Emily P. Slater

Mutual transrepression of Fos and the glucocorticoid receptor: involvement of a functional domain in Fos which is absent in FosB.

In this study, we show that Fos protein can repress transactivation by the glucocorticoid receptor (GR). In addition, we demonstrate that GR is capable of inhibiting, in a hormone‐dependent fashion, Fos‐mediated transactivation of AP‐1 dependent transcription. Moreover, repression of the serum response element by Fos is abolished by the GR in the presence of hormone. Transrepression of glucocorticoid mediated induction involves a region of Fos, located between amino acids 40 and 111, to which no function has been previously assigned, and which is poorly conserved among Fos, FosB and Fra‐1. In agreement with this finding, FosB is not capable of transrepressing GR activation of transcription, representing the first functional difference between Fos and FosB. We have mapped the domain of the GR which is required for repression of AP‐1 dependent transcription, to the region of central DNA binding domain. Our results suggest that Fos and the GR may form transcriptionally inactive complexes and point to a regulatory interrelationship between different signal transduction pathways.

Five years of prospective screening of high-risk individuals from families with familial pancreatic cancer

Objective: Familial pancreatic cancer (FPC) accounts for approximately 3% of all pancreatic cancer (PC) cases. It has been suggested that high-risk individuals (HRIs) should be offered a screening programme. Aim: To evaluate the diagnostic yield of a prospective screening programme in HRIs from families with FPC over a period of 5 years. Methods: HRIs of families with FPC of the National German Familial Pancreatic Cancer Registry (FaPaCa) were counselled and enrolled in a prospective, board-approved PC screening programme. Screening included clinical examination, laboratory tests, endoscopic ultrasound (EUS) and MRI with magnetic resonance cholangiopancreaticography (MRCP) and MR angiography. Results: Between June 2002 and December 2007, 76 HRIs of families with FPC took part in the screening programme with a total of 182 examination visits. Twenty-eight patients revealed abnormalities in EUS (n = 25) and/or MR/MRCP (n = 12). In 7 patients fine needle aspiration cytology was performed. Operative pancreatic explorations were performed in 7 individuals, resulting in limited resections in 6 cases. Histopathological examination of the resected specimens showed serous oligocystic adenomas (n = 3), pancreatic intraepithelial neoplasia 1 (PanIN1) lesions with lobular fibrosis (n = 1), PanIN2 lesions (n = 1) and PanIN1 lesion plus a gastric type intraductal papillary mucinous neoplasm (IPMN) (n = 1). Conclusions: In FPC an EUS/MR/MRCP-based screening programme leads to the detection of potential precursor lesions of PC. However, the yield of an extensive screening programme is low, especially since the tumourigenic value of low grade PanIN lesions is not yet defined. Taking into account the enormous psychological stress for the tested individual and the high costs, a general PC screening in HRIs is not justified.

Differential gene activation by glucocorticoids and progestins through the hormone regulatory element of mouse mammary tumor virus

The hormone regulatory element (HRE) of mouse mammary tumor virus can mediate activation of an adjacent promoter by glucocorticoids and progestins. A detailed comparison of the DNA binding of receptors for both hormones using DNAase I footprinting and methylation protection detects clear differences in their interactions with the HRE region between positions -130 and -100. Binding studies and gene transfer experiments with a variety of mutants covering the entire HRE demonstrate differences in the relevance of the individual sequence motifs for induction by each hormone. The influence of changes in the angular orientation of receptor binding sites is also different for glucocorticoid and progesterone induction. In transfection experiments with mutated HREs, we find a functional cooperation between the receptor binding sites that does not correlate with variations in the in vitro affinity of the receptors for the corresponding DNA fragment.

PALB2 mutations in European familial pancreatic cancer families

Slater EP, Langer P, Niemczyk E, Strauch K, Butler J, Habbe N, Neoptolemos JP, Greenhalf W, Bartsch DK. PALB2 mutations in European pancreatic cancer families.

Gene regulation by steroid hormones.

