidence of breast cancer, because they contain phytochemicals that can act as aromatase inhibitors. In our previous studies, we found that grapes and wine contain potent phytochemicals that can inhibit aromatase. We show that red wine was more effective than white wine in suppressing aromatase activity. Interestingly, our results from white wine studies suggest a weak inductive effect of alcohol on aromatase activity. On the other hand, the potent effect of anti-aromatase chemicals in red wine overcomes the weak inductive effect of alcohol in wine. Several purification procedures were performed on whole red wine to separate active aromatase inhibitors from non-active compounds. These techniques included liquid-liquid extraction, silica gel chromatography, various solid phase extraction (SPE) columns, and high performance liquid chromatography. An active Pinot Noir red wine SPE C18 column fraction (20% acetonitrile:water) was more effective than complete Pinot Noir wine in suppressing aromatase assay. This red wine extract was further analyzed in a transgenic mouse model in which aromatase was over-expressed in mammary tissue. Our gavaged red wine extract completely abrogated aromatase-induced hyperplasia and other neoplastic changes in mammary tissue. These results suggest that red wine or red wine extract may be a chemopreventive diet supplement for postmenopausal women who have a high risk of breast cancer. Further research is underway to purify and characterize the active compounds in red wine that are responsible for the inhibition of aromatase.

Cytokine regulation of cellular proliferation in endometriosis.

OBJECTIVE: This study was designed (1) to characterize the resident leukocyte population in ectopic endometrium (EE), (2) to assess proliferative activity of cellular components in EE, (3) to assess whether resident leukocytes in EE express IFN gamma mRNA and (4) to demonstrate endometrial epithelial cell IFN gamma receptors in EE. STUDY DESIGN: Biopsies of EE and normal eutopic endometrium (UE) were studied immunocytochemically using monoclonal antibodies specific for CD45 leukocyte common antigen, CD3 (a T cell marker), CD11c (a macrophage marker), and Ki67 (proliferation marker). Leukocyte types were identified immunocytochemically, followed by in situ hybridization to assess expression of IFN gamma mRNA. IFN gamma receptor expression was assessed by immunocytochemistry. RESULTS: The percentage of scattered stromal cells staining for each CD marker was greater in EE than in UE. The proliferative activity of endometrial stromal cells and epithelial cells was significantly less in EE than in UE. The overall concentration of T cells and macrophages expressing IFN gamma mRNA was significantly greater in EE than in UE. The percentage of each leukocyte type expressing IFN gamma mRNA was also greater in EE than in UE, and IFN gamma receptors were present in glandular epithelium of EE. CONCLUSIONS: These findings support a possible paracrine role for resident leukocytes and IFN gamma in regulating cell proliferation in endometriosis.

Regulation of colony stimulating factor-1 (CSF-1) in endometrial cells: glucocorticoids and oxidative stress regulate the expression of CSF-1 and its receptor c-fms in endometrial cells.

OBJECTIVE: To evaluate the regulation and expression of CSF-1 and its receptor c-fms in endometrial cells. DESIGN: In vitro study. SETTING: Research and teaching institution. PATIENT(S): None. INTERVENTION(S): In vitro experimental study. MAIN OUTCOME MEASURE(S): The effect of glucocorticoid and oxidative stress on the expression of CSF-1 and c-fms in endometrial cells. RESULT(S): Cultured nonmalignant EM42 cells not only express CSF-1 and c-fms but are also capable of responding to exogenous CSF-1. We have also seen that glucocorticoids can regulate the expression of CSF-1/c-fms in endometrial cells. Furthermore, this study shows that oxidative stress plays a significant role in the induction of CSF-1 and its receptor c-fms. CONCLUSION(S): The results suggest that CSF-1 may promote the growth of nonmalignant endometrial cells in both an autocrine and paracrine manner and that endometrial cells under oxidative stress induce CSF-1 and c-fms.

Overexpression of macrophage colony-stimulating factor (CSF-1) and its receptor, c-fms, in normal ovarian granulosa cells leads to cell proliferation and tumorigenesis.

