Ductal Dysplasia Research Articles

Optimization of the selection of the volume of surgical intervention in cases of pronounced morphological and structural changes of the parenchyma in patients with a high risk of developing pancreatic cancer

Chronic pancreatitis is a common recurrent pathology of the pancreas. The long course of the inflammatory process, accompanied by chronicity, is often attributed to the causes that lead to the development of pancreatic cancer. The aim of the work is to study the morphological changes of the pancreas in rats and the level of matrix metalloproteinases and tissue inhibitor of metalloproteinases in patients with complicated forms of chronic pancreatitis and a high risk of developing pancreatic cancer in order to optimize the selection of the volume of surgical intervention. A histological study of the pancreas of rats with chronic pancreatitis and chronic pancreatitis on the background of diabetes was carried out. 27 patients operated on during 2020-2022 were examined. In 15 of them, we were unable to clearly and convincingly differentiate of chronic pancreatitis from pancreatic cancer, since quite often the clinical symptoms and diagnostic picture of these diseases are similar. In the remaining 12 patients it was confirmed with all the inherent clinical signs of chronic pancreatitis and ductal hypertension. Matrix metalloproteinases (MMP-1, -2, -3, -8, -9, -10) and tissue inhibitor of metalloproteinases were studied. The obtained results were processed statistically. Morphological changes of the pancreas in rats correspond to high and moderate ductal dysplasia of the PanIN 2 and PanIN 3 class, which are direct predictors of pancreatic adenocarcinoma. In 22 patients moderate and severe exocrine insufficiency was observed, confirmed by a significant decrease in fecal elastase. An intraoperative biopsy of the changed tissue of the pancreas was performed. Among the 15 operated patients of the main group, 9 were diagnosed with diabetes mellitus and changes in MMTs and a peptide pool inherent in malignant tissue. Intraoperatively, after performing a punch biopsy, acinar metaplasia was detected in 3 patients, tissue changes characteristic of PanIN2-PanIN3 in 4 patients, and pancreatic cancer in the head area was confirmed in 3 patients. Changes in the protein profile of the plasma, clinical manifestations, as well as characteristic changes in the pancreas tissue, gave us grounds for performing extended resection interventions. Studying the morphological structure of the pancreas, using as many methods as possible for differential diagnosis between chronic pancreatitis and pancreatic cancer, as well as a comprehensive approach to the patient will allow for the most correct and effective intervention.

Omega-Class Glutathione Transferases of Carcinogenic Liver Fluke, Clonorchis sinensis, Modulate Apoptosis and Differentiation of Host Cholangiocytes.

The small liver fluke Clonorchis sinensis causes hepatobiliary ductal infections in humans. Clonorchiasis is characterized histopathologically by ductal dysplasia, hyperplasia and metaplasia, which closely resembles cholangiocarcinoma (CCA). The disruption of programmed cell death is critical for malignant transformation, while molecular events underlying these phenomena have poorly been understood in clonorchiasis-related CCA tumorigenesis. We incorporated recombinant C. sinensis omega-class glutathione transferase (rCsGSTo) 1 or 2 into human intrahepatic biliary epithelial cells (HIBECs) and analyzed pathophysiological alterations of HIBECs upon the application of oxidative stress. rCsGSTos partially but significantly rescued HIBECs from cell death by inhibiting oxidative stress-induced apoptosis (p < 0.01). rCsGSTos modulated transcriptional levels of numerous genes. We analyzed 13 genes involved in programmed cell death (the upregulation of five antiapoptotic and two apoptotic genes, and the downregulation of one antiapoptotic and five apoptotic genes) and 11 genes associated with cell differentiation (the increase in seven and decrease in four genes) that showed significant modifications (p < 0.05). The induction profiles of the mRNA and proteins of these differentially regulated genes correlated well with each other, and mostly favored apoptotic suppression and/or cell differentiation. We detected increased active, phosphorylated forms of Src, PI3K/Akt, NF-κB p65, MKK3/6 and p38 MAPK, but not JNK and ERK1/2. CsGSTos were localized in the C. sinensis-infected rat cholangiocytes, where cytokeratin 19 was distributed. Our results demonstrated that CsGSTos excreted to the biliary lumen are internalized and accumulated in the host cholangiocytes. When cholangiocytes underwent oxidative stressful condition, CsGSTos appeared to be critically involved in both antiapoptotic process and the differentiation of host cholangiocytes through the regulation of target genes following the activation of responsible signal molecules.