The location, orientation, and structure of the hormone regulatory elements (HRE) in nine hormonally modulated genes is described. Based on analysis of the contact points between the glucocorticoid receptor (GR) and the DNA double helix within the HREs, a model for the interaction is proposed in which a dimer of the receptor in head-to-head orientation binds to the inverted symmetry element of the HRE. The relationship between the regulatory elements for glucocorticoids and progesterone in the long terminal repeat region (LTR) of mouse mammary tumor virus (MMTV), and in the promoter region of the chicken lysozyme gene, indicates that the recognition mechanism for both receptors is similar but not identical. Curiously, the hormone ligand is not an absolute requirement for the GR to bind its HRE, though it influences the kinetics of the interaction. Other possible functions of the hormone in vivo are discussed, as well as the molecular mechanism responsible for transcriptional regulation after receptor binding to the HRE.

Bcl-2 independence of flavopiridol-induced apoptosis. Mitochondrial depolarization in the absence of cytochrome c release.

The new chemotherapeutic agent, flavopiridol, presently in clinical trials, has been extensively studied yet little is known about its mechanism of action. In this study we show that the induction of apoptosis by flavopiridol is largely independent of Bcl-2. This is indicated by the observation that neither overexpression nor the antisense oligonucleotide-mediated down-regulation of Bcl-2 had any effect on flavopiridol-induced cell killing. Our results suggest that flavopiridol can induce apoptosis through different pathways of caspase activation with caspase 8 playing a pivotal role. In human lung carcinoma cells, which contain high levels of endogenous Bcl-2 and lack procaspase 8, flavopiridol treatment leads to mitochondrial depolarization in the absence of cytochrome c release, followed by the activation of caspase 3 and cell death. These results clearly differ from observations made with other anti-tumor drugs and might explain, at least in part, the unusual anti-tumor properties of flavopiridol.

The Angiotensin-I-converting Enzyme Inhibitor Enalapril and Aspirin delay progression of Pancreatic Intraepithelial Neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer

Background and aims There are no chemopreventive strategies for pancreatic cancer or its precursor lesions, pancreatic intraepithelial neoplasia (PanINs). Recent evidence suggests that aspirin and inhibitors of angiotensin-I converting enzyme (ACE inhibitors) have potential chemopreventive properties. In this study, we used a genetically engineered mouse model of pancreatic cancer to evaluate the chemopreventive potential of these drugs. Methods Drug treatment was initiated at the age of 5 weeks. LsL-KrasG12D; Pdx1-Cre or LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice were randomly assigned to receive either mock treatment, aspirin, enalapril, or a combination of both. After 3 and 5 months, animals were killed. The effect of aspirin and enalapril was evaluated by histopathological analyses, immunostaining, and real-time PCR. Results After 3 and 5 months of treatment, enalapril and aspirin were able to significantly delay progression of mPanINs in LsL-KrasG12D; Pdx1-Cre mice. Furthermore, development of invasive pancreatic cancer in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice was partially inhibited by enalapril and aspirin. Invasive pancreatic cancer was identified in 15 of 25 (60%) LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre untreated control mice, but in only three of 17 (17.6%, p=0.01) mice treated with aspirin, in four of 17 (23.5%, p=0.03) in mice treated with enalapril alone, and in five of 16 (31.2%, p=0.11) mice treated with a combination of both drugs. Using real-time PCR we found a significant downregulation of the target genes VEGF and RelA demonstrating our ability to achieve effective pharmacological levels of aspirin and enalapril during pancreatic cancer formation in vivo. Conclusion Using a transgenic mouse model that imitates human pancreatic cancer, this study provides first evidence that aspirin and enalapril are effective chemopreventive agents by delaying the progression of PanINs and partially inhibiting the formation of murine pancreatic cancer. This study together supports the hypothesis that aspirin and ACE inhibitors might be a valid chemopreventive strategy.

