Pancreatic cancer genomics: where can the science take us?

The incidence of pancreatic ductal adenocarcinoma (PDAC) is steadily increasing and the annual death-to-incidence ratio approaches one. This is a figure that has not changed for several decades. Surgery remains the only chance of cure; however, only less than 20% of patients are amenable to operative resection. Despite successful surgical resection, the majority of the patients still succumb to recurrent metastatic disease. Therefore, there is an urgent need to develop novel therapeutic strategies and to better select patients for current therapies. In this review, we will discuss current management by highlighting the landmark clinical trials that have shaped current care. We will then discuss the challenges of therapeutic development using the current randomized-controlled trial paradigm when confronted with the molecular heterogeneity of PDAC. Finally, we will discuss strategies that may help to shape the management of PDAC in the near future.

Carriage of an ACME II variant may have contributed to methicillin-resistant Staphylococcus aureus sequence type 239-like strain replacement in Liverpool Hospital, Sydney, Australia.

Approximately 39% of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 239 (ST239)-like bloodstream isolates from Liverpool Hospital (obtained between 1997 and 2008) carry an arginine catabolic mobile element (ACME). Whole-genome sequencing revealed that an ACME II variant is located between orfX and SCCmec III, and based on pulsed-field gel electrophoresis patterns and temporal relationships of all ST239-like isolates (n = 360), ACME carriage may have contributed to subpulsotype strain replacement.

Analysis of Brca1-deficient mouse mammary glands reveals reciprocal regulation of Brca1 and c-kit.

Disruption of the breast cancer susceptibility gene Brca1 results in defective lobular-alveolar development in the mammary gland and a predisposition to breast tumourigenesis in humans and in mice. Recent evidence suggests that BRCA1 loss in humans is associated with an expansion of the luminal progenitor cell compartment in the normal breast and tumours with a luminal progenitor-like expression profile. To further investigate the role of BRCA1 in the mammary gland, we examined the consequences of Brca1 loss in mouse mammary epithelial cells in vitro and in vivo. Here, we show that Brca1 loss is associated with defective morphogenesis of SCp2 and HC11 mouse mammary epithelial cell lines and that in the MMTV-Cre Brca1(Co/Co) mouse model of Brca1 loss, there is an accumulation of luminal progenitor (CD61(+)CD29(lo)CD24(+)) cells during pregnancy. By day 1 of lactation, there are marked differences in the expression of 1379 genes, with most significantly altered pathways and networks, including lactation, the immune response and cancer. One of the most differentially expressed genes was the luminal progenitor marker, c-kit. Immunohistochemical analysis revealed that the increase in c-kit levels is associated with an increase in c-kit positivity. Interestingly, an inverse association between Brca1 and c-kit expression was also observed during mammary epithelial differentiation, and small interfering RNA-mediated knockdown of Brca1 resulted in a significant increase in c-kit mRNA levels. We found no evidence that c-kit plays a direct role in regulating differentiation of HC11 cells, suggesting that Brca1-mediated induction of c-kit probably contributes to Brca1-associated tumourigenesis via another cellular process, and that c-kit is likely to be a marker rather than a mediator of defective lobular-alveolar development resulting from Brca1 loss.

Gene expression profile of the fibrotic response in the peritoneal cavity.

The cellular response to materials implanted in the peritoneal cavity has been utilised to produce tissue for grafting to hollow smooth muscle organs (blood vessels, bladder, uterus and vas deferens). To gain insight into the regulatory mechanisms involved in encapsulation of a foreign object, and subsequent differentiation of encapsulating cells, the present study used microarray technology and real-time RT-PCR to identify the temporal changes in gene expression associated with tissue development. Immunohistochemical analysis showed that 3-7 days post-implantation of foreign objects (cubes of boiled egg white) into rats, they were encapsulated by tissue comprised primarily of haemopoietic (CD45(+)) cells, mainly macrophages (CD68(+), CCR1(+)). By day 14, tissue capsule cells no longer expressed CD68, but were positive for myofibroblast markers alpha-smooth muscle (SM) actin and SM22. In accordance with these results, gene expression data showed that early capsule (days 3-7) development was dominated by the expression of monocyte/macrophage-specific genes (CD14, CSF-1, CSF-1R, MCP-1) and pro-inflammatory mediators such as transforming growth factor (TGF-beta). As tissue capsule development progressed (days 14-21), myofibroblast-associated and pro-fibrotic genes (associated with TGF-beta and Wnt/beta-catenin signalling pathways, including Wnt 4, TGFbetaRII, connective tissue growth factor (CTGF), SMADs-1, -2, -4 and collagen-1 subunits) were significantly up-regulated. The up-regulation of genes associated with Cardiovascular and Skeletal and Muscular System Development at later time-points suggests the capacity of cells within the tissue capsule for further differentiation to smooth muscle, and possibly other cell types. The identification of key regulatory pathways and molecules associated with the fibrotic response to implanted materials has important applications not only for optimising tissue engineering strategies, but also to control deleterious fibrotic responses.