Supplementation with Fermented Barley Extract Prevents Mammary Epithelial Cell Invasion in an Early Breast Cancer Model.

Diet-based prevention of malignant transformation contributes to the maintenance of quality of life by avoiding a battle against cancer. Invasion is one of the features of malignant breast cancer, and the prevention of invasion may reduce breast cancer malignancy. A recently established early breast cancer model system showed mammary ductal dysplasia with invasion in mice. This study utilized the model system and investigated the effect of fermented barley extract (FBE), a food material. The elastic fiber layer is the outermost layer of the mammary duct. A reduction in the elastic fiber layer was observed in the mammary glands of the model system, whereas supplementation with 8% FBE containing water prevented this reduction. Moreover, we found that FBE supplementation prevented mammary epithelial cell invasion. Based on our findings, FBE might be a candidate material for a diet-based prevention of early breast cancer invasion.

Estrogen Induces Mammary Ductal Dysplasia via the Upregulation of Myc Expression in a DNA-Repair-Deficient Condition.

SummaryMammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in non-homologous end joining repair and observed dysplasia formation with cell proliferation at day 30. The protooncogene Myc is a downstream target of estrogen signaling, and we found that its expression is augmented in mammary epithelial cells in this dysplasia model. Treatment with a Myc inhibitor reduced E2-induced dysplasia formation. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insights into the mechanistic understanding of breast tumorigenesis and the development of breast cancer prevention.

Estrogen Induces Mammary Ductal Dysplasia Via the Upregulation of Myc Expression

Mammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in nonhomologous end joining repair and observed dysplasia formation with cell proliferation at day 30. The protooncogene Myc is a downstream target of estrogen signaling, and we found that its expression is augmented in mammary epithelial cells in this dysplasia model. Treatment with a Myc inhibitor reduced E2-induced dysplasia formation. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insights into the mechanistic understanding of breast tumorigenesis and the development of breast cancer prevention.

Abstract 1835: Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1)

Abstract Amplification within the 8p11-12 locus occurs in approximately 15% of breast cancer (BC) and is associated with poor survival and distant recurrence. Overexpression of many of the genes within the 8p11-12 region can confer a pathological gain of function to breast cells. We and others have previously demonstrated that NSD3 (WHSC1L1), a lysine methyltransferase, can act as a potent transforming oncogene. Predictions by the GISTIC algorithm of the Human Cancer Genome Atlas data suggest that NSD3 is a driving gene within 8p11-12 in multiple tumor types, including BC. We previously showed that NSD3 can regulate the expression of influential genes, such as estrogen receptor alpha (ESR1/ERα). To further explore the role of NSD3 in promoting BC, we generated FVB/N transgenic mice with targeted overexpression of NSD3 in the mammary gland and compared them to matched non-transgenic wild-type (WT) females. We observed that pups nursed by transgenic females were underdeveloped, regardless of genotype. Underdeveloped pups displayed delayed hair growth and eye opening, and were half the weight of pups nursed by WT females. To investigate this phenotype, we characterized thoracic and inguinal mammary glands from virgin, mid-pregnancy, lactating, and post-lactating mice by whole mount and histological analysis. Mammary glands from virgin transgenic females displayed increased branching and terminal bud formation. Alveolar buds from mid-pregnant transgenic glands were more numerous and densely packed. Glands from lactating transgenic females showed large areas in which the alveoli failed to undergo functional differentiation, resulting in a lactation defect. Mammary glands taken post-lactation exhibited areas of ductal and alveolar hyperplasia. At 40 weeks of age, multiple transgenic mice had palpable tumors which were excised at 1 cm3 size. Whole mount and histological analysis of glands from tumor bearing mice commonly demonstrated hyperplasia, dysplasia, and carcinoma in situ, contrary to age-matched WT glands. The areas of ductal dysplasia and carcinoma in situ closely resembled several patterns commonly observed in human breast cancer, including micropapillary, cribriform, and high grade areas of ductal carcinoma in situ. Mammary tumors were analogous to infiltrating ductal carcinomas, many of which were high grade tumors. In summary, overexpression of NSD3 in the mouse mammary gland elicited drastic deformation, inhibited functional differentiation, and caused tumor formation. Continued characterization of this mouse model, the oncogenic role of NSD3, and other mechanistic studies are essential to improve patient outcome in 8p11-12 altered cancers. Citation Format: Alex C. Rutkovsky, Brittany Turner-Ivey, Ericka L. Smith, Laura S. Spruill, Jamie N. Mills, Stephen P. Ethier. Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2017-1835

Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3.

NSD3 has been implicated as a candidate driver oncogene from the 8p11-p12 locus, and we have previously published evidence for its amplification and overexpression in human breast cancer. This aim of this study was to further characterize the transforming function of NSD3 in vivo. We generated a transgenic mouse model in which NSD3 gene expression was driven by the MMTV promoter and expressed in mammary epithelium of FVB mice. Mammary glands were fixed and whole mounts were stained with carmine to visualize gland structure. Mammary tumors were formalin-fixed, and paraffin embedded (FFPE) tumors were stained with hematoxylin and eosin. Pups born to transgenic females were significantly underdeveloped compared to pups born to WT females due to a lactation defect in transgenic female mice. Whole mount analysis of the mammary glands of transgenic female mice revealed a profound defect in functional differentiation of mammary gland alveoli that resulted in the lactation defect. We followed parous and virgin NSD3 transgenic and control mice to 50weeks of age and observed that several NSD3 parous females developed mammary tumors. Whole mount analysis of the mammary glands of tumor-bearing mice revealed numerous areas of mammary hyperplasia and ductal dysplasia. Histological analysis showed that mammary tumors were high-grade ductal carcinomas, and lesions present in other mammary glands exhibited features of alveolar hyperplasia, ductal dysplasia, and carcinoma in situ. Our results are consistent with our previous studies and demonstrate that NSD3 is a transforming breast cancer oncogene.

Chronic Pancreatitis with Acinar–Ductal Metaplasia and Ductal Dysplasia in a Horse

A 16-year-old Friesian gelding with relapsing colic was humanely destroyed during diagnostic laparotomy due to suspected abdominal neoplasia. On post-mortem examination, the pancreas appeared as a firm mass (20×8×8cm). The cut surface had a lobular structure with multiple cavities. Histological examination revealed severe chronic fibrosing pancreatitis with acinar-ductal metaplasia and duct dysplasia, which was considered to be the cause of the recurrent colic. Formation of tubular complexes within a background of acinar-ductal metaplasia is similar to the regressive lesions detected in the human pancreas in the context of inflammation, duct obstruction, cystic fibrosis and neoplasia. Pancreatic acinar-ductal metaplasia and ductal dysplasia are considered to be preneoplastic conditions in man and in the mouse.

Parameters of dynamic contrast-enhanced MRI as imaging markers for angiogenesis and proliferation in human breast cancer.

