Germline mutations in CDH1 are infrequent in women with early-onset or familial lobular breast cancers.

BACKGROUND: Germline mutations in CDH1 are associated with hereditary diffuse gastric cancer; lobular breast cancer also occurs excessively in families with such condition. METHOD: To determine if CDH1 is a susceptibility gene for lobular breast cancer in women without a family history of diffuse gastric cancer, germline DNA was analysed for the presence of CDH1 mutations in 318 women with lobular breast cancer who were diagnosed before the age of 45 years or had a family history of breast cancer and were not known, or known not, to be carriers of germline mutations in BRCA1 or BRCA2. Cases were ascertained through breast cancer registries and high-risk cancer genetic clinics (Breast Cancer Family Registry, the kConFab and a consortium of breast cancer genetics clinics in the United States and Spain). Additionally, Multiplex Ligation-dependent Probe Amplification was performed for 134 cases to detect large deletions. RESULTS: No truncating mutations and no large deletions were detected. Six non-synonymous variants were found in seven families. Four (4/318 or 1.3%) are considered to be potentially pathogenic through in vitro and in silico analysis. CONCLUSION: Potentially pathogenic germline CDH1 mutations in women with early-onset or familial lobular breast cancer are at most infrequent.

SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer.

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2-/- mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2-urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.

The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Loss of Cited2 affects trophoblast formation and vascularization of the mouse placenta.

Cited2 is widely expressed in the developing embryo and in extraembryonic tissues including the placenta. Gene expression can be induced by a number of factors; most notably by the hypoxia inducible transcription factor, HIF1, under low oxygen conditions. Cited2 encodes for a transcriptional co-factor that in vitro can act as both a positive and negative regulator of transcription. This function is due to its interaction with CBP/p300 and appears to depend on whether Cited2 enables CBP/p300 to interact with the basic transcriptional machinery, or if its binding prevents such an interaction from occurring. Here, we report a novel function for Cited2 in placenta formation, following gene deletion in mouse. In the absence of Cited2 the placenta and embryo are significantly small from 12.5 and 14.5 dpc respectively, and death occurs in utero. Cited2 null placentas have fewer differentiated trophoblast cell types; specifically there is a reduction in trophoblast giant cells, spongiotrophoblasts and glycogen cells. In addition, the fetal vasculature of the placenta is disorganised and there are fewer anastomosing capillaries. Given that Cited2 is expressed in both trophoblasts and the fetal vasculature, the observed defects fit well with the sites of gene expression. We conclude that Cited2 is required for normal placental development and vascularisation, and hence for embryo viability.

Prevention and control of "drunk driving": lessons for social work.

This article examines efforts to prevent and control the widespread problem of "drunk driving" in the United States. Special emphasis is placed on one institutional mechanism-the Alcohol Safety Action Project-developed to deal with the behavior of problem drinkers.

A training program for interprofessional health care teams.

Interprofessional approaches to practice have gained increased popularity. This paper describes a course in team training for professionals in health care. It is an overview of the course's content with a discussion of the problems and issues that arose as the course was designed and implemented. It concludes with a discussion of the outcomes and evaluations of the students' and faculty's experience.

Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2.

The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator.PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380-Thr381 bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2.

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.

Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop.

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.