Characteristics and attitudes of first round invitees in the Irish National Colorectal Cancer Screening Programme.

Background/objective: Colorectal cancer (CRC) screening is proven to reduce CRC-related mortality. Faecal immunochemical testing (FIT)-positive clients in the Irish National CRC Screening Programme underwent colonoscopy. Round 1 uptake was 40.2%. We sought to identify barriers to participation by assessing knowledge of CRC screening and examining attitudes towards FIT test and colonoscopy. Methods: Questionnaires based on a modified Champion's Health Belief Model were mailed to 3500 invitees: 1000 FIT-positive, 1000 FIT-negative and 1500 non-participants. 44% responded: 550 (46%) FIT-positive, 577 (48%) FIT-negative and 69 (6%) non-responders (NR). Results: 25% of respondents (n=286) did not perceive a personal risk of cancer, did not perceive CRC to be a serious disease and did not perceive benefits to screening. These opinions were more likely to be expressed by men (p=0.035). One-fifth (n=251) found screening stressful. Fear of cancer diagnosis and test results were associated with stress. FIT-positive clients, women and those with social medical insurance were more likely to experience stress. Conclusions: The CRC screening process causes stress to one-fifth of participants. Greater use of media and involvement of healthcare professionals in disseminating information on the benefits of screening may lead to higher uptake in round 2.

Establishment of a national cervical screening programme in Ireland, CervicalCheck: the first 6 years.

The national cervical screening programme, CervicalCheck, commenced in Ireland in 2008. Free cervical smear tests are offered to over 1.2 million women aged 25-60 every 3 (aged 25-44) and 5 (aged 45-60) years. The purpose of this paper is to highlight the achievements and document the experience of the first 6 years of a new cervical screening programme. Data were extracted from the programme screening register and colposcopy management systems. SAS, version 9.4 was used for statistical analysis. Over 1.98 million smear tests were performed in over 1 million women during the first 6 years of the programme. Overall 5-year coverage at the end of the sixth year was 77.0%, where coverage is presented for the target population of women aged 25-60 years and is adjusted for hysterectomy rates. The numbers of women attending colposcopy increased significantly from 10 000 new patients attending for the first time in the first year to a peak of almost 17 500 in the third year. Increased capacity in colposcopy has delivered significant improvements in waiting times; the percentage of women referred to colposcopy offered an appointment within 8 weeks increased from 41.5% in year 1 to 93.4% in year 4 and has remained above the greater than 90% standard thereafter. The number of biopsies increased markedly, with 33 768 women being diagnosed with cervical intraepithelial neoplasia-grade 2 (CIN2), CIN3 or adenocarcinoma in situ and 860 being diagnosed with invasive cancer by the end of the sixth year. Lessons from CervicalCheck include the importance of capacity planning in programme delivery. The programme continues to evolve, particularly with the increased usage of human papillomavirus testing and planning for future testing of the human papillomavirus (HPV)-vaccinated cohort.

Aging and ocular tissue stiffness in glaucoma.

Glaucoma is a progressive and chronic neurodegenerative disorder characterized by damage to the inner layers of the retina and deformation of the optic nerve head. The degeneration of retinal ganglion cells and their axons results in an irreversible loss of vision and is correlated with increasing age. Extracellular matrix changes related to natural aging generate a stiffer extracellular environment throughout the body. Altered age-associated ocular tissue stiffening plays a major role in a significant number of ophthalmic pathologies. In glaucoma, both the trabecular meshwork and the optic nerve head undergo extensive extracellular matrix remodeling, characterized by fibrotic changes associated with cellular and molecular events (including myofibroblast activation) that drive further tissue fibrosis and stiffening. Here, we review the literature concerning the role of age-related ocular stiffening in the trabecular meshwork, lamina cribrosa, sclera, cornea, retina, and Bruch membrane/choroid and discuss their potential role in glaucoma progression. Because both trabecular meshwork and lamina cribrosa cells are mechanosensitive, we then describe molecular mechanisms underlying tissue stiffening and cell mechanotransduction and how these cellular activities can drive further fibrotic changes within ocular tissues. An improved understanding of the interplay between age-related tissue stiffening and biological responses in the trabecular meshwork and optic nerve head could potentially lead to novel therapeutic strategies for glaucoma treatment.

Overview of Mammary Gland Development: A Comparison of Mouse and Human.