Benefit of Surveillance for Pancreatic Cancer in High-Risk Individuals: Outcome of Long-Term Prospective Follow-Up Studies From Three European Expert Centers

PURPOSE Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Hereditary factors play a role in the development of PDAC in 3% to 5% of all patients. Surveillance of high-risk groups, may facilitate detection of PDAC at an early stage. The aim of this study was to assess whether surveillance aids detection of early-stage PDAC or precursor lesions (PRLs) and improves the prognosis. PATIENTS AND METHODS Screening outcomes were collected from three European centers that conduct prospective screening in high-risk groups including families with clustering of PDAC (familial pancreatic cancer [FPC]) or families with a gene defect that predisposes to PDAC. The surveillance program consisted of annual magnetic resonance imaging, magnetic resonance cholangiopancreatography, and/or endoscopic ultrasound. RESULTS Four hundred eleven asymptomatic individuals participated in the surveillance programs, including 178 CDKN2A mutation carriers, 214 individuals with FPC, and 19 BRCA1/2 or PALB2 mutation carriers. PDAC was detected in 13 (7.3%) of 178 CDKN2A mutation carriers. The resection rate was 75%, and the 5-year survival rate was 24%. Two CDKN2A mutation carriers (1%) underwent surgical resection for low-risk PRL. Two individuals (0.9%) in the FPC cohort had a pancreatic tumor, including one advanced PDAC and one early grade 2 neuroendocrine tumor. Thirteen individuals with FPC (6.1%) underwent surgical resection for a suspected PRL, but only four (1.9%) had high-risk lesions (ie, high-grade intraductal papillary mucinous neoplasms or grade 3 pancreatic intraepithelial neoplasms). One BRCA2 mutation carrier was found to have PDAC, and another BRCA2 mutation carrier and a PALB2 mutation carrier underwent surgery and were found to have low-risk PRL. No serious complications occurred as consequence of the program. CONCLUSION Surveillance of CDNK2A mutation carriers is relatively successful, detecting most PDACs at a resectable stage. The benefit of surveillance in families with FPC is less evident.

Palladin Mutation Causes Familial Pancreatic Cancer: Absence in European Families

We read with interest the article published in PLoS Medicine by Pogue-Geile et al. [1] reporting an apparent mutation in the KIAA0992 splice variant of the palladin gene in a family previously reported to have a high incidence of pancreatic cancer. Pogue-Geile and others had previously established that the 4q32–34 locus segregated with pancreatic cancer in this family by screening for pre-neoplastic lesions, which could then be used as a marker for mutation carriers [2]. In the PLoS Medicine paper the authors show that the mutation in palladin is on the 4q32–34 haplotype that segregates with the disease. The European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC) and the German National Case Collection for Familial Pancreatic Cancer (FaPaCa) have recently shown that a mutation on 4q32–34 is unlikely to explain pancreatic cancer in a majority of our European families, but we did not rule out segregation with the disease in a minority of families [3]. Naturally we were keen to establish if the mutation seen in Family X from America was seen in any of our families, and so we have sequenced the locus in 74 individuals who were either affected by pancreatic cancer or who are obligate carriers (assuming autosomal dominant inheritance) of the disease mutation (in 74 families). We have also sequenced the locus in 14 affected individuals from 14 families with familial multiple mole melanoma with cases of pancreatic cancer (FAMMM-PC) [4] and nine sporadic pancreatic cancer patients of less than 50 years of age. We did not identify the mutation in any of the individuals, neither as a heterozygote or a homozygote. This does not of course mean that other mutations in coding or non-coding regions of this variant of palladin or other variants are absent from European families. However, it is noteworthy that the phenotype of Family X is significantly different from the phenotype common to the families on the EUROPAC/FaPaCa registries. In particular, the incidence of diabetes in our families is relatively low, except where the diabetes is a direct consequence of development of cancer [3]. This presentation contrasts strongly with the family harbouring the palladin mutation [1,2], where diabetes was common. It is possible that Family X (and the association with palladin mutation) is not typical of the familial pancreatic cancer syndrome.

Hormonal induction of transfected genes depends on DNA topology.

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.