OBJECTIVE: To investigate the interdependent role of macrophage colony-stimulating factor (CSF-1) and its receptor (c-fms) on their induction and their role in granulosa cell tumorigenesis. METHODS: Normal ovarian granulosa cells were used to develop stable transfectants that overexpress CSF-1 or CSF-1/c-fms. CSF-1 was expressed under the control of tissue/cell specific alpha-inhibin promoter, and c-fms was expressed constitutively using a viral promoter. Stable transfectants were used to examine the effect of overexpression of these molecules on the proliferation, induction of autocrine loop, and tumorigenesis. RESULTS: Expression vectors were developed for CSF-1 and its receptor, c-fms, and used to generate stable transfects overexpressing these genes in granulosa cells. Data show that overexpression of CSF-1 leads to the induction of its receptor. Stable transfectants that overexpress CSF-1 show about a 2.5-fold increase in cell proliferation compared with normal granulosa cells, and these cells are also converted to anchorage-independent and tumorigenic phenotype. Using an antisense RNA approach, we also demonstrated that the increased cell proliferation is CSF-1 specific. Concomitant overexpression of CSF-1 and c-fms further results in increased cell proliferation (sixfold), rapid anchorage-independent growth, and aggressive tumor formation. CONCLUSION: CSF-1 is capable of inducing its own receptor, and, similarly, the CSF-1 receptor, c-fms, can also induce its growth factor ligand. These studies also demonstrate the interdependent role of these genes in transformation of normal ovarian granulosa cells to a tumorigenic phenotype and suggest the possibility of a similar role for these genes in progression of ovarian cancer.

Reproductive tissue renin gene expression in preeclampsia.

OBJECTIVE: Using an analogy with renin gene overexpression, low-renin hypertension animal models, we wished to test the hypothesis that renin gene expression is increased in decidua basalis in human gestation with preeclampsia. METHODS: Human placentas were obtained immediately after delivery from 11 control (C) and 11 preeclamptics (PE). Tissue samples were microdissected and renin gene expression in decidua basalis (DB), chorionic villi (CV), and decidua vera (DV) was measured using dot-blot hybridization. RESULTS: Overall renin gene expression is highest in decidua basalis (mean +/- SEM, 2.66 +/- 0.69 densitometry area units) compared to chorionic villi (mean +/- SEM, 1.85 +/- 0.5) or compared to decidua vera (mean +/- SEM, 1.63 +/- 0.9) (both t-tests p = 0.001 two-tailed and analysis of variance p = 0.0001). Renin gene expression in DB and in CV was similar in both preeclamptic and normal pregnancies (DB mean +/- SEM C 2.79 +/- 0.96 versus PE 2.54 +/- 1.04, and CV mean +/- SEM C 2.11 +/- 0.91 versus PE, 1.59 +/- 0. 44). Renin gene expression in DV was approximately threefold higher in tissues from preeclamptics compared to control (mean +/- SEM PE 2. 44 +/- 1.76 versus C 0.82 +/- 0.42). Using the median value of 0.5 units for DV as cutoff, the preeclamptics displayed higher renin gene expression (chi square p = 0.033, two tailed). CONCLUSION: Our data suggest that renin gene expression is increased in preeclampsia in decidua vera. This may explain previously reported increased renin secretion in uterine circulation in preeclampsia.

Proline, glutamic acid and leucine-rich protein-1 is essential for optimal p53-mediated DNA damage response.

Proline-, glutamic acid- and leucine-rich protein-1 (PELP1) is a scaffolding oncogenic protein that functions as a coregulator for a number of nuclear receptors. p53 is an important transcription factor and tumor suppressor that has a critical role in DNA damage response (DDR) including cell cycle arrest, repair or apoptosis. In this study, we found an unexpected role for PELP1 in modulating p53-mediated DDR. PELP1 is phosphorylated at Serine1033 by various DDR kinases like ataxia-telangiectasia mutated, ataxia telangiectasia and Rad3-related or DNAPKc and this phosphorylation of PELP1 is important for p53 coactivation functions. PELP1-depleted p53 (wild-type) breast cancer cells were less sensitive to various genotoxic agents including etoposide, camptothecin or γ-radiation. PELP1 interacts with p53, functions as p53-coactivator and is required for optimal activation of p53 target genes under genomic stress. Overall, these studies established a new role of PELP1 in DDRs and these findings will have future implications in our understanding of PELP1's role in cancer progression.

Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis of breast cancer.

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Modeling ductal carcinoma in situ: a HER2-Notch3 collaboration enables luminal filling.

A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as three-dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional upregulation of multiple components of the Notch survival pathway. Importantly, luminal filling required upregulation of a signaling pathway comprising Notch3, its cleaved intracellular domain and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and cyclin D1. In line with HER2-Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report upregulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.