The mouse mammary gland is widely used as a model for human breast cancer and has greatly added to our understanding of the molecular mechanisms involved in breast cancer development and progression. To fully appreciate the validity and limitations of the mouse model, it is essential to be aware of the similarities and also the differences that exist between the mouse mammary gland and the human breast. This introduction therefore describes the parallels and contrasts in mouse mammary gland and human breast morphogenesis from an early embryonic phase through to puberty, adulthood, pregnancy, parturition, and lactation, and finally the regressive stage of involution.

Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases.

Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

Increased Global DNA Methylation and Decreased TGFß1 Promoter Methylation in Glaucomatous Lamina Cribrosa Cells.

BACKGROUND: Glaucoma is an optic neuropathy that affects 60 million people worldwide. There is an underlying fibrosis associated with the lamina cribrosa (LC) in glaucoma. DNA methylation is well established in regulating fibrosis and may be a therapeutic target for glaucoma. The purpose of this study was to compare global DNA methylation levels in primary human normal (NLC) and glaucomatous (GLC) cells, and to investigate DNA methylation in driving fibrosis through regulation of transforming growth factor ß1 (TGFß1). MATERIALS AND METHODS: LC cells were cultured from normal and glaucomatous human donors. Global methylation was assessed by ELISA. qPCR was conducted for DNA methyltransferases (DNMTs), methyl-CpG-binding protein 2 (MeCP2), TGFß 1 and 2, collagen 1α1 (COL1A1), and α-smooth muscle actin (αSMA). TGFß1 and DNMT1 were examined by immunofluorescence. Methylation of the TGFß1 promoter was determined by methylation-specific PCR (MSP). RESULTS: Global DNA methylation demonstrated an increase in GLC compared with NLC cells (P<0.05). The previously mentioned methylation and matrix genes were increased in GLC compared with NLC cells (P<0.05). Immunofluorescence showed increased TGFß1 and DNMT1 in GLC compared with NLC cells. MSP showed increased unmethylated DNA in the TGFß1 promoter of GLC compared with NLC cells. CONCLUSIONS: We found increased expression of fibrotic genes in GLC cells and demonstrated an increase in global DNA methylation and in associated enzymes in GLC cells. Furthermore, we showed decreased promoter methylation of TGFß1 in GLC cells. Determining a role for methylation in glaucoma and in regulating TGFß1 may provide a novel therapeutic approach.

Metabolomics/Proteomics strategies used to identify biomarkers for exfoliation glaucoma.

It is currently estimated that 60 to 70 million people worldwide are affected by open-angle glaucoma and the majority of patients who present to clinic have raised intraocular pressure, visual field loss, and cupping of the optic nerve. Although exfoliation glaucoma (XFG) correlates with age, it is the most common cause of secondary open-angle glaucoma in the world and, with elevated intraocular pressure at onset, this disease runs an aggressive clinical course. XFG differs from primary open-angle glaucoma, in that patients have a diminished response to medication, show accelerated rates of disease progression, and therefore have a higher need for surgery. Here we highlight some major findings in the literature, which relate to the search for biomarkers of XFG by metabolomics and proteomics strategies.

miR-187 is an independent prognostic factor in breast cancer and confers increased invasive potential in vitro.

PURPOSE: Here, we describe an integrated bioinformatics, functional analysis, and translational pathology approach to identify novel miRNAs involved in breast cancer progression. EXPERIMENTAL DESIGN: Coinertia analysis (CIA) was used to combine a database of predicted miRNA target sites and gene expression data. Using two independent breast cancer cohorts, CIA was combined with correspondence analysis and between group analysis to produce a ranked list of miRNAs associated with disease progression. Ectopic expression studies were carried out in MCF7 cells and miRNA expression evaluated in two additional cohorts of patients with breast cancer by in situ hybridization on tissue microarrays. RESULTS: CIA identified miR-187 as a key miRNA associated with poor outcome in breast cancer. Ectopic expression of miR-187 in breast cancer cells resulted in a more aggressive phenotype. In a test cohort (n = 117), high expression of miR-187 was associated with a trend toward reduced breast cancer-specific survival (BCSS; P = 0.058), and a significant association with reduced BCSS in lymph node-positive patients (P = 0.036). In a validation cohort (n = 470), high miR-187 was significantly associated with reduced BCSS in the entire cohort (P = 0.021) and in lymph node-positive patients (P = 0.012). Multivariate Cox regression analysis revealed that miR-187 is an independent prognostic factor in both cohorts [cohort 1: HR, 7.37; 95% confidence interval (CI), 2.05-26.51; P = 0.002; cohort 2: HR, 2.80; 95% CI, 1.52-5.16; P = 0.001] and in lymph node-positive patients in both cohorts (cohort 1: HR, 13.74; 95% CI, 2.62-72.03; P = 0.002; cohort 2: HR, 2.77; 95% CI, 1.32-5.81; P = 0.007). CONCLUSIONS: miR-187 expression in breast cancer leads to a more aggressive, invasive phenotype and acts as an independent predictor of outcome.