Reduced mucin sulfonation and impaired intestinal barrier function in the hyposulfataemic NaS1 null mouse.

OBJECTIVE: Sulfate (SO(4)(2-)) is an abundant component of intestinal mucins and its content is decreased in certain gastrointestinal diseases, including inflammatory bowel disease. In this study, the hyposulfataemic NaS1 sulfate transporter null (Nas1(-/-)) mice were used to investigate the physiological consequences of disturbed sulfate homeostasis on (1) intestinal sulfomucin content and mRNA expression; (2) intestinal permeability and proliferation; (3) dextran sulfate sodium (DSS)-induced colitis; and (4) intestinal barrier function against the bacterial pathogen, Campylobacter jejuni. METHODS: Intestinal sulfomucins and sialomucins were detected by high iron diamine staining, permeability was assessed by fluorescein isothiocyanate (FITC)-dextran uptake, and proliferation was assessed by 5-bromodeoxyuridine (BrdU) incorporation. Nas1(-/-) and wild-type (Nas1(+/+)) mice received DSS in drinking water, and intestinal damage was assessed by histological, clinical and haematological measurements. Mice were orally inoculated with C jejuni, and intestinal and systemic infection was assessed. Ileal mRNA expression profiles of Nas1(-/-) and Nas1(+/+) mice were determined by cDNA microarrays and validated by quantitative real-time PCR. RESULTS: Nas1(-/-) mice exhibited reduced intestinal sulfomucin content, enhanced intestinal permeability and DSS-induced colitis, and developed systemic infections when challenged orally with C jejuni. The transcriptional profile of 41 genes was altered in Nas1(-/-) mice, with the most upregulated gene being pancreatic lipase-related protein 2 and the most downregulated gene being carbonic anhydrase 1 (Car1). CONCLUSION: Sulfate homeostasis is essential for maintaining a normal intestinal metabolic state, and hyposulfataemia leads to reduced intestinal sulfomucin content, enhanced susceptibility to toxin-induced colitis and impaired intestinal barrier to bacterial infection.

A global role for zebrafish klf4 in embryonic erythropoiesis.

There are two waves of erythropoiesis, known as primitive and definitive waves in mammals and lower vertebrates including zebrafish. The founding member of the Kruppel-like factor (KLF) family of CACCC-box binding proteins, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. Morpholino knockdown experiments have shown a role for zebrafish klf4 in primitive erythropoiesis and hatching gland formation. In order to generate a global understanding of how klf4 might influence gene expression and differentiation, we have performed expression profiling of klf4 morphants, and then performed validation of many putative target genes by qRT-PCR and whole mount in situ hybridization. We found a critical role for klf4 in embryonic globin, heme synthesis and hatching gland gene expression. In contrast, there was an increase in expression of definitive hematopoietic specific genes such as larval globin genes, runx1 and c-myb from 24 hpf, suggesting a selective role for klf4 in primitive rather than definitive erythropoiesis. In addition, we show klf4 preferentially binds CACCC box elements in the primitive zebrafish beta-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions.

p53 is required for etoposide-induced apoptosis of human embryonic stem cells.

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.

Temporal and spatial transcriptional programs in murine kidney development.