BackgroundBreast cancer is the most common malignancy and the leading cause of cancer death in women worldwide; however, early diagnosis has been difficult due to its complex pathological structure. This study evaluated the value of morphological examination in conjunction with dynamic contrast-enhanced MRI (DCE-MRI) for more precise diagnosis of breast cancer, as well as their correlation with angiogenesis and proliferation biomarkers.Material/MethodsDCE-MRI parameters (including Ktrans: volume transfer coefficient reflecting vascular permeability, Kep: flux rate constant, Ve: extracellular volume ratio reflecting vascular permeability, and ADC: apparent diffusion coefficient) were obtained from 124 patients with breast cancer (124 lesions). Microvessel density (MVD) was evaluated by the immunohistochemical analysis of tumor vessels for CD31 and CD105 expression. The proliferation was assessed by analyzing Ki67.ResultsKtrans values were in the order of: malignant lesions > benign lesions > normal glands. Similar results were observed for Kep. The opposite changes were seen with Ve. Ktrans and Kep values were significantly higher in invasive ductal carcinoma (IDC) and ductal carcinoma in situ (DCIS) than in mammary ductal dysplasia (MDD; ANOVA followed by Dunnett’s test). In sharp contrast, ADC values were lower in IDC and DCIS than in MDD, and Ve was not significantly different among the three groups. The data from MIP (maximum intensity projection) showed that benign breast lesions had no or only one blood vessel, whereas malignant lesions had two or more blood vessels. In addition, expression of CD105 and Ki67, the commonly recognized markers for angiogenesis and proliferation, respectively, were closely correlated with MRI parameters as revealed by Pearson analysis.ConclusionsDetermination of Ktrans, Kep and ADC values permits estimation of tumor angiogenesis and proliferation in breast cancer and DCE-MRI parameters can be used as imaging biomarkers to predict patient prognosis and the biologic aggressiveness of the tumor.

12q21 Microdeletion in a fetus with Meckel syndrome involving CEP290/MKS4

We report on a fetus with Meckel syndrome diagnosed during the 21st gestational week, hydrocephalus and bilateral hyperechogenic kidneys were then detected on ultrasonography. Fetal pathological examination showed facial dysmorphism, occipital meningoencephalocele, characteristic renal cysts, mild hepatic ductal dysplasia, hydrocephalus in association with extreme cerebellar vermis hypoplasia and brainstem anomalies. Molecular and cytogenetic analysis identified a paternally inherited CEP290/MKS4 (MIM611134) (12q21) nonsense mutation and a maternal 12q21 microdeletion. Two cases with such a mechanism have previously been described in the literature, one of them involves an inherited microdeletion. The observation of such cases highlights the existence of a pathogenic mechanism which involves deletion and point mutation, and illustrates how homozygosity can hide hemizygosity when usual sequencing methods are used. The identification of hemizygosity enables to determine precisely the molecular mechanism and to understand some phenotypic variations. As they act as complete loss of function allele, deletions might give indication on the severity of the associated point mutation. This clinical report highlights the importance of fetal pathology following termination of pregnancies in order to guide molecular analysis and the potential role of cytogenetic cryptic disorders in autosomal recessive disease. The use of polymorphic marker analysis in association with FISH or arrayCGH provided an accurate identification of molecular mechanisms, accurate genetic counseling and optimized strategy for next pregnancies or preimplantation diagnosis.

Human pancreatic cancer fusion 2 (HPC2) 1‐B3: A novel monoclonal antibody to screen for pancreatic ductal dysplasia

BACKGROUND.: Pancreatic ductal adenocarcinoma is rarely detected early enough for patients to be cured. The objective of the authors was to develop a monoclonal antibody to distinguish adenocarcinoma and precancerous intraductal papillary mucinous neoplasia (IPMN) from benign epithelium. METHODS.: Mice were immunized with human pancreatic adenocarcinoma cells and monoclonal antibodies were screened against a panel of archived pancreatic tissue sections, including pancreatitis (23 cases), grade 1 IPMN (16 cases), grade 2 IPMN (9 cases), grade 3 IPMN (13 cases), and various grades of adenocarcinoma (17 cases). One monoclonal antibody, human pancreatic cancer fusion 2 (HPC2) 1-B3, which specifically immunostained adenocarcinoma and all grades of IPMN, was isolated. Subsequently, HPC2 1-B3 was evaluated in a retrospective series of 31 fine-needle aspiration (FNA) biopsies from clinically suspicious pancreatic lesions that had long-term clinical follow-up. RESULTS.: HPC2 1-B3 was negative in all 31 cases of chronic pancreatitis that were tested. In contrast, HPC2 1-B3 immunostained the cytoplasm and luminal surface of all 16 well- to moderately differentiated pancreatic ductal adenocarcinomas. It demonstrated only weak focal staining of poorly differentiated carcinomas. All high-grade IPMNs were found to be positive for HPC2 1-B3. The majority of low-grade to intermediate-grade IPMNs were positive (66% of cases). Immunostaining a separate series of pancreatic FNA cell blocks for HPC2 1-B3 demonstrated that the relative risk for detecting at least low-grade dysplasia (2.0 [95% confidence interval, 1.23-3.26]) was statistically significant (P = .002 by the Fisher exact test). CONCLUSIONS.: To reduce the mortality of pancreatic cancer, more effective early screening methods are necessary. The data from the current study indicate that a novel monoclonal antibody, HPC2 1-B3, may facilitate the diagnosis of early pancreatic dysplasia.

