PTPRF is disrupted in a patient with syndromic amastia

<p>Abstract</p> <p>Background</p> <p>The presence of mammary glands distinguishes mammals from other organisms. Despite significant advances in defining the signaling pathways responsible for mammary gland development in mice, our understanding of human mammary gland development remains rudimentary. Here, we identified a woman with bilateral amastia, ectodermal dysplasia and unilateral renal agenesis. She was found to have a chromosomal balanced translocation, 46,XX,t(1;20)(p34.1;q13.13). In addition to characterization of her clinical and cytogenetic features, we successfully identified the interrupted gene and studied its consequences.</p> <p>Methods</p> <p>Characterization of the breakpoints was performed by molecular cytogenetic techniques. The interrupted gene was further analyzed using quantitative real-time PCR and western blotting. Mutation analysis and high-density SNP array were carried out in order to find a pathogenic mutation. Allele segregations were obtained by haplotype analysis.</p> <p>Results</p> <p>We enabled to identify its breakpoint on chromosome 1 interrupting the <it>protein tyrosine receptor type F gene </it>(<it>PTPRF</it>). While the patient's mother and sisters also harbored the translocated chromosome, their non-translocated chromosomes 1 were different from that of the patient. Although a definite pathogenic mutation on the paternal allele could not be identified, <it>PTPRF</it>'s RNA and protein of the patient were significantly less than those of her unaffected family members.</p> <p>Conclusions</p> <p>Although <it>ptprf </it>has been shown to involve in murine mammary gland development, no evidence has incorporated <it>PTPRF </it>in human organ development. We, for the first time, demonstrated the possible association of <it>PTPRF </it>with syndromic amastia, making it a prime candidate to investigate for its spatial and temporal roles in human breast development.</p

Helicobacter pylori Infection Promotes Methylation and Silencing of Trefoil Factor 2, Leading to Gastric Tumor Development in Mice and Humans

Background & Aims Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. Methods We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130[superscript F/F] mouse model of gastric cancer. Results In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130[superscript F/F]/Tff2[superscript −/−] mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130[superscript F/F]controls. The fundus of gp130[superscript F/F]/Tff2[superscript −/−] mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. Conclusions TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.National Health and Medical Research Council (Australia

Role of TFF1 in tumor progression

2015 - 2016Trefoil Factor 1 (TFF1) is a small secreted protein, belonging to the trefoil factor family, characterized by a conserved “trefoil domain” containing six cysteine residues that form a three loop disulphide structure. It is expressed in the gastrointestinal tract, where plays an essential role in mucosal protection through mucous-barrier formation, and also in mucosal repair through promotion of restitution after injury. In recent years clinical and experimental studies have shown an active function of the trefoil peptides in the genesis of neoplastic processes. TFF1 is mainly associated with breast cancer and gastric cancer (GC), but have been described changes in expression levels also in pancreatic cancer, lung, prostate and colorectal. TFF1 had been described as a tumor suppressor gene in gastric cancer, but it is markedly elevated in gastric mucosa with atypical hyperplasia, diffuse-type gastric cancer and with lymph node metastasis. However, the distinct signalling pathways have not been fully elucidated, nor have definitive functional receptors for trefoil proteins been identified. In this PhD project, experiments were performed to understand the role of TFF1 in human GC development with particular attention to invasion and epithelial-mesenchymal transition (EMT) processes. Previously it has been demonstrated that TFF1 selectively binds copper ions, which influence homodimer formation and its biological activity. Here, by using TFF1 recombinant protein on AGS cell line and a TFF1 over-expressing clone (AGS-AC1), we demonstrated that TFF1 stimulated invasion of GC cell lines. The pro-invasive activity of TFF1 was strictly regulated by copper and was associated with a greater MMP-2 activity. We also reported that TFF1 was implicated in the occurrence of EMT, not only in the GC models but also in a prostate cancer cell line, in a same manner with a reduction of epithelial markers such as E-cadherin and cytokeratins 8 and 18 and an increase of mesenchymal ones such as vimentin. Additionally, in hypoxic condition, a significant increase of TFF1 expression was associated with hypoxia-related mesenchymal/metastatic process. Furthermore, we demonstrated a TFF1 auto-induction mechanism with the identification of a specific responsive element located between -583 bp and -212 bp region of its promoter. This region is responsive to the presence of TFF1 and able to positively regulate its expression also during hypoxia and synergistically with HIF1-α induction. Additionally, we observed that TFF1 can regulate the methylation status of its promoter. We hypothesized that it can auto-activate its own expression regulating the density of methylated CpGs. Finally, we investigated the relationship between TFF1 and the N-formyl peptide receptors (FPR1, FPR2 and FPR3), involved in innate immunity, inflammation and cancer, including GC. For the first time we reported a functional relationship between TFF1 and FPRs. In particular, we found that recombinant TFF1 protein in AGS cells induced FPR expression and FPRs influenced pro-invasive activity of TFF1. [edited by author]XV n.s. (XXIX