c-Jun N-terminal kinase activity supports multiple phases of 3D-mammary epithelial acinus formation.

Primary murine mammary epithelial cells cultured on a laminin-rich-extracellular matrix (ECM) require c-Jun N-terminal kinase (JNK) activity for acinus formation. Inhibition of JNK (using SP600125) or small interfering RNA-mediated knockdown of JNK1 blocked acinus formation, impaired cell polarisation and lumen clearance and allowed sustained extracellular signal-regulated kinase (ERK) phosphorylation, cell proliferation, adhesion-independent cell survival and expression of epithelial-mesenchymal transition markers. ERK inhibition abolished the effects of JNK blockade. Interestingly, inhibition of JNK from the time of cell seeding blocked cell polarisation and lumen clearance; later inhibition (≥ 6 h) only affected lumen clearance. ERK inhibition effectively protected cell polarisation but less so, lumen clearance. SP600125-treatment similarly affected acinus formation by the 'normal' human mammary epithelial MCF10A cell line. Expression of dominant-negative JNK1 in MCF10A cells also undermined acinus formation, generating large 'multi-acinar spheres' whose formation is probably driven by excessive luminal cell proliferation and cell survival. As JNK activity must be suppressed from the time of cell seeding to block cell polarisation, we studied the behaviour of MCF10A cells immediately after seeding in laminin rich matrix: we detected engagement of cells with the matrix, early polarisation, movement of cells into clusters and 'epithelial-cell- like' behaviour of clustered cells. Inhibition of JNK activity or expression of dominant-negative JNK1 allowed cell engagement to the matrix, but blocked cell polarisation and all subsequent 'behaviours'. While integrin activation occurred, tyrosine-phosphorylation of paxillin, Fak and Src was significantly damped by JNK inhibition. These results emphasise the multi-phase dependency of the organisation of mammary cells in 3D on JNK activity and suggest a 'permissive' support of ECM-integrin 'outside-in' signalling and a 'damping' of growth-factor ERK signalling as its two key cell physiological effects.

Molecular regulators of pubertal mammary gland development.

The pubertal mammary gland is an ideal model for experimental morphogenesis. The primary glandular branching morphogenesis occurs at this time, integrating epithelial cell proliferation, differentiation, and apoptosis. Between birth and puberty, the mammary gland exists in a relatively quiescent state. At the onset of puberty, rapid expansion of a pre-existing rudimentary mammary epithelium generates an extensive ductal network by a process of branch initiation, elongation, and invasion of the mammary mesenchyme. It is this branching morphogenesis that characterizes pubertal mammary gland growth. Tissue-specific molecular networks interpret signals from local cytokines/growth factors in both the epithelial and stromal microenvironments. This is largely orchestrated by secreted ovarian and pituitary hormones. Here, we review the major molecular regulators of pubertal mammary gland development.

PKCzeta regulates cell polarisation and proliferation restriction during mammary acinus formation.

Mammary epithelial cells organize in three dimensions and generate acini when supported on laminin-rich extracellular matrix. Acinus formation begins with the apicobasal polarisation of the outer cells of the assembly and the withdrawal of these cells from the cell cycle. Internal cells then clear out to form a hollow lumen. Here, we show that PKCζ is phosphorylated (at T410) and activated in the early stages of acinus formation in both primary cells and MCF10A cells, and during mammary tree maturation in vivo. Phospho-PKCζ colocalised with tight junction components and bound to the Par polarising complex in developing acini. To further investigate the importance of PKCζ phosphorylation in this context, acinus formation was studied in MCF10A cells overexpressing non-phosphorylatable (T410A) or 'constitutively phosphorylated' (T410E) PKCζ. In both cell types, acinus-associated cell polarisation and lumen clearance were compromised, emphasising the importance of regulated phosphorylation of PKCζ at T410 for successful acinus formation. PKCζ can be activated in a phosphorylation (at T410)-dependent and a phosphorylation-independent manner. Cells overexpressing a complete kinase-deficient PKCζ (K281W) displayed a cell polarising deficit, but also generated large 'multi-acinar' structures with associated early lumenal cell hyperproliferation. Therefore our data shows, for the first time, that two separable PKCζ activities (one phosphorylation-dependent, the other not) are required to support the cell polarisation and proliferation restriction that underpins successful acinus formation. Paralleling these contributions, we found that low levels of PKCζ mRNA expression are associated with more 'poorly differentiated' tumours and a poor outcome in a cohort of 295 breast cancer patients.