We have performed a systematic temporal and spatial expression profiling of the developing mouse kidney using Compugen long-oligonucleotide microarrays. The activity of 18,000 genes was monitored at 24-h intervals from 10.5-day-postcoitum (dpc) metanephric mesenchyme (MM) through to neonatal kidney, and a cohort of 3,600 dynamically expressed genes was identified. Early metanephric development was further surveyed by directly comparing RNA from 10.5 vs. 11.5 vs. 13.5dpc kidneys. These data showed high concordance with the previously published dynamic profile of rat kidney development (Stuart RO, Bush KT, and Nigam SK. Proc Natl Acad Sci USA 98: 5649-5654, 2001) and our own temporal data. Cluster analyses were used to identify gene ontological terms, functional annotations, and pathways associated with temporal expression profiles. Genetic network analysis was also used to identify biological networks that have maximal transcriptional activity during early metanephric development, highlighting the involvement of proliferation and differentiation. Differential gene expression was validated using whole mount and section in situ hybridization of staged embryonic kidneys. Two spatial profiling experiments were also undertaken. MM (10.5dpc) was compared with adjacent intermediate mesenchyme to further define metanephric commitment. To define the genes involved in branching and in the induction of nephrogenesis, expression profiling was performed on ureteric bud (GFP+) FACS sorted from HoxB7-GFP transgenic mice at 15.5dpc vs. the GFP- mesenchymal derivatives. Comparisons between temporal and spatial data enhanced the ability to predict function for genes and networks. This study provides the most comprehensive temporal and spatial survey of kidney development to date, and the compilation of these transcriptional surveys provides important insights into metanephric development that can now be functionally tested.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Expression of a novel mammalian epidermal growth factor-related gene during mouse neural development.

We have recently reported the preliminary characterisation of a novel EGF-related gene, Scube1 (signal peptide-CUB domain-EGF-related, gene 1), that is expressed prominently in the developing gonad, nervous system, somites, surface ectoderm and limb buds of the mouse. Here we describe the expression pattern of a closely related gene, Scube2 (also known as Cegp1), which maps to the distal region of mouse chromosome 7. Scube2 transcription is restricted to the embryonic neurectoderm but is also detectable in the adult heart, lung and testis.

Cloning, mapping, and expression analysis of a gene encoding a novel mammalian EGF-related protein (SCUBE1).

The epidermal growth factor (EGF) superfamily comprises a diverse group of proteins that function as secreted signaling molecules, growth factors, and components of the extracellular matrix, many with a role in vertebrate development. We have isolated a novel mammalian gene encoding an EGF-related protein with a CUB (C1s-like) domain that defines a new mammalian gene family. The Scube1 (signal peptide-CUB domain-EGF-related 1) gene was isolated from a developing mouse urogenital ridge cDNA library and is expressed prominently in the developing gonad, nervous system, somites, surface ectoderm, and limb buds. We have mapped Scube1 to mouse chromosome 15 and show that it is orthologous to a human gene in the syntenic region of chromosome 22q13. We discuss the possible functions of this novel gene and its role in heritable disease in light of these data.

Identification of somatic mutations of the MEN1 gene in sporadic endocrine tumours.

Endocrine tumours of the pancreas, anterior pituitary or parathyroids arise either sporadically in the general population, or as a part of inherited syndromes such as multiple endocrine neoplasia type 1 (MEN 1). The mechanisms responsible for the development of sporadic endocrine lesions are not well understood, although loss of heterozygosity (LOH) of the MEN1 locus on chromosome 11q13 and somatic mutation of the MEN1 gene have been frequently associated with the development of MEN 1-type sporadic endocrine lesions. To further investigate the role of the MEN1 gene in sporadic endocrine tumorigenesis, we analysed DNA from 14 primary parathyroid lesions, 8 anterior pituitary tumours and 3 pancreatic tumours for the presence of somatic MEN1 gene mutations and LOH of seven microsatellite markers flanking the MEN1 locus. In addition, we similarly analysed 8 secondary parathyroid lesions which arose in patients with chronic renal failure. None of the patients studied had a family history of MEN 1. Three primary parathyroid lesions and one pancreatic tumour (glucagonoma) were found to have lost one allele at the MEN1 locus. Somatic mutations were identified by SSCP and sequence analysis in one of these parathyroid lesions (P320L) and in the glucagonoma (E179V). These results support previous findings that inactivation of the MEN1 tumour suppressor gene contributes to the development of sporadic MEN 1-type endocrine lesions but is not associated with the development of parathyroid hyperplasia seen in some renal failure patients.

Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor.

Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Ras. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.

Sexually dimorphic expression of protease nexin-1 and vanin-1 in the developing mouse gonad prior to overt differentiation suggests a role in mammalian sexual development.

The mammalian sex-determining pathway is controlled by the presence or absence of SRY expression in the embryonic gonad. Expression of SRY in males is believed to initiate a pathway of gene expression resulting in testis development. In the absence of SRY, ovary development ensues. Several genes have now been placed in this pathway but our understanding of it is far from complete and several functional classes of protein appear to be absent. Sex-determining genes frequently exhibit sexually dimorphic patterns of expression in the developing gonad both before and after overt differentiation of the testis or ovary. In order to identify additional sex-determining or gonadal differentiation genes we have examined gene expression in the developing gonads of the mouse using cDNA microarrays constructed from a normalized urogenital ridge library. We screened for genes exhibiting sexually dimorphic patterns of expression in the gonad at 12.5 and 13.5 days post-coitum, after overt gonad differentiation, by comparing complex cDNA probes derived from male and female gonadal tissue at these stages on micro-arrays. Using in situ hybridization analysis we show here that two genes identified by this screen, protease nexin-1 (Pn-1) and vanin-1 (Vnn1), exhibit male-specific expression prior to overt gonadal differentiation and are detected in the somatic portion of the developing gonad, suggesting a possible direct link to the testis-determining pathway for both genes.

Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia.

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis.

A 500-kb sequence-ready cosmid contig and transcript map of the MEN1 region on 11q13.

We have generated a transcript map of an approximately 1.2-Mb region from human chromosome band 11q13 between the loci VEGFB and CAPN1, which flank the multiple endocrine neoplasia type 1 (MEN 1) locus. In total, we isolated 144 cosmids from this region and generated a sequence-ready cosmid contig of the approximately 500-kb region between the neurexin locus and D11S2196E. We identified 54 genes/ESTs by sample sequencing and have constructed a transcript map of this region. Genes were found to be clustered in three regions, and one of these genes was identical to the recently identified MEN1 locus. Relative to the latter, we have mapped the positions of 13 known genes, 18 genes which show homology to genes from humans or other organisms, and 22 genes/ESTs that appear novel. In addition, we have ascertained the directions of transcription of some of these genes and have determined intergenic distances between many loci. Full characterization of some of these genes, as well as the novel ESTs, will be useful in identifying candidate genes for other diseases known to map to this chromosomal region.

Characterization of the mouse Men1 gene and its expression during development.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.

The mouse Y chromosome interval necessary for spermatogonial proliferation is gene dense with syntenic homology to the human AZFa region.

The Delta Sxrb deletion interval of the mouse Y chromosome contains Spy, a spermatogenesis factor gene(s) whose expression is essential for the postnatal development of the mitotic germ cells, spermatogonia. The boundaries of Delta Sxrb are defined by the duplicated genes Zfy1 and Zfy2 and four further genes have previously been mapped within the interval: Ube1y and Smcy, linked with Zfy1 on a contig of 250 kb, and Dffry and Uty, which were unanchored. The interval was estimated to be >450 kb. In order to identify any further gene(s) that may underlie Spy, systematic exon trapping was performed on an extended contig, anchored on Zfy1, which covers 750 kb of the Delta Sxrb interval. Exons from two novel genes were isolated and placed together with Dffry and Uty on the contig in the order Dffry-Dby-Uty-Tspy-Eif2gammay-Smcy- Ube1y-Zfy1. All the genes, with the double exception of Tspy, are X-Y homologous and produce putatively functional, spliced transcripts. The tight linkage and order of Dffry, Dby and Uty was shown to be conserved in deletion intervals 5C/5D of the human Y chromosome by the construction of a contig of human PAC and YAC clones; this represents the first example of syntenic homology between Y chromosomes from two distinct mammalian orders. Interval 5C/5D contains the distal boundary of the AZFa interval, which, like Delta Sxrb, is believed to be necessary for spermatogonial development in the prepubertal testis. Our results therefore show that AZFa and Spy may be encoded by homologous genes.