Meckel-Gruber syndrome: Report of two cases

Meckel-Gruber syndrome (MKS) is an autosomal recessive disorder, characterized by a combination of renal cysts and variably associated with features including developmental anomalies of the central nervous system (typically encephalocele), hepatic ductal dysplasia, cysts, and polydactyly. It is a rare syndrome with highest incidence in Gujarati Indians and Finnish population. We report two such cases of MKS in non-Gujarati Indian which were diagnosed by neonatal autopsy.

Évaluation des macrobiopsies mammaires stéréotaxiques par système Vacora ® 10-gauge (541 procédures)

Purpose To evaluate stereotactic core biopsies of the breast with the 10-gauge Vacora ® biopsy device. Patients and methods Retrospective study of 541 procedures in 502 patients performed between 2007 and 2009. Results The procedure failed in 2 % of cases, non-complicated hematomas occurred in 5 % of cases and unsightly scars in two cases. A clip was deployed in 70 % of cases, successfully in 99 % of cases. The procedure was well tolerated in 88 % of cases. Core biopsies confirmed a benign lesion in 55 % of cases, borderline lesions in 19 % of cases and malignant lesions in 26 % of cases with complementary surgery performed in 40 % of cases. For surgical lesions, sensitivity, specificity, PPV and NPV were 89 %, 100 %, 100 % and 84 % respectively. Atypical ductal dysplasia was under-estimated in 8 % of cases while DCIS was under-estimated in 14 % of cases. After review of the mammograms, 3 % of Bi-Rads 4 lesions were reclassified as Bi-Rads 3 lesions, all benign at core biopsy. Half of these results were from screening mammography programs. Conclusion Results with the 10-gauge Vacora ® biopsy device are similar to reports from the literature, mainly using the Mammotome system, with regards to tolerability and reliability for a lesser cost.

Abstract 3254: Characterization of two mutant models with distinct expression profile of BRCA1 splice variant IRIS