Cyclin D1b in human breast carcinoma and coexpression with cyclin D1a is associated with poor outcome.

BACKGROUND/AIM: Cyclin D1 is a mediator of cell-cycle control that is frequently overexpressed in primary ductal breast carcinomas, but its role is controversial. A polymorphism in the CCND1 gene, G870A, results in an aberrantly spliced protein (cyclin D1b) lacking the Thr-286 phosphorylation site necessary for nuclear export. Studies of murine fibroblasts have shown that although overexpression of canonical cyclin D1 (cyclin D1a) alone is not sufficient to drive malignant transformation, expression of nuclear cyclin D1b is oncogenic. Our objectives were to determine whether cyclin D1b is expressed in human breast carcinomas and to characterize the relationship of this protein to both cyclin D1a and clinical outcome in breast cancer patients. PATIENTS AND METHODS: We performed a prospective cohort study of women with early-stage breast cancer and analyzed cyclin D1a and D1b expression in primary breast tumor sections. Expression was tested for correlation with other breast cancer prognostic factors and clinical outcome, including recurrence or death. RESULTS: A total of 118 patients were included in this analysis, with a median follow-up of 44 months. Cyclin D1b was expressed in 26% of tumors and cyclin D1a was overexpressed in 27%; co-expression occurred in 4%. Cyclin D1a and/or D1b expression were not significantly associated with estrogen or progesterone receptor negativity, Her2 overexpression, young age, lymph node positivity, high tumor grade, nor large tumor size. The risk of recurrence was higher in those co-expressing D1a and D1b compared to the expression of either alone (relative risk=5.3, 95% confidence interval 1.27 to 22.1, p=0.02). The hazard ratio for those with co-expression compared with those without was 6.05 (p=0.04). CONCLUSION: Expression of cyclin D1b occurs in primary human breast carcinomas and its coexpression with cyclin D1a may be a marker for increased recurrence risk, independently of other factors.

Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development.

Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development.

Loss of Klf4 in mice causes altered proliferation and differentiation and precancerous changes in the adult stomach.

BACKGROUND & AIMS: The epithelial zinc-finger transcription factor Klf4 (formerly GKLF) regulates cellular proliferation and differentiation in vitro. Klf4 null mice die by postnatal day 1 and show changes in epithelial differentiation of skin and colon. METHODS: We used tissue-specific gene ablation to generate mice lacking Klf4 in their gastric epithelia. Klf4 mutant mice and controls were killed for histology, immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR), and serum gastrin levels. Klf4 messenger RNA (mRNA) levels were analyzed in Foxa3-Cdx2 transgenic mice and controls. Human gastric cancers and matched normal tissue were used for qPCR and immunohistochemistry for KLF4. RESULTS: Klf4 mutant mice survive to adulthood and show increased proliferation and altered differentiation of their gastric epithelia. Klf4 mutants also display aberrant expression of acidic mucins and TFF2/SP-positive cells, findings characteristic of premalignant conditions, but no inflammation, intestinal metaplasia, dysplasia, or cancer up to 1 year of age. Expression of KLF4 is nearly absent in human gastric cancer, suggesting that failure to activate KLF4 during normal cellular differentiation may be a common feature of gastric cancers. p21 WAF1/CIP1 is an in vivo target of Klf4, but Klf4 is not a mediator of Cdx2. CONCLUSIONS: Loss of a single genetic factor, Klf4, leads to dramatic changes in the gastric epithelia of mice, and Klf4 is part of a regulatory pathway involving p21 WAF1/CIP1 but not Cdx2. Thus, Klf4 is critical for normal gastric epithelial homeostasis.

Of mice and MEN1: Insulinomas in a conditional mouse knockout.

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.