The Tff gene cluster encoding gastroprotective trefoil peptides and targeted disruption of the Tff2 gene in mouse

Kleeblattpeptide (TFF) sind kleine sezernierte Proteine, die eine wichtige Rolle bei Wund-heilungsprozessen und dem Schutz des Verdauungstraktes spielen. Das Ziel dieser Arbeit war es, Grundlagen für das Verständnis der Funktion des Tff2 Gens zu schaffen. Für die humanen Kleeblattproteine sind keine natürlich vorkommenden Mutationen, welche zu konstitutionellen Krankheiten führen könnten, bekannt. Daher sind die sogenannten 'Knockout-Mäuse' für die Grundlagenforschung von großer Bedeutung, um neue Einblicke in die Wirkungsweise der Kleeblattproteine zu erlangen. In dieser Arbeit sollte das natürlich vorhandene Tff2 Gen ausgeschaltet werden, um pathologische Situationen zu simulieren und dadurch die physiologische Funktion des Genproduktes aufzuklären. Durch homologe Rekombination wurde ein Allel des Tff2 Gens in den embryonalen Stammzellen der Maus ersetzt. Dazu wurde ein Zielvektor (pL2-?m2) konstruiert, in welchem das murine Kleeblattgen Tff2 deaktiviert vorliegt. Darüber hinaus wurde das mutierte Allel in die Keimbahn der heterozygoten (Tff2+/-) Tiere erfolgreich integriert. Basierend auf diesen Charakterisierungen werden nun die heterozygoten (Tff2+/-) Tiere untereinander gekreuzt, um letztendlich die Tff2 (-/-) Knockout-Maus zu züchten. Die Erzeugung dieser Maus wird die Familie der bereits bestehenden Kleeblatt-Knockout-Mäuse komplettieren. Als Grundlage für diese Arbeit wurde die genomische Struktur der murinen Kleeblattfamilie bestimmt. Drei bakterielle und eine von der Hefe abstammende Rekombinante (BACs und YAC) wurden identifiziert und für die detaillierte Charakterisierung durch PCR, Restriktionskartierung, Hybridisierung, und Fluoreszenz in situ Hybridisierung (FISH) verwendet. Daraus ergab sich, dass in der Maus die drei Tff Gene ähnlich wie im menschlichen TFF Gen-Cluster angeordnet sind. Sie erstrecken sich über eine Länge von ca. 40 kb in der transkriptionellen Anordnung Tff1-Tff2-Tff3 auf Chromosom 17.Trefoil peptides (TFFs) are a group of small secreted proteins, which play an important role in the protecting and healing processes of the stomach and intestine. The aim of these experiments was to create a basic foundation for understanding the exact function and/or the mode of action of the Tff2 gene. Since in the case of human trefoil peptides, no naturally occurring chance mutations leading to constitutional diseases had been discovered, a straight forward experimental approach for new insight into this trefoil peptide function is to engineer a strain of so-called knock-out mice, in which Tff2 is missing. By employing a replacement strategy, one allele of Tff2 was substituted in ES cells by homologous recombination in order to create the Tff2 knock-out mouse. The primary aim of this project was to construct the targeting vector (pL2-?m2), whereby the mouse trefoil gene Tff2 was disrupted, and to characterise the germline transmission of the mutant allele in heterozygous (Tff2+/-) animals. Indeed, this goal was realised within the scope of this work. In the near future, the mice will be mated inter se to generate the mutant Tff2 (-/-) knockout mice, completing the family of already existing trefoil knockout mice. To obtain fundamental information about the mouse trefoil gene cluster, the genomic structure was determined. Three bacterial and one yeast artificial chromosome recombinants (BACs and YAC) were identified and used for detailed characterisation by PCR, restriction mapping, hybridisation, and fluorescence in situ hybridisation (FISH). In a similar fashion to the TFF gene cluster in humans, the mouse Tff genes cover a region of approximately 40 kb in the transcriptional order Tff1-Tff2-Tff3 and are localised on chromosome 17

Functional and Mechanical Role of Splice Variant of Mucin4 (MUC4/X) and Trefoil Factors in Pancreatic Cancer Pathogenesis