Abstract More than 400 cancer-associated Brca1 mutations have been discovered and it is reported that different mutations in the Brca1 gene confer different cancer-related phenotypes. An important aspect that has complicated proper interpretation of the various Brca1 mutants in human and mouse model is the fact that Brca1 transcripts are subject to alternative splicing. In humans, two splice variants (Brca1-Δ11 and Brca1-IRIS) have been functionally analyzed to date. Unlike full-length Brca1, BRCA1-IRIS although largely encoded by identical sequences behaves more like a proto-oncogene. To investigate the function of the BRCA1-IRIS allele in vivo, we studied growth and tumorigenesis in Brca1F22–24 mice which express a BRCA1-IRIS and compared the phenotype directly to the Brca1F5–13 model that is functionally null for all BRCA1 activity. Brca1−/−;p53+/−;K14Cre mice generated from both Brca1 alleles showed a phenotype of premature aging. Alopecia was developed at average 12 and 20 wks of age in Brca1F5–13/F5–13;p53+/−;K14Cre and Brca1F22–24/F22–24;p53+/−;K14Cre genotypes respectively. Mammary glands from Brca1F5–13/F5–13;p53+/−;K14Cre mice displayed ductal dysplasia. This ductal abnormality was also observed in Brca1F22–24/F22–24;p53+/−;K14Cre mice. But in contrast to the Brca1F5–13 allele, gland from these mice showed ductal hyperplasia. Mammary tumors developed only in the Brca1F22–24/F22–24;p53+/−;K14Cre mice with high frequency. However, Brca1F5–13/ F5–13;p53+/−;K14Cre mice never developed tumors possibly due to severe aging process taken place before full mammary development. MEF cells generated from two transgenic models also showed different features in their growth and cell cycle. Brca1 abrogation retarded cell growth by arresting cells in G2/M phase. Arrested cell fraction was higher in MEF cells obtained from Brca1F22–24/ F22–24 mouse compared to one from Brca1F5–13/ F5–13 mouse. Consequently Brca1F22–24/ F22–24 MEF cells were more sensitive to M phase specific inhibitor vinorelbine. Different Brca1 mutations that encode truncated version of full length protein could, in theory, still able to produce other Brca1 splice variants. Various genotype-phenotype correlations found in different Brca1 mutation carrier are likely to be caused by different expression profiles of Brca1 splice variants. Our study, using two different Brca1 mutant models that show distinct expression of Brca1-IRIS, is first attempt to distinguish different Brca1 knock out models regarding Brca1 splice variant. Further investigation of this new aspect of BRCA1 biology will help us to understand different functions exerted by distinct BRCA1 splice variants. Our study could have important implications for genetic counseling and treatment of carriers having different type of Brca1 mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3254.

Mouse Mammary Tumor Expressing MMTV-SmoM2 as a Tool To Study Lung Metastasis.

Abstract Background: The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, ductal dysplasias, and increase the proportion of mammosphere-forming cells, with reduction of stem cell pool, in virgin mice.Material and Methods: We isolated and established a tumor line from this mouse line (named TU505). To investigate the role of hedgehog signaling in mammary tumorigenesis and metastasis we transplanted TU505 fragments (1-2mm3) to a cleared fat pad of 3 week-old mice. After 10 days we removed the primary tumors and let the mice recover for another 3 weeks, which at that time the animals were sacrificed and the lungs removed under stereomicroscope. The lung metastases visible at naked eyes were snap frozen for RNA isolation, and the remaining lungs fixed in 4%PFA.Results: The TU505 is a tumor expressing MMTV-SmoM2, triple-negative (ER-, PR-, and HEB2-), hormone independent, highly metastatic to the lung, and with a robust expression of Ptch1, Ptch2, Smo, Gli2, and Gli3. The gene expression of primary tumor and metastasis are compared.Discussion: The hedgehog signaling networks has been associated with metastasis. The TU505 is a strong tool to study mammary gland tumorigenesis and metastasis. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2165.

Abstract A26: Aromatase over-expression results in the development of ductal growth abnormalities in the mammary gland of a conditional transgenic mouse model

Abstract Estrogens are required for the development of the normal mammary gland; however, studies suggest that estrogen and its metabolites may have mutagenic and carcinogenic potential in the mammary gland. Aromatase is key to estrogen biosynthesis, but its over-expression may play a role in the development of breast cancer by increasing mitotic activity in breast epithelial cells. Therefore, it is highly valuable to have mouse models with increased local estrogen production to use to develop optimal therapeutic and chemopreventive strategies to treat these lesions. The purpose of this study was to evaluate if increased local estrogen production led to the development of mammary gland preneoplasia. To test our hypothesis we developed a conditional transgenic mouse model to control the spatial and temporal aromatase expression in the mammary gland. Mammary glands were collected at 4 and 12 months of age for morphological, histological, gene and protein expression studies in both MMTV-rtTA/tet-op-Aromatase and wild-type mice. RT-PCR analysis of aromatase mRNA confirmed that the gene was conditionally expressed in the mammary gland as compared to control mice. Aromatase activity assays confirmed a significantly higher enzyme activity in the mammary glands of MMTV-rtTA/tet-op-Aromatase compared to wild-type. Mammary glands were evaluated for morphological changes using confocal microscopy, whole-mount and hematoxylin and eosin staining. Aromatase over-expression resulted in a higher incidence of morphological and histological abnormalities such as hyperplastic alveolar nodules, ductal dysplasia, ductal carcinoma in situ, and the unexpected persistence of terminal end buds at 12 months of age. These changes were associated with ERK1/2 and AKT activation. In summary, we generated a novel transgenic mouse system to investigate the role of aromatase over-expression in mammary epithelial cells during mammary gland development. Increased aromatase activity resulted in the development of ductal growth abnormalities and increased ductal density. This model represents a valuable tool to investigate the effect of estrogen over-production in the mammary gland microenvironment at specific times during development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A26.