Pancreatic Cancer (PC) is one of the vicious cancers as it ranks third in the race of leading cause of cancer-related death. Lack of early diagnostic marker, poor understanding of molecular mechanism of the disease and failure to conventional chemotherapy makes this disease dreadful. Mucin 4 (MUC4), a high molecular weight glycoprotein is one of the top differentially expressed molecules in PC while not expressed in normal pancreas. Accumulating evidence from our lab suggested its tumorigenic role in PC by increasing cell proliferation, invasion, chemotherapy resistance, tumor growth, and metastasis. Previously, our lab and other has identified 24 different splice variant of MUC4 among them MUC4/X is devoid of exon 2 and 3 and MUC4/Y is devoid of exon 2. Exon 2 encodes for the largest domain of MUC4 suggesting that MUC4/X is devoid of the largest domain of MUC4 which variable tandem repeat. Though lots of effort has been made to identify its role in PC, there is still a gap on understanding its splice variant in PC as splice variant has an invaluable role in tumor pathogenesis. Recently splice variant has emerged as one of the key players for tumorigenesis and MUC4 is one of the key players for PC pathogenesis, we aim to identify the functional and mechanical role of MUC4/X, a splice variant which is devoid of the largest domain of MUC4 yet contains all other functional domain, in PC pathogenesis. Thus, in this part of dissertation, we sought to identify the role of splice variant MUC4/X, a unique splice variant of wild-type MUC4 which contain all functional domain except largest tandem repeat. First, we identified that, MUC4/X in aberrantly expressed in poorly differentiated PC clinical sample. Then our invitro experimental evidence suggested overexpression of MUC4/X in PC cells is involved in increased cell proliferation, invasion and metastasis. Moreover, our orthotopic transplantation system also corroborated our in-vitro findings which showed increased volume of tumor and metastasis to distant organ. Using inducible tet-on system to overexpress MUC4/X in the presence of WT-MUC4 in CAPAN-1 cells, we identified that MUC4/X has increased cell proliferation and invasion suggesting their role as tumorigenic alone as well as in the presence of WT-MUC4. Our mechanical investigation indicate that overexpression of MUC4/X led to upregulation of integrin β1-FAK-ERK pathway which might be potential mechanism for MUC4/X mediated PC tumorigenesis. Lack of early effective diagnostic marker and resistance to chemotherapy are the major reasons for poor PC patient outcome. There is a pressing need to identify highly specific and sensitive biomarker as well as precise understanding of chemoresistance of PC. Trefoil factors (TFFs) are small secretory molecules mostly associated with mucin. Their primary role is to protect gastrointestinal tract partnering with mucin. Report on aberrant expression, potential as biomarker and role in tumorigenicity has conveyed for many cancers, however, their role in PC is still elusive. Recently they have emerged as a part of gene signature of classical subtype of PC, a subtype which showed gemcitabine resistance towards PC. As it is high time to identify effective biomarker and understanding the role of chemoresistance in PC, in this part of my thesis, we focused to evaluate TFFs diagnostic potential using a training and validation cohort of PC clinical sample. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 (TFFs) in combination with CA19.9 for detection of PC. In silico analysis of publicly available datasets and expression analysis from human and spontaneous PC mouse model revealed a significantly increased expression of TFFs in precursor lesions and PC cases. Additionally, we performed a comprehensive analysis in the sample set (n= 377) comprising of independent training and validation set using ELISA consisted of benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Our analysis revealed that TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to BC (P Additionally, we also aim to identify the molecular landscape of TFFs role in gemcitabine resistance of PC which integrates analyzing publicly available cancer genome dataset, dissecting transcriptomic and signaling pathways and identification of biochemical interaction. From TCGA database analysis revealed a significant positive correlation between TFF1 and GR predictor of PC (P=0.0001). Our in vitro studies showed that SW1990-TFF1-KD cells induced apoptosis, reduced colony formation capacity and modulated many apoptotic regulators such increase of cleaved caspases and decrease of CIAP in the presence of gemcitabine. Furthermore, TFF1 was observed to be colocalized with MUC5AC, in human and mouse PC tissues suggesting their partnering are critical for PC pathogenesis. Interestingly, our chromatin immunoprecipitation indicates that 16 fold enrichment of GATA-6, an overexpressed transcription factor in classical subtype of PC, was observed on two distinct TFF1 promoter sites and GATA-6-siRNA repressed expression of TFF1. Moreover, protein-protein docking studies revealed the interaction of TFF1 with CXCR4 at Phe-172, Ser-122 and Glu-1 and TFF1 recombinant protein treatment in SW1990 cells increased CXCR4 mediated downstream signaling critical for GR. In this part, our overall data demonstrate that TFF1 may play a crucial role in gemcitabine resistance which is regulated by GATA6 and by interacting with MUC5AC