Ptch1is required locally for mammary gland morphogenesis and systemically for ductal elongation

Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1(mes)), we found that Ptch1(mes) homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1(mes) homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness.

P53 heterozygosity leads to deregulated apoptosis and cell proliferation in the mammary gland of ERα over-expressing mice.

Abstract Abstract #2008 Background: Transgenic mouse models are a common experimental tool for unraveling gene function. Breast cancer develops as genetic changes accumulate in the ductal epithelium giving rise to precursor lesions such as atypical ductal hyperplasias, which may progress to ductal carcinoma in situ (DCIS) and eventually invasive breast cancer. This process is associated with mutations in tumor suppressor genes such as p53 resulting in the deregulation of essential genes. p53 plays a role in mediating cell response to various stresses by inducing or repressing genes involved in cell cycle arrest, senescence, apoptosis and DNA repair. Studies have shown that p53 is a predictor for local recurrence of DCIS. About 65% of breast cancers are estrogen receptor alpha (ERα) positive and estrogen dependent. Deregulated expression of ERα in normal breast epithelium has been found in conjunction with breast cancer, leading to the concept that loss of the normal regulatory mechanisms that control expression levels of ERα in normal breast epithelium may increase the risk for the development of breast cancer. This study tests the hypothesis that p53 heterozygosity may lead to an aberrant growth response to hormonal stimuli and development of mammary gland preneoplasia.&amp;#x2028; Methodology: To examine if p53 heterozygosity promotes disease in mammary glands with deregulated expression of ERα, a conditional ERα in mammary tissue mouse model (MMTV-rtTA/tet-op-ERα or CERM) was bred to a p53 homozygous mutant (p53-/-) mouse model. For progression studies, mammary glands were collected at 12 months of age for morphological and histological studies both in intact nulliparous mice. A group of mice was ovariectomized at 14 weeks of age and 2 weeks later treated with either placebo or 17β-estradiol for 48 hours to dissect the molecular events attributable to specific hormonal stimulation as compared to normal ovarian function.&amp;#x2028; Results: Loss of p53 resulted in a statistically significant higher percentage of hyperplastic alveolar nodules, both by itself and in mice with ERα deregulation in the mammary gland. p53 heterozygosity resulted in a higher incidence of morphological and histological abnormalities such as dilated ducts, ductal dysplasia, ductal carcinoma in situ, and abnormal stroma. These lesions showed higher rates of cell proliferation and lower rates of apoptosis. Phospho-p53 immunohistochemistry showed evidence of DNA damage in the p53 heterozygous mice. There was no significant change in the terminal ductal structures in mammary glands of ovariectomized mice when compared to mice treated with exogenous 17β-estradiol after 48 hrs.&amp;#x2028; Conclusions: p53 heterozygosity predisposed mammary tissue to preneoplastic changes both by itself and in collaboration with deregulated ERα expression. The combination of deregulated ERα expression and p53 heterozygosity was associated with more disease than either genetic change by itself. Loss of p53 leads to a deregulation in apoptosis and cell proliferation in the mammary gland of ERα over-expressing mice suggesting that both processes are involved in the development of preneoplasia. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2008.

Therapeutic targeting of the c-fms oncogene diminishes the growth of breast cancer cells and ductal hyperplasia in the mammary gland of the CSF-1 transgenic mouse model.

Abstract Abstract #3120 The macrophage colony-stimulating factor (CSF-1) is a cytokine produced by macrophages as well as epithelial cancer cells. By signaling via its receptor c-fms, CSF-1 induces growth/survival pathways in breast cancer cells. Defects in mammary gland development were observed in mice deficient in CSF-1/c-fms signaling, implicating this pathway in normal mammary development as well. In breast cancer, elevated levels of both CSF-1 and c- fms have been detected, with 80% of invasive breast carcinomas abnormally expressing c-fms. To study the role of CSF-1 signaling in breast cancer, we have previously generated mammary specific transgenic mouse models that over-express these factors (termed MMTV-CSF-1 and MMTV-c-fms). Both these transgenic mouse strains developed ductal hyperplasia and dysplasia as well as tumor formation. Our previous evidence also showed increase in cellular proliferation factors such as Cyclin D1 and PCNA in these mice. To examine which signaling pathways are altered in these mice, we have compared in this study the expression profile of target genes of signaling pathways in MMTV-CSF-1 mice and wild type mice using the Signal Transduction Pathway Finder RT-PCR array (Superarrray). Wnt signaling pathway was induced in the mammary gland of the MMTV-CSF-1 mouse strain compared to wild type, suggesting that this pathway may mediate CSF-1 signaling. In a proof-of-principle experiment, an MMTV-CSF-1 mouse was injected subcutanuously with neutralizing anti-c-fms antibody (Santacruz Biotech, 2-4A5) at a dose of 5 μg daily for 7 days. The treated mouse exhibited a decrease in the elevated ductal branching and hyperplasia that are characteristics of untreated MMTV-CSF-1 mice. This treatment also resulted in reversing many of expression alterations in MMTV-CSF-1 mice. For example, expression of the CSF-1 responsive gene Cyclin D1 was reduced by over 3-fold due to antibody treatment. Fas, the receptor for the pro-apoptotic factor Fas ligand, decreased by 5-folds in the MMTV-CSF-1 strain relative to wild type but increased 14-folds due to c-fms antibody treatment. The expression of the proapoptotic factor Bax increased about 4-fold, whereas the angiogenic factor Angiopoietin 1 was reduced by 60% due to anti-cfms antibody treatment. The treatment also reversed alterations in the expression of the wnt transcription factors Lef-1/TCF7. Because small molecule tyrosine kinase inhibitors that target c-fms, Gleevecâ (Imatinib, obtained from Novartis) and GW2580 (Calbiochem), are emerging, we examined their effects on the growth of SKBR3 breast cancer cells that express both CSF-1 and c-fms. Our data showed both these drugs inhibited the growth of SKBR3 cells, providing the rationale to test their effects on tumor formation and underlying biochemical effects in our transgenic preclinical models. The current studies warrant further investigation of treating breast cancer using antibody and small molecule drugs targeting CSF-1/c-fms signaling. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3120.

Pdx-1-Driven Overexpression of Aurora A Kinase Induces Mild Ductal Dysplasia of Pancreatic Ducts Near Islets in Transgenic Mice

To further explore the oncogenic activity of Aurora A kinase while attempting to develop a useful mouse model for pancreatic cancer, Aurora A kinase was targeted to pancreatic duodenal homeobox gene-1 (Pdx-1)-positive cells. Aurora A kinase overexpression was targeted to mouse pancreas tissues using the Pdx-1 promoter in a transgenic model. The pancreas tissues of 7- to 11-month-old transgenic animals were evaluated for metastatic adenocarcinomas, preinvasive ductal neoplasia, or other histological anomalies. Examination of pancreatic tissue from Pdx-1-Aurora A transgenic mice revealed abnormalities, such as mild islet cell hyperplasia, lymphocytic infiltration, and general dysplasia between ductal/islet cell interfaces. However, most tissues from these transgenic mice were normal. The overexpression of Aurora A can potentially initiate the development of mild abnormalities in pancreatic tissue; however, neither preinvasive ductal neoplasia nor fully metastatic adenocarcinomas were observed. Combining the Pdx-1-Aurora A transgenic model with other genetic alterations may provide additional insight.